CA3118327A1 - Nucleic acids for inhibiting expression of aldh2 in a cell - Google Patents

Nucleic acids for inhibiting expression of aldh2 in a cell Download PDF

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CA3118327A1
CA3118327A1 CA3118327A CA3118327A CA3118327A1 CA 3118327 A1 CA3118327 A1 CA 3118327A1 CA 3118327 A CA3118327 A CA 3118327A CA 3118327 A CA3118327 A CA 3118327A CA 3118327 A1 CA3118327 A1 CA 3118327A1
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strand
nucleic acid
nucleotides
modified
rna
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Inventor
Sibylle DAMES
Ute SCHAEPER
Steffen Schubert
Stephan TENBAUM
Lucas Bethge
Christian Frauendorf
Judith HAUPTMANN
Adrien Weingartner
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Silence Therapeutics GmbH
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Silence Therapeutics GmbH
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Priority claimed from EP17201447.4A external-priority patent/EP3483269A1/en
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    • 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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    • 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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    • C12N2310/30—Chemical structure
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
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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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    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol

Abstract

The present invention relates to products and compositions and their uses. In particular the invention relates to nucleic acid products that interfere with the ALDH2 gene expression or inhibits its expression and therapeutic uses such as for the treatment or prevention of alcohol use disorder.

Description

2 PCT/EP2018/081105 Nucleic acids for inhibiting expression of ALDH2 in a cell The present invention relates to products and compositions and their uses. In particular 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.
Background 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 19;391(6669):806-11 and Elbashir et al., 2001, Nature. 2001 May 24;411(6836):494-8) by a mechanism that has been termed RNA interference (RNAi). 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 (RISC), a sequence-specific, multi-component nuclease that degrades messenger RNAs homologous to the silencing trigger loaded into the RISC
complex. Interfering RNA (termed herein iRNA) 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. 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.
Compulsive, excessive alcohol consumption beyond social drinking (aka.
alcoholism, alcohol abuse, alcohol dependence) is a worldwide increasing health problem with severe personal and socioeconomic impact. Current medications that aid alcohol dishabituation are either not very effective or they have considerable undesired side effects. As a therapeutic approach, reduction of activity of a rate-limiting key enzyme of the hepatic alcohol metabolism pathway, the aldehyde dehydrogenase-2 (ALDH2), leads to accumulation of the toxic metabolite acetaldehyde. This accumulation results in unpleasant physiological effects in alcohol-consuming individuals. The association of unpleasant to severe feelings affects the patient's alcohol seeking behaviour, reduces alcohol consumption and, thereby, assists dishabituation. This therapeutic avenue has been exploited successfully for decades using small molecule ALDH-inhibiting compounds that act systemically (Kragh et al., 2008, Bull. Hist. Chem., Vol. 33, 2). The safety profile of those systemically acting inhibitors is worsened by the unwanted side effects even in individuals that do not consume alcohol or are produced by involuntary exposure to environmental aldehydes present in e.g. food, perfumes or other forms harmless to the organism in normal circumstances.
The current antidipsotropic medications that are aimed at keeping alcohol dependent patients away from drinking have low molecular and tissue specificity and show, therefore, a bad safety profile with considerable effects even in non-alcohol consuming patients.
Treatment failure of those therapies is high due to high abandonment and cheating (not taking the medicine in order to be able to resume the consumption of alcohol).
So there is a medical need to improve such therapeutic approach.
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, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 55.
In one embodiment, 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: 11, 17,3, 1, 5, 7, 9, 13, 15, 19, 21, 23, 25 27 or 55.
In one embodiment, 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
3 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:11 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
4 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.
In the nucleic acid of the invention, 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 phosphoramidate modification, 5'-phosphorothioate group modification, a 5' phosphate or
5' phosphate mimic modification and a cholesteryl derivative or a dodecanoic acid bisdecylamide group modification and/or 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 complementary to at least a portion of an 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, 11, 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 5 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 2'0Me and the second modification may be 2'F.
1.13 In the nucleic acid of the second aspect, the first strand may comprise the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:11 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.
The sequence and modifications may be as shown in the Table below; which shows preferred sequences based on an extract of Table 1 as provided herein:
Sequence siRNA ID sequence modifications ID No 3 5'-AAUGUUUUCCUGCUGACGG-3' 6254515173547182748 ALDH2-hcm-2 4 5'-CCGUCAGCAGGAAAACAUU-3' 3745364728462627251 11 5'-UCUUCUUAAACUGAGUUUC-3' 5351715262718281517 ALDH2-hcm-6 12 5'-GAAACUCAGUUUAAGAAGA-3' 4626353645152646282 17 5'-AUGUAGCCGAGGAUCUUCU-3' 6181647382846171535 ALDH2-hcm-9 18 5'-AGAAGAUCCUCGGCUACAU-3' 2826461735384716361 wherein the specific modifications are depicted by the following numbers 1=2"F-dU, 2=2`F-dA, 3=2"F-dC, 4=2"F-dG, 5=2'-0Me-rU;
6=2'-0Me-rA;
7=2'-0Me-rC;
8=2'-0Me-rG.
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 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, 11, 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 (GaINAc) moieties and derivatives thereof, and (ii) a linker, wherein the linker conjugates the GaINAc 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-X1-P-X93-A-X3- (I) wherein:
S represents a saccharide, wherein the saccharide is N-acetyl galactosamine;
X1 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);
X2 is alkylene or an alkylene ether of the formula (-CH2)n-O-CH2- where n = 1-6;
A is a branching unit;
X3 represents a bridging unit;
wherein a nucleic acid according to the present invention is conjugated to X3via a phosphate or modified phosphate (preferably a thiophosphate).

The present invention therefore additionally provides a conjugated nucleic acid having one of the following structures OH
OH

AcH A:FITCH

H:)0 S=L0-I OH
(D
s.0 /7c-0H
PcH

/ ___________________ 0 0 H
S ______________ Jr s 011_0--b-OH

OH OH

HO ,./.1...,... AcHN

NHAc Ll*sl 1 e 0 =P ¨S
&l 0 -) 1 e 0 =P¨S

0 --/¨j AcHN OH
i / I /

If Z ¨0¨ P ¨0 le S L.1 0 o-A-cr le S I

OH
HO\,_,.. 0H
OH OH

HOND AcHN

NHAc L1*1 0 =P ¨S

I e 0 =P ¨S
i OH
0 AcHN
_______________________________________ / 0/' OH
/

II /
Z-0 ¨P-0 -__ S
? /
0¨P¨O
to S
9 OH
HO\,...,011 OH OH

AcHN

NHAc Li) i 0 0=P¨S
i 0.1 0 i 0 0=P¨S

/I OH

/ AcHN C
0¨ OH
'N
_______________________________________ / r().-R'OH
_Ex J-0/ 0 -., II
I¨O¨P-0 LI,µ 1? f ,.
S 0¨P-0 so S, OH
H0µ,_ OH
OH OH

AcHN

NHAc 1') 0 =P ¨S
i 0 = P ¨S

/ I OH
0,, o j OH
/
AcHN
, r i c ( 1 -FON
_x_rxi¨ 0 _____________________ õ, 0 0 .--II , z-0¨P¨O 0¨p-0 i 0 10 S S

OH
HO k,___01.1 AcHN

NHAc 04¨Se \
i 04¨Se i AcHN OH
OH
0 ,,, 0 --/¨/ (12/)(OH

II
Z ¨0 ¨P ¨0 ¨rj---le s o 0¨p-0 le s OH
HO OH
OH OH
AcH 0N

NHAc i 0 0=P ¨S
\

0 =P ¨ S
I
0 AcHN OH
OH
0 0 ¨r0 OH
/

/ I

2 ¨0 ¨11-0 ¨T.-1j¨ LI,. 0 /
10 0 S 0 ¨P ¨0 S
11 DH
HL
,,.---OH
OH OH b t f AcHN

r H ; \,....-\.....\.....\0 \

0 =P -S
i 0%1 0 1....1 t S0 =
t AcHiv H
i.......\ OH

_____________________________________________ / 0 0/ co 11 li /
2 -0 -P -0 LI,,, 0 -P -0 t 0 t 0 S s wherein Z represents a nucleic acid as defined herein before.
Alternatively, a nucleic acid according to the present invention may be conjugated to a ligand of the following structure OH
0H H FlA
H
H o c 1N
NNc 01 OH
O-P=0 H

H
H

HAc Hae7.0/ANNN
H
H
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
12 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:
(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 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, 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:
(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 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
13 (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.
Also provided is a nucleic acid or conjugated nucleic acid according to any aspect of the invention 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.
After subcutaneous application, 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.
A method of making the nucleic acid or conjugated nucleic acid according to the invention is also included.
14 Detailed Description of Invention 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, 11, 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: 11, 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.
In one aspect, 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: 11, 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.
In one aspect, the first strand of the nucleic acid comprises a sequence of at least 19 nucleotides of any of the reference sequences SEQ ID NO: 11, 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.

By 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 10 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.
15 The nucleic acid may comprise ribonucleotides, modified ribonucleotides, deoxynucleotides, deoxyribonucleotides, or nucleotide analogues non-nucleotides that are able to mimic nucleotides such that they may 'pair' with the corresponding base on the target sequence or complementary strand. 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 complementary oligonucleotides that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a duplex between oligonucleotide strands that are complementary or substantially complementary. For example, an 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. Further, within the duplex region, 100% complementarity is not required;
substantial complementarity is allowable within a duplex region. Substantial 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.
16 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.
Depending on the length of a nucleic acid, a perfect match in terms of base complementarity between the first strand and the second strand is not necessarily required. However, the 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.
In the context of this invention, "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. Alternatively, 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 nucleotides from any one of the sequences listed in Table 1.
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: 11, and optionally wherein the second strand comprises a nucleotide
17 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: 6; or a first strand that comprises a nucleotide sequence of SEQ ID NO: 7, and optionally wherein the second strand comprises a nucleotide sequence of SEQ ID
NO: 8;
or a first strand that comprises a nucleotide sequence of SEQ ID NO: 9, and optionally wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 10; or a first strand that comprises a nucleotide sequence of SEQ ID NO: 13, and optionally wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 14; or a first strand that comprises a nucleotide sequence of SEQ ID NO: 15, and optionally wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 16; or a first strand that comprises a nucleotide sequence of SEQ ID NO: 19, and optionally wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 20; or a first strand that comprises a nucleotide sequence of SEQ ID NO: 21, and optionally wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 22; or a first strand that comprises a nucleotide sequence of SEQ ID NO: 23, and optionally wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 24; or a first strand that comprises a nucleotide sequence of SEQ ID NO:25, and optionally wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 26; or a first strand that comprises a nucleotide sequence of SEQ ID NO: 27, and optionally wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 28.
Use of a nucleic acid according to the present invention 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,
18 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 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:11 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.

Preferably, the nucleic acid mediates RNA interference.
In one embodiment, 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: 11, 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 1186 to 1217 of the ALDH2 reference sequence NM_000690.3 (SEQ ID NO: 55 ¨ AUGUAGCCGAGGAUCUUCUUAAACUGAGUUUC).
This locus is particularly well suited as a target for RNA interference because siRNAs with sequences SEQ ID NO: 11 and SEQ ID NO: 17, which both allow efficiently reducing the ALDH2 mRNA level, both bind to parts of this short locus.
In a further aspect the 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. In particular, 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.
5 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. Optionally, an overhang may consist of 1 or 2 nucleotides, which may or may not be modified.
Unmodified polynucleotides, particularly ribonucleotides, 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.
One or more nucleotides 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.
One or more nucleotides of a 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-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. In each case 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. All of the pyrimidines 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 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), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5'-carboxymethylaminomethy1-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethy1-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid and 2-thiocytidine.
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. 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.
Many of the modifications described below that occur within a nucleic acid will be repeated within a polynucleotide molecule, such as a modification of a base, or a phosphate moiety, or a non-linking 0 of a phosphate moiety. In some cases the modification will occur at all of the possible positions/nucleotides in the polynucleotide but in many cases it will not. 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 0 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 phosphorothioate modifications. 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 oligoribonucleotides more stable to nucleases.
Modified nucleic acids, as used herein, can include one or more of:
(i) 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);
(ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar;

(iii) replacement of the phosphate moiety with "dephospho" linkers;
(iv) modification or replacement of a naturally occurring base;
(v) replacement or modification of the ribose-phosphate backbone;
(vi) 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 fluorescently labelled moiety, to either the 3' or 5' end of RNA.
The terms replacement, modification, alteration, indicate a difference from a naturally occurring molecule.
Specific modifications are discussed in more detail below.
Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
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.
Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar);
polyethyleneglycols (PEG), 0(CH2CH20)nCH2CH2OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl is connected, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; 0-AMINE (AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino) and aminoalkoxy, 0(CH2)nAMINE, (e.g., AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino).

"Deoxy" modifications include hydrogen, halogen, amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH2CH2NH)nCH2CH2-AMINE (AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl 5 amino), ¨NHC(0)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano;
mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality. Other substitutents of certain embodiments include 2'-methoxyethyl, 2'-OCH3, 2'-0-allyl, 2'-C-allyl, and 2'-fluoro.
10 The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
Thus, a modified nucleotides may contain a sugar such as arabinose.
Modified nucleotides can also include "abasic" sugars, which lack a nucleobase at C-1'.
15 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).
The phosphate groups can individually be replaced by non-phosphorus containing connectors.
Examples of moieties which can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. In certain embodiments, replacements may include the methylenecarbonylamino and methylenemethylimino groups.
The phosphate linker and ribose sugar may be replaced by nuclease resistant nucleotides.
Examples include the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. In certain embodiments, PNA surrogates may be used.

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. For example, the 3' and 5' ends of 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). 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' 0, N, S
or C group of the sugar. Alternatively, the linker can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs). These spacers or linkers can include e.g., ¨(CH2)n¨, ¨(CH2)nN¨, ¨(CH2)n0¨, ¨(CH2)nS¨, ¨ (CH2CH20)nCH2CH20¨ (e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents. The 3' end can be an -OH group.
Other examples of terminal modifications include dyes, intercalating agents (e.g., acridines), cross-linkers (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases, EDTA, lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG
(e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles).
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 ((H0)2(0)P-0-5'); 5'-diphosphate ((H0)2(0)P¨O¨P(H0)(0)-0-5'); 5'-triphosphate ((H0)2(0)P-0¨(H0)(0)P¨O¨
P(H0)(0)-0-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-0-5'-(H0)(0)P-0¨(H0)(0)P¨O¨P(H0)(0)-0-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-0-5'-(H0)(0)P-0¨(H0)(0)P¨O¨
P(H0)(0)-0-5'); 5'-monothiophosphate (phosphorothioate; (H0)2(S)P-0-5'); 5'-monodithiophosphate (phosphorodithioate; (H0)(HS)(S)P-0-5'), 5'-phosphorothiolate ((H0)2(0)P¨S-5'); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g., 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((H0)2(0)P¨NH-5', (H0)(NH2)(0)P-0-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(0)-0-5'-, (OH)2(0)P-5'-CH2-), 51vinylphosphonate, 5'-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(0)-0-5'-).
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.
Optionally, each or either end of the first strand may comprise one or two or three phosphorothioate modified nucleotides. Optionally, 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.
E.g., 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.
Alternatively, 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 ally!
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-substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethy1-2-thiouracil, 5-methylaminomethy1-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N<4>-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or 0-alkylated bases.
As used herein, the terms "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. In some embodiments the non-base pairing nucleotide analogue is a ribonucleotide.
In other embodiments it is a deoxyribonucleotide.
As used herein, the term, "terminal functional group" includes without limitation a halogen, alcohol, amine, carboxylic, ester, amide, aldehyde, ketone, ether groups.
Certain 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' 0 alkyl modifications;
inverted abasic ribose and abasic deoxyribose moieties and modifications thereof, C6-imino-Pi;
a mirror nucleotide including L-DNA and L-RNA; 5'0Me nucleotide; and nucleotide analogues including 4',5'-methylene nucleotide; 1-(6-D-erythrofuranosyl)nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecyl phosphate;
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).
As used herein, 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). Such 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.
The term "abasic" as used herein, refers to moieties lacking a base or having other chemical groups in place of a base at the 1' 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, 11 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 5 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'.
10 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 15 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 20 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 25 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
30 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.
In the nucleic acid of the invention, 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 nucleotides at 5' end. 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, 11, 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, 11, 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. The nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23 may be modified by a second modification on the first strand.
The nucleotides numbered 1, 3, 5, 7, 9, 11, 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.
Clearly, if the 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. 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. In other words, 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. Another option is to have dissimilar modifications in each of the strands.
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 phosphoramidate modification, 5'-phosphorothioate group modification, a 5' phosphate or 5' phosphate mimic modification and a cholesteryl derivative or a dodecanoic acid bisdecylamide group modification and/or 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. Such inverted nucleotides provide stability to the nucleic acid.
Preferably, 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.
Throughout the description of the invention, "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. For example, 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'-0Me-rU, 2'-0Me-rA; 2'-0Me-rC; 2'-0Me-rG. A
2'F modification is a different modification to a 2'0Me modification.
Some representative modified nucleic acid sequences of the present invention are shown in the examples. These examples are meant to be representative and not limiting.
Preferably, 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 (2'0Me) 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, 11, 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 5 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.
10 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 15 modification on the even numbered nucleotides. The first modification may be 2'0Me and the second modification may be 2' F. The first strand may comprise the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:11 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. There is a need to 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.
One example is the asialoglycoprotein receptor complex (ASGP-R) composed by varying ratios of multimers of membrane ASGR1 and ASGR2 receptors, which is highly abundant on hepatocytes and has high affinity to the here described GaINAc moiety. One of the first disclosures of the use of triantennary cluster glycosides as conjugated ligands was in US
patent number US 5,885,968. Conjugates having three GaINAc ligands and comprising phosphate groups are known and are described in Dubber et al. (Bioconjug.
Chem. 2003 Jan-Feb;14(1):239-46.). The ASGP-R complex shows a 50-fold higher affinity for N-Acetyl-D-Galactosylamine (GaINAc) than D-Gal.
The asialoglycoprotein receptor complex (ASGP-R), which recognizes specifically terminal 8-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 (Ishibashi,S.; et. al., J
Biol. Chem. 1994 Nov 11;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 8-galactosyl containing glycoproteins to the cell's endosomes. Thus, 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).
More generally the ligand can comprise a saccharide that is selected to have an affinity for at least one type of receptor on a target cell. In particular, 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 (GaINAc).
A ligand for use in the present invention may therefore comprise (i) one or more N-acetyl galactosamine (GaINAc) moieties and derivatives thereof, and (ii) a linker, wherein the linker conjugates the GaINAc 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.
"GaINAc" refers to 2-(Acetylamino)-2-deoxy-D- galactopyranose, commonly referred to in the literature as N-acetyl galactosamine. Reference to "GaINAc" or "N-acetyl galactosamine" includes both the 13- form: 2-(Acetylamino)-2-deoxy-8 -D-galactopyranose and the a-form: 2-(Acetylamino)-2-deoxy-a-D- galactopyranose. Both the 13-form: 2-(Acetylarnino)-2-deoxy-8-D-galactopyranose and a-form: 2-(Acetylamino)-2-deoxy-a-D-galactopyranose may be used interchangeably. Preferably, the compounds of the invention comprise the 13-form, 2-(Acetylarnino)-2-deoxy-13-D-galactopyranose.
The ligand may therefore comprise GaINAc.
The ligand may comprise a compound of formula I:
[S-X1-P-X93-A-X3- (I) wherein:
S represents a saccharide, wherein the saccharide is N-acetyl galactosamine;
X1 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);
X2 is alkylene or an alkylene ether of the formula (-CH2)n-O-CH2- where n = 1-6;
A is a branching unit;
X3 represents a bridging unit;
wherein a nucleic acid according to the present invention is conjugated to X3 via a phosphate or modified phosphate (preferably a thiophosphate).
In formula I, 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:
I I
Al.._ -4,. At 4in All A1,..,....¨Al/
Ai 1 / and I
¨....,õ
wherein each A1 independently represents 0, S, C=0 or NH; and each n independently represents an integer from 1 to 20.
The branching unit may have a structure selected from:

Ad 41 1:in FAJ! )nf , (iA=A
n ¨ A and )0 wherein each A1 independently represents 0, S, C=0 or NH; and each n independently represents an integer from 1 to 20.
The branching unit may have a structure selected from:
rris siss )n 111 1ln 1%.
111.1: n n and n n wherein A1 is 0, S, C=0 or NH; and each n independently represents an integer from 1 to 20.
The branching unit may have the structure:
0, -7, The branching unit may have the structure:

An".
=
The branching unit may have the structure:

/
\
_______________ /1\
\ss s' Optionally, the branching unit consists of only a carbon atom.
The "X3" portion is a bridging unit. The bridging unit is linear and is covalently bound to the branching unit and the nucleic acid.
X3 may be selected from -C1-C20 alkylene-, -C2-C20 alkenylene-, an alkylene ether of formula -(C1-C20 alkylene)-0¨(Ci-C20 alkylene)-, -C(0)-C1-C20 alkylene-, -Co-Ca alkylene(Cy)Co-Ca alkylene- wherein Cy represents a substituted or unsubstituted 5 or 6 membered cycloalkylene, arylene, heterocyclylene or heteroarylene ring, -Ci-Ca alkylene-NHC(0)-Ci-C4 alkylene-, -Ci-Ca alkylene-C(0)NH-C1-C4 alkylene-, -Ci-Ca alkylene-SC(0)-Ci-alkylene-, -Ci-Ca alkylene-C(0)S-Ci-C4 alkylene-, -Ci-Ca alkylene-OC(0)-Ci-C4 alkylene-, -Ci-Ca alkylene-C(0)0-Ci-C4 alkylene-, and -C1-C6 alkylene-S-S-Ci-Co alkylene-.
X3 may be an alkylene ether of formula -(C1-C20 alkylene)-0¨(Ci-C20 alkylene)-. X3 may be an alkylene ether of formula -(C1-C20 alkylene)-0¨(C4-C20 alkylene)-, wherein said (C4-C20 alkylene) is linked to Z. X3 may be selected from the group consisting of -CH2-0-C3H6-, -CH2-0-C4F-18-, -CH2-0-C6F-112- and -CH2-0-C8H16-, especially -CH2-0-C4F-18-, -and -CH2-0-C81-116-, wherein in each case the -CH2- group is linked to A.
The ligand may comprise a compound of formula (II):
[S-X1-P-X2]3-A-X3- (I I ) wherein:
S represents a saccharide;
X1 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);
X2 is Ci-Co alkylene;
A is a branching unit selected from:

K
Al n ) n Al AlyA¨Al n n Al Al Al A2-1 Al = 0, NH Al = 0, NH A2 = NH, CH2, 0 n = 1 to 4 n = 1 to 4 X3 is a bridging unit;
wherein a nucleic acid according to the present invention is conjugated to X3via a phosphate or modified phosphate (preferably a thiophosphate) Branching unit A may have the structure:
AoxoN

Branching unit A may have the structure:
-40¨v-0N
0 __ /IIN A
X , wherein X3 is attached to the nitrogen atom.
X3 may be C1-C20 alkylene. Preferably, X3 is selected from the group consisting of -C3H6-, -C4H8-,-C6F-112- and -C81-116-, especially -C4H8-, -C6F-112- and -C81-116-=
The ligand may comprise a compound of formula (Ill):
[S-X1-P-X93-A-X3- (Ill) wherein:
S represents a saccharide;
X1 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);
X2 is an alkylene ether of formula -C3H6-0-CH2-;
A is a branching unit;
X3 is an alkylene ether of formula selected from the group consisting of -CH2-0-CH2-, -CH2-0-C2H4-, -CH2-0-C3H6-, -CH2-0-C4H8-, -CH2-0-C6H10-, -CH2-0-C6F-112-, -CH2-C7I-114-, and -CH2-0-C81-116-, wherein in each case the -CH2- group is linked to A, and wherein X3 is conjugated to a nucleic acid according to the present invention by a phosphate or modified phosphate (preferably a thiophosphate).
The branching unit may comprise carbon. Preferably, the branching unit is carbon.

X3 may be selected from the group consisting of -CH2-0-C4H8-, -CH2-0-05H10-, -C6H12-, -CH2-0-C7H14-, and -CH2-0-C8H16-. Preferably, X3 is selected from the group consisting of -CH2-0-C4H8-, -CH2-0-C6H12- and -CH2-0-C8H16.
For any of the above aspects, when P represents a modified phosphate group, P
can be represented by:

Y

E0_P- 01 Y-wherein Y1 and Y2 each independently represent =0, =S, -0-, -OH, -SH, -BH3, -OCH2CO2, -OCH2CO2Rx, -OCH2C(S)0Rx, and ¨0Rx, wherein Rx represents Ci-C6 alkyl and wherein ¨I indicates attachment to the remainder of the compound.
By modified phosphate it is meant a phosphate group wherein one or more of the non-linking oxygens is replaced. Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. 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 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.
For example, Y1 may represent -OH and Y2 may represent =0 or =S; or Y1 may represent -0- and Y2 may represent =0 or =S;
Y1 may represent =0 and Y2 may represent ¨CH3, -SH, -0Rx, or ¨BH3 Y1 may represent =S and Y2 may represent ¨CH3, ORx or ¨SH.
It will be understood by the skilled person that in certain instances there will be delocalisation between Y1 and Y2.

Preferably, the modified phosphate group is a thiophosphate group.
Thiophosphate groups include bithiophosphate (i.e. where Y1 represents =S and Y2 represents ¨S-) and monothiophosphate (i.e. where Y1 represents -0- and Y2 represents =S, or where represents =0 and Y2 represents ¨S-). Preferably, 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.
P may also be an ethylphosphate (i.e. where Y1 represents =0 and Y2 represents OCH2CH3).
The saccharide may be selected to have an affinity for at least one type of receptor on a target cell. In particular, the receptor is on the surface of a mammalian liver cell, for example, the hepatic asialoglycoprotein receptor complex (ASGP-R).
For any of the above aspects, the saccharide may be selected from N-acetyl with one or more of galactosamine, mannose, galactose, glucose, glucosamine and fructose.
Typically a ligand to be used in the present invention may include N-acetyl galactosamine (GaINAc). Preferably the compounds of the invention may have 3 ligands, which will each preferably include N-acetyl galactosamine.
"GaINAc" refers to 2-(Acetylamino)-2-deoxy-D- galactopyranose, commonly referred to in the literature as N-acetyl galactosamine. Reference to "GaINAc" or "N-acetyl galactosamine" includes both the 13- form: 2-(Acetylamino)-2-deoxyl3 -D-galactopyranose and the a-form: 2-(Acetylamino)-2-deoxy-a-D- galactopyranose. In certain embodiments, both the 13-form: 2-(Acetylarnino)-2-deoxy13-D-galactopyranose and a-form: 2-(Acetylamino)-2-deoxy-a-D-galactopyranose may be used interchangeably.
Preferably, the compounds of the invention comprise the 13-form, 2-(Acetylarnino)-2-deoxy13-D-galactopyranose.
.-:
FF'44.41%...e," ''=-,.-FtOH

1-1C= . r~-, 1 1 2-(Acetylamino)-2-deoxy-D-galactopyranose OH
HO
',j [ i Ac 2-(Acetylamino)-2-deoxy-P-D-galactopyranose OH
HO
.....,.....\____.-0 HO \\6 \\,....
I !A:
.......\---__ ......'s/
2-(Acetylamino)-2-deoxy-a-D-galactopyranose For any of the above compounds of formula (III), X1 may be (-CH2-CH2-0)(-CH2)2-. X' may be (-CH2-CH2-0)2(-CH2)2-. X1 may be (-CH2-CH2-0)3(-CH2)2-. Preferably, X1 is (-0)2(-CH2)2-. Alternatively, X' represents C3-C6 alkylene. X1 may be propylene.
X1 may be butylene. X' may be pentylene. X1 may be hexylene. Preferably the alkyl is a linear alkylene. In particular, X1 may be butylene.
For compounds of formula (III), X2 represents an alkylene ether of formula -i.e. C3 alkoxy methylene, or ¨CH2CH2CH2OCH2-.

The invention provides a conjugated nucleic acid having one of the following structures:
OH
OH
OH H7:) AcH OH
N.01-1 AcH

.0 ( it AcHN7-c-00:

(:) 0¨/¨/
/

(!)- s OH
HOµ,_4Dii OH OH

HO,:L._.... AcHN

NHAc LI) 1 e 0 =P-S

0 0_f AcHNF
OH
/
criOH
/
0 ..,,, II
le S
N, 9 /

OH
H0µ,. OH
OH OH

HON.14.1....õ AcHN

NHAc 1 e 0 =P -S
i --,, I 0 O=P-S
Nsi i 0 ___/¨1 0 AcHN OH
_______________________________________ /
r(i)OH
/
O _j__ j-0 =,,o /0 Z -0 -P-0 L, .., II

I
S

OH
HO\,...,011 OH OH

AcHN

NHAc Li) i 0 0=P¨S
i 0.1 0 i 0 0=P¨S

/I OH

/ AcHN C
0¨ OH
'N
_______________________________________ / r().-R'OH
_Ex J-0/ 0 -., II
I¨O¨P-0 LI,µ 1? f ,.
S 0¨P-0 so S, OH
H0µ,_ OH
OH OH

AcHN

NHAc 1') 0 =P ¨S
i 0 = P ¨S

/ I OH
0,, o j OH
/
AcHN
, r i c ( 1 -FON
_x_rxi¨ 0 _____________________ õ, 0 0 .--II , z-0¨P¨O 0¨p-0 i 0 10 S S

OH
HO k,,,_01.1 OH OH

AcHN

NHAc 0 = -Se \
i o4-s i AcHN OH
OH

1 0 ¨OH(2) II
Z-0 -P -0 ¨rj---le s o le s OH
HO OH

AcHN

\
NHAc 0 =I) -S
i 1/4-) 0 -4 -Se I

AcHN OH
/..-6 )0H

I /
/

0 T 0 /o I0 it S 0 -1, -0 t 0 S

DH
H` <,..i.---OH
OH OH b t f AcHti \

0.1,-s 0,1 0 oP-1...1 I S0 =
I

AcH i iv H
.......\ OH

_____________________________________________ / 0 j. j..../...... Jr- 0/ c) II ?I /
2 ¨0 ¨P ¨0 CL,,, 0 ¨P ¨0 t 0 t 0 S s wherein 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.
Preferably, 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. Preferably therefore, 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.
In one embodiment, 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 conjugated to any ligand or ligands 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 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.
In an embodiment of the present invention, the second RNA strand (i.e. the sense strand) is conjugated at the 5' end to a targeting ligand, the first RNA strand (i.e.
the antisense strand) is conjugated at the 3' end to the targeting ligand and the 3' 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 59A is formed.
In an embodiment of the present invention, 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.

In an embodiment of the present invention, both the second RNA strand (i.e.
the sense strand) and 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 5 formed.
In an embodiment of the present invention, 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 10 3' ends to the targeting ligand, such that a conjugate with the schematic structure as shown in Figure 59D is formed.
In any one of the above embodiments, ."-",-,-r indicates the linker which conjugates the ligand to the ends of the nucleic acid portion; the ligand may be a GaINAc moiety such as 15 GaINAc; and the schematic structure as shown in Figure 59E represents the nucleic acid portion.
These schematic diagrams are not intended to limit the number of nucleotides in the first or second strand, nor do the diagrams represent any kind of limitation on complementarity 20 of the bases or any other limitation.
The ligands may be monomeric or multimeric (e.g. dimeric, trimeric, etc.).
Suitably, the ligands are monomeric, thus containing a single targeting ligand moiety, e.g.
25 a single GaINAc moiety.
Alternatively, 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:

, HN, OH HN
wherein n is 1 or 2 and Y is S or 0.
Preferably, the ligands are monomeric.
Suitably, 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 C1_15 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 0, N, S(0)p, wherein p is 0, 1 or 2 (for example a CH2 group is replaced with 0, or with NH, or with S, or with SO2 or a ¨CH3 group at the terminus of the chain or on a branch is replaced with OH or with NH2) wherein said chain is optionally substituted by one or more oxo groups (for example 1 to 3, such as 1 group).
Suitably, the linker moiety is a serinol-derived linker moiety.
More suitably, the further linker comprises a saturated, unbranched C1_15 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.
More suitably, the further linker comprises a PEG-chain.
More suitably, the further linker comprises a saturated, unbranched C1_15 alkyl chain.
More suitably, the further linker comprises a saturated, unbranched C1_6 alkyl chain.
More suitably, the further linker comprises a saturated, unbranched C4 or C6 alkyl chain, e.g. a C4 alkyl chain.
In an embodiment, ,n-rtrtf is a linking moiety of formula (V):

7 \II \ Y
ll .
-i¨i¨Li¨O¨P-0--Li¨O¨P-0¨:¨
I I
\ OH i / OH
(V) wherein n, Y and L1 are defined below and the 0 of the phosphoro-group is attached to the terminal oligonucleoside of the RNA strands.
Thus in an embodiment, the targeting ligand portion is a linking moiety of formula (VI):
7 11 \ Y

\
GaINAc ______ L1 0 Pi 0 L1 0 11 0 OH 1 OH
/
(VI) wherein n, Y and L1 are defined below and the 0 of the phosphoro-group is attached to the terminal oligonucleoside of the RNA strands.
Suitably, sr%"-"f is a linking moiety of formula (XIV):
L L
/ /
HN HN
/ Y Y
_______________ \ II \ ___ \ II .
HO _____________ 0 P 0 0¨P-0-1,¨

I I
\ Ri OH /n R1 OH
(XIV) wherein n, Y, R1 and L are defined below, L is connected to the targeting ligand e.g.
GaINAc and the 0 of the phosphoro-group is attached to the terminal oligonucleoside of the RNA strands.
Suitably, the targeting ligand portion is a linking moiety of formula (IV):
GaINAc GaINAc L/ L/
/ /

Y
\ II Y
__________________________________ \ II .
HO ________________________ 0 P 0 I ) 0¨P-0¨
. I
\ R1 OH R1 OH
n (IV) wherein n, Y, R1 and L are defined below and the 0 of the phosphoro-group is attached to the terminal oligonucleoside of the RNA strands.
Suitably, sr%"-"f is a linking moiety of formula (VII):
-( Y \ Y
II II
I
OH /
in OH
(VII) wherein n, Y and L2 are defined below and the 0 of the phosphoro-group is attached to the terminal oligonucleoside of the RNA strands.
Suitably, the targeting ligand portion is a linking moiety of formula (VIII):

1(1 Y
ll _ .
GaINAc _______ L2 0 P 0 ___ L2 0 1:,) u-I I
\ OH / OH
VIII
wherein n, Y and L2 are defined below and the 0 of the phosphoro-group is attached to the terminal oligonucleoside of the RNA strands.
Suitably, -ruN-ry is a linking moiety of formula (IX):
Y
Il ' I
, -,'-L 0¨P¨O-H
= 1¨F/ 1- I
OH (IX) wherein F, Y and L are defined below and the 0 of the phosphoro-group is attached to the terminal oligonucleoside of the RNA strands.
Suitably, the targeting ligand portion is a linking moiety of formula (IXa):
Y
II .
GaINAc¨L 0¨P¨O-H
1¨F/ 1- I
OH (IXa) wherein F, Y and L are defined below and the 0 of the phosphoro-group is attached to the terminal oligonucleoside of the RNA strands.

Suitably, L is:

, .
In any of the above structures, suitably the ligands are selected from GaINAc and galactose moieties, especially GaINAc moieties. Alternatively, GalNac may be replaced by another targeting ligand, e.g. a saccharide.
In an embodiment of the invention, the first RNA strand is a compound of formula (X):
_ ¨
Y Y \
II II
5'Z1-3' O¨P-0 L1 ________________________ 0 P 0 L1--O _______ H
I I
OH OH
/
¨ ¨ b (x) wherein b is 0 or 1; and the second RNA strand is a compound of formula (XI):
-Y \ Y
II Y
5' 3' II Y
-( \
H-0 Li-0-111-0-'-1_1-0¨P-0-4-0¨P-0¨Li _________________ 0-111-0¨L1-LO¨H
I I I I
OH / OH OH OH
/
- -c - - d (Xi);
wherein:
c and d are independently 0 or 1;
Z1 and Z2 are the RNA portions of the first and second RNA strands respectively;
Y is 0 or S;
n is 0, 1, 2 or 3; and L1 is a linker to which a ligand is attached;
and wherein b + c + d is 2 or 3.
Suitably, the first RNA strand is a compound of formula (XV):

¨ _ GaINAc GaINAc \ \
L\ L
\
NH NH
/ _____________________________________ / II /
Z1-0¨P-0 0 P 0 0¨H
I I
OH R1 \ OH Ri /
n b¨ ¨ (XV) wherein b is 0 or 1; and the second RNA strand is a compound of formula (XVI):
_ _ _ _ GaINAc ,GaINAc GaINAc GaINAc /µ µ
L L L L
HN HN NH NH
\
Yil \ Y Y / Yil H-0-/R OPO OPO Z20P0 OP d R1 -)-0¨H
I I I I I
\ Ri i OH Ri OH OH R1 \ OH
n n c _ ¨ d ¨ ¨
5 (XVI);
wherein c and d are independently 0 or 1;
wherein:
Z1 and Z2 are the RNA portions of the first and second RNA strands respectively;
Y is 0 or S;
10 R1 is H or methyl;
n is 0, 1,2 or 3; and L is the same or different in formulae (XV) and (XVI) and is selected from the group consisting of:
-(CH2)r-C(0)-, wherein r = 2-12;
15 -(CH2-CH2-0)s-CH2-C(0)-, wherein s = 1-5;
-(CH2)1-CO-NH-(CH2)1-NH-C(0)-, wherein t is independently is 1-5;
-(CH2).-CO-NH-(CH2).-C(0)-, wherein u is independently is 1-5; and -(CH2),-NH-C(0)-, wherein v is 2-12; and wherein the terminal C(0) (if present) is attached to the NH group;
20 and wherein b + c + d is 2 or 3.
Suitably, the first RNA strand is a compound of formula (XII):

Y Y
II II
5,Z1-3, 0¨P-0 L2 ________________________ 0 P¨O¨L2 O¨H
I I
OH OH
_ n ¨ b (XII) wherein b is 0 or 1; and the second RNA strand is a compound of formula (XIII):
¨
_ Y \ Y Y Y
5' 3' II Il H-0 L2-0JI-0¨H_2-0-11:1-0¨Z2-0¨P¨O¨L2 0¨PI-0¨L2 0 H
I I
OH / OH OH OH
n ¨ / _ c ¨
n _d (MO;
wherein:
c and d are independently 0 or 1;
Z1 and Z2 are the RNA portions of the first and second RNA strands respectively;
Y is 0 or S;
n is 0, 1,2 or 3; and L2 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:
0 y F. ,L, il GaINAc H
H NI, GaINAc QN
FLGaINAc F L
(.)g, õ...k........L......),µ,.
.....I.....:3õ, = . ; and 1 ; or , , n is 0 and L2 is:
, H
-1 ,NõGaINIAc iF 1_-and the terminal OH group is absent such that the following moiety is formed:
Y
II .
GaINAc¨L 0¨P-0-1 I-1\1¨F/ I
= OH , wherein F is a saturated branched or unbranched (such as unbranched) Ci_salkyl (e.g.

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 0 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:
-(CH2)r-C(0)-, wherein r = 2-12;
-(CH2-CH2-0)s-CH2-C(0)-, wherein s = 1-5;
-(CH2)1-CO-NH-(CH2)1-NH-C(0)-, wherein t is independently is 1-5;
-(CH2).-CO-NH-(CH2).-C(0)-, wherein u is independently is 1-5; and -(CH2)v-NH-C(0)-, wherein v is 2-12; and wherein the terminal C(0) (if present) is attached to the NH group;
and wherein b + c + d is 2 or 3.
In any one of the above formulae where GaINAc is present, the GaINAc may be substituted for any other targeting ligand, such as those mentioned herein.
Suitably, 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.
More suitably, 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.
Most suitably, b is 0, c is 1 and d is 1.
In one embodiment, Y is 0. In another embodiment, Y is S.
In one embodiment, R1 is H or methyl. In one embodiment, R1 is H. In another embodiment, R1 is methyl.
In one embodiment, n is 0, 1, 2 or 3. Suitably, n is 0.
In one embodiment, L is selected from the group consisting of:
-(CH2)r-C(0)-, wherein r = 2-12;
-(CH2-CH2-0)s-CH2-C(0)-, wherein s = 1-5;
-(CH2)1-CO-NH-(CH2)1-NH-C(0)-, wherein t is independently is 1-5;
-(CH2).-CO-NH-(CH2).-C(0)-, wherein u is independently is 1-5; and -(CH2)v-NH-C(0)-, wherein v is 2-12;
wherein the terminal C(0) is attached to the NH group.
Suitably, L is -(CH2)r-C(0)-, wherein r = 2-12. Suitably, r = 2-6. More suitably, r = 4 or 6 e.g.
4.

Suitably, L is:

%,..",..,............/.7....N......A./...
, .
Example F moieties include (CH2)1_6 e.g. (CH2)1-4 e.g. CH2, (CH2)4, (CH2)5 or (CH2)6, or CH20(CH2)2_3, e.g. CH20(CH2)CH3.
Suitably, L2 is:
H
N, GaINAc L
Suitably, L2 is:
H
aON I_GaINAc . , Suitably, L2 is:
N'L GaINAc H
i .
. .
Suitably, L2 is:
L 0,),......--W .õ ..,..
N GaINAc H
N

.
Suitably, n is 0 and L2 is:
H
c..N, LGaINAc , , and the terminal OH group is absent such that the following moiety is formed:
GaINAc \
L¨NH
\ Y
' 20 OH , wherein Y is as defined elsewhere herein.
Within the moiety bracketed by b, c and d, L2 is typically the same. Between moieties bracketed by b, c and d, L2 may be the same or different. In an embodiment, L2 in the moiety bracketed by c is the same as the L2 in the moiety bracketed by d. In an embodiment, L2 in the moiety bracketed by c is not the same as L2 in the moiety bracketed by d. In an embodiment, the L2 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.
derived from L-serine isomer, D-serine isomer, a racemic serine or other combination of isomers. In a preferred aspect of the invention, the serinol-GaINAc moiety (SerGN) has the following stereochemistry:
yiPr2 DMTo oPoCN
0 \ NH
ss, HOOH *
NH2 H .\
DMT0 oN =L-Serine 0 Serinol derived linker moieties 2\
(S)-Serinol building blocks .
i.e. is based on an (S)-serinol-amidite or (S)-serinol succinate solid supported building block derived from L-serine isomer.
In one embodiment, the targeted cells are hepatocytes.
General synthesis schemes 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(III) 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). GaINAc conjugation may be achieved by peptide bond formation of a GaINAc-carboxylic acid building block to the prior assembled and purified oligonucleotide 5 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-H PLC 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.
10 Identity of the respective single stranded products was proved by LC-MS
analysis.
Synthesis of Synthons Scheme 1: Synthesis of DMT-serinol(TFA) linker synthons NH3C1 HN 0 ii HN 0 iii NH2 HO - C) HO - ¨1" DMTOyO DMTOOH

i DMTO0y)LOH
DMTOOH

DMT-Serinol(TFA)-succinate 6 5 vil iv DMTOO

N DMTOOõOCN

NiPr2 DMT-Serinol(TFA)-CEP 7 i) ethyl trifluoroacetate, NEt3, Me0H, 0 C, 16h, 2: 86% 5: 90%, ii) DMTCI, pyridine, 0 C, 16h, 74%, iii) LiBH4, Et0H/THF (1/1, v/v), 0 C, 1h, 76%, iv) 2-cyanoethyl-N,N-diisopropylchloro phosphoramidite, EtNPr2, CH2Cl2, 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. 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 Et0H. 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.
Scheme 2: Synthesis of GaINAc synthon 9 OAc OAc OAc OAc CL vii, viii 1&.....T2..\___ Ac01&"\'`\--0Ac ¨"- Ac0 OrOH
NHAc NHAc 8 GaIN(Ac4)-04-acid 9 (vii) TMSOTf, DCM, hexenol, viii) RuC13, Nalat, DCM, CH3CN, H20, 46% over two steps.
Synthesis of the GaINAc synthon 9 can be prepared according to methods as described in Nair et al. (2014), starting from commercially available per-acetylated galactose amine 8.
Synthesis of single stranded serinol-derived GalNAc conjugates Scheme 3: General procedure of oligonucleotide synthesis for serinol-derived linkers HN Steps ).LCF3 0 Ny12 ' H2N \
(see Figure 25) _ P
DMTOOL ND ¨I-- HO - 0õ0 5oligonucleotide strand3' H

im n n=0,m=1:11 =A0264 n = 1, m = 0: 11 =A0220 n=1,m=1:11 =A0329 Oligonucleotide synthesis of 3' mono-GaINAc conjugated oligonucleotides (such as compound A0264) 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 5 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. Further, oligonucleotide synthesis of 5' mono-GaINAc conjugated oligonucleotides may be commenced from a solid support 10 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. Upon completion of chain elongation, the protective DMT group of the last coupled amidite building block is removed, as in step 1) of the phosphoramidite synthesis cycle.
Upon completion of the last synthesizer step, 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-H PLC and SEC to yield the precursor oligonucleotide for further GaINAc conjugation.
Scheme 4: GaINAc conjugation synthesis of serinol-derived precursor oligonucleotides (Ac OAc Ac0 NHAc OH _I-1 OH OH OH

HO
HO
NHAc NHAc 1. HBTU, DIPEA, DMSO, 2min 2. 11 in DMSO/H20, 30 min 3. 40% MeNH aq., 15min /I-IN 0 5' 3' H2N
\ 0 HOO,D,0 oligonucleotide strand 0,p,0 OH
ir\

im n = 0, m = 1:12 =A0268 n = 1, m = 0:12 =A0241 n = 1, m = 1:12 =A0330 Post solid phase synthesis GaINAc-conjugation was achieved by pre-activation of the GaIN(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 GaIN(Ac4)-conjugates. The acetyl groups protecting the hydroxyl groups in the GaINAc-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.
Synthesis of single stranded non-serinol-derived GalNAc conjugates 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 GaINAc conjugation. For example 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.
For example, to make 14 (A0653) 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. In a similar fashion 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).
Table 6: Commercially available building blocks C3Am-CPG (10-1) is: GlyC3Am-CPG (10-2) is:

NHFnnoc 0.NHTFA
.0 CPG .0 ,CPG
DMTr0 0 DMTr0 0 C7Am-CPG (10-3) is: PipAm-CPG (10-5) is:
01,......======,..../..,.....====,.
NHTFA
0C.0NHFmoc r DMTr CPG 0.0 TMTr CPG
C3Am-Phos (13-1) is: GlyC3Am-Phos (13-2) is:
NHFmoc 0.-.NHFmoc .0 .0 CEP
DMTr0 0CEP DMTr0 0 C6Am-Phos (13-4) is: PipAm-Phos (13-5) is:
NHTFA 0.).,õ,-w NHTFA
CEP N

c<0 TMTrO CEP

Scheme 5: General procedure for oligonucleotide synthesis A) Ga Ga HN, I
HN, Oa 0 Steps L.,a 0 5' 3' DMTOL5a L3 N HO-oligonucleotide strand-0Põ0L5a, p).r)-LNO
' L3 H (see Figure 25) o 'cN 0 10-1, 10-2, 10-3 1 1. 13, BIT, Acetonitrile 10-1, L5a = CH2, L3a absent, Lsa = CH2, G = Fmoc 2. further steps 10-2, L5a = CH2, L3a absent, Lsa = CH20(CH2)3, G = TFA
10-3, L5a = CH2, L3a = CH2, Oa = (CH2)4, G = Fmoc H2N,Lsb H2N.Lsa 5' 3' HO, -1 p L5b ,0-oligonucleotide strand-0õ L5a L3 0, pH
L3 'F) P

1 Sb = ru nif-u \
A0653: L5a, L5b = CH2, L3a, L3b absent, Lsa , , L,n2,-,ks-,..2i3 A0563: L5a, L5b = CH2, L3a, L3b absent, Lsa , Lsb = CH2 or H2N,Lsb H2N.Lsa 5' 3' 3 p ,0-oligonuc1eotide strand-0õ0, L3 pH
L 'P P L5a I/ \

A0561: Oa = CH2, L3a, L3b absent, Lsa = CH20(CH2)3, Lsb = (CH2)5 A0651: L5a, Ca = CH2, L3b absent, Lsa = (CH2)4, Lsb =(CH2)5 Gb Gb I I
HN, HN, Lsb Lsb DMT0-.L513-LL3P--P-- -.....--"-cN L- q Pip ,C1 ' CN
I I
N
13-1, 13-2 rr 13-1, L5b = CH2, L3b absent, Lscb = CH2, G = Fmoc 13-4, L3b = absent, Lscb = (CH2)5, G = TEA
13-2, L5b = CH2, L3b absent, Lscb = CH20(CH2)3, G = TEA

0yl_aNH2 B) Ga N
41sa , Steps 0 L 0 3' HO-Oligonucleotide strand-0Põ0 3, p TMT13'L5ajL3PIN L5a L )-HN 0 H (see Figure 25) 10-5 1. 13, BTT, Acetonitrile 10-5, Oa = CH2, L3a = CH2, Lsa = (CH2)5, G = TEA 2. further steps 0 Ls,a y Ga 0yl_bNH2 0yl_aNH2 N
--- :-...
N N
..-- --.
TMTO X p OCN 5' 3' I HO, X põ0-oligonucleotide strand-0,p-O,LXL3pH
-.TNT- L b L3 P
6 \SH /, \

13-5, Lsb = CH2, L3b = CH2, Lscb = (CH2)5, G = TFA A0655:
L5a, L5b = CH2, L38, L3b = CH2, LSa , LSb = (CH2)5 The resulting precursor oligonucleotides 14 can then be conjugated with GaIN(Ac4)-C4-acid (9) to yield the desired example compounds 15 (Scheme 6).

Scheme 6: GaINAc conjugation synthesis of precursor oligonucleotides OH _OH OH _OH

HO ===\--0 HO
====\o NHAc NHAc HNSID, HN, L
3' HO,L5b-LL3,p,p,CA' ligonucleotide strand¨O..p,0,0gLopH
6 \SH 6 \SH
OAc OAc A0654: Oa, L5b = CH2, Oa, L3b absent, Lsa i , _Sb = _ - , - 0(CH
2,3 Ac0 OrOH A0564: Oa, L5b = CH2, Oa, L3b absent, Lsa , L Sb = c NHAc 9 0 OL _H OH OH
or 1. HBTU, DIPEA, DMSO, 2min HO HO
NHAc 2. 14 in DMSO/H20, 30 min NHAc 3. 40% MeNH aq., 15min IN- '15 HN, HN, LSb 5' 3' Lop,p_O-oligonucleotide strand-0,p,0,0a-LopH
OSH 6 \SH
A0562: L5a = CH2, L3a, L3b absent, Lsa = CH20(CH2)3, Lsb = (CH2)5 A0652: L5a, L3a = CH2, L3b absent, Lsa = (CH2)4, Lsb =(CH2)5 or OH _OH OH /DH

HO HO
NHAc0 LT' NHAc0 y N y N 0 5' 3' HO, 51,cZ 3b0õ0-oligonucleotide strand-0õ0, 3pH
L L P P L L
6 \SH OSH
A0656: L5a, L5b = CH2, L3a, L3b = CH2, Lsa , _sb I = (CH 2)5 Synthesis of the single stranded tri-antennary GaINAc conjugates in reference conjugates Oligonucleotides synthesis of tri-antennary GaINAc-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(III) 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. For the on column conjugation of a trivalent tri-antennary GaINAc cluster the same synthesis cycle was applied with using the necessary trivalent branching amidite C4XLT-phos followed by another round of the synthesis cycle using the GaINAc amidite ST23-phos. Upon completion of this last synthesizer step, the oligonucleotide was cleaved from the solid support and additional protecting groups may be removed by methylamine treatment. The crude products were then purified each by AEX-H PLC and SEC.
General procedure of double strand formation In order to obtain the double stranded conjugates, individual single strands are dissolved in a concentration of 60 OD/mL in H20. 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 (including Schemes 1-6 and Figures 25 and 26) may be easily adapted to replace GalNac with another targeting ligand e.g. a saccharide.
In any of the above aspects, instead of post solid phase synthesis conjugation it is possible to make a preformed Serinol(GN)-phosphoramidite and use this for on-column conjugation.
General synthesis schemes: 2 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. GaINAc conjugation may be achieved by peptide bond formation of a GaINAc-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 synthons ..(4?
NH3ci HN 0 ii HN 0 iii NH2 HO - C) HO - ¨1" DMTOyO DMTOOH

i A
DMTO0y)LOH HN CF3 DMTOOH

DMT-Serinol(TFA)-succinate 6 5 vil iv y N 4111 DMTO- 0,p, CN

NiPr2 DMT-Serinol(TFA)-CEP 7 i) ethyl trifluoroacetate, NEt3, Me0H, 0 C, 16h, 2: 86% 5: 90%, ii) DMTCI, pyridine, 0 C, 16h, 74%, iii) LiBH4, Et0H/THF (1/1, v/v), 0 C, 1h, 76%, iv) 2-cyanoethyl-N,N-diisopropylchloro phosphoramidite, EtNPr2, CH2Cl2, 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 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 5 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. The DMT-Serinol(TFA)-succinate 6 and a coupling reagent such as HBTU is dissolved in a solvent such as CH3CN. A base, such as 10 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 Et0H. 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.
Scheme 2: Synthesis of GaINAc synthon 9 _Ac OAc OAc OAc vii, viii Ac0 OAc Ac0 OH
NHAc NHAc 8 GaIN(Ac4)-04-acid 9 5 (vii) TMSOTf, DCM, hexenol, viii) RuC13, Nalat, DCM, CH3CN, H20,46% over two steps.
Synthesis of the GaINAc 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.
Scheme 3: General procedure of oligonucleotide synthesis HNACF3 o Steps NH2 DMTOONX-) HOO
oligonucleotide strand 0 (see Figure 29) All 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' GaINAc conjugated oligonucleotides precursors (such as compound X0385B-prec) is outlined in Figure 29 and summarised in Scheme 3.
15 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.
To complete synthesis of example compound X0385B-prec (which has a serinol-derived 20 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.
Upon completion of the last synthesizer step, 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 25 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 IEX
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.
Scheme 4: General procedure for GaINAc conjugation OAc OAc OH (OH
NH2 (I) Ac0"\=2-\--0 OH + HOO,oligo NHAc NHAc (i) 9, HBTU, DIPEA, DMSO; 11, H20, DMSO, DIPEA; then activated 9, 11; then 40%

MeNH2, H20 Conjugation of the GaINAc 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. For example, the respective amino-modified precursor molecule 11 is dissolved in H20 and a polar solvent such as DMSO (e.g.
DMSO/H20, 2/1, v/v) is added, followed by a base such as DIPEA (e.g. 2.5% of total volume). In a separate reaction vessel pre-activation of the GaINAc synthon 9 can be performed by reacting 2 eq. (per amino function in the amino-modified precursor oligonucleotide) of the carboxylic acid component with 2 eq. of a coupling reagent such as HBTU in presence of 8 eq. of a base, such as DIPEA, in a polar solvent such as DMSO.
After 2 min the activated compound 9 is added to the solution of the respective amino-modified precursor molecule 11. The reaction progress can be monitored by LCMS
or AEX-HPLC. Upon completion of the conjugation reaction (e.g. 30 minutes) the crude product can be precipitated by addition of 10x PrOH 0.1x 2M NaCI and harvested by centrifugation decantation. The acetyl hydroxy-protecting groups are removed under basic conditions, such as 40% MeNH2 (1mL per 500 OD). After 15 min at RT H20 (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.
General procedure of double strand formation Individual single strands are dissolved in a concentration of 60 OD/mL in H20.
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 (including Schemes 1-4 and Figure 29) 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 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; or (ii) 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 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, 11, 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.

Preferably, the nucleic acid comprises SEQ ID NO:3, SEQ ID NO:11 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 2'0Me 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 2'0Me on the even numbered nucleotides and modified with a 2'F on the odd numbered nucleotides.
More preferably, the nucleic acid comprises SEQ ID NO:3, SEQ ID NO:11 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.
Sequence siRNA ID sequence modifications ID No 3 5'-AAUGUUUUCCUGCUGACGG-3' 6254515173547182748 ALDH2-hcm-2 4 5'-CCGUCAGCAGGAAAACAUU-3' 3745364728462627251 11 5'-UCUUCUUAAACUGAGUUUC-3' 5351715262718281517 ALDH2-hcm-6 12 5'-GAAACUCAGUUUAAGAAGA-3' 4626353645152646282 17 5'-AUGUAGCCGAGGAUCUUCU-3' 6181647382846171535 ALDH2-hcm-9 18 5'-AGAAGAUCCUCGGCUACAU-3' 2826461735384716361 wherein the specific modifications are depicted by numbers 1=2"F-dU, 2=2`-F-dA, 3=2"F-dC, 4=2"F-dG, 5=2'-0Me-rU;
6=2'-0Me-rA;
7=2'-0Me-rC;
8=2'-0Me-rG.
The ligand may comprise GaINAc 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.
For example, 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. Other agents such as preservatives and stabilizers can also be added. Methods for the delivery of nucleic acids are known in the art and within the knowledge of the person skilled in the art.
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. In one embodiment, 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. Alternatively, 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.
The pharmaceutical 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. On this basis, 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-5 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. Inhibition may last 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks or up to 3 months, when used in a subject, such as a human patient. 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.
In cells and/or subjects treated with or receiving the nucleic acid or conjugated nucleic acid of the present invention, 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.
Also included in the invention is 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.
The nucleic acid or conjugated nucleic acid of the present invention can also be administered for use in combination with other therapeutic compounds, either administered separately or simultaneously, e.g., as a combined unit dose. 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. For example, using solid phase chemical synthesis or using a nucleic acid-based expression vector including viral derivates or partially or completely synthetic expression systems. In one embodiment, the expression vector 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 (GaINAc) sugar moiety that ensures hepatocyte-specific cellular uptake, though specific binding to the asialoglycoprotein receptor complex (ASGPR). 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.
Low substrate specificity of known drugs inhibiting up to 20 different enzymes in addition to ALDH2 leads to severe undesired physiological effects in several tissues such as heart muscle and the central and peripheral nervous system even in non-drinking patients. 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.
In one embodiment, the first strand may include more than 1 phosphodiester linkage.
In one embodiment, the first strand may comprise phosphodiester linkages between at least the terminal three 5' nucleotides.
In one embodiment, the first strand may comprise phosphodiester linkages between at least the terminal four 5' nucleotides.
In one embodiment, the first strand may comprise formula (XVII):
(Vp)-N(po)[N(po)]n-(XVII) where '(vp)-' is the 5' (E)-vinylphosphonate, 'N' is a nucleotide, 'po' is a phosphodiester linkage, and 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 -4).
In one embodiment, the first strand may include at least one phosphorothioate (ps) linkage.
In one embodiment, the first strand may further comprise a phosphorothioate linkage between the terminal two 3' nucleotides or phosphorothioate linkages between the terminal three 3' nucleotides.
In one embodiment, the linkages between the other nucleotides in the first strand are phosphodiester linkages.
In one embodiment, the first strand may include more than 1 phosphorothioate linkage.
In a further embodiment, the second strand may comprise a phosphorothioate linkage between the terminal two 3' nucleotides or phosphorothioate linkages between the terminal three 3' nucleotides.
In another further embodiment, the second strand may comprise a phosphorothioate linkage between the terminal two 5' nucleotides or phosphorothioate linkages between the terminal three 5' nucleotides.
In an embodiment, the terminal 5' (E)-vinylphosphonate nucleotide is an RNA
nucleotide.
A terminal 5' (E)-vinylphosphonate nucleotide is a nucleotide wherein the natural phosphate group at the 5'-end has been replaced with a E-vinylphosphonate, in which the bridging 5'-oxygen atom of the terminal nucleotide of the 5' phosphorylated strand is replaced with a methynyl (-CH=) group:

Nucleotides with a natural phosphate at the 5'-end -0' "~1 Rase 6 OMe C=P-0 Nucleotide with a E-vinylphosphonate at the 5'-end Base "8-0=P-C

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. In the context of the present invention, 5' (E) vinylphosphonate mimics the function of a normal 5' phosphate, e.g. enabling efficient RISC
loading. In addition, because of its slightly altered structure, 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 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.
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 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' 0-methyl modification, and the nucleotide on the second strand which corresponds to position 13 of the first strand is not modified with a 2' 0-methyl modification.
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.

In one aspect 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.
In one aspect the nucleotide on the second strand which corresponds to position 11 of the 5 first strand is the nucleotide that forms a base pair with position 11 of the first strand.
In one aspect 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.
10 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 11 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. Likewise position 11 of the second strand is suitably the 11th 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.
In one aspect, in the case of a partially complementary first and 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.
Also preferred are:
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' 0-methyl modification, and the nucleotide on the second strand which corresponds to position 11 of the first strand is not modified with a 2' 0-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' 0-methyl modification, and the nucleotides on the second strand which corresponds to position 11 and 13 of the first strand are not modified with a 2' 0-methyl modification.
In one aspect the nucleotide on the second strand which corresponds to position 12 of the first strand is not modified with a 2' 0-methyl modification. This limitation on the nucleic acid may be seen with any other limitation described herein.
Therefore another aspect of the invention is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the Send of the first strand are not modified with a 2' 0-methyl modification, and the nucleotides on the second strand which corresponds to position 11-13 of the first strand are not modified with a 2' 0-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' 0-methyl modification, and the nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand are modified with a 2' fluoro modification.
A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified with a 2' fluoro modification, and the nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand are not modified with a 2' 0-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 modified with a 2' fluoro modification, and the nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand are modified with a 2' fluoro modification.
A nucleic acid as disclosed herein wherein greater than 50% of the nucleotides of the first and/or second strand comprise a 2' 0-methyl modification, such as greater than 55%, 60%, 65%, 70%, 75%, 80%, or 85%, or more, of the first and/or second strand comprise a 2' 0-methyl modification, preferably measured as a percentage of the total nucleotides of both the first and second strands.

A nucleic acid as disclosed herein wherein greater than 50% of the nucleotides of the first and/or second strand comprise a naturally occurring RNA modification, such as wherein greater than 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more of the first and/or second strands comprise such a modification, preferably measured as a percentage of the total nucleotides of both the first and second strands. Suitable naturally occurring modifications include, as well as 2 0' methyl, other 2' sugar modifications, in particular a 2' H
modification resulting in a DNA nucleotide.
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' 0 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.
A nucleic acid as disclosed herein, wherein all nucleotides are modified with a 2' 0-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 11, or 13, or 11 and 13, or 11-13 of the first strand. Preferably the nucleotides that are not modified with 2' 0-methyl are modified with fluoro at the 2' position.
Preferred is a nucleic acid as disclosed herein wherein all nucleotides of the nucleic acid are modified at the 2' position of the sugar. Preferably these nucleotides are modified with a 2'- fluoro modification where the modification is not a 2' 0-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 11, or 13, or 11 and 13, or 11-13 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 11, or 13, or 11 and 13, or 11-13 of the first strand.
In one aspect 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. Preferably the second strand is modified with 2' fluoro modifications at nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand. Preferably the second strand is modified with 2' fluoro modifications at positions 11-13 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' 0-methyl.
In one aspect the 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.
In one aspect the nucleic acid comprises one or more phosphorodithioate linkages, such as 1, 2, 3 or 4 phosphorodithioate linkages. Preferably there are up to 4 phosphorodithioate linkages, one each at the 5' and 3' ends of the first and second strands.
All the features of the nucleic acids can be combined with all other aspects of the invention disclosed herein.
In particular, preferred are 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' 0-methyl modification, and the nucleic acid comprises one or more or all of:
(i) an inverted nucleotide, preferably a 3'-3' linkage at the 3' end of the second strand;
(ii) one or more phosphorodithioate linkages;
(iii) the second strand nucleotide corresponding to position 11 or 13 of the first strand is not modified with a 2' 0-methyl modification, preferably wherein one or both of these positions comprise a 2' fluoro modification;
(iv) 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.
Also provided by the present invention is a 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:
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' 0-methyl modification, and the nucleotide at position 7 from the 5' end of the second strand is not modified with a 2' 0-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' 0-methyl modification, and the nucleotide at position 9 from the 5' end of the second strand is not modified with a 2' 0-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' 0-methyl modification, and the nucleotides at position 7 and 9 from the 5' end of the second strand are not modified with a 2' 0-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' 0-methyl modification, and the nucleotides at positions 7 - 9 from the 5' end of the second strand are not modified with a 2' 0-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' 0-methyl modification, 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.

A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified with a 2' fluoro modification, and the nucleotides at positions 7 and/or 9, or 7 - 9 from the 5' end of the second strand are not modified with a 2' 0-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 modified with a 2' fluoro modification, 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.
A nucleic acid as disclosed herein wherein greater than 50% of the nucleotides of the first and/or second strand comprise a 2' 0-methyl modification, such as greater than 55%, 60%, 65%, 70%, 75%, 80%, or 85%, or more, of the first and/or second strand comprise a 2' 0-methyl modification, preferably measured as a percentage of the total nucleotides of both the first and second strands.
A nucleic acid as disclosed herein wherein greater than 50% of the nucleotides of the first and/or second strand comprise a naturally occurring RNA modification, such as wherein greater than 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more of the first and/or second strands comprise such a modification, preferably measured as a percentage of the total nucleotides of both the first and second strands. Suitable naturally occurring modifications include, as well as 2 0' methyl, other 2' sugar modifications, in particular a 2' H
modification resulting in a DNA nucleotide.
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' 0 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.
A nucleic acid as disclosed herein, wherein all nucleotides are modified with a 2' 0-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.
Preferably the nucleotides that are not modified with 2' 0-methyl are modified with fluoro at the 2' position.
A nucleic acid as disclosed herein, wherein all nucleotides are modified with a 2' 0-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.
Preferably the nucleotides that are not modified with 2' 0-methyl are modified with fluoro at the 2' position.
For a nucleic acid comprising a 20 base pair duplex region, the second strand preferably does not have a 2' 0-methyl group at nucleotides 8 or 9 or 10 counting from the 5' end of the duplex corresponding to positions 13, 12, and 11 of the first strand respectively.
For a nucleic acid comprising a 21 base pair duplex region, the second strand preferably does not have a 2' 0-methyl group at nucleotides 9 or 10 or 11 counting from the 5' end of the duplex corresponding to positions 13, 12, and 11 of the first strand respectively.
The present invention also relates to the unmodified sequences of all modified sequences disclosed herein.
The invention will now be described with reference to the following non-limiting Figures and Examples.
Figures 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 GaINAc-coupled siRNAs targeting Aldh2 mRNA in primary mouse hepatocytes.
Figure 5 shows the dose response for Gal NAc-coupled siRNAs against ALDH2 mRNA
in primary human hepatocytes.
Figure 6 shows the reduction of ALDH2 mRNA by 15nM of GaINAc-coupled ALDH2-hcm-2 siRNA in 10 human, cynomolgus and mouse hepatocytes.

Figure 7 shows the comparison in dose-response on ALDH2 mRNA knockdown by GaINAc-conjugated ALDH2 siRNAs hcm2, hcm6, hcm9 in conjugation to L1 linker vs.
conjugation to the L6 GaINAc-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 days upon a single dose application in mice using as an example GaINAc-ALDH2-hcm6 and hcm9 conjugated to the L6 linked GaINAc moiety.
Figures 10a and 10b show the structure of the GaINAc L1 and L6 ligands, respectively, to which the example oligonucleotides were conjugated.
Figure 11 shows different siRNA duplexes containing inverted RNA nucleotides at both T-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 GaINAc-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 GaINAc-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 GaINAc-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 GaINAc-siRNA
conjugates of ALDH2-hcm6 and ALDH2-hcm9 (Table 5) on knockdown efficacy on Aldh2 mRNA expression levels in vivo in mice.
Figure 18 depicts Conjugate 1.
Figure 19 depicts Conjugate 2.
Figure 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.
In each of Figures 18-24 and 37-49, the top strand is the antisense strand and the bottom strand is the sense strand. In addition, to show more clearly the connection between the nucleic acid and ligand portions, 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-GaINAc 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 GaINAc 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. In particular, 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";
and 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 GaINAc siRNA
and "untreated" ("UT") represents untreated cells. Both RC3 and UT are negative controls.
mRNA levels were normalised against Ptenll.
Figure 28 shows a time course of serum TTR in c57BL/6 mice cohorts of n=4 at 7, 14, and 27 days post s.c. treatment with lmg/kg - Conjugates 1-3, Reference Conjugates (RC) 1, 2 and 4 and mock treated (PBS) individuals.
Figure 29 shows oligonucleotide synthesis of 3' and 5' GaINAc conjugated oligonucleotides precursors (such as compound X0385B-prec).
Figure 30 discloses GaINAc conjugates with each one serinol-linked GaINAc 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 GaINAc 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 GaINAc 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 GaINAc 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 GaINAc 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 GaINAc 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 1mg/kg ALDH2-hcm9 siRNA conjugate 16 with 5' & 3' serinol-GaINAc second strand conjugation as compared to conjugate 14 with 5' second strand triantennary GaINAc-conjugation.
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-GaINAc-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-GaINAc-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-GaINAc-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-GaINAc-linkers are conjugated via a phosphorothioate bond to the 3' end and the 5' end of the sense strand. The serinol-GaINAc-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 GaINAc-C6-amino-modifier linker is conjugated at the 5' end of the sense strand and a GaINAc-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 GaINAc-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 GaINAc-piperidyl-amino-modifier linker is conjugated at the 5' and 3' ends of 5 the sense strand.
Figure 44 depicts Conjugate 11. 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 GaINAc-C3-amino-modifier linker is conjugated at the 5' and 3' ends of the sense strand.
10 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 GaINAc-C6-amino-modifier linker is conjugated at the 5' end of the sense strand and a GaINAc-GlyC3-amino-modifier linker is conjugated at the 3' end of the sense strand.
15 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-GaINAc-linkers are conjugated via a phosphorothioate bond to the 3' end and the 5' end of the sense strand.
20 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 GaINAc-linker is conjugated via a phosphorothioate bond to the 5' end of the sense strand in both conjugates.
25 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 GaINAc-linker is conjugated via a phosphorothioate bond to the 5' end of the sense strand in both conjugates.
30 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 GaINAc-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. In particular, Figure 50A
35 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 (RC3) represents a non-targeting GaINAc 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. In particular, Figure 51A
shows the in vitro determination of TTR knockdown by Conjugates 8, 9, 10, 11 and 2 compared to "Luc" (Reference Conjugate 3) as well as the untreated control "UT"; Figure 51B 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 GaINAc 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 LPA mRNA knockdown by Conjugate 19 compared to controls. Ctr represents a non-targeting GaINAc siRNA and "untreated" ("UT") represents untreated cells. Both Ctr and UT are negative controls. mRNA level were normalised against ACTB.
Figure 53 shows a time course of liver ALDH2 mRNA in c57BL/6 mice cohorts of n=6 at 14, 28 and 42 days post s.c. treatment with lmg/kg - Conjugate 15, Reference Conjugate (RC) 6 and mock treated (PBS) individuals. mRNA levels were normalised against Pten.
Figure 54 shows a time course of liver ALDH2 mRNA in c57BL/6 mice cohorts of n=6 at 14, 28 and 42 days post s.c. treatment with lmg/kg - Conjugate 16, Reference Conjugate (RC) 7 and mock treated (PBS) individuals. mRNA level were normalised against Pten.
Figure 55 shows a time course of liver TMPRSS6 mRNA in c57BL/6 mice cohorts of n=6 at 14, 28 and 42 days post s.c. treatment with 1mg/kg - Conjugate 18, Reference Conjugate (RC) 8 and mock treated (PBS) individuals. mRNA level were normalised against Pten.
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, 11, 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-targeting GaINAc conjugates with an optimised 2'-0Me/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.

Examples The numbering referred to in each example is specific for said example.
Example 1 Nucleic acids in accordance with the invention were synthesised, using the 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:
ALDH2-hcm-2 GaINAc-ALDH2-hcm-2-L1:
First strand (SEQ ID NO: 84, based on SEQ ID NO: 3) 5'0MeA(ps)- FA-(ps)-0MeU-FG-0MeU-FU-OMeU-FU-OMeC-FC-OMeU-FG-OMeC-FU-OMeG-FA-OMeC-(ps)-FG-(ps)-0MeG 3' Second strand (SEQ ID NO: 85, based on SEQ ID NO: 4) 5'[5-123 (ps)]3 long trebler (ps) FC-0MeC-FG-0MeU-FC-OMeA-FG-OMeC-FA-OMeG-FG-OMeA-FA-OMeA-FA-OMeC-FA-(ps)-0MeU-(ps)-FU 3' GaINAc-ALDH2-hcm-2-L6:
First strand (SEQ ID NO: 86, based on SEQ ID NO: 3) 5'0MeA(ps)- FA-(ps)-0MeU-FG-0MeU-FU-OMeU-FU-OMeC-FC-OMeU-FG-OMeC-FU-OMeG-FA-OMeC-(ps)-FG-(ps)-0MeG 3' Second strand (SEQ ID NO: 87, based on SEQ ID NO: 4) 5'[5-123 (ps)]3 5T43 (ps) FC-0MeC-FG-0MeU-FC-OMeA-FG-OMeC-FA-OMeG-FG-OMeA-FA-0MeA-FA-0MeC-FA-(ps)-0MeU-(ps)-FU 3' FN (N=A, C, G, U) denotes 2'Fluoro, 2' DeoxyNucleosides OMeN (N=A, C, G, U) denotes 2'0 Methyl Nucleosides (ps) indicates a phosphorothioate linkage ST23 and ST43 are as below.
A further example is ALDH2-hcm-6 GaINAc-ALDH2-hcm6-L1 First strand (SEQ ID NO: 88, based on SEQ ID NO: 11) 5' OMeU-(ps)-FC-(ps)-0MeU-FU-OMeC-FU-OMeU-FA-OMeA-FA-OMeC-FU-OMeG-FA-OMeG-FU-OMeU-(ps)-FU-(ps)-0MeC
Second strand (SEQ ID NO: 89, based on SEQ ID NO: 12) 5'[5-123 (ps)]3 long trebler (ps) FG-0MeA-FA-0MeA-FC-OMeU-FC-OMeA-FG-OMeU-FU-OMeU-FA-0MeA-FG-OMeA-FA-(ps)-0MeG-(ps)-FA 3' GaINAc-ALDH2-hcm6-L6 First strand (SEQ ID NO: 90, based on SEQ ID NO: 11) 5' OMeU-(ps)-FC-(ps)-0MeU-FU-OMeC-FU-OMeU-FA-OMeA-FA-OMeC-FU-OMeG-FA-OMeG-FU-OMeU-(ps)-FU-(ps)-0MeC
Second strand (SEQ ID NO: 91, based on SEQ ID NO: 12) 5'[5-123 (ps)]3 5T43 (ps) FG-0MeA-FA-0MeA-FC-OMeU-FC-OMeA-FG-OMeU-FU-OMeU-FA-OMeA-FG-OMeA-FA-(ps)-0MeG-(ps)-FA 3' FN (N=A, C, G, U) denotes 2'Fluoro, 2' DeoxyNucleosides OMeN (N=A, C, G, U) denotes 2'0 Methyl Nucleosides (ps) indicates a phosphorothioate linkage 5T23 and 5T43 are as below.
A further example is ALDH2-hcm-9 GaINAc-ALDH2-hcm9-L1 First strand (SEQ ID NO: 92, based on SEQ ID NO: 17) 5'0MeA-(ps)-FU-(ps)-0MeG-FU-OMeA-FG-0MeC-FC-OMeG-FA-OMeG-FG-OMeA-FU-OMeC-FU-OMeU-(ps)-FC-(ps)-0MeU 3' Second strand (SEQ ID NO: 93, based on SEQ ID NO:18) 5.[ST23 (ps)]3 long trebler (ps) FA-0MeG-FA-0MeA-FG-OMeA-FU-OMeC-FC-OMeU-FC-OMeG-FG-OMeC-FU-OMeA-FC-(ps)-0MeA-(ps)-FU 3' GaINAc-ALDH2-hcm9-L6 First strand (SEQ ID NO: 94, based on SEQ ID NO: 17) 5'0MeA-(ps)-FU-(ps)-0MeG-FU-OMeA-FG-0MeC-FC-OMeG-FA-OMeG-FG-OMeA-FU-OMeC-FU-OMeU-(ps)-FC-(ps)-0MeU 3' Second strand (SEQ ID NO: 95, SEQ ID NO:18) 5'[5-123 (ps)]3 5T43 (ps) FA-0MeG-FA-0MeA-FG-OMeA-FU-OMeC-FC-OMeU-FC-OMeG-FG-0MeC-FU-OMeA-FC-(ps)-0MeA-(ps)-FU 3' GaINAc-ALDH2-hcm9-L4 First strand (SEQ ID NO: 96, based on SEQ ID NO: 17) 5'0MeA-(ps)-FU-(ps)-0MeG-FU-OMeA-FG-0MeC-FC-OMeG-FA-OMeG-FG-OMeA-FU-OMeC-FU-OMeU-(ps)-FC-(ps)-0MeU 3' Second strand (SEQ ID NO: 97, based on SEQ ID NO:18) 5'[5-123 (ps)]3 5T41 (ps) FA-0MeG-FA-0MeA-FG-OMeA-FU-OMeC-FC-OMeU-FC-OMeG-FG-OMeC-FU-OMeA-FC-(ps)-0MeA-(ps)-FU 3' FN (N=A, C, G, U) denotes 2'Fluoro, 2' DeoxyNucleosides OMeN (N=A, C, G, U) denotes 2'0 Methyl Nucleosides (ps) indicates a phosphorothioate linkage 5T23, ST41and 5T43 are as below.
5T23-phos is a GalNac C4 phosphoramidite (structure components as below) OAc N
Ac0 Ac0 (:)/\
NHAc -5T23-phos ODMT
r/DOCOODMT
NCC' NOP02 \ __ \
\ _________________________________________________________ ODMT
long trebler (STKS) ST41-phos is as follows:
DMTr 0 0 )LN'L
DMIr 0 0 Oc:115Ø,CN
DMTr.

5 ST43-phos is as follows and as described in W02017/174657:
DMTr.
DMTr, 0 0 0015,10,,CN
DMTr, ST43-phos 10 All 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 2"0-Methyl RNA phosphoramidites, 2"Fluoro DNA phosphoramidites (all standard protection) and commercially available long trebler phosphoramidite (Glen research) were 15 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., 5T23, 5T41 or 5T43) was achieved 20 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 25 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.
For annealing, 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.
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.
Sequence siRNA ID sequence modifications ID No 1 5'-UUGUUUAUGAAAAUCUGGU-3' 5181516182626171845 ALDH2-h-1 2 5'-ACCAGAUUUUCAUAAACAA-3' 2736461515361626362 3 5'-AAUGUUUUCCUGCUGACGG-3' 6254515173547182748 ALDH2-hcm-2 4 5'-CCGUCAGCAGGAAAACAUU-3' 3745364728462627251 5 5'-UUGAACUUCAGGAUCUGCA-3' 5182635172846171836 ALDH2-hc-3 6 5'-UGCAGAUCCUGAAGUUCAA-3' 1836461735462815362 7 5'-UUUCACUUCAGUGUAUGCC-3' 5153635172818161837 ALDH2-hc-4 8 5'-GGCAUACACUGAAGUGAAA-3' 4836163635462818262 9 5'-UUCUUAUGAGUUCUUCUGA-3' 5171525464517153546 ALDH2-hc-5 10 5'-UCAGAAGAACUCAUAAGAA-3' 1728264627172526462 11 5'-UCUUCUUAAACUGAGUUUC-3' 5351715262718281517 ALDH2-hcm-6 12 5'-GAAACUCAGUUUAAGAAGA-3' 4626353645152646282 13 5'-UCCUUGAUCAGGUUGGCCA-3' 5371546172845184736 ALDH2-hc-7 14 5'-UGGCCAACCUGAUCAAGGA-3' 1847362735461726482 5'-UUGAAGAACAGGGCGAAGU-3' 5182646272848382645 ALDH2-hc-8 16 5'-ACUUCGCCCUGUUCUUCAA-3' 2715383735451715362 17 5'-AUGUAGCCGAGGAUCUUCU-3' 6181647382846171535 ALDH2-hcm-9 18 5'-AGAAGAUCCUCGGCUACAU-3' 2826461735384716361 19 5'-AUCUGGUUGCAGAAGACCU-3' 6171845183646282735 ALDH2-hmr-10 5'-AGGUCUUCUGCAACCAGAU-3' 2845351718362736461
21 5'-AAAUCCACCAGGUAGGAGA-3' 6261736372845284646 ALDH2-hr-11
22 5'-UCUCCUACCUGGUGGAUUU-3' 1717352735481846151
23 5'-ACUCUCUUGAGGUUGCUGC-3' 6353535182845183547 ALDH2-hm-12
24 5'-GCAGCAACCUCAAGAGAGU-3' 4728362735362828281 5'-AUGUCCAAAUCCACCAGGU-3' 6181736261736372845 ALDH2-hcmr-13 26 5'-ACCUGGUGGAUUUGGACAU-3' 2735481846151846361 ALDH2-hm-14 5'-CCAUGCUUGCAUCAGGAGC-3' 7361835183617284647 1 28 15'-GCUCCUGAUGCAAGCAUGG-3' 14717354618362836184 I
Table 1 Nucleotides modifications are depicted by the following numbers (column 4), 1=2"F-dU, 2=2'F-dA, 3=2"F-dC, 4=2"F-dG, 5=2'-0Me-rU; 6=2'-0Me-rA; 7=2'-0Me-rC; 8=2'-0Me-rG.
Example 2 Screening for inhibition ALDH2 mRNA expression in human Hep3B cells was carried out as follows.
Cells were plated at a density of 150,000 cells per well in 6-well format. 24 hours post seeding, cells were transfected with 20 nM of indicated non-conjugated siRNAs using 1 pg/ml of AtuFECT as a liposomal transfection reagent. 48 hours after transfection, RNA
was isolated using the Spin Cell Mini Kit 250 (Stratec). ALDH2 mRNA levels were determined by gRT-PCR relative to PTEN mRNA expression as housekeeper. Values were normalized to the amount of ALDH2 mRNA in untreated cells (UT). Means and SD
of normalized triplicate measurements are shown. Results are shown in Figure 1.
Table 2: Sequences of ALDH2, ApoB, beta-Actin and PTEN qPCR amplicon sets that were used to measure mRNA levels are shown below.
SEQ ID
NO:: Gene Species Sequences 29 ALDH2 (upper) 5' GGCAAGCCCTATGTCATCTCCT 3' human, ALDH2 (lower) cynomolgus 30 5' GGATGGTTTTCCCGTGGTACTT 3' ALDH2 (probe) 31 5' TGGTCCTCAAATGTCTCCGGTATTATGCC 3' ALDH2 (upper) 32 5' GGCAAGCCTTATGTCATCTCGT 3' ALDH2 (lower) 33 mouse 5' GGAATGGTTTTCCCATGGTACTT 3' ALDH2 (probe) 34 5' TGAAATGTCTCCGCTATTACGCTGGCTG 3' 35 ApoB (upper) 5' TCATTCCTTCCCCAAAGAGACC 3' 36 ApoB (lower) human 5' CACCTCCGTTTTGGTGGTAGAG 3' 37 ApoB (probe) 5' CAAGCTGCTCAGTGGAGGCAACACATTA 3' 38 ApoB (upper) 5' TCATTCCTTCCCCAAAGAAACC 3' 39 ApoB (lower) cynomolgus 5' CACCTCCGTTTTGGTGGTAGAG 3' 40 ApoB (probe) 5' TCAAGCTGTTAAGTGGCAGCAACACGTT 3' 41 beta-Actin (upper) 5' GCATGGGTCAGAAGGATTCCTAT 3' human 42 beta-Actin (lower) 5' TGTAGAAGGTGTGGTGCCAGATT 3' 43 beta-Actin (probe) 5' TCGAGCACGGCATCGTCACCAA 3' 44 beta-Actin (upper) 5' AAGGCCAACCGCGAGAAG 3' 45 beta-Actin (lower) cynomolgus 5' AGAGGCGTACAGGGACAGCA 3' 46 beta-Actin (probe) 5' TGAGACCTTCAACACCCCAGCCATGTAC 3' - _______________________________________ 47 beta-Actin (upper) 5' CCTAAGGCCAACCGTGAAAAG 3' 48 beta-Actin (lower) mouse 5' AGGCATACAGGGACAGCACAG 3' 49 beta-Actin (probe) 5' TGAGACCTTCAACACCCCAGCCATGTAC 3' 50 PTEN (upper) 5' CACCGCCAAATTTAACTGCAGA 3' 51 PTEN (lower) human 5' AAGGGTTTGATAAGTTCTAGCTGT 3' 52 PTEN (probe) 5' TGCACAGTATCCTTTTGAAGACCATAACCCA
3' Table 2 Table 3: Non-conjugated derivatives of ALDH2-hcm2 with the indicated modification pattern are shown:
Duplex sequence and chemistry SEQ ID
ID top: first strand, bottom: second strand, both 5'-3 NO:
ALD01 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC(ps)fG(ps)mG 98 fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfA(ps)mU(ps)fU 99 ALD02 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG ivA 100 fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmUfU ivA 101 ALD03 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG ivU 102 fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmUfU ivU 103 ALD04 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG ivC 104 fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmUfU ivC 105 ALD05 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG ivG 106 fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmUfU ivG 107 ALD06 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfG ivA 108 fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmU ivA 109 ALD07 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfG ivU 110 fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmU ivU 111 ALD08 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfG ivC 112 fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmU ivC 113 ALD09 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfG ivG 114 fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmU ivG 115 mA, mU, mC, mG ¨ 2`-0Me RNA
fA, fU, fC, fG ¨ 2`-F RNA
ivA, ivU, ivC, ivG - inverted RNA (3'-3') (ps) ¨ phosphorothioate Table 3 Table 4: L6-GaINAc linker conjugated derivatives of ALDH2-hcm2 with the indicated modification pattern are shown SEQ
Duplex sequence and chemistry ID
ID top: first strand, bottom: second strand, both 5'-3 NO:

mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC(ps)fG(ps)mG 116 ST23(ps)3 ST

43(ps)fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfA(ps)mU(ps)fU
STS22002V1L6 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC(ps)fG(ps)mG 118 5T23(ps)3 ST

43(ps)fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmUivA
5T522002V2L6 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC(ps)fG(ps)mG 120 5T23(ps)3 ST 121 43(ps)fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmUivG
5T522002V3L6 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmGivA

5T23(ps)3 ST

43(ps)fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfA(ps)mU(ps)fU
5T522002V4L6 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmGivG

5T23(ps)3 ST

43(ps)fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfA(ps)mU(ps)fU
mA, mU, mC, mG ¨ 2`-0Me RNA
fA, fU, fC, fG ¨ 2`-F RNA
ivA - inverted RNA (3'-3') (ps) ¨ phosphorothioate Table 4 Table 5: L6-GalNac linker conjugated derivatives of ALDH2-hcm6 and ALDH2-hcm9 with the indicated modification pattern tested in vivo in mice are shown SEQ
Duplex sequence and chemistry ID
ID top: first strand, bottom: second strand, both 5%3' NO:
S1S22006L6 mU(ps)fC(ps)mUfUmCfUmUfAmAfAmCfUmGfAmGfUmU(ps)fU(ps)mC

S123(ps)3 ST 43(ps)fGmAfAmAfCmUfCmAfGmUfUmUfAmAfGmAfAmGfA

STS22006V1L6 mU(ps)fC(ps)mUfUmCfUmUfAmAfAmCfUmGfAmGfUmU(ps)fU(ps)mC

S123(ps)3 ST 43(ps)mGmAmAmAmCmUfCfAfGmUmUmUmAmAmGmAmAmGivA 129 S1S22009L6 mA(ps)fU(ps)mGfUmAfGmCfCmGfAmGfGmAfUmCfUmU(ps)fC(ps)mU

S123(ps)3 ST 43(ps)fAmGfAmAfGmAfUmCfCmUfCmGfGmCfUmAfCmAfU

STS22009V1L6 mA(ps)fU(ps)mGfUmAfGmCfCmGfAmGfGmAfUmCfUmU(ps)fC(ps)mU

S123(ps)3 ST 43(ps)mAmGmAmAmGmAfUfCfCmUmCmGmGmCmUmAmCmAivA 133 S1S22009V2L6 mA(ps)fU(ps)mGmUmAmGmCmCmGmAmGmGmAfUmCmUmU(ps)mC(ps)mU

S123(ps)3 ST 43(ps)mAmGmAmAmGmAfUfCfCmUmCmGmGmCmUmAmCmAivA 135 mA, mU, mC, mG ¨ 2'-0Me RNA
fA, fU, fC, fG ¨ 2`-F RNA
ivA - inverted RNA (3'-3') (ps) ¨ phosphorothioate Table 5 Example 3 Dose responses of ALDH2-siRNA molecules in human hepatocarinoma HepG2 cells were measured as follows.
Cells 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 pg/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. IC50 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 1nM of ALDH2-hcm-2 siRNA or an siRNA
against firefly luciferase as a non-targeting control (Fluc) using 1pg/m1 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.
For Western Blotting, cells were lysed in 500 pl lysis buffer (Tris/HCI, 15%
Glycerin, 1%
Triton). Protein amount was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific), and 30 pg per sample were used for electrophoresis in Laemmli buffer.
Membranes were probed with antibodies against ALDH2 (Abcam #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 Mitochondria! Aldehyde Dehydrogenase Activity Assay Kit (Abcam) according to manufacturer's protocol. Results are shown in Figure 3.
Example 5 Dose-response for GaINAc- L1-linker-coupled siRNAs against Aldh2 mRNA in primary mouse hepatocytes was measured as follows.
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-GaINAc-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 gRT-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 GaINAc- 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). GaINAc-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 gRT-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.
Example 7 Reduction of ALDH2-mRNA by 15 nM of GaINAc-L1 linker-coupled ALDH2-hcm-2 siRNA
in primary hepatocytes of different species indicated 6 days post treatment was measured as follows.

Hepatocytes (Thermo Fisher) were plated at a density of 250.000 (human and cynomolgus) or 150.000 (mouse) cells per well on collagen-coated 24-well plates (Life Technologies). L1-GaINAc conjugated ALDH2-hcm-2 or non-silencing control siRNA
were added at 15 nM concentration in triplicates immediately after plating. 6 days post treatment, cells were lysed with InviTrap Spin Cell RNA Mini Kit (Stratec).
ALDH2 mRNA
levels of the different species were determined by qRT-PCR relative to geometric means of ACTIN and APOB expression (human and cynomolgus) or to Actin alone (mouse) as housekeeper. Means and SD of normalized triplicate values are shown. Results are shown in Figure 6.
Example 8 Comparison of ALDH2 mRNA knockdown by GaINAc-conjugated ALDH2 siRNAs with linkers L1 and L6 in human primary hepatocytes was measured as follows.
Human hepatocytes (Thermo Fisher) were seeded at 30.000 cells per well in collagen-coated 96-well plates (Life Technologies). Immediately after seeding, GaINAc-conjugated siRNAs were added as indicated in graphs. 24 hours post treatment, cells were lysed with with InviTrap Spin Cell RNA Mini Kit (Stratec). ALDH2 mRNA levels were determined by qRT-PCR relative to geometric means of ACTIN and APOB expression as housekeepers.
Values were normalized to the amount of ALDH2 mRNA in untreated cells. Means and SD
of normalized triplicate values are shown. IC50 values and 90% confidence intervals were calculated. Results are shown in Figure 7.
Example 9 Activity of GaINAc-siRNAs against ALDH2 in vivo was measured as follows.
Male mice (C5761/6J) 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).
A -RNA was extracted from 10 mg of liver tissue using an InviTrap Spin Tissue RNA Mini Kit (Stratec). Aldh2 mRNA levels were determined by qRT-PCR relative to the geometric means of Actin and ApoB (mouse) as housekeepers. Values were normalized to the amount of Aldh2 mRNA in mice treated with the negative control siRNA (GN-Dmcn).

Means and SD of normalized values are shown as well as values for individual animals.
Measurements of untreated control mice (PBS) is shown. Results are shown in Figure 8A.
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 pg per sample were used for electrophoresis in Laemmli buffer. Membranes were probed with antibodies against ALDH2 (Abcam #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 Mitochondria! Aldehyde Dehydrogenase Activity Assay Kit (Abcam) according to manufacturer's protocol. Results are shown in Figure 8B.
C - Livers from mice untreated (PBS) or treated with the indicated GaINAc-siRNAs (n=6) at the indicated doses for 10 days were analysed for its amount of hepatic acetaldehyde.
Acetaldehyde content was measured in whole liver tissue homogenates (100 mg) in aqueous solution using an Agilent gas chromatography (GC) 6890 and 7890 series system coupled with a 5975 C MS detector (Agilent), MSD Chemstation (Agilent) and Maestro software for data analyses. Internal standard was 2-propanol and the following settings were applied: Incubation: 70 C for 30 min; Syringe temp.: 80 C;
Injector temp.:
200 C; Injection mode: SHS, Split 1/5; Injection volume: 1 ml (gas syringe);
Column: DB-WAX MS, 30m x 0.25 mm; 1pm; Helium Flow: 1 ml/min; Oven : 37 C for 5 min then 30 C/min to 120 C for 1 min; MS Transfer Line: 240 C; Incubation solvent:
Purified Water;
MS detection: El - Scan from 10 to 300 uma. Individual measurements as well as mean +/-SD are depicted. Results are shown in Figure 8C.
Example 10 Activity of GaINAc-siRNAs against Aldh2 in vivo ¨ duration and dose-response.
Male mice (C5761/6J) 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 Tissue RNA Mini Kit (Stratec). Aldh2 mRNA levels were determined by gRT-PCR
relative to the geometric means of Actin and ApoB expression (mouse) as housekeepers.
Values were normalized to the amount of Aldh2 mRNA in mice treated with the non-silencing control siRNA. Means and SD of normalized values are shown as well as values for individual animals. Results are shown in Figure 9.
Example 11 Different siRNA duplexes containing inverted RNA nucleotides at both 3`-ends were tested for serum stability. 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 pM
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 11.
Example 12:
The influence of inverted A, U, C and G RNA nucleotides at 3'-overhang positions on the efficacy of ALDH2 mRNA knockdown was analyzed using derivatives of the siRNA
ALDH2-hcm2 (see Table 3). ALD01 contains phosphorothioates at all termini, whereas 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 pg/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 gRT-PCR.
Each bar represents mean SD of three technical replicates normalized to the house keeping gene expression (ACTIN). Results are shown in Figure 12.

Example 13 The influence of inverted A, U, C and G RNA nucleotides at terminal 3' positions on the efficacy of ALDH2 mRNA knockdown was analyzed using derivatives of the siRNA
ALDH2-hcm2 (see Table 3). ALD01 contains phosphorothioates at all termini, whereas 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 pg/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 gRT-PCR. Each bar represents mean SD of three technical replicates normalized to the house keeping gene expression (ACTIN). Results are shown in Figure 13.
Example 14 Different GaINAc-L6-linker conjugates of ALDH2-hcm2 containing inverted RNA
nucleotides (see Table 4) were tested for serum stability. 5T522002L6 contains phosphorothioates at all non-conjugated ends, whereas 5T522002V1-L6 and 5T522002V2-L6 contain inverted RNA nucleotides at the second strand's 3`-end, where the respective inverted nucleotide is present instead of the last nucleotide.

L6 and 5T522002V4-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 5T522002V1-L6 and 5T522002V3-L6, whereas ivG was used in 5T522002V2-L6 and 5T522002V4-L6. All inverted RNA nucleotides substitute for terminal phosphorothioates used in other derivatives for stabilization.

and 5T522002V2-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 GaINAc-siRNA conjugates which were incubated at 37 C at 5 pM 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 14.

Example 15 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, 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 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. Cells were seeded at a density of 20,000 cells per 96-well, treated with the indicated doses from 125 nM to 0.04 nM of the respective indicated siRNA conjugates directly after plating. Non-treated cells and a control siRNA
targeting firefly luciferase at 10 nM (Luc) were included. Cells were lysed after 24 h.
Total RNA was extracted and Aldh2 and Actin mRNA levels were determined by Taqman gRT-PCR.
Each bar represents mean SD of three biological replicates normalized to the house keeping gene expression (Actin). Results are shown in Figure 15.
Example 16 The influence of ivA at the first strand's 3`-end on the efficacy of ALDH2 mRNA
knockdown was analyzed in vivo. Therefore, GaINAc-L6 derivatives of ALDH2-hcm6 and ALDH2-hcm9 siRNA conjugates targeting Aldh2 were used in an in vivo efficacy study.
5T522002L6 contains phosphorothioates at all non-conjugated termini, whereas 5T522002V3-L6 contains an ivA at the first strand's 3`-end in addition to the last nucleotide. C57BL/6 male mice (n=6) were treated subcutaneously with vehicle (PBS), 10 mg/kg and 3 mg/kg of the indicated GaINAc-L6 conjugate, respectively. Seven days after treatment, at necropsy liver tissue was harvested and total RNA was extracted.
Aldh2 and ApoB mRNA levels were analyzed by Taqman gRT-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 Different modified derivatives of the GaINAc-siRNA conjugates of ALDH2-hcm6 and ALDH2-hcm9 (Table 5) were analyzed for knockdown efficacy on ALDH2 mRNA
expression levels in vivo in mice. "V1" variants contain a different 2`-0Me/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`-0Me/2`-F modification pattern in the first strand as specified in Table 5. C57BL/6 male mice (n=6) were subcutaneously treated with vehicle (PBS) or 3 mg/kg and 1 mg/kg GaINAc-L6 conjugate derivatives, respectively.
Nine days after treatment, at necropsy liver tissue was harvested and total RNA was extracted. Aldh2 and ApoB mRNA levels were analyzed by Taqman gRT-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.
Statistical analysis is based on Kruskal-Wallis test with Dunn's multiple comparisons test respective to the control group (PBS). Results are shown in Figure 17.
Example 18 - Synthesis of conjugates 1 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. GaINAc conjugation was achieved by peptide bond formation of a GaINAc-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 2"0-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:
0.1M 12 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.
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 Hoevelmann etal. (Chem. Sci., 2016, 7, 128-135)). Tri-antennary GaINAc clusters (ST23/C4XLT) were introduced by successive coupling of the respective trebler amidite derivatives (C4XLT-phos) followed by the GaINAc 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.
Individual single strands were dissolved in a concentration of 60 OD/mL in H20. Both individual oligonucleotide solutions were added together in a reaction vessel.
For easier reaction monitoring a titration was performed. The first strand was added in
25% excess over the second strand as determined by UV-absorption at 260nm. The reaction mixture was heated to 80 C for 5min and then slowly cooled to RT. Double strand formation was monitored by ion pairing reverse phase HPLC. From the UV-area of the residual single strand the needed amount of the second strand was calculated and added to the reaction mixture. The reaction was heated to 80 C again and slowly cooled to RT. This procedure was repeated until less than 10% of residual single strand was detected.
Synthesis of compounds 2-10 Compounds 2 to 5 and (S)-DMT-Serinol(TFA)-phosphoramidite 7 were synthesised according to literature published methods (Hoevelmann et al. Chem. Sci., 2016,7, 128-135).
(S)-4-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(2,2,2-trifluoroacetamido)propoxy)-4-oxobutanoic acid (6).
To a solution of 5 in pyridine was added succinic anhydride, followed by DMAP.
The resulting mixture was stirred at room temperature overnight. All starting material was consumed, as judged by TLC. The reaction was concentrated. The crude material was chromatographed in silica gel using a gradient 0% to 5% methanol in DCM (+ 1%
triethylamine) to afford 1.33 g of 6 (yield = 38%). m/z (ESI-): 588.2 (100%), (calcd. for C30H29F3N08- [M-H] 588.6). 1 H-NMR: (400 MHz, CDCI3) 6 [ppm] = 7.94 (d, 1H, NH), 7.39 - 7.36 (m, 2H, CHary1), 7.29 - 7.25 (m, 7H, CHary1), 6.82-6.79 (m, 4H, CHary1), 4.51 ¨
4.47 (m, 1H), 4.31 ¨4.24 (m, 2H), 3.77 (s, 6H, 2xDMTr-OMe), 3.66 - 3.60 (m, 16H, HNEt3+), 3.26 - 3.25 (m, 2H), 2.97 ¨ 2.81 (m, 20H, NEt3), 2.50-2.41 (4H, m), 1.48¨ 1.45 (m, 26H, HNEt3+), 1.24 - 1.18 (m, 29H, NEt3).
(S)-DMT-Serinol(TFA)-succinate-lcaa-CPG (10) The (S)-DMT-Serinol(TFA)-succinate (159 mg, 270 umol) and HBTU (113 mg, 299 umol) were dissolved in CH3CN (10 mL). Diisopropylethylamine (DIPEA, 94 pL, 540 umol) was added to the solution, and the mixture was swirled for 2 min followed by addition native amino-lcaa-CPG (500 A, 3 g, amine content: 136 umol/g). The suspension was gently shaken at room temperature on a wrist-action shaker for 16h then filtered, and washed with DCM and Et0H. The solid support was dried under vacuum for 2 h. The unreacted amines on the support were capped by stirring with acetic anhydride/lutidine/N-methylimidazole at room temperature. The washing of the support was repeated as above. The solid was dried under vacuum to yield solid support 10 (3 g, 26 umol/g loading).
GaINAc Synthon (9) Synthesis of the GaINAc 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.
The characterising data matched the published data.
Synthesis of Oligonucleotides All single stranded oligonucleotides were synthesised according to the reaction conditions described above and in Figure 25 and 26.
All final single stranded products were analysed by AEX-H PLC 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 (non-modified, amino-modified precursors or GaINAc conjugated oligonucleotides) was proved by LC-MS
analysis.
Table 7: Single stranded un-conjugated oligonucleotides Product Name MW MW (ESI-) YoFLP

(11) calc. found (AEX-HPLC) A0002 STS16001A 6943.3 Da 6943.0 Da 86.6%
A0006 STS16001BL4 8387.5 Da 8387.5 Da 94.1%
A0130 STS18001A 6259.9 Da 6259.8 Da 76.5%
A0131 STS18001BL4 7813.2 Da 7813.1 Da 74.3%
A0220 STS16001B-5'1xNH2 6982.2 Da 6982.1 Da 95.7%
A0237 STS16001A 6943.3 Da 6943.3 Da 95.6%
A0244 STS16001BV1 6845.2 Da 6844.9 Da 98.2%
A0264 STS16001AV4-3'1xN H2 7112.4 Da 7112.2 Da 95.4%
A0329 STS16001BV6-3'5'1xNH2 7183.3 Da 7183.2 Da 88.8%
51 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. For example, 1x3'NH2 on A0264 means there is free amino group which can be reacted with GaINAc synthon 9 at the 3' end of the strand A0264. 3'5'1xNH2 means there is one serinol-derived free amino group which can be reacted with GaINAc linker 9 at the 3' end and the 5' end of the strand.
Synthesis of conjugates 1-3 and reference conjugates 1-2 Conjugated singles strands for conjugates 1-2 and reference conjugates 1-2 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 H20 (500 OD/mL) and DMSO (DMSO/H20, 2/1, v/v) was added, followed by DIPEA (2.5% of total volume). In a separate reaction vessel pre-activation of the GaIN(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. of HBTU in presence of 8 eq. DIPEA in DMSO. After 2 min the pre-activated compound 9 was added to the solution of the respective amino-modified precursor molecule. After 30 min the reaction progress was monitored by LCMS or AEX-HPLC. Upon completion of the conjugation reaction the crude product was precipitated by addition of 10x PrOH and 0.1x 2M NaCI and harvested by centrifugation and decantation. To set free the acetylated hydroxyl groups in the GaINAc moieties the resulting pellet was dissolved in 40% MeNH2 (1mL per 500 OD) and after 15 min at RT diluted in H20 (1:10) and finally purified again by anion exchange and size exclusion chromatography and lyophilised to yield the final product 12.
Table 8: Single stranded GaINAc-conjuqated oliqonucleotides Product Starting Name MW MW (ESI-) YoFLP
(12) Material calc. found (AEX-HPLC) A0241 A0220 STS16001BL20 7285.5 Da 7285.3 Da 91.8%
A0268 A0264 STS16001AV4L33 7415.7 Da 7415.4 Da 96.9%
A0330 A0329 STS16001BV6L42 7789.8 Da 7789.8 Da 95.5%
Double strand formation 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.
Table 9: Nucleic acid conjugates Product Starting Materials Name % double strand First Strand Second Strand Ref. Conj. 1 A0237 A0241 STS16001L20 97.7%
Ref. Conj. 2 A0268 A0244 STS16001L33 97.8%
Ref. Conj. 3 A0130 A0131 STS18001L4 96.8%
Ref. Conj. 4 A0002 A0006 STS16001L4 90.1%
Conjugate 1 A0268 A0241 STS16001L24 96.0%
Conjugate 2 A0237 A0330 STS16001V1L42 98.5%
Conjugate 3 A0268 A0330 STS16001V1L43 98.2%
Sequences Modifications key for the following sequences:
f denotes 2"Fluoro 2"deoxyribonucleotide or 2'-fluoro ribonucleotide (the terms are interchangeable) m denotes 2'0 Methyl ribonucleotide (ps) denotes phosphorothioate linkage Ser(GN) is a GaINAc-C4 building block attached to serinol derived linker moiety:
OH
HO ONH
NHAc wherein the 0--- is the linkage between the oxygen atom and e.g. H, phosphordiester linkage or phosphorothioate linkage.
C4XLT is:

s'soo ST23 is:
OAc OAc AcO
NHAc Synthesis of the phosphoramidite derivatives of C4XLT (C4XLT-phos) as well as (5T23-phos) can be performed as described in W02017/174657.
C4XLT-phos:
DMTroo JN1 CN

ST23-phos:
OAc OAc NHAc Conjugate 1 Antisense strand - 5T516001AL33 (SEQ ID NO: 136) 5' 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 (ps) Ser(GN) 3' Sense strand - STS16001BL20 (SEQ ID NO: 137) 5' Ser(GN) (ps) fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU (ps) mA
(ps) fA 3' Conjugate 2 Antisense strand - STS16001A (SEQ ID NO: 138) 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
Sense strand - STS16001BV1L42 (SEQ ID NO: 139) Ser(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA
fU
(ps) mA (ps) fA (ps) Ser(GN) Conjugate 3 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 fA mC fU mG (ps) fU
(ps) mU (ps) Ser(GN) 3' Sense strand - STS16001BV1L42 (SEQ ID NO: 139) 5' Ser(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU
mA fU
(ps) mA (ps) fA (ps) Ser(GN) 3' Reference conjugate 1 Antisense strand - STS16001A (SEQ ID NO: 138) 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
Sense strand ¨ STS16001BL20 (SEQ ID NO: 137) Ser(GN) (ps) fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU (ps) mA
(ps) fA
Reference conjugate 2 Antisense strand - 5T516001AL33 (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
(ps) Ser(GN) Sense strand - STS16001V1B (SEQ ID NO: 140) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU (ps) mA
(ps) fA
Reference Conjugate 3 Antisense strand - STS18001A (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 - STS18001BL4 (A0131; SEQ ID NO: 142) [(5T23) (ps)13 C4XLT (ps) fC mG fU mA fC mG fC mG fG mA fA mU fA mC fU mU fC
(ps) mG (ps) fA

Reference Conjugate 4 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
Sense strand - STS16001BL4 (SEQ ID NO: 143) 5"[(5T23) (ps)13 C4XLT(ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC
fU
mC fU mA fU (ps) mA (ps) fA
Example 19 ¨ In vitro determination of TTR knockdown of various TTR siRNA
GaINAc conjugates Murine primary hepatocytes were seeded into collagen pre-coated 96 well plates (Thermo Fisher Scientific, #A1142803) at a cell density of 30,000 cells per well and treated with siRNA-conjugates at concentrations ranging from 10nM to 0.0001nM. 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 (Ptenl I) were quantified by TaqMan analysis.
Target gene expression in primary murine hepatocytes 24h following treatment with the conjugates of the invention, Conjugates 1-3, showed that target gene expression decreases as the dose of the conjugate increased compared to the negative controls (see "UT" column and Reference Conjugate 3), as shown in Figure 27. This indicates that the first strand is binding to the target gene, thus lowering gene expression. Figure 27 also shows the target gene expression levels of Reference Conjugates 1 and 2 which act as comparator conjugates. As can be seen from a comparison between the data presented in Figures 27A
and 27C, and 27B and 27C, the conjugates of the invention (Conjugates 1-3) 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. The most effective conjugate at 0.1 nM, 0.5 nM, 1 nM and 10 nM appears to be Conjugate 3.
Example 20 ¨ In vivo time course of serum TTR in mice C57BL/6 mice were treated s.c. with 1mg/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, PALMS/lot 22, 2008003B) according to the manufacturers protocol (sample dilution 1:8000 or 1:800).

The results of the time course of serum TTR in c57BL/6 mice cohorts of n=4 at 7, 14, and 27 days post s.c. treatment with 1 mg/kg Conjugates 1-3, Reference Conjugates 1, 2 and 4, and mock treated (PBS) individuals is shown in Figure 28. As indicated by the data in Figure 28, 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.
Thus, it may be expected that 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 21 - Synthesis of conjugates 2 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. GaINAc conjugation was achieved by peptide bond formation of a GaINAc-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 2"0-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:
0.1M 12 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.

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 GaINAc clusters (ST23/C4XLT or ST23/C6XLT) were introduced by successive coupling of the respective trebler amid ite derivatives (C4XLT-phos or C6XLT-phos) followed by the GaINAc 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.
Individual single strands were dissolved in a concentration of 60 OD/mL in H20. Both individual oligonucleotide solutions were added together in a reaction vessel.
For easier reaction monitoring a titration was performed. The first strand was added in 25% excess over the second strand as determined by UV-absorption at 260nm. The reaction mixture was heated to 80 C for 5min and then slowly cooled to RT. Double strand formation was monitored by ion pairing reverse phase HPLC. From the UV-area of the residual single strand the needed amount of the second strand was calculated and added to the reaction mixture. The reaction was heated to 80 C again and slowly cooled to RT. This procedure was repeated until less than 10% of residual single strand was detected.
Synthesis of compounds 2-10 Compounds 2 to 5 and (S)-DMT-Serinol(TFA)-phosphoramidite 7 were synthesised according to literature published methods (Hoevelmann et al. Chem. Sci., 2016,7, 128-135).
(S)-4-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(2,2,2-trifluoroacetamido)propoxy)-4-oxobutanoic acid (6).
To a solution of 5 in pyridine was added succinic anhydride, followed by DMAP.
The resulting mixture was stirred at room temperature overnight. All starting material was consumed, as judged by TLC. The reaction was concentrated. The crude material was chromatographed in silica gel using a gradient 0% to 5% methanol in DCM (+ 1%
triethylamine) to afford 1.33 g of 6 (yield = 38%). m/z (ESI-): 588.2 (100%), (calcd. for C30H29F3N08- [M-H] 588.6). 1 H-NMR: (400 MHz, CDCI3) 6 [ppm] = 7.94 (d, 1H, NH), 7.39 - 7.36 (m, 2H, CHary1), 7.29 - 7.25 (m, 7H, CHary1), 6.82-6.79 (m, 4H, CHary1), 4.51 ¨
4.47 (m, 1H), 4.31 ¨4.24 (m, 2H), 3.77 (s, 6H, 2xDMTr-OMe), 3.66 - 3.60 (m, 16H, HNEt3+), 3.26 - 3.25 (m, 2H), 2.97 ¨ 2.81 (m, 20H, NEt3), 2.50-2.41 (4H, m), 1.48¨ 1.45 (m, 26H, HNEt3+), 1.24 - 1.18 (m, 29H, NEt3).
(S)-DMT-Serinol(TFA)-succinate-lcaa-CPG (10) The (S)-DMT-Serinol(TFA)-succinate (159 mg, 270 umol) and HBTU (113 mg, 299 umol) were dissolved in CH3CN (10 mL). Diisopropylethylamine (DIPEA, 94 pL, 540 umol) was added to the solution, and the mixture was swirled for 2 min followed by addition native amino-lcaa-CPG (500 A, 3 g, amine content: 136 umol/g). The suspension was gently shaken at room temperature on a wrist-action shaker for 16h then filtered and washed with DCM and Et0H. The solid support was dried under vacuum for 2 h. The unreacted amines on the support were capped by stirring with acetic anhydride/lutidine/N-methylimidazole at room temperature. The washing of the support was repeated as above. The solid was dried under vacuum to yield solid support 10 (3 g, 26 umol/g loading).
GaINAc Synthon (9) Synthesis of the GaINAc 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.
The characterising data matched the published data.
Synthesis of Oligonucleotides All single stranded oligonucleotides were synthesised according to the reaction conditions described above and in Figure 25 and 26.
All final single stranded products were analysed by AEX-H PLC 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 (non-modified, amino-modified precursors, C4XLT/5T23 or C6XLT/5T23 GaINAc conjugated oligonucleotides) was proved by LC-MS analysis.
Table 10: Single stranded un-conjugated and on-column conjugated oligonucleotides Product MW MW (ESI-) YoFLP
(11) calc. Found (AEX-HPLC) X0385A 6315.0 Da 6314.6 Da 91.0%
X0385B-prec 6593.1 Da 6593.1 Da 87.5%
X038BA 6315.0 Da 6314.6 Da 91.0%
X0386B-prec 6547.1 Da 6546.9 Da 87.5%

X0383A 6315.0 Da 6314.5 Da 91.9%
X0383B-prec 6508.8 Da 6508.6 Da 84.6%
X0371A 6416.1 Da 6416.1 Da 88.4%
X0371B-prec 6522.0 Da 6521.8 Da 91.9%
X0320A 6143.8 Da 6143.7 Da 94.6%
X0320B-prec 6665.0 Da 6664.8 Da 87.0%
X0477A 6143.8 Da 6143.4 Da 85.6%
X0477B-prec 6749.3 Da 6749.2 Da 83.1%
X0027A 6416.1 Da 6415.8 Da 92.8%
X0027B 7642.0 Da 7641.8 Da 88.2%
Synthesis of conjugates 1-3 and reference conjugates 1-2 Conjugated single strands for conjugates 1-2 and reference conjugates 1-2 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 H20 (500 OD/mL) and DMSO (DMSO/H20, 2/1, v/v) was added, followed by DIPEA (2.5% of total volume). In a separate reaction vessel pre-activation of the GaIN(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. of HBTU in presence of 8 eq. DIPEA in DMSO. After 2 min the pre-activated compound 9 was added to the solution of the respective amino-modified precursor molecule. After 30 min the reaction progress was monitored by LCMS or AEX-HPLC. Upon completion of the conjugation reaction the crude product was precipitated by addition of 10x PrOH and 0.1x 2M NaCI and harvested by centrifugation and decantation. To set free the acetylated hydroxyl groups in the GaINAc moieties the resulting pellet was dissolved in 40% MeNH2 (1mL per 500 OD) and after 15 min at RT diluted in H20 (1:10) and finally purified again by anion exchange and size exclusion chromatography and lyophilised to yield the final product 12.
Table 11: Single stranded GaINAc-conjugated oligonucleotides Product Starting MW MW (ESI-) YoFLP
(12) Material (11) calc. found (AEX-HPLC) X0385B X0385B-prec 7199.8 Da 7199.3 Da 93.2%
X0386B X0386B-prec 7153.8 da 7153.0 Da 86.2%
X0383B X0383B-prec 7115.5 Da 7115.4 Da 93.7%
X0320B X0320B-prec 7271.7 Da 7271.7 Da 90.0%
X0371B X0371B-prec 7128.8 Da 7128.3 Da 95.0%
X0477B X0477B-prec 7356.0 Da 7355.7 Da 91.4%

Double strand formation 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.
Table 12: Nucleic acid conjugates Product Starting Materials cyo First Second double Strand Strand strand X0385 X0385A X0385B 97.5%
X0386 X0386A X0386B 96.9%
X0383 X0383A X0383B 91.9%
X0371 X0371A X0371B 97.7%
X0027 X0027A X0027B 93.4%
X0320 X0320A X0320B 98.6%
X0477 X0477A X0477B 96.0%
Sequences Modifications key for the following sequences:
f denotes 2"Fluoro 2"deoxyribonucleotide or 2'-fluoro ribonucleotide (the terms are interchangeable) m denotes 2'0 Methyl ribonucleotide (ps) denotes phosphorothioate linkage Ser(GN) is a GaINAc-C4 building block attached to serinol derived linker moiety:
OH
HO ONH
NHAc wherein the 0--- is the linkage between the oxygen atom and e.g. H, phosphordiester linkage or phosphorothioate linkage.
C4XLT is:
C6XLT is:

---oo ST23 is:
OAc OAc 11(f Ac0 NHAc Synthesis of the phosphoramidite derivatives of C4XLT (C4XLT-phos), C6XLT
(C6XLT-phos) as well as 5T23 (5T23-phos) can be performed as described in W02017/174657.
C4XLT-phos:
DMTroc) )1\lj C6XLT-phos:
)1\1 DMIN,0,-...,...õ--,0,-,......õ 1 0-,....."..õ.õ-- ,P,.. ..-".......-CN

DMTr-,0,--........õ..--..õ0õ--5T23-phos:
OAc OAc ,...\,..C..)...\___ i Ac0 0(:),PCN
NHAc Example 22 GaINAc conjugates with each one serinol-linked GaINAc moiety at both termini of the second strand and with 2'-F at positions 7-9 of the second strand reduce ALDH2 mRNA
levels in vitro.
Different siRNA conjugates targeting human ALDH2 were tested for in vitro activity in mouse primary hepatocytes. Both conjugates X0320 and X0477 contain a similar first strand, which is modified with alternating 2'-0Me/2'-F. In both conjugates X0320 and X0477, serinol-linked GaINAc moieties are conjugated to both ends of the second strand.
In X0320, the second strand is modified with alternating 2'-F/2'-0Me. In X0477, the second strand is modified with 2'-0Me at positions 1-6 and 10-19 and with 2'-F
at positions 7-9. Both conjugates mediate dose-dependent reduction of Aldh2 target gene levels. "UT" indicates an untreated sample all other samples were normalized to. "Luc"
indicates a GaINAc-conjugated siRNA (X0028) targeting Luciferase, which was used as non-targeting control and does not reduce target mRNA levels.
The experiment was conducted in mouse primary hepatocytes. The cells were seeded at a density of 20,000 cells per 96-well and treated with 1 nM, 10 nM, and 100 nM
GaINAc conjugates directly after plating. Transfections with 10 nM GaINAc-siRNA and 1 pg/ml Atufect liposomal transfection reagent served as control. Cells were lysed after 24 h, total RNA was extracted and ALDH2 and ACTB mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean SD from three technical replicates.
Data are shown in Figure 30.
Sequence Table Single Sequence (A, first strand; B, second strand, both SEQ ID
Duplex strands 5'-3`) NO:
X0028 X0028A mU (ps) fC (ps) mG fA mA fG mU fA mU fU mC fC 144 mG fC mG fU mA (ps) fC (ps) mG
X0028B [5T23(ps)]3 5T41(ps)fC mG fU mA fC mG fC mG fG 145 mA fA mU fA mC fU mU fC (ps) mG (ps) fA
X0320 X0320A mU (ps) fC (ps) mU fU mC fU mU fA mA fA mC fU 146 mG fA mG fU mU (ps) fU (ps) mC
X0320B Ser(GN) (ps) fG (ps) mA (ps) fA mA fC mU fC mA fG 147 mU fU mU fA mA fG mA fA (ps) mG (ps) fA (ps) Ser(GN) X0477 X0477A mU (ps) fC (ps) mU fU mC fU mU fA mA fA mC fU 148 mG fA mG fU mU (ps) fU (ps) mC
X0477B Ser(GN) (ps) mG (ps) mA (ps) mA mA mC mU fC fA 149 fG mU mU mU mA mA mG mA mA (ps) mG (ps) mA
(ps) Ser(GN) mA, mU, mC, mG 2`-0-Methyl RNA
fA, fU, fC, fG 2'-deoxy-2'-fluoro RNA
irA, irC, irU, irG inverted RNA (3'-3' or 5'-5') (ps) phosphorothioate Example 23 GaINAc 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.
All tested conjugates contain each one Serinol-linked GaINAc moiety at the 5' end and at the 3' end of the second strand. The siRNAs are modified with alternating 2'-0Me/2'-F and contain each two phosphorothioate internucleotide linkages at their 5' and 3' termini, if not stated differently. X0319 contains two phosphorothioate internucleotide linkages at the 5' end of the first strand. X0362 contains a vinylphosphonate modification at the first nucleotide and no phosphorothioate internucleotide linkages at the 5' end of the first strand. Both siRNA conjugates reduce Aldh2 target gene levels in vitro. "ut"
indicates an untreated sample, which the other samples were normalised to. "Luc" indicates an siRNA
targeting Luciferase (X0028), which was used as non-targeting control and does not reduce target mRNA levels.
The experiment was conducted in mouse primary hepatocates. 25,000 cells were seeded per 96-well and treated with 0.1 ¨ 100 nM GaINAc-conjugated siRNA directly after plating.
Cells were lysed after 24 h, total RNA was extracted and Aldh2 and ApoB mRNA
levels were determined by Taqman gRT-PCR. Each bar represents mean SD from three technical replicates.
Data are shown in Figure 31.
Example 24 GaINAc 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.
All tested conjugates contain each one Serinol-linked GaINAc moiety at the 5' end and at the 3' end of the second strand. The siRNAs are modified with alternating 2'-0Me/2'-F and contain each two phosphorothioate internucleotide linkages at their 5' and 3' termini, if not stated differently. X0320 contains two phosphorothioate internucleotide linkages at the 5' end of the first strand. X0363 contains a vinylphosphonate modification at the first nucleotide and no phosphorothioate internucleotide linkages at the 5' end of the first strand. Compared to X0320, X0363 shows improved reduction of Aldh2 target gene levels in vitro. "ut" indicates an untreated sample, which the other samples were normalised to.
"Luc" indicates an siRNA targeting Luciferase (X0028), which was used as non-targeting control and does not reduce target mRNA levels.
The experiment was conducted in mouse primary hepatocates. 25,000 cells were seeded per 96-well and treated with 0.1 ¨ 100 nM GaINAc-conjugated siRNA directly after plating.
Cells were lysed after 24 h, total RNA was extracted and Aldh2 and ApoB mRNA
levels were determined by Taqman gRT-PCR. Each bar represents mean SD from three technical replicates.
Data are shown in Figure 32.
Example 25 GaINAc 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.
Both tested siRNA conjugates contain each one Serinol-linked GaINAc moiety at the 5' end and at the 3' end of the second strand. The siRNAs are modified with alternating 2'-OMe/2'-F and contain each two phosphorothioate internucleotide linkages at their 5' and 3' termini, if not stated differently. X0319 contains two phosphorothioate internucleotide linkages at the 5' end of the first strand. X0362 contains a vinylphosphonate modification at the first nucleotide and no phosphorothioate internucleotide linkages at the 5' end of the first strand. Both GaINAc siRNA conjugates are stable for at least 72 hours.
To assess stability, 5 pM siRNA conjugate was incubated with acidic rat tritosome extract (pH 5) at 37 C for 0, 4, and 72 hours. After incubation, RNA was purified, separated on 20% TBE polyacrylamide gels and visualised by ethidium bromide staining.
Data are shown in Figure 33.

Example 26 GaINAc 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.
Both tested siRNA conjugates contain each one Serinol-linked GaINAc moiety at the 5' end and at the 3' end of the second strand. The siRNAs are modified with alternating 2'-OMe/2'-F and contain each two phosphorothioate internucleotide linkages at their 5' and 3' termini, if not stated differently. X0320 contains two phosphorothioate internucleotide linkages at the 5' end of the first strand. X0363 contains a vinylphosphonate modification at the first nucleotide and no phosphorothioate internucleotide linkages at the 5' end of the first strand. Both GaINAc siRNA conjugates are stable for at least 72 hours.
To assess stability, 5 pM siRNA conjugate was incubated with acidic rat tritosome extract (pH 5) at 37 C for 0, 4, and 72 hours. After incubation, RNA was purified, separated on 20% TBE polyacrylamide gels and visualised by ethidium bromide staining.
Data are shown in Figure 34.
Example 27 GaINAc 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.
All tested conjugates contain each one Serinol-linked GaINAc moiety at the 5' end and at the 3' end of the second strand. The siRNAs contain each two phosphorothioate internucleotide linkages at their 5' and 3' termini, if not stated differently. X0320 and X363 are modified with alternating 2'-0Me/2'-F. X0477 and X0478 are modified with alternating 2'-0Me/2'-F in the first strand and with 2'-0Me at positions 1-6 and 10-19 of the second strand and with 2'-F at positions 7-9 of the second strand. X0320 and X0477 contain two phosphorothioate internucleotide linkages at the 5' end of their first strands. X0363 and X0478 contains a vinylphosphonate modification at the first nucleotide and no phosphorothioate internucleotide linkages at the 5' end of the first strand.
Compared to X0320, X0363 reduced Aldh2 mRNA levels more. Compared to X0477, X0478 reduced Aldh2 mRNA levels more. "ut" indicates an untreated sample, which the other samples were normalised to. "Luc" indicates an siRNA targeting Luciferase (X0028), which was used as non-targeting control and does not reduce target mRNA levels.
The experiment was conducted in mouse primary hepatocates. 20,000 cells were seeded per 96-well and treated with 1 ¨ 100 nM GaINAc-conjugated siRNA directly after plating.
Cells were lysed after 24 h, total RNA was extracted and Aldh2 and Actb mRNA
levels were determined by Taqman gRT-PCR. Each bar represents mean SD from three technical replicates.
Data are shown in Figure 35.
Material & Methods:
Primer:
SEQ ID NO:
fw GGCAAGCCTTATGTCATCTCGT 32 ALDH2 rev GGAATGGTTTTCCCATGGTACTT 33 probe BHQ1-TGAAATGTCTCCGCTATTACGCTGGCTG-FAM 150 fw AAAGAGGCCAGTCAAGCTGTTC 53 ApoB rev GGTGGGATCACTTCTGTTTTGG 54 probe BHQ1-CAGCAACACACTGCATCTGGTCTCTACCA-VIC 151 fw CACCGCCAAATTTAACTGCAGA 50 PTEN rev AAGGGTTTGATAAGTTCTAGCTGT 51 probe VIC
Sequences SEQ ID
duplex Strand Sequence (A first stand; B, second stand, both 6-3`) NO:
X0319A mA(ps)fA(ps)mUfG mUfU mUfU mCfC mUfG mCfU mGfAmC 152 (ps)fG(ps)mG

Ser(GN)(ps)fC (ps) mC (ps)fG mU fC mAfG mCfA mGfG mAfA mA 153 fA mCfA (ps)mU (ps)fU (ps)Ser(GN) (vp)-mUfAmUfG mUfU mUfU mCfCmUfG mCfU mGfAmC(ps) 154 fG(ps)mG

Ser(GN)(ps)fC (ps) mC (ps)fG mU fC mAfG mCfA mGfG mAfA mA 155 fA mCfA (ps)mU (ps)fA (ps)Ser(GN) X0320A mU (ps)fC (ps)mUit1 mCU mUfAmAfAmCfU mGfAmGU mU 156 (ps)1U (ps)mC

Ser(GN)(ps)fG (ps)mA (ps)fA mAfC mU C mAfG mUIU mU fA mA 147 fG mAfA (ps) mG (ps)fA (ps)Ser(GN) (vp)-mUfCmUit1 mCU mUfAmAfAmCU mGfAmGIU mU (ps)1U 157 X0363 (ps)mC
Ser(GN)(ps)fG (ps)mA (ps)fA mAfC mU C mAfG mUIU mU fA mA 147 fG mAfA (ps) mG (ps)fA (ps)Ser(GN) X0028 X0028A mU (ps)fC (ps) mGfA mAfG mUfAmUit1 mCfCmGfCmGU mA 141 (ps)fC(ps)mG

X0028B [ST23(ps)]3 ST41(ps)fC mGfU mAfC mGfC mGfG mAfA mU fA mC 142 11_1 mUfC (ps)mG (ps)fA
X0477A mU (ps)fC (ps) mU 11_1 mCU mUfAmAfAmCfU mGfAmGU mU 153 X0477 (ps)1U (ps)mC
X0477B Ser(GN)(ps)mG (ps)mA (ps)mA mA mC mUfCfAfG mU mU mU 158 mAmAmG mAmA(ps)mG (ps)mA(ps)Ser(GN) (vp)-mUfCmUit1 mCU mUfAmAfAmCU mGfAmGIU mU (ps)1U X0478A 157 X0478 (ps)mC
X0478B Ser(GN)(ps)mG (ps)mA (ps)mA mA mC mUfCfAfG mU mU mU 158 mAmAmG mAmA(ps)mG (ps)mA(ps)Ser(GN) Legend mA, mU, mC, mG 2`-0-Methyl RNA
fA, fU, fC, fG 2'-deoxy-2'-fluoro RNA
(ps) phosphorothioate (vp)-mU (E)-vinylphosphonate mU
Example 28 Improved duration of Aldh2 mRNA knockdown in mice following s.c.
administration of 1mg/kg ALDH2-hcm9 siRNA conjugate 16 with 5' & 3' serinol-GaINAc second strand conjugation as compared to conjugate 14 with 5' second strand triantennary GaINAc-conjugation.
The siRNAs are modified with alternating 2'-0Me/2'-F and contain each two phosphorothioate (PS) internucleotide linkages at their 5' and 3' terminal two internucleotide linkages. In conjugate 16 two serinol-GaINAc units are attached via a PS-bonds to the Sand 3' of the second strand. In conjugate 14 the two terminal 5' internucleotide linkages of the second are phosphodiesters and a triantennary GaINAc linker is attached via a PS bond to this 5' end.
In vivo activity was assessed by s.c. application of 1mg/kg siRNA to c57BL/6 mice. The vehicle control was PBS. At indicated time points following treatment animals were sacrificed, RNA isolated from liver tissue and transcript levels quantified by MultiPLEX RT-qPCR. The Aldh2 transcript ct-value for each treatment group was normalized to the Pten transcript ct value (Act) and to the PBS cohort (8, A ct).
Data are shown in Figure 36.

Material & Methods:
siRNAs SEQ
name batch strand sequence ID NO:
X0025A mA (ps) fU (ps) mG fU mA fG mC fC mG fA 159 Reference mG fG mA fU mC fU mU (ps) fC (ps) mU
Conjugate X0025 [ST23 (ps)]3 ltrb (ps) fA mG fA mA fG mA fU

7 X0025B mC fC mU fC mG fG mC fU mA fC (ps) mA
(ps) fU
X0321A mA (ps) fU (ps) mG fU mA fG mC fC mG fA 161 mG fG mA fU mC fU mU (ps) fC (ps) mU
Conjugate X0321 Ser(GN) (ps) fA (ps) mG (ps) fA mA fG mA 162 X0321B fU mC fC mU fC mG fG mC fU mA fC (ps) mA (ps) fU (ps) Ser(GN) Legend mA, mU, mC, mG 2`-0-Methyl RNA
fA, fU, fC, fG 2'-deoxy-2'-fluoro RNA
(ps) phosphorothioate (po) phosphodiester Primer:
SEQ ID NO:
fw GGCAAGCCTTATGTCATCTCGT 32 ALDH2 rev GGAATGGTTTTCCCATGGTACTT 33 probe BHQ1-TGAAATGTCTCCGCTATTACGCTGGCTG-FAM 150 General Methods In vitro experiments Primary murine hepatocytes (Thermo Scientific: GIBCO Lot: #MC798) were thawed and cryo-preservation medium exchanged for Williams E medium supplemented with 5%
FBS, 1 pM dexamethasone, 2 mM GlutaMax, 1% PenStrep, 4mg/m1 human recombinant insulin, 15mM Hepes. Cell density was adjusted to 250000 cells per lml. 100p1 per well of this cell suspension were seeded into collagen pre-coated 96 well plates. The test article was prediluted in the same medium (5 times concentrated) for each concentration and 25p1 of this prediluted siRNA or medium only were added to the cells. Cells were cultured at 37 C
and 5% CO2. 24 h post treatment the supernatant was discarded, and cells were washed in cold PBS and 250 pl RNA- Lysis Buffer S (Stratec) was added. Following 15 min incubation at room temperature plates were storage at -80 C until RNA
isolation according to the manufacturers protocol.

TaqMan analysis For mTTR & PTEN MultiPlex TaqMan analysis 10p1 isolated RNA for each treatment group were mixed with 10 pl PCR mastermix (TAKYON low Rox) containing 600 nM mTTR-primer, 400 nM ApoB-primer and 200nM of each probe as well as 0.5 units Euroscript 11 RT
polymerase with 0.2 units RNAse inhibitor. TaqMan analysis was performed in 384-well plate with a 10 min RT step at 48 C, 3 min initial denaturation at 95 C and 40 cycles of 95 C
for 10 sec and 60 C for 1 min. The primers contain two of BHQ1, FAM and YY, one at each end of the sequence.
For TMPRSS6 & ApoB MultiPlex TaqMan analysis 10 pl isolated RNA for each treatment group were mixed with 10 pl PCR mastermix (TAKYON low Rox) containing 800 nM
TMPRSS6 primer, 100 nM ApoB primer and 200 nM of either probe as well as 0.5 units Euroscriptll RT polymerase with 0.2 units RNAse inhibitor. TaqMan analysis was performed in 384-well plate with a 10min RT step at 48 C, 3min initial denaturation at 95 C and 40 cycles of 95 C for 10 sec and 60 C for 1 min.
In vivo experiments To compare in vivo potency of different siRNA conjugates 1 mg/kg siRNA
dissolved in PBS
was administered sub cutaneous in the scapular region of c57BL/6 mice. Cohorts of of n=6 for were treated with siRNA targeting Aldh2 or Tmprss6 at day 1 and sacrificed at selected times points post treatment. Liver samples were snap frozen in liquid nitrogen and stored at -80 C until extraction RNA with InviTrap Spin Tissue RNA Mini Kit (stratec) according to the manufacturers manual. Following, transcript level of Aldh2, Tmprss6 and Pten were quantified as described above.
Tritosome stability assay To probe for RNAase stability in the endosomal / lysosomal compartment of hepatic cells in vitro siRNA was incubated for 0 h, 4 h, 24 h or 72 h in Sprague Dawley Rat Liver Tritosomes (Tebu- Bio, CatN.: R0610.LT, lot: 1610405, pH: 7.4, 2.827 Units/m1). To mimic the acidified environment the Tritosomes were mixed 1:10 with low pH buffer (1.5M acetic acid, 1.5M
sodium acetate pH 4.75). 30 pl of this acidified Tritosomes. Following 10p1 siRNA (20pM) were mixed with and incubated for the indicated times at 37 C. Following incubation RNA
was isolated with the Clarity OTX Starter Kit-Cartriges (Phenomenex CatNo: KSO-8494) according to the manufactures protocol for biological fluids. Lyophilized RNA
was reconstituted in 30 pl H20, mixed with 4xloading buffer and 5 pl were loaded to a 20% TBE-polyacrylamide gel electrophoresis (PAGE) for separation qualitative semi-quantitative analysis. PAGE was run at 120 V for 2 h and RNA visualized by Ethidum-bromide staining with subsequent digital imaging with a Biorad Imaging system.
Example 29 - Synthesis of conjugates 3 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. GaINAc conjugation was achieved by peptide bond formation of a GaINAc-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 2"0-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), Fmoc-Amino-DMT C-7 CE phosphoramidite (GlyC3Am), 3'-Amino Modifier C-3 lcaa CPG 500A (C3Am), Fmoc-Amino-DMT C-3 CED
phosphoramidite (C3Am) and TFA-Amino C-6 CED phosphoramidite (C6Am) (Chemgenes), 3'-Amino-Modifier C7 CPG (C7Am) (Glen Research), Non-nucleosidic TFA amino Phosphoramidite (Pip), Non-nucleosidic TFA amino Solid Support (PipAm) (AM Chemicals) 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:
0.1M 12 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.
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 Hoevelmann et al. (2016)). Tri-antennary GaINAc clusters (ST23/C4XLT) were introduced by successive coupling of the respective trebler amid ite derivatives (C4XLT-phos) followed by the GaINAc amid ite (ST23-phos).
Attachment of amino modified moieties (non-serinol-derived linkers) was achieved by use of either the respective commercially available amino modified building block CPG or amidite.
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.
Individual single strands were dissolved in a concentration of 60 OD/mL in H20. Both individual oligonucleotide solutions were added together in a reaction vessel.
For easier reaction monitoring a titration was performed. The first strand was added in 25% excess over the second strand as determined by UV-absorption at 260nm. The reaction mixture was heated to 80 C for 5min and then slowly cooled to RT. Double strand formation was monitored by ion pairing reverse phase HPLC. From the UV-area of the residual single strand the needed amount of the second strand was calculated and added to the reaction mixture. The reaction was heated to 80 C again and slowly cooled to RT. This procedure was repeated until less than 10% of residual single strand was detected.
Synthesis of compounds 2-10 Compounds 2 to 5 and (S)-DMT-Serinol(TFA)-phosphoramidite 7 were synthesised according to literature published methods (Hoevelmann et al. Chem. Sci., 2016,7, 128-135).
(S)-4-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(2,2,2-trifluoroacetamido)propoxy)-4-oxobutanoic acid (6).
To a solution of 5 in pyridine was added succinic anhydride, followed by DMAP.
The resulting mixture was stirred at room temperature overnight. All starting material was consumed, as judged by TLC. The reaction was concentrated. The crude material was chromatographed in silica gel using a gradient 0% to 5% methanol in DCM (+ 1%
triethylamine) to afford 1.33 g of 6 (yield = 38%). m/z (ESI-): 588.2 (100%), (calcd. for C30H29F3N08- [M-H] 588.6). 1 H-NMR: (400 MHz, CDCI3) 6 [ppm] = 7.94 (d, 1H, NH), 7.39 - 7.36 (m, 2H, CHary1), 7.29 - 7.25 (m, 7H, CHary1), 6.82-6.79 (m, 4H, CHary1), 4.51 ¨
4.47 (m, 1H), 4.31 ¨4.24 (m, 2H), 3.77 (s, 6H, 2xDMTr-OMe), 3.66 - 3.60 (m, 16H, HNEt3+), 3.26 - 3.25 (m, 2H), 2.97 ¨ 2.81 (m, 20H, NEt3), 2.50-2.41 (4H, m), 1.48¨ 1.45 (m, 26H, HNEt3+), 1.24 - 1.18 (m, 29H, NEt3).
(S)-DMT-Serinol(TFA)-succinate-lcaa-CPG (10) The (S)-DMT-Serinol(TFA)-succinate (159 mg, 270 umol) and HBTU (113 mg, 299 umol) were dissolved in CH3CN (10 mL). Diisopropylethylamine (DIPEA, 94 pL, 540 umol) was added to the solution, and the mixture was swirled for 2 min followed by addition native amino-lcaa-CPG (500 A, 3 g, amine content: 136 umol/g). The suspension was gently shaken at room temperature on a wrist-action shaker for 16h then filtered, and washed with DCM and Et0H. The solid support was dried under vacuum for 2 h. The unreacted amines on the support were capped by stirring with acetic anhydride/lutidine/N-methylimidazole at room temperature. The washing of the support was repeated as above. The solid was dried under vacuum to yield solid support 10 (3 g, 26 umol/g loading).
GaINAc Synthon (9) Synthesis of the GaINAc 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.
The characterising data matched the published data.
Synthesis of Oligonucleotides All single stranded oligonucleotides were synthesised according to the reaction conditions described above and in Figure 25 and 26, and are outlined in Tables 13 and 14.
All final single stranded products were analysed by AEX-H PLC 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 (non-modified, amino-modified precursors or GaINAc conjugated oligonucleotides) was proved by LC-MS
analysis.
Table 13: Single stranded un-conjugated oligonucleotides Product Name MW MW (ESI-) YoFLP
(11) calc. found (AEX-HPLC) A0002 STS16001A 6943.3 Da 6943.0 Da 86.6%
A0006 STS16001BL4 8387.5 Da 8387.5 Da 94.1%
A0114 STS22006A 6143.8 Da 6143.7 Da 94.3%
A0115 STS22006BL1 7855.1 Da 7855.1 Da 92.8%
A0122 STS22009A 6260.9 Da 6260.6 Da 92.8%

A0123 STS22009BL1 7783.0 Da 7782.9 Da 87.1%
A0130 STS18001A 6259.9 Da 6259.8 Da 76.5%
A0131 STS18001BL4 7813.2 Da 7813.1 Da 74.3%
A0220 STS16001B-5'1xNH2 6982.2 Da 6982.1 Da 95.7%
A0237 STS16001A 6943.3 Da 6943.3 Da 95.6%
A0244 STS16001BV1 6845.2 Da 6844.9 Da 98.2%
A0264 STS16001AV4-3'1xN H2 7112.4 Da 7112.2 Da 95.4%
A0329 STS16001BV6-3'5'1xNH2 7183.3 Da 7183.2 Da 88.8%
A0560 STS16001A 6943.3 Da 6943.3 Da 96.7%
A0541 STS16001BV1-3'5'NH2 7151,3 Da 7151,0 Da 85,6%
A0547 STS16001BV16-3'5'NH2 7119,3 Da 7119,1 Da 89,9%
A0617 STS16001BV20-3'5'NH2 7087,3Da 7086,7 Da 90,1%
A0619 STS16001BV1-3'5'2xNH2 7521,3 Da 7521,3 Da 93.4%
A0680 STS16001A 6943.3 Da 6942.9 Da 91.2%
A0514 STS22006A 6143.8 Da 6143.7 Da 94.6%
A0516 STS22009BV11-3'5'NH2 6665.0 Da 6664.8 Da 87.0%
A0517 STS22009BV11-3'5'NH2 6593.0 Da 6593.0 Da 86.0%
A0521 STS12009BV1-3'5'NH2 6437,7 Da 6437.8 Da 91.1%
A0303 STS12209BL4 7665.0 Da 7664.9 Da 90.4%
A0304 STS12209A 6393.1 Da 6392.9 Da 77.6%
A0319 STS22009A 6260,9 Da 6260.5 Da 86.9%
A0353 STS12009A 6416.1 Da 6416.1 Da 94.1%
A0216 STS17001A 6178.8 Da 6178.7 Da 87.2%
A0217 STS17001BL6 7937.2 Da 7937.2 Da 78.3%
51 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. For example, 1x3'NH2 on A0264 means there is free amino group which can be reacted with GaINAc 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 GaINAc linker 9 at the 3' end and the 5' end of the strand.
Table 14: Single stranded oligonucleotides with 5' and 3' modifications Product Name 5'mod 3'mod MW MW (ESI-) YoFLP
calc. found (AEX-HPLC) A0561 STS16001BV1-3'5'1xNH2 C6Am GlyC3Am 7267.5 Da 7267.5 Da 66.7%
A0563 STS16001BV1-3'5'1xNH2 C3Am C3Am 7183.4 Da 7183.1 Da 75.1%
A0651 STS16001BV1-3'5'1xNH2 C6Am C7Am 7265.6 Da 7265.2 Da 99.8%
A0653 STS16001BV1-3'5'1xNH2 GlyC3Am GlyC3Am 7299.5 Da 7299.3 Da 88.1%
A0655 STS16001BV1-3'5'1xNH2 pipAm PipAm 7517.7 Da 7517.5 Da 89.8%
Similarly, 351 x NH2 refers to the position (3' and 5' end) and number (1 x NH2 each) of free amino groups which are available for conjugation. For example, 3'5'1xNH2 on A0561 means there are 2 free amino group (1 at the 3' AND 1 at the 5' end) which can be reacted with GaINAc synthon 9 at the 3' end of the strand A0561.
Synthesis of certain conjugates of the invention and reference conjugates 1-2 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 H20 (500 OD/mL) and DMSO (DMSO/H20, 2/1, v/v) was added, followed by DIPEA (2.5% of total volume). In a separate reaction vessel pre-activation of the GaIN(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. of HBTU in presence of 8 eq. DIPEA in DMSO. After 2 min the pre-activated compound 9 was added to the solution of the respective amino-modified precursor molecule. After 30 min the reaction progress was monitored by LCMS or AEX-HPLC. Upon completion of the conjugation reaction the crude product was precipitated by addition of 10x PrOH and 0.1x 2M NaCI and harvested by centrifugation and decantation. To set free the acetylated hydroxyl groups in the GaINAc moieties the resulting pellet was dissolved in 40% MeNH2 (1mL per 500 OD) and after 15 min at RT diluted in H20 (1:10) and finally purified again by anion exchange and size exclusion chromatography and lyophilised to yield the final product 12 (Table 15).
Table 15: Single stranded GaINAc-conjugated oligonucleotides Product Starting Name MW MW (ESI-) YoFLP
(12) Material calc. found (AEX-HPLC) A0241 A0220 STS16001BL20 7285.5 Da 7285.3 Da 91.8%
A0268 A0264 STS16001AV4L33 7415.7 Da 7415.4 Da 96.9%
A0330 A0329 STS16001BV6L42 7789.8 Da 7789.8 Da 95.5%
A0544 A0541 STS16001BV1L75 7757,9 Da 7757,7 Da 93.3%
A0550 A0547 STS16001BV16L42 7725,9 Da 7725.7 Da 88.5%
A0620 A0617 STS16001BV20L75 7693,91 Da 7693,2 Da 90.9%
A0622 A0619 STS16001BV1L94 8734,3 Da 8734,6 Da 82.9%
A0519 A0516 STS22006BV11L42 7271.7 Da 7271.7 Da 90.0%
A0520 A0517 STS22009BV11L42 7199.6 Da 7199.7 Da 92.9%
A0522 A0521 STS12009BV1L42 7044.4 Da 7044.4 Da 96.0%
A0603 A0602 STS20041BV1L42 7280.7 Da 7280.4 Da 93.4%
Synthesis of certain conjugates of the invention Conjugation of the GalNac synthon (9) was achieved by coupling to the amino function of the respective oligonucleotide strand 14 using a peptide coupling reagent.
Therefore, the respective amino-modified precursor molecule 14 was dissolved in H20 (500 OD/mL) and DMSO (DMSO/H20, 2/1, v/v) was added, followed by DIPEA (2.5% of total volume).
In a separate reaction vessel pre-activation of the GaIN(Ac4)-C4-acid (9) was performed by reacting 2 eq. (per amino function in the amino-modified precursor oligonucleotide 14) of the carboxylic acid component with 2 eq. of HBTU in presence of 8 eq. DIPEA in DMSO.
After 2 min the pre-activated compound 9 was added to the solution of the respective amino-modified precursor molecule. After 30 min the reaction progress was monitored by LCMS
or AEX-HPLC. Upon completion of the conjugation reaction the crude product was precipitated by addition of 10x PrOH and 0.1x 2M NaCI and harvested by centrifugation and decantation. To set free the acetylated hydroxyl groups in the GaINAc moieties the resulting pellet was dissolved in 40% MeNH2 (1mL per 500 OD) and after 15 min at RT
diluted in H20 (1:10) and finally purified again by anion exchange and size exclusion chromatography and lyophilised to yield the final product 15 (Table 16).
Table 16: Single stranded GaINAc-conjuqated olicionucleotides Product Starting Name MW MW (ESI-) YoFLP
(15) Material calc. found (AEX-HPLC) A0562 A0561 STS16001BV1L87 7874.2 Da 7874.0 Da 82.7%
A0564 A0563 STS16001BV1L88 7790.0 Da 7789.4 Da 90.4%
A0652 A0651 STS16001BV1L96 7872.2 Da 7871.8 Da 94.6%
A0654 A0653 STS16001BV1L97 7906.2 Da 7905.6 Da 89.9%
A0656 A0655 STS16001BV1L98 8124.3 Da 8124.0 Da 93.6%
Double strand formation 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 (Table 17).
Table 17: Nucleic acid conjugates Product Starting Materials Name % double strand First Strand Second Strand Ref. Conj. 1 A0237 A0241 STS16001L20 97.7%
Ref. Conj. 2 A0268 A0244 STS16001L33 97.8%
Ref. Conj. 3 A0130 A0131 STS18001L4 96.8%
Ref. Conj. 4 A0002 A0006 STS16001L4 90.1%
Ref. Conj. 5 A0216 A0217 STS17001L6 88.4%
Conjugate 1 A0268 A0241 STS16001L24 96.0%
Conjugate 2 A0237 A0330 STS16001V1L42 98.5%
Conjugate 3 A0268 A0330 STS16001V1L43 98.2%

Conjugate 4 A0560 A0544 STS16001V1L75 92.5%
Conjugate 5 A0560 A0550 STS16001V16L42 95.3%
Conjugate 6 A0237 A0620 STS16001V20L75 97.8%
Conjugate 7 A0237 A0622 STS16001V1L94 93.7%
Conjugate 8 A0680 A0652 STS16001V1L96 98.4%
Conjugate 9 A0680 A0654 STS16001V1L97 95.8%
Conjugate 10 A0680 A0656 STS16001V1L98 97.6%
Conjugate 11 A0560 A0564 STS16001V1L88 95.0%
Conjugate 12 A0237 A0562 STS16001V1L87 96.8%
Conjugate 13 A0114 A0115 STS22006L1 85.6%
Conjugate 14 A0122 A0123 STS22009L1 96.4%
Conjugate 15 A0514 A0519 STS22006V11L42 98.6%
Conjugate 16 A0319 A0520 STS22009V11L42 97.9%
Conjugate 17 A0304 A0303 STS12209L4 93.0%
Conjugate 18 A0353 A0522 STS12009V1L42 98.9%
Conjugate 19 A0601 A0603 STS20041BL42 97.6%
Sequences Modifications key for the following sequences:
f denotes 2"Fluoro 2"deoxyribonucleotide or 2'-fluoro ribonucleotide (the terms are interchangeable) m denotes 2'0 Methyl ribonucleotide (ps) denotes phosphorothioate linkage FAM = 6-Carboxyfluorescein BHQ = Black Hole Quencher 1 YY = Yakima Yellow Definitions Ser(GN) is a GaINAc-C4 building block attached to serinol derived linker moiety:
OH
HO.
ONH
HO"\I"====¨.\/
NHAc wherein the 0--- is the linkage between the oxygen atom and e.g. H, phosphordiester linkage or phosphorothioate linkage.
GN is:
OH
HO
NHAc C4XLT (also known as ST41) is:

---000 ..........õ,¨....õ..¨..õ.0,õ-ST23 is:
OAc OAc Ac0&1111L-00õ-NHAc Synthesis of the phosphoramidite derivatives of C4XLT (C4XLT-phos) as well as (5T23-phos) can be performed as described in W02017/174657.
C4XLT-phos:
DMTroo LI\I
DMTr(:)0 1 0õ,õ0,--.õ....

DMTroe 5T23-phos:
OAc OAc Ac0,. __...\,..C.)...\_ I
0(:),PCN
NHAc C3Am is: Itrb is: GlyC3Am is:
--'00 H
H
C6Am is: Pip Am is: C7Am is:
H 01,w, NG N'G
.7..N,G
H H

,00.
wherein G = H (pre conjugation) or G = GN (post conjugation).
Conjugate 1 Antisense strand - 5T516001AL33 (SEQ ID NO: 136) 5' 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 (ps) Ser(GN) 3' Sense strand - STS16001BL20 (SEQ ID NO: 137) 5' Ser(GN) (ps) fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU (ps) mA
(ps) fA 3' Conjugate 2 Antisense strand - STS16001A (SEQ ID NO: 138) 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
Sense strand - STS16001BV1L42 (SEQ ID NO: 139) Ser(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA
fU
(ps) mA (ps) fA (ps) Ser(GN) Conjugate 3 Antisense strand - 5T516001AL33 (SEQ ID NO: 136) 5' 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 (ps) Ser(GN) 3' Sense strand - STS16001BV1L42 (SEQ ID NO: 139) 5' Ser(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU
mA fU
(ps) mA (ps) fA (ps) Ser(GN) 3' Conjugate 4 Antisense strand - STS16001A (SEQ ID NO: 138) 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
Sense strand - STS16001BV1L75 (SEQ ID NO: 163) 5' Ser(GN) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU
(ps) mA (ps) fA Ser(GN) 3' Conjugate 5 Antisense strand - STS16001A (SEQ ID NO: 138) 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

Sense strand - 5T516001BV16L42 (SEQ ID NO: 164) 5' Ser(GN) (ps) fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU mA fA

(ps) Ser(GN) 3' Conjugate 6 Antisense strand - STS16001A (SEQ ID NO: 138) 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
Sense strand - 5T516001BV20L75 (SEQ ID NO: 165) 5' Ser(GN)fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU mA fA
Ser(GN) 3' Conjugate 7 Antisense strand - STS16001A (SEQ ID NO: 138) 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
Sense strand - STS16001BV1L94 (SEQ ID NO: 166) 5' Ser(GN) (ps) Ser(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG
mC fU
mC fU mA fU (ps) mA (ps) fA (ps) Ser(GN) (ps) Ser(GN) 3' Conjugate 8 Antisense strand - STS16001A (SEQ ID NO: 138) 5' 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 3' Sense strand - STS16001V1BL96 (SEQ ID NO: 167) 5' C6Am(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU
mA
fU (ps) mA (ps) fA (ps) C7Am(GN) 3' Conjugate 9 Antisense strand - STS16001A (SEQ ID NO: 138) 5' 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 3' Sense strand - STS16001V1BL97 (SEQ ID NO: 168) 5' GlyC3Am(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fU mC
fU
mA fU (ps) mA (ps) fA (ps) GlyC3Am(GN) 3' Conjugate 10 Antisense strand - STS16001A (SEQ ID NO: 138) 5' 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 3' Sense strand (SEQ ID NO: 169) 5' PipAm(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU
mA
fU (ps) mA (ps) fA (ps) PipAm(GN) 3' Conjugate 11 Antisense strand - STS16001A (SEQ ID NO: 138) 5' 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 3' Sense strand - STS16001V1BL88 (SEQ ID NO: 170) 5' C3Am(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU
mA
fU (ps) mA (ps) fA (ps) C3Am(GN) 3' Conjugate 12 Antisense strand - STS16001A (SEQ ID NO: 138) 5' 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 3' Sense strand - STS16001V1BL87 (SEQ ID NO: 171) 5' C6Am(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU
mA
fU (ps) mA (ps) fA (ps) GlyC3Am(GN) 3' Conjugate 15 Antisense strand (SEQ ID NO: 146) mU (ps) fC (ps) mU fU mC fU mU fA mA fA mC fU mG fA mG fU mU (ps) fU (ps) mC
Sense strand (SEQ ID NO: 147) Ser(GN) (ps) fG (ps) mA (ps) fA mA fC mU fC mA fG mU fU mU fA mA fG mA fA (ps) mG
(ps) fA (ps) Ser(GN) Conjugate 16 Antisense strand (SEQ ID NO: 159) mA (ps) fU (ps) mG fU mA fG mC fC mG fA mG fG mA fU mC fU mU (ps) fC (ps) mU
Sense strand (SEQ ID NO: 162) Ser(GN) (ps) fA (ps) mG (ps) fA mA fG mA fU mC fC mU fC mG fG mC fU mA fC (ps) mA
(ps) fU (ps) Ser(GN) Conjugate 18 Antisense strand (SEQ ID NO: 172) mA (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (ps) fG (ps) mA
Sense strand (SEQ ID NO: 173) Ser(GN) (ps) fU (ps) mC (ps) fA mC fC mU fG mC fU mU fC mU fU mC fU mG fG (ps) mU
(ps) fU (ps) Ser(GN) Conjugate 19 Antisense strand (SEQ ID NO: 174) mA (ps) fU (ps) mA fA mC fU mC fU mG fU mC fC mA fU mU fA mC (ps) fC (ps) mG
Sense strand (SEQ ID NO: 175) Ser(GN) (ps) fC (ps) mG (ps) fG mU fA mA fU mG fG mA fC mA fG mA fG mU fU (ps) mA
(ps) fU (ps) Ser(GN) Reference conjugate 1 Antisense strand - STS16001A (SEQ ID NO: 138) 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
Sense strand ¨ STS16001BL20 (SEQ ID NO: 137) Ser(GN) (ps) fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU (ps) mA
(ps) fA

Reference conjugate 2 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
(ps) Ser(GN) Sense strand - STS16001BV1 (SEQ ID NO: 140) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU (ps) mA
(ps) fA
Reference Conjugate 3 ¨ "Luc"
Antisense strand - STS18001A (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 - STS18001BL4 (A0131; SEQ ID NO: 142) [(5T23) (p5)13 C4XLT (ps) fC mG fU mA fC mG fC mG fG mA fA mU fA mC fU mU fC
(ps) mG (ps) fA
Reference Conjugate 4 Antisense strand - 5T516001AL33 (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
Sense strand - STS16001BL4 (SEQ ID NO: 143) 5"[(5T23) (p5)13 C4XLT(ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC
fU
mC fU mA fU (ps) mA (ps) fA
Reference Conjugate 5 ¨ "Ctr"
Antisense strand (SEQ ID NO: 176) mC (ps) fU (ps) mU fA mC fU mC fU mC fG mC fC mC fA mA fG mC (ps) fG (ps) mA
Sense strand (SEQ ID NO: 177) [(5T23) (ps)]3 (C6XLT) (ps) fU mC fG mC fU mU fG mG fG mC fG mA fG mA fG mU fA
(ps) mA (ps) fG
Reference Conjugate 6 Antisense strand (SEQ ID NO: 146) mU (ps) fC (ps) mU fU mC fU mU fA mA fA mC fU mG fA mG fU mU (ps) fU (ps) mC
Sense strand (SEQ ID NO: 178) [5T23 (ps)]3 Itrb (ps) fG mA fA mA fC mU fC mA fG mU fU mU fA mA fG mA fA (ps) mG
(ps) fA
Reference Conjugate 7 Antisense strand (SEQ ID NO: 159) mA (ps) fU (ps) mG fU mA fG mC fC mG fA mG fG mA fU mC fU mU (ps) fC (ps) mU
Sense strand (SEQ ID NO: 160) [5T23 (ps)]3 Itrb (ps) fA mG fA mA fG mA fU mC fC mU fC mG fG mC fU mA fC (ps) mA
(ps) fU
Reference Conjugate 8 Antisense strand (SEQ ID NO: 179) mU (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (ps) fG (ps) mA
Sense strand [5T23 (ps)]3 5T41 (ps)fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG fG (ps) mU
(ps) fA
Reference Conjugate 9 Antisense strand (SEQ ID NO: 174) mA (ps) fU (ps) mA fA mC fU mC fU mG fU mC fC mA fU mU fA mC (ps) fC (ps) mG
Sense strand (SEQ ID NO: 181) [5T23 (ps)]3 C6XLT (ps) fC mG fG mU fA mA fU mG fG mA fC mA fG mA fG mU fU
(ps) mA (ps) fU
Example 30 ¨ In vitro determination of TTR knockdown of various TTR siRNA
GaINAc conjugates Conjugates 1 to 3 Murine primary hepatocytes were seeded into collagen pre-coated 96 well plates (Thermo Fisher Scientific, #A1142803) at a cell density of 30,000 cells per well and treated with siRNA-conjugates at concentrations ranging from 10nM to 0.0001nM. 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 (Ptenl I) were quantified by TaqMan analysis.
Target gene expression in primary murine hepatocytes 24h following treatment at 0.01 nM, 0.1 nM, 0.5 nM, 1nM and 10nM with the conjugates of the invention, Conjugates 1-3, showed that target gene expression decreases as the dose of the conjugate increased compared to the negative controls (see "UT" column and Reference Conjugate 3), as shown in Figure 27. This indicates that the first strand is binding to the target gene, thus lowering gene expression. Figure 27 also shows the target gene expression levels of Reference Conjugates 1 and 2 which act as comparator conjugates. As can be seen from a comparison between the data presented in Figures 27A and 27C, and 27B and 27C, the conjugates of the invention (Conjugates 1-3) 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.
The most effective conjugate at 0.1 nM, 0.5 nM, 1 nM and 10 nM appears to be Conjugate 3.
Conjugates 4 to 7 The method described above under "In vitro experiments" in the General Method section was followed.
Target gene expression in primary murine hepatocytes 24h following treatment at 0.01 nM, 0.1 nM, 0.5 nM, 1nM and 10nM with the conjugates of the invention, Conjugates 4-7, showed that target gene expression decreases as the dose of the conjugate increased compared to the negative controls (see "UT" column and Luc [Reference Conjugate 3]), as shown in Figure 50. This indicates that the first strand is binding to the target gene, thus lowering gene expression.
The in vitro data show that in the context of one or two serinol-derived linker moieties being provided at 5' and 3' ends of the sense strand in Conjugates 4-7, the number of phosphorothioate (PS) bonds between the terminal nucleotide and the linker, and/or between the terminal three nucleotides in the sense strand, can be varied whilst maintaining efficacy for decreasing target gene expression.
Conjugates 8 to 12 and 19 The method described above under "In vitro experiments" in the General Method section was followed.

Target gene expression in primary murine hepatocytes 24h following treatment at 0.01 nM, 0.1 nM, 0.5 nM, 1nM and 10nM with the conjugates of the invention, Conjugates 8-12, showed that target gene expression decreases as the dose of the conjugate increased compared to the negative controls (see "UT" column and Luc [Reference Conjugate 3]), as shown in Figure 51. This indicates that the first strand is binding to the target gene, thus lowering gene expression. In particular, Conjugates 8, 9, 10 and 11 appear to be comparable to or better than Conjugate 2 which was previously shown to be the most effective conjugate at 0.01 nM.
Conjugate 19 was also shown to decrease target gene expression compared to the negative controls (see "UT" column and Ctr which is a non-targeting siRNA and also referred to as Reference Conjugate 5), as shown in Figure 52. This indicates that the first strand is binding to the target gene, thus lowering gene expression.
The in vitro data for Conjugates 8-12 and 19 show that a number of linkers which are structurally diverse and which are conjugated at both termini of the sense strand are effective at decreasing target gene expression. Conjugates 8-12 and 19 decrease target gene expression more effectively than "Luc" which is Reference Conjugate 3 (for Conjugates 8-12), "Ctr" which is Reference Conjugate 5 (for Conjugate 19) and untreated control.
Example 31 ¨ In vivo time course of serum TTR, ALDH2 and TMPRSS6 in mice Conjugates 1 to 3 C57BL/6 mice were treated s.c. with 1mg/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, PALMS/lot 22, 2008003B) according to the manufacturers protocol (sample dilution 1:8000 or 1:800).
The results of the time course of serum TTR in c57BL/6 mice cohorts of n=4 at 7, 14, and 27 days post s.c. treatment with lmg/kg Conjugates 1-3, Reference Conjugates 1, 2 and 4, and mock treated (PBS) individuals is shown in Figure 28. As indicated by the data in Figure 28, 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.
Thus, it may be expected that the dosing level of Conjugate 2 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.
More specifically, Conjugate 2 resulted in 3-fold lower target protein level in serum at day seven and 4-fold lower target protein level in serum at day 27 compared to Reference Conjugate 4 at equimolar dose in wild type mice. Furthermore, Conjugate 2 resulted in 85%
reduction of target serum protein level at day 27 after single injection, compared to 36%
reduction by equimolar amount of Reference Conjugate 4.
Conjugates 15 to 18 The method described above under "In vivo experiments" in the General Method section was followed.
The results of the time course of serum Aldh2 in c57BL/6 mice cohorts of n=6 at 14,28 and 42 days post s.c. treatment with 1 mg/kg Conjugates 15 and 16, Reference Conjugates 6 and 7, and mock treated (PBS) individuals is shown in Figures 53 and 54. As indicated by the data in Figures 53 and 54, the conjugates of the invention are particularly effective at reducing target gene expression compared to the negative control (PBS) and Reference Conjugates 6 and 7 respectively.
The results of the time course of serum Tmprss6 in c57BL/6 mice cohorts of n=6 at 14, 28 and 42 days post s.c. treatment with 1 mg/kg Conjugate 18, Reference Conjugate 8, and mock treated (PBS) individuals is shown in Figure 55. As indicated by the data in Figure 55, the conjugates of the invention are particularly effective at reducing target gene expression compared to the negative control (PBS) and Reference Conjugate 8.
Overall, the in vivo data show that a variety of example linkers which are conjugated at both termini of the second strand are effective at decreasing target gene expression in vivo. The positioning of the linker improves in vivo potency conjugates, as compared to a triantenary GaINAc-linker control at the 5' terminus of the second strand (Reference Conjugates 6, 7 and 8).
Example 32 ¨ Serum Stability Studies The method described above under "Tritosome stability assay" in the General Method section was followed.
Figure 56 shows the results from the serum stability studies in respect of Conjugates 2, 4, 5, 6 and 7. Figure 57 shows the serum stability of Conjugates 2, 8, 9, 10, 11 and 12.

All conjugates of the invention that were tested are more stable in serum compared to control.
All tested conjugates contain each one GaINAc linker unit at the 5' end and another at the 3' end of the second strand. The siRNAs are modified with alternating 2'-0Me/2'-F and contain each two phosphorothioate (PS) internucleotide linkages at their 5' and 3' terminal two internucleotide linkages, unless stated differently.
In Conjugate 4 the serinol-GaINAc units are attached via a phosphodiester bond. In Conjugate 5 the serinol-GaINAc units are conjugated via PS, whereas all internucleotide linkage in the second strand are phosphodiesters. In Conjugate 6 the second strand contains no PS. In Conjugate 7 two serinol-GaINAc units are attached to each second strand terminus and to each other via a PS-bonds at the respective ends. In Conjugate 8 a C6-amino-modifier at 5' and a C7-amino-modifier at the 3' end of the second strand were applied for ligand attachment. In Conjugate 9 Gly-C3-amino-modifiers, in Conjugate 10 piperidyl-amino-modifiers, in Conjugate 11 C3-amino-modifiers and in Conjugate 2 serinol-GaINAc units were used as linkers for conjugation to both ends of the second strand. In Conjugate 2 both terminal internucleotides as well as the nucleotide-serinol bonds are PS.
In Conjugate 12 a C6-amino-modifier at the 5' and a GlyC3-amino-modifier at the 3' end of second strand were applied for ligand attachment. "ut" indicates an untreated sample which the other samples were normalised to. "Luc" indicates an siRNA targeting Luciferase (Reference Conjugate 3), which was used as non-targeting control and does not reduce target mRNA levels.
The data show that in context of a serinol-derived linker moiety being provided at 5' and 3' ends of the sense strand, the number of phosphorothioate (PS) bonds between the terminal nucleotide and the linker, and/or between the terminal three nucleotides in the sense strand, can be varied whilst maintaining stability in serum.
Example 33 ALDH2-targeting GaINAc conjugates with an optimized 2'-0Me/2'-F modification pattern in the second strand and with end-stabilising inverted RNA at the 3' end of the second strand show extended reduction of ALDH2 mRNA levels in mice. Modified variants of the GaINAc-siRNA conjugates 5T522006 and 5T522009 were analyzed for knockdown activity in vivo.
5T522006L6 and 5T522009L6 contain alternating 2'-0Me/2'-F modifications in both first and second strand. 5T522006V1L6 and 5T522009V1L6 contain alternating 2'-0Me/2'-F
only in the first strand. In the second strand, these conjugates contain 2'-0Me at positions 1-6 and 10-18 and 2'-F at positions 7-9. An inverted RNA nucleotide is present at the 3' end of the second strand. There, it substitutes for the last nucleotide of the second strand. The 3' end of the second strand does not contain any phosphorothioate linkages, whereas all other, non-conjugated ends are stabilized by two terminal phosphorothioates.
All conjugates reduce ALDH2 target gene levels. Among the timepoints analysed here, strongest reduction is achieved 10 days after treatment. STS22006V1L6 has improved initial knockdown activity and duration of action compared to STS22006L6.

and STS22009V1L6 show equal levels of activity.
C57BL/6 male mice were subcutaneously treated with 3 mg/kg GaINAc conjugate.
Liver sections were prepared 10, 20, and 30 days after treatment (labeled "d11", "d21" and "d31"), total RNA was extracted from the tissue and ALDH2 and ApoB mRNA levels were analyzed by Taqman qRT-PCR. Each bar represents mean SD of six animals.
Results are shown in Figure 58.
Statements of Invention 1. 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 RNA
transcribed from the ALDH2 gene, wherein said first strand comprises a nucleotide sequence selected from the following sequences: SEQ ID NOs: 11, 1, 3, 5, 7, 9, 13, 15, 17, 19, 21, 23, 25 or 27 or any sequence disclosed herein.
2. A nucleic acid of statement 1, wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 12, 2, 4, 6, 8, 10, 14, 16, 18, 20, 22, 24, 26 or 28, or any sequence disclosed herein.
3. A nucleic acid of statement 1 or statement 2, wherein said first strand comprises a nucleotide sequence of SEQ ID NO:11, SEQ ID NO:3 or SEQ ID NO:17.
4. A nucleic acid of any one of statements 1 to 3, wherein said second strand comprises the nucleotide sequence of SEQ ID NO:12, SEQ ID NO:4 or SEQ ID
NO:18.
5. A nucleic acid according to any one of statements 1 to 4, wherein said first strand and/or said second strand are each from 17-35 nucleotides in length.

6. A nucleic acid of any one of statements 1 to 5, wherein the at least one duplex region consists of 19-25 consecutive nucleotide base pairs.
7. A nucleic acid of any preceding statement, which a) is blunt ended at both ends; or b) has an overhang at one end and a blunt end at the other; or c) has an overhang at both ends.
8. A nucleic acid according to any preceding statement, wherein one or more nucleotides on the first and / or second strand are modified, to form modified nucleotides.
9. A nucleic acid of statement 8, wherein one or more of the odd numbered nucleotides of the first strand are modified.
10. A nucleic acid according to statement 9, wherein one or more of the even numbered nucleotides of the first strand are modified by at least a second modification, wherein the at least second modification is different from the modification of statement 9.
11. A nucleic acid of statement 10, wherein at least one of the one or more modified even numbered nucleotides is adjacent to at least one of the one or more modified odd numbered nucleotides.
12. A nucleic acid of any one of statements 9 to 11, wherein a plurality of odd numbered nucleotides are modified.
13. A nucleic acid of statement 10 to 12, wherein a plurality of even numbered nucleotides are modified by a second modification.
14. A nucleic acid of any of statements 8 to 13, wherein the first strand comprises adjacent nucleotides that are modified by a common modification.
15. A nucleic acid of any of statements 9 to 14, wherein the first strand comprises adjacent nucleotides that are modified by a second modification that is different to the modification of statement 9.

16. A nucleic acid of any of statements 9 to 15, wherein one or more of the odd numbered nucleotides of the second strand are modified by a modification that is different to the modification of statement 9.
17. A nucleic acid according to any of statements 9 to 15, wherein one or more of the even numbered nucleotides of the second strand are modified by the modification of statement 9.
18. A nucleic acid of statement 16 or 17, wherein at least one of the one or more modified even numbered nucleotides of the second strand is adjacent to the one or more modified odd numbered nucleotides of the second strand.
19. A nucleic acid of any of statements 16 to 18, wherein a plurality of odd numbered nucleotides of the second strand are modified by a common modification.
20. A nucleic acid of any of statements 16 to 19, wherein a plurality of even numbered nucleotides are modified by a modification according to statement 9.
21. A nucleic acid of any of statements 16 to 20, wherein a plurality of odd numbered nucleotides on the second strand are modified by a second modification, wherein the second modification is different from the modification of statement 9.
22. A nucleic acid of any of statements 16 to 21, wherein the second strand comprises adjacent nucleotides that are modified by a common modification.
23. A nucleic acid of any of statements 16 to 22, wherein the second strand comprises adjacent nucleotides that are modified by a second modification that is different from the modification of statement 9.
24. A nucleic acid according to any one of statements 8 to 23, wherein each of the odd numbered nucleotides in the first strand and each of the even numbered nucleotides in the second strand are modified with a common modification.
25. A nucleic acid of statement 24, wherein each of the even numbered nucleotides are modified in the first strand with a second modification and each of the odd numbered nucleotides are modified in the second strand with a second modification.
26. A nucleic acid according to any one of statements 8 to 25, wherein the modified nucleotides of the first strand are shifted by at least one nucleotide relative to the unmodified or differently modified nucleotides of the second strand.
27. A nucleic acid according to any one of statements 8 to 26, wherein the modification and/or modifications are each and individually 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 phosphoramidate modification, 5'-phosphorothioate group modification, a 5' phosphate or 5' phosphate mimic modification and a cholesteryl derivative or a dodecanoic acid bisdecylamide group modification.
28. A nucleic acid according to any one of statements 8 to 27, wherein the modification is any one of a locked nucleotide, an abasic nucleotide or a non-natural base comprising nucleotide.
29. A nucleic acid according to any one of statements 8 to 28, wherein at least one modification is 2'-0-methyl.
30. A nucleic acid according to any one of statements 8 to 29, wherein at least one modification is 2'-F.
31. 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: 11, 1, 3, 5, 7, 9, 13, 15, 17, 19, 21, 23, 25 or 27, wherein the nucleotides of first strand are modified by 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.
32. A nucleic acid of statement 31, wherein second sequence comprises a nucleotide sequence of SEQ ID NO: 12, 2, 4, 6, 8, 10, 14, 16, 18, 20, 22, 24, 26 or 28.
33. A nucleic acid of statement 31 or 32, wherein the fourth modification and the second modification are the same.
34. A nucleic acid of any one of statements 31 to 33, wherein the first modification and the third modification are the same.
35. A nucleic acid of any one of statements 31 to 34, wherein the first modification is 2'0-Me and the second modification is 2'F.
36. A nucleic acid of any one of statements 31 to 35, wherein the first strand comprises the nucleotide sequence of SEQ ID NO:11, SEQ ID NO:3 or SEQ ID NO:17 and the second strand comprises the nucleotide sequence of SEQ ID NO:12, SEQ
ID
NO:4 or SEQ ID NO:18.
37. A nucleic acid of any one of statements 31 to 36, comprising a sequence and modifications as shown below:
Sequence siRNA ID sequence modifications ID No 3 5'-AAUGUUUUCCUGCUGACGG-3' 6254515173547182748 ALDH2-hcm-2 4 5'-CCGUCAGCAGGAAAACAUU-3' 3745364728462627251 11 5'-UCUUCUUAAACUGAGUUUC-3' 5351715262718281517 ALDH2-hcm-6 12 5'-GAAACUCAGUUUAAGAAGA-3' 4626353645152646282 17 5'-AUGUAGCCGAGGAUCUUCU-3' 6181647382846171535 ALDH2-hcm-9 18 5'-AGAAGAUCCUCGGCUACAU-3' 2826461735384716361 wherein, the specific modifications are depicted by numbers 1=2"F-dU, 2=2`F-dA, 3=2"F-dC, 4=2"F-dG, 5=2'-0Me-rU;
6=2'-0Me-rA;
7=2'-0Me-rC;
8=2'-0Me-rG.
38. A nucleic acid according to any one of statements 1 to 37, conjugated to a ligand.
39. A nucleic acid according to any one of statements 1 to 38, comprising a phosphorothioate linkage between the terminal one, two or three 3' nucleotides and/or 5' nucleotides of the first and/or the second strand.
40. A nucleic acid according to any one of statements 1 to 39 comprising two phosphorothioate linkage 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.
41. 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: 11, 1, 3, 5, 7, 9, 13, 15, 17, 19, 21, 23, 25 or 27, and wherein the nucleic acid is conjugated to a ligand.
42. A nucleic acid according to statement 41, wherein the ligand comprises (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 nucleic acid as defined in statement 41.
43. A nucleic acid according to any of statements 41 or 42, wherein linker may be a bivalent or trivalent or tetravalent branched structure.
44. A nucleic acid of any of statements 41 to 43, wherein the nucleotides are modified as defined in any preceding statements.
45. A nucleic acid of any preceding statement, which is conjugated to a ligand comprising the formula I:
[S-X1-P-X2]3-A-X3- (I) wherein:
S represents a saccharide, wherein the saccharide is N-acetyl galactosamine;
X1 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);

X2 is alkylene or an alkylene ether of the formula (-CH2)n-O-CH2- where n = 1-6;
A is a branching unit;
X3 represents a bridging unit;
wherein a nucleic acid as defined in any of statements 1 to 40 is conjugated to X3 via a phosphate or modified phosphate (preferably a thiophosphate).
46. A conjugated nucleic acid having one of the following structures OH
OH
OH
PcH Ft¨OH
\OH

L

S¨LO-t OH
OH
0- Pd-1 0¨/¨/

OH
HOI,&_.0H
OH OH
HO õ1....) ...... AcHNks NHAc 1'1') 0=P ¨S
$

..$) $ 9 0 =P¨S
(!) OH
0 o.1¨/ OH
AcHN
_______________________________________ /
Cr1(10H
/

0 /......"--0 .., II
Z-0¨P-0 S
L 1 9 if 0 ¨P-0 $0 S :
OH
HO\,_ 0H
OH OH

HO,,,L....) .... AcHN

NHAc L.I1 0 =P ¨ S
i 0 =P ¨S

/ I OH
0,,,. o¨/ OH
AcHN
_______________________________________ /
$CrIOH
/
_ j_ j--0 0 '...ct $1 Z ¨0¨P-0 S
? /
0¨P-0 S

OH
HO\,...,011 OH OH

AcHN

NHAc Li) i 0 0=P¨S
i 0.1 0 i 0 0=P¨S

/I OH

/ AcHN C
0¨ OH
'N
_______________________________________ / r().-R'OH
_Ex J-0/ 0 -., II
I¨O¨P-0 LI,µ 1? f ,.
S 0¨P-0 so S, OH
H0µ,_ OH
OH OH

AcHN

NHAc 1') 0 =P ¨S
i 0 = P ¨S

/ I OH
0,, o j OH
/
AcHN
, r i c ( 1 -FON
_x_rxi¨ 0 _____________________ õ, 0 0 .--II , z-0¨P¨O 0¨p-0 i 0 10 S S

OH
HO k,,,_01.1 AcHN

NHAc 04¨Se \
i 04¨Se i AcHN OH
OH
0 ,,, 0 --/¨/ (12/)(OH

Z ¨0 ¨P ¨0 ¨rj---le s o 0¨p-0 le s OH
HO OH
OH OH
AcH 0N

NHAc i 0 0=P ¨S
\

0 =P ¨ S
I
0 AcHN OH
OH
0 0 ¨r0 OH
/

/ I

2 ¨0 ¨11-0 ¨T.-1j¨ LI,. 0 /

S 0 ¨P ¨0 S

OH
H\,,_ 0 li OH OH , 0 AcHN
HO..õ...&.,,) 0 r1HAc \
i 0 =P -Se 0,1 0 OH
0 =P -S
of AcHN;;OH

r-17C.. ? /
II

1 < 10 S s wherein Z is a nucleic acid according to any of statements 1 to 40.
47. A nucleic acid according to any of statements 1 to 40, which is conjugated to a ligand of the following structure HO
c04. H (:) H FiAc 1 r\INc 01 OH
0-P=0 n( (---*V
µAcH orliN 0 H
H
ir.1 k 0 HAc H H Ce 0 /AN==vNN
H
48. A nucleic acid or conjugated nucleic acid of any preceding statement, wherein the duplex comprises separate strands.
49. A nucleic acid or conjugated nucleic acid of any preceding statement, wherein the duplex comprises a single strand comprising a first strand and a second strand.
50. A composition comprising a nucleic acid or conjugated nucleic acid of any preceding statement and a physiologically acceptable excipient.
51. A nucleic acid or conjugated nucleic acid according to any preceding statement for use in the prevention or treatment of a disease, disorder or syndrome.
52. Use of a nucleic acid or conjugated nucleic acid according to any preceding statement in the manufacture of a medicament for preventing or treating a disease, disorder or syndrome.
53. A method of preventing or treating a disease, disorder or syndrome comprising administration of a composition comprising a nucleic acid or conjugated nucleic acid according to any preceding statement to an individual in need of treatment.
54. The method of statement 53, wherein the nucleic acid or conjugated nucleic acid is administered to the subject subcutaneously, intravenously or using any other application routes such as oral, rectal or intraperitoneal.
55. A use or method according to statements 51 to 54, wherein said disease or disorder is alcohol use disorder.
56. A use or method according to statement 55, wherein the alcohol use disorder is acute alcohol sensitivity, alcoholic neuropathy, alcohol abuse or fetal alcohol disorder or other pathologies associated to acute or prolonged excessive alcohol consumption.
57. A process for making a nucleic acid or conjugated nucleic acid of any one of statements 1 to 49.
58. A nucleic acid for inhibiting expression of ALDH2, 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 RNA transcribed from the ALDH2 gene, wherein the expression of ALDH2 is reduced to levels which are at least 15% lower than expression levels observed in same test conditions but in the absence of the nucleic acid or conjugated nucleic acid or in the presence of a non-silencing control.
59. A nucleic acid for inhibiting expression of ALDH2, 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: 11, 1, 3, 5, 7, 9, 13, 15, 17, 19, 21, 23, 25 or 27, wherein the expression of ALDH2 is reduced to levels which are at least 15%
lower than expression levels observed in same test conditions but in the absence of the nucleic acid or conjugated nucleic acid or in the presence of a non-silencing control.
Other clauses of the invention include:
1. 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 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 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 or 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 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, optionally wherein the linker moiety is a serinol-derived linker moiety or one of the other linker types described herein.
2. The conjugate according to clause 1 wherein the second RNA strand is conjugated at the 5' end to the targeting ligand, 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.
3. The conjugate according to clause 1 wherein the second RNA strand is conjugated at the 5' end to the targeting ligand, 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.
4. The conjugate according to clause 1 wherein the second RNA strand is conjugated at the 5' end to the targeting ligand and both the second RNA strand and the first RNA
strand are also conjugated at the 3' ends to the targeting ligand.

5. The conjugate according to clause 1 wherein 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.
6. The conjugate according to any one of clauses 1 to 5 wherein the ligands are monomeric ligands.
7. The conjugate according to any one of clauses 1 to 6 wherein the ligands are selected from GaINAc and galactose moieties, especially GaINAc moieties.
8. The conjugate according to any one of clauses 1 to 7 wherein the conjugated RNA
strands are conjugated to a targeting ligand via a serinol-derived linker moiety including a further linker wherein the further linker is or comprises a saturated, unbranched or branched C1-16 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 0, N, S(0)p wherein p is 0, 1 or 2, (for example a CH2 group is replaced with 0, or with NH, or with S, or with SO2 or a ¨CH3 group at the terminus of the chain or on a branch is replaced with OH
or with NH2) wherein said chain is optionally substituted by one or more oxo groups (for example 1 to 3, such as 1 group).
9. The conjugate according to clause 8 wherein the further linker comprises a saturated, unbranched C1_15 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.
10. The conjugate according to clause 9 wherein the further linker comprises a PEG-chain.
11. The conjugate according to clause 8 wherein the further linker comprises a saturated, unbranched Ci_16 alkyl chain.
12. The conjugate according to clause 11 wherein the further linker comprises a saturated, unbranched C1_6 alkyl chain.
13. The conjugate according to clause 12 wherein the further linker comprises a saturated, unbranched C4 or C6 alkyl chain, e.g. a C4 alkyl chain.

14. The conjugate according to clause 1 wherein the first RNA strand is a compound of formula (XV):
_ GaINAc GaINAc µ \
L L
\ \
NH NH
Y
Zi¨O¨P-0 0 P 0 _______ 0 __ H
I I
OH R1 \ OH Ri /
n b¨ ¨ (XV) wherein b is 0 or 1; and the second RNA strand is a compound of formula (XVI):
GaINAc GaINAc GaINAc GaINAc /
/ \ \
L L L L
/ / \ \
HN HN NH NH

0 P 0 Z2 0 P 0 0 __ I I I
n n \ Ri OH i R1 OH OH R1 \ OH R1 i c _ ¨ d ¨ ¨
(XVI);
wherein c and d are independently 0 or 1;
wherein:
Z1 and Z2 are the RNA portions of the first and second RNA strands respectively;
Y is 0 or S;
R1 is H or methyl;
n is 0, 1,2 or 3; and L is the same or different in formulae (XV) and (XVI) and is selected from the group consisting of:
-(CH2)q, wherein q = 2-12;
-(CH2),-C(0)-, wherein r = 2-12;
-(CH2-CH2-0)s-CH2-C(0)-, wherein s = 1-5;
-(CH2)1-CO-NH-(CH2)1-NH-C(0)-, wherein t is independently is 1-5;
-(CH2).-CO-NH-(CH2).-C(0)-, wherein u is independently is 1-5; and -(CH2),-NH-C(0)-, wherein v is 2-12; and wherein the terminal C(0) (if present) is attached to the NH group;
and wherein b + c + d is 2 or 3.
15. The conjugate according to clause 14 wherein b is 0, c is 1 and d is 1.
16. The conjugate according to clause 14 wherein b is 1, c is 0 and d is 1.

17. The conjugate according to clause 14 wherein b is 1, c is 1 and d is O.
18. The conjugate according to clause 14 wherein b is 1, c is 1 and d is 1.
19. The conjugate according to any one of clauses 14-18 wherein Y is 0.
20. The conjugate according to any one of clauses 14-18 wherein Y is S.
21. The conjugate according to any one of clauses 14-20 wherein R1 is H.
22. The conjugate according to any one of clauses 14-20 wherein R1 is methyl.
23. The conjugate according to any one of clauses 14-22 wherein n is 0.
1.5 24. The conjugate according to any one of clauses 14-23 wherein L is -(CH2)r-C(0)-, wherein r = 2-12.
25. The conjugate according to clause 24 wherein r = 2-6.
26. The conjugate according to clause 25 wherein r = 4 or 6 e.g. 4.
27. The conjugate according to any preceding clause, with any feature or combination of features disclosed herein.

Summary SEQUENCE TABLE

t..) o SEQ ID Sequence (5'-3') Unmodified Sequence counterpart (5'-3') Name O-NO
t..) t..) oe ALDH2-h-1 1st t..) 1 strand UUGUUUAUGAAAAUCUGGU
UUGUUUAUGAAAAUCUGGU
ALDH2-h-1 2nd ACCAGAUUUUCAUAAACAA
strand ALDH2-hcm-2 3 1st strand AAUGUUUUCCUGCUGACGG
AAUGUUUUCCUGCUGACGG
ALDH2-hcm-2 4 2nd strand CCGUCAGCAGGAAAACAUU
CCGUCAGCAGGAAAACAUU
ALDH2-hc-3 1st P
UUGAACUUCAGGAUCUGCA UUGAACUUCAGGAUCUGCA .
strand , , ALDH2-hc-3 2nd UGCAGAUCCUGAAGUUCAA o ,"
strand 4. "

ALDH2-hc-4 1st N), UUUCACUUCAGUGUAUGCC , c, strand .
, ALDH2-hc-4 2nd .

GGCAUACACUGAAGUGAAA
strand ALDH2-hc-5 1st UUCUUAUGAGUUCUUCUGA
strand ALDH2-hc-5 2nd UCAGAAGAACUCAUAAGAA UCAGAAGAACUCAUAAGAA
strand ALDH2-hcm-6 11 1st strand UCUUCUUAAACUGAGUUUC
UCUUCUUAAACUGAGUUUC od n ALDH2-hcm-6 GAAACUCAGUUUAAGAAGA t=1 2nd strand od t..) ALDH2-hc-7 1st UCCUUGAUCAGGUUGGCCA
oe strand O-oe ALDH2-hc-7 2nd UGGCCAACCUGAUCAAGGA
strand o u, ALDH2-hc-8 1st UUGAAGAACAGGGCGAAGU t..) strand o ALDH2-hc-8 2nd ,z ACUUCGCCCUGUUCUUCAA O-strand ,z t..) t..) ALDH2-hcm-9 oe AUGUAGCCGAGGAUCUUCU t..) 1st strand ALDH2-hcm-9 18 2nd strand AGAAGAUCCUCGGCUACAU
AGAAGAUCCUCGGCUACAU
ALDH2-hmr-10 AUCUGGUUGCAGAAGACCU
1st strand ALDH2-hmr-10 AGGUCUUCUGCAACCAGAU
2nd strand ALDH2-hr-11 1st AAAUCCACCAGGUAGGAGA P
strand -, ALDH2-hr-11 2nd , .3 UCUCCUACCUGGUGGAUUU
strand "
u, ALDH2-hm-12 N)-ACUCUCUUGAGGUUGCUGC
1st strand ,I, ALDH2-hm-12 2nd strand ALDH2-hcmr-13 AUGUCCAAAUCCACCAGGU
1st strand ALDH2-hcmr-13 ACCUGGUGGAUUUGGACAU
2nd strand ALDH2-hm-14 CCAUGCUUGCAUCAGGAGC
1st strand od ALDH2-hm-14 n GCUCCUGAUGCAAGCAUGG
2nd strand m od ALDH2 (upper) t..) o 29 Human, GGCAAGCCCTATGTCATCTCCT
GGCAAGCCCTATGTCATCTCCT
cio O-Cynomolgus oe o vi ALDH2 (lower) t..) 30 Human, GGATGGTTTTCCCGTGGTACTT
GGATGGTTTTCCCGTGGTACTT o Cynomolgus O-ALDH2 (probe) t..) t..) 31 Human, TGGTCCTCAAATGTCTCCGGTATTATGCC
TGGTCCTCAAATGTCTCCGGTATTATGCC cio t..) Cynomolgus 32 ALDH2 (upper) GGCAAGCCTTATGTCATCTCGT
GGCAAGCCTTATGTCATCTCGT
Mouse 33 ALDH2 (lower) GGAATGGTTTTCCCATGGTACTT
GGAATGGTTTTCCCATGGTACTT
Mouse 34 ALDH2 (probe) TGAAATGTCTCCGCTATTACGCTGGCTG
TGAAATGTCTCCGCTATTACGCTGGCTG
Mouse ApoB (upper) P TCATTCCTTCCCCAAAGAGACC CATTCCTTCCCCAAAGAGACC
Human , , ApoB (lower) CACCTCCGTTTTGGTGGTAGAG
CACCTCCGTTTTGGTGGTAGAG .3 "
Human o, ,, .
ApoB (probe) CAAGCTGCTCAGTGGAGGCAACACATTA " 37 CAAGCTGCTCAGTGGAGGCAACACATTA , , Human .
' ApoB (upper) TCATTCCTTCCCCAAAGAAACC
TCATTCCTTCCCCAAAGAAACC

Cynomolgus ApoB (lower) Cynomolgus ApoB (probe) TCAAGCTGTTAAGTGGCAGCAACACGTT
TCAAGCTGTTAAGTGGCAGCAACACGTT
Cynomolgus beta-Actin GCATGGGTCAGAAGGATTCCTAT
GCATGGGTCAGAAGGATTCCTAT
(upper) Human oo n 42 beta-Actin (lower) TGTAGAAGGTGTGGTGCCAGATT
TGTAGAAGGTGTGGTGCCAGATT
t=1 Human oo t..) beta-Actin TCGAGCACGGCATCGTCACCAA
TCGAGCACGGCATCGTCACCAA cio (probe) Human O-oe o u, beta-Actin 44 (upper) AAGGCCAACCGCGAGAAG
AAGGCCAACCGCGAGAAG t..) o Cynomolgus O-45 beta-Actin (lower) AGAGGCGTACAGGGACAGCA
AGAGGCGTACAGGGACAGCA t..) t..) Cynomolgus oe t..) beta-Actin 46 (probe) TGAGACCTTCAACACCCCAGCCATGTAC
TGAGACCTTCAACACCCCAGCCATGTAC
Cynomolgus beta-Actin CCTAAGGCCAACCGTGAAAAG
(upper) Mouse 48 beta-Actin (lower) AGGCATACAGGGACAGCACAG
AGGCATACAGGGACAGCACAG
Mouse beta-Actin P 49 TGAGACCTTCAACACCCCAGCCATGTAC TGAGACCTTCAACACCCCAGCCATGTAC
(probe) Mouse .
, , 50 PTEN (upper) CACCGCCAAATTTAACTGCAGA
CACCGCCAAATTTAACTGCAGA
Human o, , ,, PTEN (lower) .

AAGGGTTTGATAAGTTCTAGCTGT
N) , , Human ' TGCACAGTATCCTTTTGAAGACCATAACCC
52 PTEN (probe) TGCACAGTATCCTTTTGAAGACCATAACCCA
A

53 ApoB fw Mouse AAAGAGGCCAGTCAAGCTGTTC
AAAGAGGCCAGTCAAGCTGTTC
54 ApoB rev Mouse GGTGGGATCACTTCTGTTTTGG
GGTGGGATCACTTCTGTTTTGG
ALDH2 siRNA
AUGUAGCCGAGGAUCUUCUUAAACUGAG

binding locus UUUC
Modified SEQ ID
56 NO: 1 5181516182626171845 UUGUUUAUGAAAAUCUGGU oo n Modified SEQ ID
57 NO: 2 2736461515361626362 ACCAGAUUUUCAUAAACAA m oo t..) Modified SEQ ID
58 NO: 3 6254515173547182748 AAUGUUUUCCUGCUGACGG oe O-oe Modified SEQ ID
59 NO: 4 3745364728462627251 CCGUCAGCAGGAAAACAUU
u, Modified SEQ ID
60 NO: 5 5182635172846171836 UUGAACUUCAGGAUCUGCA t..) o ,-, Modified SEQ ID
61 NO: 6 1836461735462815362 UGCAGAUCCUGAAGUUCAA O-t..) t..) Modified SEQ ID
oe
62 NO: 7 5153635172818161837 UUUCACUUCAGUGUAUGCC t..) Modified SEQ ID
63 NO: 8 4836163635462818262 GGCAUACACUGAAGUGAAA
Modified SEQ ID
64 NO: 9 5171525464517153546 UUCUUAUGAGUUCUUCUGA
Modified SEQ ID
65 NO: 10 1728264627172526462 UCAGAAGAACUCAUAAGAA
Modified SEQ ID
66 5351715262718281517 UCUUCUUAAACUGAGUUUC P
NO: 11 , Modified SEQ ID
, .3
67 NO: 12 4626353645152646282 GAAACUCAGUUUAAGAAGA ,-, oe Modified SEQ ID
"
-
68 5371546172845184736 UCCUUGAUCAGGUUGGCCA
NO: 13 ,I, Modified SEQ ID
69 NO: 14 1847362735461726482 UGGCCAACCUGAUCAAGGA
Modified SEQ ID
70 NO: 15 5182646272848382645 UUGAAGAACAGGGCGAAGU
Modified SEQ ID
71 NO: 16 2715383735451715362 ACUUCGCCCUGUUCUUCAA
Modified SEQ ID
72 NO: 17 6181647382846171535 AUGUAGCCGAGGAUCUUCU
od Modified SEQ ID
n
73 NO: 18 2826461735384716361 AGAAGAUCCUCGGCUACAU
m od Modified SEQ ID
t..)
74 NO: 19 6171845183646282735 AUCUGGUUGCAGAAGACCU o ,-, cio Modified O-SEQ ID
oe
75 NO: 20 2845351718362736461 AGGUCUUCUGCAACCAGAU
,-, o u, Modified SEQ ID
76 6261736372845284646 AAAUCCACCAGGUAGGAGA t..) NO: 21 o ,-, Modified SEQ ID
77 1717352735481846151 UCUCCUACCUGGUGGAUUU NO: 22 t..) t..) Modified SEQ ID
cio
78 6353535182845183547 ACUCUCUUGAGGUUGCUGC t..) NO: 23 Modified SEQ ID
79 4728362735362828281 GCAGCAACCUCAAGAGAGU
NO: 24 Modified SEQ ID
80 6181736261736372845 AUGUCCAAAUCCACCAGGU
NO: 25 Modified SEQ ID
81 2735481846151846361 ACCUGGUGGAUUUGGACAU
NO: 26 Modified SEQ ID
82 7361835183617284647 CCAUGCUUGCAUCAGGAGC P
NO: 27 o , Modified SEQ ID
, .3
83 4717354618362836184 GCUCCUGAUGCAAGCAUGG ,-, " NO: 28 o, , ,, GaINAc-ALDH2- OMeA(ps)-FA-(ps)-0MeU-FG-0MeU-FU-OMeU-FU-OMeC--,, , ' 84 hcm-2-L1 1st FC-0MeU-FG-0MeC-FU-OMeG-FA-OMeC-(ps)-FG-(ps)-AAUGUUUUCCUGCUGACGG .
' strand OMeG
.
GaINAc-ALDH2- [5T23 (ps)]3 long trebler (ps) FC-0MeC-FG-0MeU-FC-OMeA-85 hcm-2-L1 2nd FG-0MeC-FA-OMeG-FG-OMeA-FA-OMeA-FA-OMeC-FA-(ps)-CCGUCAGCAGGAAAACAUU
strand OMeU-(ps)-FU
GaINAc-ALDH2- OMeA(ps)- FA-(ps)-0MeU-FG-0MeU-FU-OMeU-FU-OMeC-86 hcm-2-L6 1st FC-0MeU-FG-0MeC-FU-OMeG-FA-OMeC-(ps)-FG-(ps)-AAUGUUUUCCUGCUGACGG
strand OMeG
GaINAc-ALDH2- [5T23 (ps)]3 5T43 (ps) FC-0MeC-FG-0MeU-FC-OMeA-FG-oo 87 hcm-2-L6 2nd OMeC-FA-OMeG-FG-0MeA-FA-0MeA-FA-0MeC-FA-(ps)-CCGUCAGCAGGAAAACAUU n 1-i strand OMeU-(ps)-FU
m oo GaINAc-ALDH2- OMeU-(ps)-FC-(ps)-0MeU-FU-OMeC-FU-OMeU-FA-OMeA-t..) o 88 hcm6-L1 1st FA-0MeC-FU-OMeG-FA-OMeG-FU-OMeU-(ps)-FU-(ps)-UCUUCUUAAACUGAGUUUC
oe strand OMeC
O-oo ,-, ,-, o u, GaINAc-ALDH2- [ST23 (ps)]3 long trebler (ps) FG-0MeA-FA-0MeA-FC-OMeU-89 hcm6-L1 2nd FC-0MeA-FG-OMeU-FU-OMeU-FA-OMeA-FG-OMeA-FA-(ps)-GAAACUCAGUUUAAGAAGA t..) o ,-, strand OMeG-(ps)-FA
O-GaINAc-ALDH2- OMeU-(ps)-FC-(ps)-0MeU-FU-OMeC-FU-OMeU-FA-OMeA-t..) 90 hcm6-L6 1st FA-0MeC-FU-OMeG-FA-OMeG-FU-OMeU-(ps)-FU-(ps)-UCUUCUUAAACUGAGUUUC t..) oe t..) strand OMeC
GaINAc-ALDH2- [ST23 (ps)]3 ST43 (ps) FG-0MeA-FA-0MeA-FC-OMeU-FC-91 hcm6-L6 2nd OMeA-FG-OMeU-FU-OMeU-FA-0MeA-FG-0MeA-FA-(ps)-GAAACUCAGUUUAAGAAGA
strand OMeG-(ps)-FA
GaINAc-ALDH2- OMeA-(ps)-FU-(ps)-0MeG-FU-OMeA-FG-OMeC-FC-OMeG-92 hcm9-L1 1st FA-0MeG-FG-0MeA-FU-OMeC-FU-OMeU-(ps)-FC-(ps)-AUGUAGCCGAGGAUCUUCU
strand OMeU
GaINAc-ALDH2- [ST23 (ps)]3 long trebler (ps) FA-0MeG-FA-0MeA-FG-OMeA-P
93 hcm9-L1 2nd FU-OMeC-FC-OMeU-FC-OMeG-FG-OMeC-FU-OMeA-FC-AGAAGAUCCUCGGCUACAU .
strand (ps)-0MeA-(ps)-FU
, , .3 GaINAc-ALDH2- OMeA-(ps)-FU-(ps)-0MeG-FU-OMeA-FG-OMeC-FC-OMeG-o 94 hcm9-L6 1st FA-0MeG-FG-0MeA-FU-OMeC-FU-OMeU-(ps)-FC-(ps)-AUGUAGCCGAGGAUCUUCU 0"
IV
strand OMeU
, , GaINAc-ALDH2- [ST23 (ps)]3 ST43 (ps) FA-0MeG-FA-0MeA-FG-OMeA-FU- ' 95 hcm9-L6 2nd OMeC-FC-OMeU-FC-OMeG-FG-OMeC-FU-OMeA-FC-(ps)-AGAAGAUCCUCGGCUACAU
strand OMeA-(ps)-FU
GaINAc-ALDH2- OMeA-(ps)-FU-(ps)-0MeG-FU-OMeA-FG-OMeC-FC-OMeG-96 hcm9-L4 1st FA-0MeG-FG-0MeA-FU-OMeC-FU-OMeU-(ps)-FC-(ps)-AUGUAGCCGAGGAUCUUCU
strand OMeU
GaINAc-ALDH2- [ST23 (ps)]3 ST41 (ps) FA-0MeG-FA-0MeA-FG-OMeA-FU-97 hcm9-L4 2nd OMeC-FC-OMeU-FC-OMeG-FG-OMeC-FU-OMeA-FC-(ps)-AGAAGAUCCUCGGCUACAU oo strand OMeA-(ps)-FU
n 1-i mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC(ps)f m 98 ALD01 1st strand AAUGUUUUCCUGCUGACGG oo G(ps)mG
t..) o ALD01 2nd fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfA(ps)mU
,-, CCGUCAGCAGGAAAACAUU O-strand (ps)fU
.
,-, ,-, o u, mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG

100 ALD02 1st strand AAUGUUUUCCUGCUGACGGA t..) ivA
o ,-, ALD02 2nd fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmUfU
101 CCGUCAGCAGGAAAACAUUA strand ivA
ivA
t..) t..) mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG
oe 102 ALD03 1st strand AAUGUUUUCCUGCUGACGGU t..) ivU
ALD03 2nd fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmUfU

CCGUCAGCAGGAAAACAUUU
strand ivU
mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG
104 ALD04 1st strand AAUGUUUUCCUGCUGACGGC
ivC
ALD04 2nd fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmUfU

CCGUCAGCAGGAAAACAUUC
strand ivC
mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG
106 ALD05 1st strand AAUGUUUUCCUGCUGACGGG P
ivG
.
, ALD05 2nd fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmUfU
, CCGUCAGCAGGAAAACAUUG ,-, strand ivG
,-, mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfG
IV

108 ALD06 1st strand AAUGUUUUCCUGCUGACGA
ivA
0' ALD06 2nd 109 fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmU ivA

strand mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfG
110 ALD07 1st strand AAUGUUUUCCUGCUGACGU
ivU
ALD07 2nd 111 fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmU ivU
CCGUCAGCAGGAAAACAUU
strand mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfG
112 ALD08 1st strand AAUGUUUUCCUGCUGACGC
ivC
oo ALD08 2nd n 1-i 113 fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmU ivC
CCGUCAGCAGGAAAACAUC
strand m oo mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfG
t..) 114 ALD09 1st strand AAUGUUUUCCUGCUGACGG o ,-, ivG
oe O-ALD09 2nd oe 115 fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmU ivG
CCGUCAGCAGGAAAACAUG
strand o u, STS22002L6 1st mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC(ps)f AAUGUUUUCCUGCUGACGG
t..) o strand G(ps)mG
,o ST23(ps)3 ST
O-5T522002L6 2nd ,o 117 43(ps)fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfA(ps)mU( CCGUCAGCAGGAAAACAUU t..) t..) strand oo ps)fU
t..) 118 STS22002V1L6 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC(ps)f AAUGUUUUCCUGCUGACGG
1st strand G(ps)mG
119 STS22002V1L6 5T23(ps)3 ST
CCGUCAGCAGGAAAACAUA
2nd strand 43(ps)fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmUivA
120 5T522002V2L6 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC(ps)f AAUGUUUUCCUGCUGACGG
1st strand G(ps)mG
121 5T522002V2L6 5T23(ps)3 ST
CCGUCAGCAGGAAAACAUG
2nd strand 43(ps)fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmUivG
P
5T522002V3L6 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG
AAUGUUUUCCUGCUGACGGA
.

, , 1st strand ivA
a' ,-, " 5T23(ps)3 ST
-4 , t..) ,, 123 43(ps)fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfA(ps)mU( CCGUCAGCAGGAAAACAUU
" , 2nd strand , ps)fU
.
' 124 5T522002V4L6 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG
AAUGUUUUCCUGCUGACGGG
',;
1st strand ivG
S
5T522002V4L6 T23(ps)3 ST
125 2nd strand 43(ps)fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfA(ps)mU( CCGUCAGCAGGAAAACAUU
ps)fU

5T522006L6 1st mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC(ps)f UGUUUUCCUGCUGACGG
strand G(ps)mG
5T23(ps)3 ST
oo 5T522006 L6 2nd n 127 43(ps)fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfA(ps)mU( CCGUCAGCAGGAAAACAUU
strand m ps)fU
oo t..) STS22006V1L6 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC(ps)f AAUGUUUUCCUGCUGACGG
,-, oe 1st strand G(ps)mG

O-STS22006V1L6 5T23(ps)3 ST
CCGUCAGCAGGAAAACAUA
oe ,-, ,-, 2nd strand 43(ps)fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmUivA
o u, 130 STS22009L6 1st mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC(ps)f AAUGUUUUCCUGCUGACGG
o strand G(ps)mG
131 STS22009L6 2nd ST23(ps)3 ST
CCGUCAGCAGGAAAACAUG
O-strand 43(ps)fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmUivG
t..) t..) oe 132 STS22009V1L6 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG
AAUGUUUUCCUGCUGACGGA
t..) 1st strand ivA
5T23(ps)3 ST

43(ps)fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfA(ps)mU( CCGUCAGCAGGAAAACAUU
2nd strand ps)fU
AA 134 5T522009V2L6 mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG
UGUUUUCCUGCUGACGGG
1st strand ivG
5T23(ps)3 ST

43(ps)fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfA(ps)mU( CCGUCAGCAGGAAAACAUU
p 2nd strand ps)fU
-, , 136 5T516001AL33 mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC
,-, UUAUAGAGCAAGAACACUGUU
.3 fA mC fU mG (ps) fU (ps) mU (ps) Ser(GN) "
(...) ,, .
137 STS16001BL20 Ser(GN) (ps) fA mA fC mA fG mU fG mU fU mC fU mU fG mC
AACAGUGUUCUUGCUCUAUAA
N), , fU mC fU mA fU (ps) mA (ps) fA
.
' mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC
UUAUAGAGCAAGAACACUGUU

fA mC fU mG (ps) fU (ps) mU
139 STS16001BV1L4 Ser(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU
AACAGUGUUCUUGCUCUAUAA
2 mU fG mC fU mC fU mA fU (ps) mA (ps) fA (ps) Ser(GN) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG mC fU
AACAGUGUUCUUGCUCUAUAA

mC fU mA fU (ps) mA (ps) fA
141 STS18001A mU (ps) fC (ps) mG fA mA fG mU fA mU fU mC fC mG fC mG
UCGAAGUAUUCCGCGUACG
fU mA (ps) fC (ps) mG
oo n [(5T23) (ps)]3 C4XLT (ps) fC mG fU mA fC mG fC mG fG mA
CGUACGCGGAAUACUUCGA

fA mU fA mC fU mU fC (ps) mG (ps) fA
m oo t..) 143 STS16001BL4 [(5T23) (ps)]3 C4XLT(ps) fA (ps) mA (ps) fC mA fG mU
fG mU
AACAGUGUUCUUGCUCUAUAA
' ,-, fU mC fU mU fG mC fU mC fU mA fU (ps) mA (ps) fA
cio O-cio mU (ps) fC (ps) mG fA mA fG mU fA mU fU mC fC mG fC mG
UCGAAGUAUUCCGCGUACG

fU mA (ps) fC (ps) mG
o u, [ST23(ps)]3 ST41(ps) fC mG fU mA fC mG fC mG fG mA fA

CGUACGCGGAAUACUUCGA
mU fA mC fU mU fC (ps) mG (ps) fA
t..) o ,-, mU (ps) fC (ps) mU fU mC fU mU fA mA fA mC fU mG fA mG

UCUUCUUAAACUGAGUUUC fU mU mU (ps) fU (ps) mC
t..) Ser(GN) (ps) fG (ps) mA (ps) fA mA fC mU fC mA fG mU fU
t..) cio GAAACUCAGUUUAAGAAGA t..) mU fA mA fG mA fA (ps) mG (ps) fA (ps) Ser(GN) mU (ps) fC (ps) mU fU mC fU mU fA mA fA mC fU mG fA mG

UCUUCUUAAACUGAGUUUC
fU mU (ps) fU (ps) mC
149 X0477B Ser(GN) (ps) mG (ps) mA (ps) mA mA mC mU fC fA fG mU
mU
GAAACUCAGUUUAAGAAGA
mU mA mA mG mA mA (ps) mG (ps) mA (ps) Ser(GN) ALDH2 probe 150 based on SEQ ID BHQ1-TGAAATGTCTCCGCTATTACGCTGGCTG-FAM
TGAAATGTCTCCGCTATTACGCTGGCTGA
NO: 34 P
ApoB probe .

CAGCAACACACTGCATCTGGTCTCTACCA
, Mouse , .3 mA (ps) fA (ps) mU fG mU fU mU fU mC fC mU fG mC fU mG
,-, "

AAUGUUUUCCUGCUGACGG
fA mC (ps) fG (ps) mG
"
,, Ser(GN) (ps) fC (ps) mC (ps) fG mU fC mA fG mC fA mG fG
, ' CCGUCAGCAGGAAAACAUU .
' mA fA mA fA mC fA (ps) mU (ps) fU (ps) Ser(GN) .
(vp)-mU fA mU fG mU fU mU fU mC fC mU fG mC fU mG fA

UAUGUUUUCCUGCUGACGG
mC (ps) fG (ps) mG
Ser(GN) (ps) fC (ps) mC (ps) fG mU fC mA fG mC fA mG fG

CCGUCAGCAGGAAAACAUA
mA fA mA fA mC fA (ps) mU (ps) fA (ps) Ser(GN) mU (ps) fC (ps) mU fU mC fU mU fA mA fA mC fU mG fA mG

UCUUCUUAAACUGAGUUUC
fU mU (ps) fU (ps) mC
(vp)-mU fC mU fU mC fU mU fA mA fA mC fU mG fA mG fU
oo UCUUCUUAAACUGAGUUUC
mU (ps) fU (ps) mC
n 1-i Ser(GN) (ps) mG (ps) mA (ps) mA mA mC mU fC fA fG mU mU
t=1 GAAACUCAGUUUAAGAAGA oo mU mA mA mG mA mA (ps) mG (ps) mA (ps) Ser(GN) t..) o mA (ps) fU (ps) mG fU mA fG mC fC mG fA mG fG mA fU mC
,-, oe AUGUAGCCGAGGAUCUUCU
fU mU (ps) fC (ps) mU
O-oe ,-, ,-, o u, [ST23 (ps)]3 ltrb (ps) fA mG fA mA fG mA fU mC fC mU fC mG

AGAAGAUCCUCGGCUACAU t..) fG mC fU mA fC (ps) mA (ps) fU
o ,-.
mA (ps) fU (ps) mG fU mA fG mC fC mG fA mG fG mA fU mC

AUGUAGCCGAGGAUCUUCU fU mU mU (ps) fC (ps) mU
t..) t..) Ser(GN) (ps) fA (ps) mG (ps) fA mA fG mA fU mC fC mU fC
tc!.

AGAAGAUCCUCGGCUACAU
mG fG mC fU mA fC (ps) mA (ps) fU (ps) Ser(GN) STS16001BV1L7 Ser(GN) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mU fG

AACAGUGUUCUUGCUCUAUAA
mC fU mC fU mA fU (ps) mA (ps) fA Ser(GN) STS16001BV16L Ser(GN) (ps) fA mA fC mA fG mU fG mU fU mC fU mU fG mC

AACAGUGUUCUUGCUCUAUAA
42 fU mC fU mA fU mA fA (ps) Ser(GN) STS16001BV2OL Ser(GN) fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU

AACAGUGUUCUUGCUCUAUAA
75 mC fU mA fU mA fA Ser(GN) Ser(GN) (ps) Ser(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU

P
166 fU mC fU mU fG mC fU mC fU mA fU (ps) mA (ps) fA (ps) , Ser(GN) (ps) Ser(GN) , .3 ,-.
STS16001V1BL9 C6Am(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU

AACAGUGUUCUUGCUCUAUAA
6 mU fG mC fU mC fU mA fU (ps) mA (ps) fA (ps) C7Am(GN) .
,, , GlyC3Am(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC
, .
168 fU mU fG mC fU mC fU mA fU (ps) mA (ps) fA (ps) AACAGUGUUCUUGCUCUAUAA
7 .
GlyC3Am(GN) Conjugate 10 PipAm(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC
fU

AACAGUGUUCUUGCUCUAUAA
sense strand mU fG mC fU mC fU mA fU (ps) mA (ps) fA (ps) PipAm(GN) STS16001V1BL8 C3Am(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU

AACAGUGUUCUUGCUCUAUAA
8 mU fG mC fU mC fU mA fU (ps) mA (ps) fA (ps) C3Am(GN) STS16001V1BL8 C6Am(GN) (ps) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU

AACAGUGUUCUUGCUCUAUAA
7 mU fG mC fU mC fU mA fU (ps) mA (ps) fA (ps) GlyC3Am(GN) oo Conjugate 18 mA (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG
n AACCAGAAGAAGCAGGUGA
antisense strand fG mU (ps) fG (ps) mA
m oo Conjugate 18 Ser(GN) (ps) fU (ps) mC (ps) fA mC fC mU fG mC fU mU
fC t..) UCACCUGCUUCUUCUGGUU ' sense strand mU fU mC fU mG fG (ps) mU (ps) fU (ps) Ser(GN) re -a Conjugate 19 mA (ps) fU (ps) mA fA mC fU mC fU mG fU mC fC mA fU mU
oe AUAACUCUGUCCAUUACCG
antisense strand fA mC (ps) fC (ps) mG
o u, Conjugate 19 Ser(GN) (ps) fC (ps) mG (ps) fG mU fA mA fU mG fG mA
fC 0 CGGUAAUGGACAGAGUUAU t..) sense strand mA fG mA fG mU fU (ps) mA (ps) fU (ps) Ser(GN) o ,-, Reference mC (ps) fU (ps) mU fA mC fU mC fU mC fG mC fC mC fA mA
O-176 Conjugate 5 CUUACUCUCGCCCAAGCGA
fG mC (ps) fG (ps) mA
t..) t..) antisense oe t..) Reference [(ST23) (ps)]3 (C6XLT) (ps) fU mC fG mC fU mU fG mG fG mC
177 Conjugate 5 UCGCUUGGGCGAGAGUAAG
fG mA fG mA fG mU fA (ps) mA (ps) fG
sense Reference [ST23 (ps)]3 ltrb (ps) fG mA fA mA fC mU fC mA fG mU fU mU
178 Conjugate 6 GAAACUCAGU U UAAGAAGA
fA mA fG mA fA (ps) mG (ps) fA
sense Reference mU (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG
179 Conjugate 8 UACCAGAAGAAGCAGGUGA
fG mU (ps) fG (ps) mA
P
antisense .
, Reference , [ST23 (ps)]3 ST41 (ps)fU mC fA mC fC mU fG mC fU mU fC
,-, 180 Conjugate 8 UCACCUGCUUCUUCUGGUA
mU fU mC fU mG fG (ps) mU (ps) fA
o, IV
sense .
IV
F' Reference , [ST23 (ps)]3 C6XLT (ps) fC mG fG mU fA mA fU mG fG mA fC
,?.
181 Conjugate 9 CGGUAAUGGACAGAGUUAU ' mA fG mA fG mU fU (ps) mA (ps) fU -sense Key 1 = 2'F-dU
2 = 2'F-dA
oo 3 = 2'F-dC
n 1-i 4 = 2'F-dG
m oo t..) o = 2'0Me-rU
oe O-6 = 2'0Me-rA
oe ,-, ,-, o u, 7 = 2'0Me-rC

t..) o 8 = 2'0Me-rG
,¨

,o O-,o t..) t..) mA, mU, mC, mG, OMeA, OMeU, OMeC, OMeG ¨ 2`-0Me RNA
oe t..) fA, fU, fC, fG, FA, FU, FG, FC ¨ 2'deoxy-2'-F RNA
(ps) ¨ phosphorothioate (vp) - Vinyl-(E)-phosphonate ivA, ivC, ivU, ivG - inverted RNA (3'-3') P
.
w , , A single sequence may have more than one name. In those cases, one of those names is given in the summary sequence table. .3 I, L' IV
,j IV

IV
F' I
Where specific linkers and or modified linkages are taught within an RNA
sequence, such as PS and [ST23 (ps)]3 ST41 (ps) etc, these are optional , parts of the sequence, but are a preferred embodiment of that sequence.
The following abbreviations may be used:
, _ FAM 6-Carboxyfluorescein BHQ Black Hole Quencher 1 Iv n ST23 oi._ ( _Ac OAc M
IV
n.) Ac0 ---400,.-o NHAc oe oe 1-.
1-.
o vi ST41/C4XLT ''-oo t..) ..........õ..¨..._õ..¨...._o-,--o ---.00 -a-, t.., t..) ST43-phos/C6XLT DMTr oo N
DMTrN
0 0 001:1.0,CN
DMTrN

Long trebler/Itrb/STKS (phosphoramidite) ODMT
IDCDOCOODMT
NC() I
N(/P02 \
P
\-0DMT
w I, L' Ser(GN) (phosphoramidite) OH

, oo HO.\...o.
,, .
,, , HO NH
, , NHAc w 0 .
C3Am(GN) 0 0.,y,"....õ.õ--...õ,. -, GaINAc NH
õ.0j --od n GlyC3Am(GN) 0 0.y....õ..."...,..../.. .., GaINAc m od t..) ONH
o oo -a-, ,oj --o' .
=
u, C-) <
Z
TO

< \

c73 <ct) 0 \

To /

=
0 :
, /
=
\O
R R
\ , , E E E
< < <
CD N=-= 0-O 0 il

Claims (21)

Claims
1. 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 RNA
transcribed from the ALDH2 gene, wherein said first strand comprises a nucleotide sequence selected from the following sequences: SEQ ID NOs: 11, 17, 1, 3, 5, 7, 9, 13, 15, 17, 19, 21, 23, 25 or 27.
2. The nucleic acid of claim 1, wherein said first strand comprises a nucleotide sequence of SEQ ID NO:11, and optionally wherein said second strand comprises a nucleotide sequence of SEQ ID NO: 12; or said first strand comprises a nucleotide sequence of SEQ ID NO:17, and optionally wherein said second strand comprises a 1.5 nucleotide sequence of SEQ ID NO: 18; or said first strand comprises a nucleotide sequence of SEQ ID NO:3 and optionally wherein said second strand comprises a nucleotide sequence of SEQ ID NO: 4.
3. The nucleic acid of any of the preceding claims, wherein said first strand and/or said second strand are each from 17-35 nucleotides in length.
4. The nucleic acid of any of the preceding claims, wherein the at least one duplex region consists of 17-25, preferably 19-25, consecutive nucleotide base pairs.
5. The nucleic acid of any of the preceding claims, wherein the nucleic acid:
a) is blunt ended at both ends; or b) has an overhang at one end and a blunt end at the other; or c) has an overhang at both ends.
6. The nucleic acid of any of the preceding claims, wherein one or more nucleotides on the first and/or second strand are modified, to form modified nucleotides.
7. The nucleic acid of any of the preceding claims, wherein the nucleic acid comprises a phosphorothioate linkage between the terminal one, two or three 3' nucleotides and/or 5' nucleotides of one or both ends of the first and/or the second strand.
8. The nucleic acid of any of the preceding claims, wherein the nucleic acid is conjugated to a ligand.
9. The nucleic acid of claim 8, wherein the ligand comprises (i) one or more N-acetyl galactosamine (GaINAc) moieties or derivatives thereof, and (ii) a linker, wherein the 1.13 linker conjugates the at least one GaINAc moiety or derivative thereof to the nucleic acid.
10. The nucleic acid of any of claims 8-9, wherein the nucleic acid is conjugated to a ligand comprising a compound of formula (I):
1.5 [S-X1-P-X2]3-A-X3- (1) wherein:
S represents a saccharide, preferably wherein the saccharide is N-acetyl galactosamine;
20 X1 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;
X2 is alkylene or an alkylene ether of the formula (-CH2)n-O-CH2- where n = 1-6;
A is a branching unit;
25 X3 represents a bridging unit;
wherein a nucleic acid as defined in any of claims 1 to 7 is conjugated to X3 via a phosphate or modified phosphate, preferably a thiophosphate.
11. The nucleic acid of any of claims 8-9, wherein the first RNA strand is a compound of 30 formula (X):

Y / Y \
5' 3' ll II
Z1-0¨P-0 Li _____________________________ 0 P 0 L1 ______ 0 H
I I
OH \ OH
/
- - b (x) wherein b is 0 or 1; and the second RNA strand is a compound of formula (XI):
_ Y Y Y
-( \ Y \
Il II II II
H-0 Li¨O¨P-0-'-Li¨O¨P-0-5Z2 3' 0 P 0 Li _______________ 0 P 0 Li-LO ___ H
I I I I
OH / i OH OH OH - c -- / -d (XI);
wherein:
c and d are independently 0 or 1;
Z1 and Z2 are the RNA portions of the first and second RNA strands respectively;
Y is 0 or S;
n is 0, 1, 2 or 3; and L1 is a linker to which a ligand is attached; and wherein b + c + d is 2 or 3.
12. 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 RNA
transcribed from the ALDH2 gene, wherein said first strand comprises, and preferably consists of, the nucleotide sequence of SEQ ID NO: 11 and optionally, wherein said second strand comprises, and preferably consists of, the nucleotide sequence of SEQ
ID NO. 12.
13. The nucleic acid of claim 12, wherein the nucleic acid is conjugated to a ligand and has the following structure OH
<:\ 0 iü L....\., Aci iN , I 0 =P¨s0 -1) INN1 i 0 0=P¨S

..,,, f I
o ri II
LI, lei? s 0- P-0 s .
wherein Z is a nucleic acid according ot claim 12.
14. The nucleic acid of claim 13, wherein the nucleic acid comprises 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 and wherein the ligand is conjugated to the 5' end of the second strand.
15. The nucleic acid of claim 12, wherein the nucleic acid is conjugated to a ligand, wherein the first RNA strand is a compound of formula (XV):
GaINAc\ GaINAc L " L
\ \
NH NH
5 3' 1(1 / Y / ) I I
OH R1 \ OH R1 n b ¨ ¨ (XV) wherein b is 0 or 1; and the second RNA strand is a compound of formula (XVI):
GaINAc HN/ /GaINAc GaINAc GaINAc Hd / . , L L L\NH L
\
NH

\ II \ IIll / Y /
H-0 O¨P-0 0¨P-0¨Z2-0¨P-0 O¨P-0 \ 0¨H
OH OH OH \ I
-(-R1 R1 R1 \ OH R1 /n n c _ ¨ d ¨ ¨ (XVI);
wherein c and d are independently 0 or 1;
wherein:
Z1 and Z2 are the RNA portions of the first and second RNA strands respectively;
Y is 0 or S;
R1 is H or methyl;
n is 0, 1, 2 or 3; and L is the same or different in formulae (XV) and (XVI) and is selected from the group consisting of:
-(CH2)q, wherein q = 2-12;
-(CH2)r-C(0)-, wherein r = 2-12;
-(CH2-CH2-0)s-CH2-C(0)-, wherein s = 1-5;
-(CH2)t-CO-NH-(CH2)t-NH-C(0)-, wherein t is independently is 1-5;
-(CH2).-CO-NH-(CH2)u-C(0)-, wherein u is independently is 1-5; and -(CH2),-NH-C(0)-, wherein v is 2-12; and wherein the terminal C(0), if present, is attached to the NH group;
and wherein b + c + d is 2 or 3.
16. The nucleic acid of any of claims 12-15, comprising a sequence and modifications as shown below:
SEQ ID NO: sequence modifications 11 5'-UCUUCUUAAACUGAGUUUC-3' 5351715262718281517 12 5'-GAAACUCAGUUUAAGAAGA-3' 4626353645152646282 wherein, the specific modifications are depicted by numbers 1=2"F-dU, 2=2`F-dA, 3=2"F-dC, 4=2"F-dG, 5=2'-0Me-rU;
6=2'-0Me-rA;
7=2'-0Me-rC;
8=2'-0Me-rG.
17. The nucleic acid of any of claims 12-15, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified with a 2' fluoro modification, and the nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand are modified with a 2' fluoro modification.
18. A composition comprising a nucleic acid of any of claims 1-17 and optionally a delivery vehicle and/or a physiologically acceptable excipient and/or a carrier and/or a diluent and/or a buffer and/or a preservative, for use as a medicament, preferably for the prevention or treatment of a disease, disorder or syndrome, wherein the disease, disorder or syndrome preferably is an alcohol use disorder.
19. A pharmaceutical composition comprising a nucleic acid of any of claims 1-17 and further comprising a delivery vehicle, preferably liposomes and/or a physiologically acceptable excipient and/or a carrier and/or a diluent.
20. Use of a nucleic acid of any of claims 1-17 or a pharmaceutical composition of claim 19 in the prevention or treatment of a disease, disorder or syndrome, wherein the disease, disorder or syndrome preferably is an alcohol use disorder.
21. A method of preventing or treating a disease, disorder or syndrome comprising administering a composition comprising a nucleic acid of any of claims 1-17 or a composition according to claims 1 8-1 9 to an individual in need of treatment, preferably wherein the nucleic acid or composition is administered to the subject subcutaneously, intravenously or using any other application routes such as oral, rectal or intraperitoneal.
CA3118327A 2017-11-13 2018-11-13 Nucleic acids for inhibiting expression of aldh2 in a cell Pending CA3118327A1 (en)

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GB1815921.0 2018-09-28
GB201815921 2018-09-28
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