Polynucleotide molecule for suppressing DMPK expression and use thereof

Abstract

The present invention is suitable for the technical field of molecular biology, and specifically provides a polynucleotide molecule binding mRNA of a DMPK gene. The polynucleotide molecule comprises any nucleotide sequence among SEQ ID NO: 1 to SEQ ID NO: 200 and SEQ ID NO: 281 to SEQ ID NO: 490 or at least 12 contiguous nucleotides in a nucleotide sequence complementary thereto, or a sequence differing by no more than 3 nucleotides from the at least 12 contiguous nucleotides; and the nucleotides are in a modified or unmodified state. The polynucleotide molecule is complementary to a target sequence of DMPK and mediates RNA interference against the DMPK, thereby treating muscular atrophy or myotonic dystrophy in a subject.

Description

thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methylphosphonates and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidate          and          aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, morpholino linkages, and boranophosphates; 2'-5' linked analogs of these substances, and those with reversed polarity, where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. In other embodiments, the modified internucleoside linkage or backbone contains no phosphorus atom. Modified internucleoside linkages or backbones without a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl sugar linkages, mixed heteroatom and alkyl or cycloalkyl sugar linkages, and one or more short chain heteroatom or heterocyclic sugar linkages. In some embodiments, modified internucleoside backbones include, but are not limited to, siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH2 component parts.

[0146] Polynucleotide Synthesis

[0147] In some embodiments, the polynucleotide molecules described herein are constructed using chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. For example, the polynucleotide molecules are chemically synthesized using naturally occurring nucleotides or various modified nucleotides. Alternatively, the polynucleotide molecules are produced biologically using an expression vector into which the polynucleotide molecule has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted polynucleotide molecule will be in an antisense orientation to a target polynucleotide molecule of interest).

[0148] In some embodiments, the polynucleotide molecule is synthesized by a tandem synthesis method, wherein the two strands are synthesized as a single contiguous oligonucleotide fragment or chain separated by a cleavable linker, which is subsequently cleaved to provide separate fragments or strands that hybridize and allow purification of the duplex.

[0149] In some embodiments, the polynucleotide molecule is also assembled from two distinct nucleic acid strands or fragments, wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the molecule.

[0150] Other modification methods for incorporating modifications such as sugar, base, and phosphate modifications are described in the prior art, and reference is made to methods in the prior art.

[0151] In some embodiments, although chemical modification of the internucleoside linkages of the polynucleotide molecule using phosphorothioate, phosphorodithioate, and / or 5'-methylphosphonate linkages improves stability, excessive modification can sometimes cause toxicity or reduced activity. Therefore, when designing nucleic acid molecules, in some embodiments, the amount of these internucleoside linkages is minimized. In such cases, reducing the concentration of these linkages results in reduced toxicity, improved efficacy, and higher specificity of these molecules.

[0152] Pharmaceutical Composition

[0153] The present application provides a pharmaceutical composition comprising the aforementioned polynucleotide molecule and optionally a pharmaceutically acceptable excipient.

[0154] In some embodiments, the pharmaceutical composition or medicament may comprise a pharmacologically effective amount of at least one said polynucleotide molecule and one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (excipient) is a substance that has undergone appropriate safety evaluation and is intentionally included in a drug delivery system other than the active pharmaceutical ingredient (API, therapeutic product, such as the polynucleotide molecule). An excipient does not exert or is not intended to exert a therapeutic effect at the intended dosage. Excipients may be used to a) aid in the processing of the drug delivery system during manufacture, b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API, c) assist in product identification, and / or d) enhance any other attribute that contributes to the overall safety and effectiveness of the delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0155] Excipients include: absorption enhancers, anti-adherents, defoamers, antioxidants, binders, buffering agents, carriers, coating agents, coloring agents, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickening agents, tonicity agents, vehicles, waterproofing agents, and wetting agents.

[0156] In some embodiments, the pharmaceutical composition may further comprise any other suitable therapeutic agent for treating a subject (e.g., a human subject with DM1). In some embodiments, other therapeutic agents may enhance or supplement the efficacy of the polynucleotide molecules described herein. In some embodiments, other therapeutic agents may function to treat symptoms or diseases different from those targeted by the polynucleotide molecules described herein.

[0157] In some embodiments, the polynucleotide molecule is combined with one or more additional therapeutic agents or treatments, including but not limited to: small molecule drugs, antibodies, antibody fragments, and / or vaccines. In some embodiments, the therapeutic agent or treatment includes an anti-transferrin receptor antibody.

[0158] In some embodiments, the pharmaceutical composition can be in solid form, aqueous form, or liquid form. In some embodiments, the aqueous or liquid form can be nebulized or lyophilized. In some embodiments, the nebulized or lyophilized form can be reconstituted with an aqueous solution or liquid solution.

[0159] In some embodiments, the pharmaceutical composition (e.g., polynucleotide molecule or antibody) is lyophilized to extend its shelf life and subsequently reconstituted into a solution before use (e.g., administration to a subject). Thus, the excipient can be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).

[0160] In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration. Some examples of routes of administration include parenteral administration, e.g., intravenous, intradermal, subcutaneous. Typically, the route of administration is intravenous or subcutaneous.

[0161] The pharmaceutical composition of the present application can be used to inhibit the expression of the DMPK gene in a cell, tissue, or organism. In some embodiments, the pharmaceutical composition is used to treat a subject suffering from a disease, disorder, or condition that would benefit from reduced or inhibited DMPK expression. In some embodiments, the disease, disorder, or condition that would benefit from reduced or inhibited DMPK expression includes, but is not limited to: muscle atrophy or myotonic dystrophy, where muscle atrophy is caused by and / or associated with cachexia (e.g., cancer cachexia), denervation, myopathy, motor neuron disease, diabetes, chronic obstructive pulmonary disease, liver disease, congestive heart failure, chronic renal failure, chronic infection, sepsis, fasting, sarcopenia, glucocorticoid-induced atrophy, disuse, or spaceflight. In some embodiments, the muscle atrophy includes diabetes-associated muscle atrophy or cancer cachexia-associated muscle atrophy. In some embodiments, the myotonic dystrophy is DM1. In some embodiments, the subject is a mammal, including but not limited to a human.

[0162] Cells, tissues, and non-human organisms comprising at least one polynucleotide molecule described herein are contemplated. The cells, tissues, or non-human organisms are prepared by delivering the polynucleotide molecule to the cell, tissue, or non-human organism by any means available in the art. In some embodiments, the cell is a mammalian cell, including but not limited to a human cell. The cells, tissues, or non-human organisms can be used for research or as research tools (e.g., drug testing or diagnostics).

[0163] Methods of Use / Treatment

[0164] The present application provides a method for preventing and / or treating a disease or disorder, the method comprising administering to a target subject an effective amount of the aforementioned polynucleotide molecule and / or the aforementioned pharmaceutical composition.

[0165] In some embodiments, the polynucleotide molecules and / or pharmaceutical compositions described herein can be used to treat muscle atrophy or myotonic dystrophy (DM1) by reducing DMPK expression.

[0166] In some embodiments, a therapeutically effective amount of one or more of said polynucleotide molecules is administered to a subject, thereby inhibiting DMPK expression in the subject (e.g., an amount effective to inhibit DMPK expression in the subject). In some embodiments, the subject can be a human subject, a non-human primate subject, a rodent subject, or any suitable mammalian subject. In some embodiments, the subject has a DMPK allele that may optionally contain a disease-associated repeat. In some embodiments, the subject may have a DMPK allele with an expanded disease-associated repeat, said expanded disease-associated repeat comprising about 2 to 10 repeat units, about 2 to 50 repeat units, about 2 to 100 repeat units, about 50 to 1,000 repeat units, about 50 to 500 repeat units, about 50 to 250 repeat units, about 50 to 100 repeat units, about 500 to 10,000 repeat units, about 500 to 5,000 repeat units, about 500 to 2,500 repeat units, about 500 to 1,000 repeat units, or about 1,000 to 10,000 repeat units.

[0167] In some embodiments, the pharmaceutical composition comprising the aforementioned polynucleotide molecule can be administered by a suitable route, by which the polynucleotide molecule is brought into contact with the body. The route may include intravenous administration, e.g., as a bolus or by continuous infusion over a period of time. In some embodiments, intravenous administration can be performed by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, or intrathecal routes.

[0168] In some embodiments, the polynucleotide molecule and / or pharmaceutical composition is administered at an effective concentration that confers a therapeutic effect on the subject. As recognized by those skilled in the art, the effective amount varies depending on the severity of the disease, the unique characteristics of the subject being treated (e.g., age, physical condition, health, or weight), the duration of treatment, the nature of any concurrent therapy, the route of administration, and related factors. These related factors are known to those skilled in the art and can be resolved by routine experimentation. In some embodiments, the effective concentration is the maximum dose considered safe for the patient. In some embodiments, the effective concentration will be the lowest possible concentration that provides maximum efficacy.

[0169] Generally, for administration of any of the polynucleotide molecules and / or pharmaceutical compositions described herein, depending on the above factors, e.g., safety or efficacy, the initial candidate dose may be about 1 to 100 mg / kg or higher. In some embodiments, treatment will be administered once. In some embodiments, treatment will be administered daily, bi-weekly, weekly, bimonthly, monthly, or at any time interval that provides maximum efficacy while minimizing safety risk to the subject. Typically, efficacy and treatment as well as safety risks can be monitored throughout the course of treatment.

[0170] In some embodiments, a single administration or dose of the pharmaceutical composition to the subject is sufficient to inhibit the activity or expression of the target gene for at least 1 to 5 days, 1 to 10 days, 5 to 15 days, 10 to 20 days, 15 to 30 days, 20 to 40 days, 25 to 50 days, or more days. In some embodiments, a single administration or dose of the pharmaceutical composition to the subject is sufficient to inhibit the activity or expression of the target gene for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In some embodiments, a single administration or dose of the pharmaceutical composition to the subject is sufficient to inhibit the activity or expression of the target gene for at least 1, 2, 3, 4, 5, or 6 months.

[0171] In some embodiments, the pharmaceutical composition is administered to the subject at an effective concentration sufficient to inhibit target gene activity or expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% relative to a control (e.g., baseline level of gene expression before treatment).

[0172] In some embodiments, the polynucleotide molecules or compositions described herein can be delivered to cells, cell populations, tissues, or subjects using polynucleotide molecule delivery techniques known in the art. Generally, any suitable method recognized in the art for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for the polynucleotide molecules described herein. For example, delivery can be by local administration (e.g., direct injection, implantation, or topical administering), systemic administration, or subcutaneous, intravenous, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), intranasal, rectal, or topical (including buccal and sublingual) administration. In some embodiments, the composition is administered by subcutaneous or intravenous infusion or injection.

[0173] In some embodiments, the polynucleotide molecule can be combined with lipids, nanoparticles, polymers, liposomes, micelles, or other delivery systems available in the art. The polynucleotide molecule can also be chemically conjugated to targeting groups, lipids (including but not limited to cholesterol and cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, or other delivery systems available in the art. The polynucleotide molecule can be conjugated to a delivery polymer. In some embodiments, the delivery polymer is a reversibly masked / modified amphipathic membrane-active polyamine.

[0174] Uses

[0175] In another aspect, the present application provides a use of the aforementioned polynucleotide molecule or the aforementioned pharmaceutical composition in the manufacture of a medicament for the prevention and / or treatment of the risk of muscle atrophy or myotonic dystrophy.

[0176] In some embodiments, the muscle atrophy includes muscle atrophy associated with insulin deficiency, associated with chronic renal failure, associated with congestive heart failure, associated with chronic respiratory disease, associated with chronic infection, associated with fasting, associated with denervation, associated with sarcopenia, or associated with glucocorticoid therapy. In some embodiments, the myotonic dystrophy is DM1.

[0177] Kits

[0178] In some embodiments, the present application discloses kits and articles of manufacture for use with one or more compositions and methods described herein. Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers, such as vials, tubes, etc., each container containing a separate element to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the container is formed from various materials such as glass or plastic.

[0179] In one embodiment, the container contains a polynucleotide molecule described herein. Such kits optionally contain an identifying description or label or instructions for its use in a method described herein.

[0180] The present application is further described below by means of specific examples.

[0181] siRNA Sequences and Synthesis

[0182] Initially designed siRNAs consist of fully complementary sense and antisense strands with a strand length of 19 or 21 nucleotides, wherein the antisense strand must be perfectly matched to the target mRNA to exert catalytic activity. In some cases, to increase cellular preference for the antisense strand, 2 nucleotides can be added as an overhang to the 3' end of the antisense strand, with the overhang not pairing with the sense strand. Thus, 19 / 21mer can be used to describe an siRNA design where 2 nucleotides are added to the 3' end of the antisense strand of an initially 19-nucleotide siRNA, where 19 and 21 refer to the lengths of the sense and antisense strands, respectively. 21 / 23mer can be used to describe an siRNA design where a 2-nucleotide overhang is added to the 3' end of the antisense strand of an initially 21-nucleotide siRNA, where 21 and 23 refer to the lengths of the sense and antisense strands, respectively.

[0183] In some cases, the base sequence of the 3' overhang of the antisense strand can be perfectly matched to the target mRNA or consist of other unrelated dinucleotide sequences, such as "UU", without affecting siRNA activity. In some cases, when the antisense strand is loaded into Ago2, the first base at the 5' end binds to Ago2 but not to the target mRNA, and "U" is thermodynamically most preferred for binding to Ago2. Therefore, for all antisense strands, the first base at the 5' end can be replaced with "U", and correspondingly, the last base at the 3' end (position 19) of all sense strands can be replaced with "A", without affecting siRNA activity.

[0184] Both single strands of the siRNAs in the present application were synthesized separately on solid supports using standard phosphoramidite chemistry. The specific sequences are shown below. The single strands were purified by HPLC, and the purity was controlled above 95%. Subsequently, the two complementary single strands were annealed and paired at an approximate molar ratio to form siRNA duplexes, which were then lyophilized for later use.

[0185] Example 1: siRNA Design and Preliminary Analysis and Screening

[0186] 1A: Design and Screening of 19 / 21mer siRNA Naked Sequences

[0187] In this example, 100 pairs of 19 / 21mer siRNAs (siRNA ID# 0001-0100) were designed for the human DMPK transcript variant NM_001081563.2 or NM_001081560.3, wherein SEQ ID NO: 001-SEQ ID NO: 100 are the sense strands, and SEQ ID NO: 101-200 are the antisense strands. The first 19 / 21mer siRNA is siRNA ID# 0001, with its sense strand being SEQ ID NO: 001 and its antisense strand being SEQ ID NO: 101, and so on. In the sequence design of the present application, non-functional siRNAs, sequences with high off-target risk, humanmonkey mismatches, or low scores were removed (see Figure 1 for the specific design process). Detailed sequence information is shown in the table.

[0188] Table 1: Specific sequences of 19 / 21mer siRNAs siRNA ID# Sense Strand Sequence (5'-3') - 19nt Seq ID NO: Antisense Strand Sequence (5'-3') -21nt Seq ID NO: 0001 CCAUGAAGAUCAUGAACAA 1 UUGUUCAUGAUCUUCAUGGUU 101 0002 GCCAUGAAGAUCAUGAACA 2 UGUUCAUGAUCUUCAUGGCUU 102 0003 GACACAUGCAACUUCGACA 3 UGUCGAAGUUGCAUGUGUCUU 103 0004 CAUGCAACUUCGACUUGGA 4 UCCAAGUCGAAGUUGCAUGUU 104 0005 CCUUCCAGGAUGAGAACUA 5 UAGUUCUCAUCCUGGAAGGUU 105 0006 GAAGGUGCCACCGACACAA 6 UUGUGUCGGUGGCACCUUCUU 106 0007 GUAUUCGCCUAUGAAAUGA 7 UCAUUUCAUAGGCGAAUACUU 107 0008 CAGGAUGAGAACUACCUGA 8 UCAGGUAGUUCUCAUCCUGUU 108 0009 CUGACACUGCUGAGCAAGA 9 UCUUGCUCAGCAGUGUCAGUU 109 0010 GGCAAGAUCGUCCACUACA 10 UGUAGUGGACGAUCUUGCCUU 110 0011 GUGAGGAGAGGGACGUGUA 11 UACACGUCCCUCUCCUCACUU 111 0012 CUUCUACCUGGCGGAGAUA 12 UAUCUCCGCCAGGUAGAAGUU 112 0013 GCAAGAUCGUCCACUACAA 13 UUGUAGUGGACGAUCUUGCUU 113 0014 GGAGACACUGUCGGACAUA 14 UAUGUCCGACAGUGUCUCCUU 114 0015 GUAGUGAAGAUGAAGCAGA 15 UCUGCUUCAUCUUCACUACUU 115 0016 GCCUUCCAGGAUGAGAACA 16 UGUUCUCAUCCUGGAAGGCUU 116 0017 GUGUAUUCGCCUAUGAAAA 17 UUUUCAUAGGCGAAUACACUU 117 0018 GGUGUAUUCGCCUAUGAAA 18 UUUCAUAGGCGAAUACACCUU 118 0019 CGCCUAUGAAAUGUUCUAA 19 UUAGAACAUUUCAUAGGCGUU 119 0020 GUGUAUGCCAUGAAGAUCA 20 UGAUCUUCAUGGCAUACACUU 120 0021 CGGUAGUGAAGAUGAAGCA 21 UGCUUCAUCUUCACUACCGUU 121 0022 CCUUUUGUGGGCUACUCCA 22 UGGAGUAGCCCACAAAAGGUU 122 0023 CUUUUGUGGGCUACUCCUA 23 UAGGAGUAGCCCACAAAAGUU 123 0024 CCGACAUUCCUCGGUAUUA 24 UAAUACCGAGGAAUGUCGGUU 124 0025 CCGACACAUGCAACUUCGA 25 UCGAAGUUGCAUGUGUCGGUU 125 0026 CAGGUGUAUGCCAUGAAGA 26 UCUUCAUGGCAUACACCUGUU 126 0027 GACGACUUCGAGAUUCUGA 27 UCAGAAUCUCGAAGUCGUCUU 127 0028 GACAUCAAACCCGACAACA 28 UGUUGUCGGGUUUGAUGUCUU 128 0029 CCAUCGUGGUGAGGCUUAA 29 UUAAGCCUCACCACGAUGGUU 129 0030 GCCUCGACUGGGAUGGUCA 30 UGACCAUCCCAGUCGAGGCUU 130 0031 GGGUGUAUUCGCCUAUGAA 31 UUCAUAGGCGAAUACACCCUU 131 0032 GACAUUCCUCGGUAUUUAA 32 UUAAAUACCGAGGAAUGUCUU 132 0033 CCCCCUUUACACCGGAUUA 33 UAAUCCGGUGUAAAGGGGGUU 133 0034 CCCUUCUACGCGGAUUCCA 34 UGGAAUCCGCGUAGAAGGGUU 134 0035 CCGAGAUGGCGCGCUUCUA 35 UAGAAGCGCGCCAUCUCGGUU 135 0036 CGUUUUGGAUGCACUGAGA 36 UCUCAGUGCAUCCAAAACGUU 136 0037 CCCCAAUCCACGUUUUGGA 37 UCCAAAACGUGGAUUGGGGUU 137 0038 GACACUGCUGAGCAAGUUA 38 UAACUUGCUCAGCAGUGUCUU 138 0039 GGGACGACUUCGAGAUUCA 39 UGAAUCUCGAAGUCGUCCCUU 139 0040 CGGUUUGGAUAUUUAUUGA 40 UCAAUAAAUAUCCAAACCGUU 140 0041 CCACCGACACAUGCAACUA 41 UAGUUGCAUGUGUCGGUGGUU 141 0042 CCCGACAUUCCUCGGUAUA 42 UAUACCGAGGAAUGUCGGGUU 142 0043 CCCAUCGUGGUGAGGCUUA 43 UAAGCCUCACCACGAUGGGUU 143 0044 CUCGGUAUUUAUUGUCUGA 44 UCAGACAAUAAAUACCGAGUU 144 0045 GACAGCGUGCCCCCCUUUA 45 UAAAGGGGGGCACGCUGUCUU 145 0046 GACUGCAGAGGGACGACUA 46 UAGUCGUCCCUCUGCAGUCUU 146 0047 CGUGCACAGGGACAUCAAA 47 UUUGAUGUCCCUGUGCACGUU 147 0048 GCCAGGUGUAUGCCAUGAA 48 UUCAUGGCAUACACCUGGCUU 148 0049 CCCCAACAACCCCAAUCCA 49 UGGAUUGGGGUUGUUGGGGUU 149 0050 CCCUGAACCCUAGAACUGA 50 UCAGUUCUAGGGUUCAGGGUU 150 0051 GCAGACGGGCCAGGUGUAA 51 UUACACCUGGCCCGUCUGCUU 151 0052 CCCAAUCCACGUUUUGGAA 52 UUCCAAAACGUGGAUUGGGUU 152 0053 CUGAGACCCCGACAUUCCA 53 UGGAAUGUCGGGGUCUCAGUU 153 0054 CUGUACCUGGUCAUGGAGA 54 UCUCCAUGACCAGGUACAGUU 154 0055 GUAGCGGUAGUGAAGAUGA 55 UCAUCUUCACUACCGCUACUU 155 0056 GCACUGAGACCCCGACAUA 56 UAUGUCGGGGUCUCAGUGCUU 156 0057 GAAGAACUGGGUCUUAUUA 57 UAAUAAGACCCAGUUCUUCUU 157 0058 CCGCUUCGGCGGUUUGGAA 58 UUCCAAACCGCCGAAGCGGUU 158 0059 GAUCACAGACCAUUUCUUA 59 UAAGAAAUGGUCUGUGAUCUU 159 0060 GGCGGUUUGGAUAUUUAUA 60 UAUAAAUAUCCAAACCGCCUU 160 0061 GGAAGAACUGGGUCUUAUA 61 UAUAAGACCCAGUUCUUCCUU 161 0062 GGACACAUCCCUUCUUCUA 62 UAGAAGAAGGGAUGUGUCCUU 162 0063 GACACAUCCCUUCUUCUUA 63 UAAGAAGAAGGGAUGUGUCUU 163 0064 CCUAGAACUGUCUUCGACA 64 UGUCGAAGACAGUUCUAGGUU 164 0065 GGAUCACGCAGCUGCACUA 65 UAGUGCAGCUGCGUGAUCCUU 165 0066 CAAAUCCAGGGUUUUCCAA 66 UUGGAAAACCCUGGAUUUGUU 166 0067 GGAUCACAGACCAUUUCUA 67 UAGAAAUGGUCUGUGAUCCUU 167 0068 GUACCUGGUCAUGGAGUAA 68 UUACUCCAUGACCAGGUACUU 168 0069 CUGAACCCUAGAACUGUCA 69 UGACAGUUCUAGGGUUCAGUU 169 0070 CCCCGUUGGAAGACUGAGA 70 UCUCAGUCUUCCAACGGGGUU 170 0071 CCUUCUCCCCUUGUUCUUA 71 UAAGAACAAGGGGAGAAGGUU 171 0072 GCUACAGGACCCCCAACAA 72 UUGUUGGGGGUCCUGUAGCUU 172 0073 GAGGUAGCGGUAGUGAAGA 73 UCUUCACUACCGCUACCUCUU 173 0074 CCGAACUGGCCCAGGACAA 74 UUGUCCUGGGCCAGUUCGGUU 174 0075 CGUGGAUGGGCAAACUGCA 75 UGCAGUUUGCCCAUCCACGUU 175 0076 CCUUACACGGUGUUUAUGA 76 UCAUAAACACCGUGUAAGGUU 176 0077 CCUGAUUGUCAUCUGUAAA 77 UUUACAGAUGACAAUCAGGUU 177 0078 CUGUUCGCCGUUGUUCUGA 78 UCAGAACAACGGCGAACAGUU 178 0079 CUGAUUGUCAUCUGUAAAA 79 UUUUACAGAUGACAAUCAGUU 179 0080 GAUUGUCAUCUGUAAAUAA 80 UUAUUUACAGAUGACAAUCUU 180 0081 GCGAGGUAGCGGUAGUGAA 81 UUCACUACCGCUACCUCGCUU 181 0082 CCCGCUUCGGCGGUUUGGA 82 UCCAAACCGCCGAAGCGGGUU 182 0083 CGCUUCGGCGGUUUGGAUA 83 UAUCCAAACCGCCGAAGCGUU 183 0084 GCUUCGGCGGUUUGGAUAA 84 UUAUCCAAACCGCCGAAGCUU 184 0085 CAAGCUGCGGGCAGAUGGA 85 UCCAUCUGCCCGCAGCUUGUU 185 0086 CGCUUUCCCUGCUCCUGUA 86 UACAGGAGCAGGGAAAGCGUU 186 0087 GCUCCUGUUCGCCGUUGUA 87 UACAACGGCGAACAGGAGCUU 187 0088 GAACUGGCCCAGGACAAGA 88 UCUUGUCCUGGGCCAGUUCUU 188 0089 CCAGUUUAACAUUCCUAGA 89 UCUAGGAAUGUUAAACUGGUU 189 0090 GGGAUCACAGACCAUUUCA 90 UGAAAUGGUCUGUGAUCCCUU 190 0091 GCCUGAUUGUCAUCUGUAA 91 UUACAGAUGACAAUCAGGCUU 191 0092 CUUCGGCGGUUUGGAUAUA 92 UAUAUCCAAACCGCCGAAGUU 192 0093 GAACCACCUGCAUCUGAAA 93 UUUCAGAUGCAGGUGGUUCUU 193 0094 GAACCUUACACGGUGUUUA 94 UAAACACCGUGUAAGGUUCUU 194 0095 CAUGGUGCUGCCUGUCCAA 95 UUGGACAGGCAGCACCAUGUU 195 0096 CUCUAUGGGAGGGCAUUUA 96 UAAAUGCCCUCCCAUAGAGUU 196 0097 CCAAAGUGUGGUUCAAGAA 97 UUCUUGAACCACACUUUGGUU 197 0098 CCAAAUCCAGGGUUUUCCA 98 UGGAAAACCCUGGAUUUGGUU 198 0099 CGGACACAUCCCUUCUUCA 99 UGAAGAAGGGAUGUGUCCGUU 199 0100 GCUACGGGCCCGAGUGUGA 100 UCACACUCGGGCCCGUAGCUU 200

[0189] 1B: Design of 21 / 23mer siRNA Naked Sequences

[0190] For the human DMPK transcript variant NM_004409.5, 105 21 / 23mer siRNAs (siRNA ID# 0141-0245) were designed. Among these, SEQ ID NO: 281 385 are the sense strands, and SEQ ID NO: 386-490 are the antisense strands. The first 21 / 23mer siRNA is siRNA ID# 0141, with its sense strand being SEQ ID NO: 281 and its antisense strand being SEQ ID NO: 386, and so on. Non-functional siRNAs, sequences with high off-target risk, human-monkey mismatches, or low scores were removed during sequence design (see Figure 2 for the specific design process). The sequence information is shown in Table 2.

[0191] Table 2: Design of 21 / 23mer siRNAs siRNA ID# Sense Strand Sequence (5'3') - 21nt Seq ID NO: Antisense Strand Sequence (5'3') - 23nt Seq ID NO: 0141 AUGGCAAGAUCGUCCA CUACA 281 UGUAGUGGACGAUCUUGC CAUAG 386 0142 UGGCAAGAUCGUCCAC UACAA 282 UUGUAGUGGACGAUCUU GCCAUA 387 0143 GGCAAGAUCGUCCACU ACAAG 283 CUUGUAGUGGACGAUCUU GCCAU 388 0144 AAGAUCGUCCACUACA AGGAG 284 CUCCUUGUAGUGGACGAU CUUGC 389 0145 UCCACUACAAGGAGCA CCUCU 285 AGAGGUGCUCCUUGUAGU GGACG 390 0146 CACUACAAGGAGCACC UCUCU 286 AGAGAGGUGCUCCUUGUA GUGGA 391 0147 UACAAGGAGCACCUCU CUCUG 287 CAGAGAGAGGUGCUCCUU GUAGU 392 0148 CACGCCGGCCAACUCAC CGCA 288 UGCGGUGAGUUGGCCGGC GUGGG 393 0149 CGCCGGCCAACUCACCG CAGU 289 ACUGCGGUGAGUUGGCCG GCGUG 394 0150 GCCGGCCAACUCACCGC AGUC 290 GACUGCGGUGAGUUGGCC GGCGU 395 0151 CCGGCCAACUCACCGCA GUCU 291 AGACUGCGGUGAGUUGGC CGGCG 396 0152 CGGCCAACUCACCGCA GUCUG 292 CAGACUGCGGUGAGUUGG CCGGC 397 0153 CCCGCGCUCCCUGAACC CUAG 293 CUAGGGUUCAGGGAGCGC GGGCG 398 0154 CCGCGCUCCCUGAACCC UAGA 294 UCUAGGGUUCAGGGAGCG CGGGC 399 0155 CGCGCUCCCUGAACCCU AGAA 295 UUCUAGGGUUCAGGGAGC GCGGG 400 0156 GCGCUCCCUGAACCCU AGAAC 296 GUUCUAGGGUUCAGGGA GCGCGG 401 0157 CGCUCCCUGAACCCUA GAACU 297 AGUUCUAGGGUUCAGGG AGCGCG 402 0158 CUCCCUGAACCCUAGA ACUGU 298 ACAGUUCUAGGGUUCAGG GAGCG 403 0159 UCCCUGAACCCUAGAA CUGUC 299 GACAGUUCUAGGGUUCAG GGAGC 404 0160 CCCUGAACCCUAGAAC UGUCU 300 AGACAGUUCUAGGGUUCA GGGAG 405 0161 CCUGAACCCUAGAACU GUCUU 301 AAGACAGUUCUAGGGUUC AGGGA 406 0162 CUGAACCCUAGAACUG UCUUC 302 GAAGACAGUUCUAGGGU UCAGGG 407 0163 GAACCCUAGAACUGUC UUCGA 303 UCGAAGACAGUUCUAGGG UUCAG 408 0164 ACCCUAGAACUGUCUU CGACU 304 AGUCGAAGACAGUUCUAG GGUUC 409 0165 CCCUAGAACUGUCUUC GACUC 305 GAGUCGAAGACAGUUCUA GGGUU 410 0166 CAGAAGCCGCGCCCACC GCCU 306 AGGCGGUGGGCGCGGCUU CUGUG 411 0167 AGCCGCGCCCACCGCCU GCCA 307 UGGCAGGCGGUGGGCGCG GCUUC 412 0168 GCCGCGCCCACCGCCUG CCAG 308 CUGGCAGGCGGUGGGCGC GGCUU 413 0169 CCGCGCCCACCGCCUGC CAGU 309 ACUGGCAGGCGGUGGGCG CGGCU 414 0170 CGCGCCCACCGCCUGCC AGUU 310 AACUGGCAGGCGGUGGGC GCGGC 415 0171 GCGCCCACCGCCUGCCA GUUC 311 GAACUGGCAGGCGGUGGG CGCGG 416 0172 CGCCCACCGCCUGCCAG UUCA 312 UGAACUGGCAGGCGGUGG GCGCG 417 0173 GCCCACCGCCUGCCAGU UCAC 313 GUGAACUGGCAGGCGGUG GGCGC 418 0174 CCCACCGCCUGCCAGUU CACA 314 UGUGAACUGGCAGGCGGU GGGCG 419 0175 ACCUCGUCCUCCGACUC GCUG 315 CAGCGAGUCGGAGGACGA GGUCA 420 0176 CUCGUCCUCCGACUCGC UGAC 316 GUCAGCGAGUCGGAGGAC GAGGU 421 0177 UCGUCCUCCGACUCGC UGACA 317 UGUCAGCGAGUCGGAGGA CGAGG 422 0178 GUCCUCCGACUCGCUG ACAGG 318 CCUGUCAGCGAGUCGGAG GACGA 423 0179 AACCCCAAUCCACGUU UUGGA 319 UCCAAAACGUGGAUUGGG GUUGU 424 0180 ACCCCAAUCCACGUUU UGGAU 320 AUCCAAAACGUGGAUUGG GGUUG 425 0181 CCCCAAUCCACGUUUU GGAUG 321 CAUCCAAAACGUGGAUUG GGGUU 426 0182 AUCCACGUUUUGGAUG CACUG 322 CAGUGCAUCCAAAACGUG GAUUG 427 0183 UCCACGUUUUGGAUGC ACUGA 323 UCAGUGCAUCCAAAACGU GGAUU 428 0184 CACGUUUUGGAUGCAC UGAGA 324 UCUCAGUGCAUCCAAAAC GUGGA 429 0185 ACGUUUUGGAUGCACU GAGAC 325 GUCUCAGUGCAUCCAAAA CGUGG 430 0186 UUGGAUGCACUGAGAC CCCGA 326 UCGGGGUCUCAGUGCAUC CAAAA 431 0187 ACAUUCCUCGGUAUUU AUUGU 327 ACAAUAAAUACCGAGGAA UGUCG 432 0188 UUCCUCGGUAUUUAUU GUCUG 328 CAGACAAUAAAUACCGAG GAAUG 433 0189 UCCUCGGUAUUUAUUG UCUGU 329 ACAGACAAUAAAUACCGA GGAAU 434 0190 UCGGUAUUUAUUGUCU GUCCC 330 GGGACAGACAAUAAAUAC CGAGG 435 0191 UUUAUUGUCUGUCCCC ACCUA 331 UAGGUGGGGACAGACAA UAAAUA 436 0192 UUGUCUGUCCCCACCU AGGAC 332 GUCCUAGGUGGGGACAGA CAAUA 437 0193 CCCCACCCCCGACCCUC GCGA 333 UCGCGAGGGUCGGGGGUG GGGGU 438 0194 CCCACCCCCGACCCUCG CGAA 334 UUCGCGAGGGUCGGGGGU GGGGG 439 0195 CCACCCCCGACCCUCGC GAAU 335 AUUCGCGAGGGUCGGGGG UGGGG 440 0196 CACCCCCGACCCUCGCG AAUA 336 UAUUCGCGAGGGUCGGGG GUGGG 441 0197 CCCCGACCCUCGCGAAU AAAA 337 UUUUAUUCGCGAGGGUCG GGGGU 442 0198 CCCGACCCUCGCGAAU AAAAG 338 CUUUUAUUCGCGAGGGUC GGGGG 443 0199 CCGACCCUCGCGAAUA AAAGG 339 CCUUUUAUUCGCGAGGGU CGGGG 444 0200 GACCCUCGCGAAUAAA AGGCC 340 GGCCUUUUAUUCGCGAGG GUCGG 445 0201 ACCCUCGCGAAUAAAA GGCCC 341 GGGCCUUUUAUUCGCGAG GGUCG 446 0202 CCCUCGCGAAUAAAAG GCCCU 342 AGGGCCUUUUAUUCGCGA GGGUC 447 0203 UCGCGAAUAAAAGGCC CUCCA 343 UGGAGGGCCUUUUAUUCG CGAGG 448 0204 CGCGAAUAAAAGGCCC UCCAU 344 AUGGAGGGCCUUUUAUUC GCGAG 449 0205 GCGAAUAAAAGGCCCU CCAUC 345 GAUGGAGGGCCUUUUAU UCGCGA 450 0206 CGAAUAAAAGGCCCUC CAUCU 346 AGAUGGAGGGCCUUUUA UUCGCG 451 0207 AUGAAGCAGACGGGCC AGGUG 347 CACCUGGCCCGUCUGCUU CAUCU 452 0208 GAAGCAGACGGGCCAG GUGUA 348 UACACCUGGCCCGUCUGC UUCAU 453 0209 AUGCCAUGAAGAUCAU GAACA 349 UGUUCAUGAUCUUCAUGG CAUAC 454 0210 UGCCAUGAAGAUCAUG AACAA 350 UUGUUCAUGAUCUUCAUG GCAUA 455 0211 GCCAUGAAGAUCAUGA ACAAG 351 CUUGUUCAUGAUCUUCAU GGCAU 456 0212 CCAUGAAGAUCAUGAA CAAGU 352 ACUUGUUCAUGAUCUUCA UGGCA 457 0213 AUGAAGAUCAUGAACA AGUGG 353 CCACUUGUUCAUGAUCUU CAUGG 458 0214 AUCAUGAACAAGUGGG ACAUG 354 CAUGUCCCACUUGUUCAU GAUCU 459 0215 AUGAACAAGUGGGACA UGCUG 355 CAGCAUGUCCCACUUGUU CAUGA 460 0216 GAACAAGUGGGACAUG CUGAA 356 UUCAGCAUGUCCCACUUG UUCAU 461 0217 AACAAGUGGGACAUGC UGAAG 357 CUUCAGCAUGUCCCACUU GUUCA 462 0218 ACAAGUGGGACAUGCU GAAGA 358 UCUUCAGCAUGUCCCACU UGUUC 463 0219 AUGCUGAAGAGGGGCG AGGUG 359 CACCUCGCCCCUCUUCAG CAUGU 464 0220 UGCUGAAGAGGGGCGA GGUGU 360 ACACCUCGCCCCUCUUCA GCAUG 465 0221 GGGGCGAGGUGUCGUG CUUCC 361 GGAAGCACGACACCUCGC CCCUC 466 0222 GGCGAGGUGUCGUGCU UCCGU 362 ACGGAAGCACGACACCUC GCCCC 467 0223 AGGUGUCGUGCUUCCG UGAGG 363 CCUCACGGAAGCACGACA CCUCG 468 0224 GUCGUGCUUCCGUGAG GAGAG 364 CUCUCCUCACGGAAGCAC GACAC 469 0225 GUGCUUCCGUGAGGAG AGGGA 365 UCCCUCUCCUCACGGAAG CACGA 470 0226 UGGUGAAUGGGGACCG GCGGU 366 ACCGCCGGUCCCCAUUCA CCAAC 471 0227 GACCGGCGGUGGAUCA CGCAG 367 CUGCGUGAUCCACCGCCG GUCCC 472 0228 CCGGCGGUGGAUCACG CAGCU 368 AGCUGCGUGAUCCACCGC CGGUC 473 0229 GCGGUGGAUCACGCAG CUGCA 369 UGCAGCUGCGUGAUCCAC CGCCG 474 0230 CUUCGCCUUCCAGGAU GAGAA 370 UUCUCAUCCUGGAAGGCG AAGUG 475 0231 UUCGCCUUCCAGGAUG AGAAC 371 GUUCUCAUCCUGGAAGGC GAAGU 476 0232 UCGCCUUCCAGGAUGA GAACU 372 AGUUCUCAUCCUGGAAGG CGAAG 477 0233 CGCCUUCCAGGAUGAG AACUA 373 UAGUUCUCAUCCUGGAAG GCGAA 478 0234 CGGUGGGCCUGGGACA GGCAG 374 CUGCCUGUCCCAGGCCCA CCGCC 479 0235 UGGGCCUGGGACAGGC AGCUA 375 UAGCUGCCUGUCCCAGGC CCACC 480 0236 GGGCCUGGGACAGGCA GCUAC 376 GUAGCUGCCUGUCCCAGG CCCAC 481 0237 GGCCUGGGACAGGCAG CUACG 377 CGUAGCUGCCUGUCCCAG GCCCA 482 0238 GUGACUGGUGGGCGCU GGGUG 378 CACCCAGCGCCCACCAGU CACAC 483 0239 UAUUCGCCUAUGAAAU GUUCU 379 AGAACAUUUCAUAGGCGA AUACA 484 0240 AUUCGCCUAUGAAAUG UUCUA 380 UAGAACAUUUCAUAGGCG AAUAC 485 0241 AUGGGCAGACGCCCUU CUACG 381 CGUAGAAGGGCGUCUGCC CAUAG 486 0242 CAGACGCCCUUCUACG CGGAU 382 AUCCGCGUAGAAGGGCGU CUGCC 487 0243 GACGCCCUUCUACGCG GAUUC 383 GAAUCCGCGUAGAAGGGC GUCUG 488 0244 GCCCUUCUACGCGGAU UCCAC 384 GUGGAAUCCGCGUAGAAG GGCGU 489 0245 CCCUUCUACGCGGAUU CCACG 385 CGUGGAAUCCGCGUAGAA GGGCG 490

[0192] Example 2. Preliminary Screening of siRNA

[0193] The following in vitro evaluation methods for siRNA were used to conduct preliminary screening of siRNAs (different methods were selected for different types of siRNAs, as detailed below).

[0194] 1. In Vitro Evaluation Method 1 for siRNA

[0195] Human SJCRH30 rhabdomyosarcoma myoblasts were grown in DMEM supplemented with 10% v / v heat-inactivated fetal bovine serum, 4.5 mg / mL glucose, 4 mM L-glutamine, 10 mM HEPES, and 1 mM sodium pyruvate. To facilitate transfection, cells were seeded at a density of 5,000-10,000 cells per well in 24-well plates and transfected within 24 hours.

[0196] Using RNAiMax (ThermoFisher), siRNAs diluted to the specified concentrations were mixed with the cells to be transfected according to the manufacturer’s instructions. The transfected cells were incubated for 2 days at 37°C in 5% CO2, then washed with PBS, harvested in TRIzol (ThermoFisher), and stored at -80°C. RNA was isolated using the Direct-zol-96 RNA Kit (Zymo Research) following the manufacturer’s instructions. RNA was reverse transcribed into cDNA using ReverTra Ace™ qPCR RT Master Mix (TOYOBO) according to the manufacturer’s instructions. The cDNA samples were evaluated by qPCR using TaqMan Fast Advanced Master Mix (Applied Biosystems) with DMPK-specific and GAPDH-specific primers and the SYBR Green method. Detailed information on the primers used is provided in Table 3. In the two-dose screening experiment, DMPK values were normalized to GAPDH gene expression within each sample. Quantification of DMPP knockdown was performed using the standard 2-AACT method. All experiments were carried out in triplicate, and the average of three repeats was calculated. In the multi-dose validation experiment, data were analyzed by nonlinear regression using a three-parameter dose-response inhibition function (GraphPad Prism 8.3.0) to calculate IC50 and KDmax.

[0197] Table 3. Human DMPK Primer Information Primer Sequence (5’-3’) Human DMPK-F CACTGTCGGACATTCGGGAAGGTGC(Seq ID NO: 621) Human DMPK-R GCTTGCACGTGTGGCTCAAGCAGCTG(Seq ID NO: 622) Human GADPH-F ATGGGGAAGGTGAAGGTCG(Seq ID NO: 623) Human GADPH-R GGGGTCATTGATGGCAACAATA(Seq ID NO: 624)

[0198] 2. In Vitro Evaluation Method 2 for siRNA

[0199] Human SJCRH30 rhabdomyosarcoma myoblasts were grown in DMEM supplemented with 10% v / v heat-inactivated fetal bovine serum, 4.5 mg / mL glucose, 4 mM L-glutamine, 10 mM HEPES, and 1 mM sodium pyruvate. siRNAs were prepared using electroporation according to the manufacturer's instructions. Cells were seeded onto 48-well tissue culture plates at 100,000 cells per well 24 hours before transfection. DMPK siRNAs were transfected into SJCRH30 cells. At 48 hours post-transfection, cells were washed with PBS and harvested using reagents (Life Technologies).

[0200] RNA was isolated using the Direct-zol-96 RNA Kit (Zymo Research) following the manufacturer's instructions. RNA was reverse transcribed into cDNA using ReverTra Ace™ qPCR RT Master Mix (TOYOBO) according to the manufacturer's instructions. The cDNA samples were evaluated by qPCR using AceQ Universal U+ Probe Master Mix V2 (Vazyme) with DMPK-specific and GAPDH-specific primers and human gene probes, with three replicates per gene. Information on the primers and probes used is provided in Table 4.

[0201] Table 4. Human DMPK Primer and Probe Information Primer Sequence (5’-3’) human DMPK-F GAGACTTCATTCAGCGGTT(Seq ID NO: 625) human DMPK-R CTTCGAAATCCGGTGTAAAG(Seq ID NO: 626) human DMPK-Probe 5’ -FAM-CCTTCTTCTTTGGCCTCGACTGGGA-BHQ 1(Seq ID NO: 627) human GADPH-F ACCCAGAAGACTGTGGATGG(Seq ID NO: 628) human GADPH-R TCAGCTCAGGGATGACCTTG(Seq ID NO: 629) human GADPH-Probe 5’-FAM-CCCACAGCCTTGGCAGCGCC-BHQ1(Seq ID NO: 630)

[0202] 3. In Vitro Evaluation Method 3 for siRNA

[0203] Human SJCRH30 rhabdomyosarcoma myoblasts were grown in DMEM supplemented with 10% v / v heat-inactivated fetal bovine serum, 4.5 mg / mL glucose, 4 mM L-glutamine, 10 mM HEPES, and 1 mM sodium pyruvate. siRNAs were prepared using the transfection reagent Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer's instructions. Cells were seeded in triplicate onto 96-well tissue culture plates at 4,000 cells per well 24 hours before transfection. DMPK siRNAs were transfected into SJCRH30 cells. At 48 hours posttransfection, cells were washed with PBS and harvested using reagents (Life Technologies).

[0204] RNA was extracted from the cells using FastPure Cell / Tissue Total RNA Isolation Kit V2 (Vazyme) according to the manufacturer's instructions. RNA was reverse transcribed into cDNA using ReverTra Ace™ qPCR RT Master Mix (TOYOBO) following the manufacturer's instructions. The cDNA samples were evaluated by qPCR using AceQ Universal U+ Probe Master Mix V2 (Vazyme) with DMPK-specific and GAPDH-specific human gene probes, with three replicates per gene. Information on the primers and probes used is the same as in Table 4 above.

[0205] 4. In Vitro Evaluation Method 4 for siRNA

[0206] Human SJCRH30 rhabdomyosarcoma myoblasts were grown in DMEM supplemented with 10% v / v heat-inactivated fetal bovine serum, 4.5 mg / mL glucose, 4 mM L-glutamine, 10 mM HEPES, and 1 mM sodium pyruvate. For transfection, cells were seeded at a density of 4,000 cells per well in 96-well plates and transfected within 24 hours. siRNAs were prepared using the transfection reagent Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer's instructions. DMPK siRNAs were transfected into SJCRH30 cells. At 48 hours post-transfection, cells were washed with PBS and harvested using reagents (Life Technologies).

[0207] Cells were lysed and reverse transcribed using the SuperScript™ IV CellsDirect™ cDNA Synthesis Kit according to the manufacturer's instructions to obtain cDNA. The cDNA samples were evaluated by qPCR using TaqMan Fast Advanced Master Mix (Applied Biosystems) with DMPK-specific and GAPDH-specific TaqMan human gene expression probes (Thermo Fisher), with three replicates per gene. The primers and probes used are shown in Table 5.

[0208] Table 5. Human DMPK Primer Assay Kit human DMPK Catalog Number Hs01094329_m 1 human GADPH Catalog Number Hs02786624_g 1

[0209] 5. In Vitro Evaluation Method 5 for siRNA (Mouse Cells, WX)

[0210] C2C12 myoblasts (ATCC) were grown in DMEM supplemented with 10% v / v FBS. For transfection, cells were seeded at a density of 2,000 cells per well in 96-well plates and transfected within 24 hours. siRNAs were prepared using the transfection reagent Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer's instructions. DMPK siRNAs were transfected into C2C12 cells. At 48 hours post-transfection, cells were washed with PBS and harvested using reagents (Life Technologies).

[0211] Cells were lysed and reverse transcribed using the SuperScript™ IV CellsDirect™ cDNA Synthesis Kit according to the manufacturer's instructions to obtain cDNA. The cDNA samples were evaluated by qPCR using TaqMan Fast Advanced Master Mix (Applied Biosystems) with DMPK-specific and GAPDH-specific TaqMan mouse gene expression probes (Thermo Fisher), with three replicates per gene. The primers and probes used are shown in Table 6.

[0212] Table 6. Mouse DMPK Primer Assay Kit mouse DMPK Catalog Number Mm00446261_m 1 mouse GADPH Catalog Number Mm99999915_g 1

[0213] Example 3. siRNA Screening Validation and Screening of Modified siRNAs

[0214] siRNA screening validation mainly consists of three parts: screening and validation of naked sequences, screening and validation of modified sequences, and druggability studies of preferred siRNAs. Among these, the screening of naked sequences and modified sequences for 19 / 21mer siRNA and 21 / 23mer siRNA were conducted separately, while head-to-head comparisons were performed in a mixed manner during the final sequence activity validation and druggability studies. The detailed workflow is shown in Figure 3. A total of seven commonly used siRNA modification patterns were employed in the siRNA screening validation, as shown in Figure 4. Specifically, in Figure 4, light gray text with a white background indicates 2'-O-methyl modification; black text with an open circle indicates T-deoxy-2'-fluoro modification; a solid black circle indicates ethylene glycol nucleic acid (EGNA); and a bold short dash indicates a phosphorothioate linkage. The activity of modified siRNAs is sequence-dependent, meaning that the optimal modification pattern often varies depending on the sequence.

[0215] A1: Primary Screening of 19 / 21mer siRNA Naked Sequences

[0216] Using the in vitro evaluation method 2 for siRNA from Example 2, a predetermined amount of siRNA was transfected into SJCRH30 cells. The DMPK mRNA levels were calculated as described in the method and are shown in Table 7. Values are expressed as the percentage of mRNA levels relative to the control group, along with the coefficient of variation (CV).

[0217] Table 7. Primary Screening Results of 19 / 21mer siRNA Naked Sequences siRNA ID# 2pmol CV% 0.02pmol CV% 0001 34.38 13.81 105.03 2.30 0002 36.50 8.55 68.77 8.91 0003 40.68 10.06 79.27 9.68 0004 47.89 4.00 102.08 0.48 0005 57.57 9.70 77.59 3.56 0006 54.05 2.76 98.22 1.42 0007 55.57 3.75 59.21 4.03 0008 42.32 3.81 71.75 6.49 0009 20.01 10.51 59.74 4.82 0010 30.12 12.54 62.22 1.69 0011 27.57 1.81 387.17 25.27 0012 35.77 12.82 63.15 2.50 0013 32.20 10.49 81.37 4.00 0014 44.39 7.56 69.38 3.00 0015 32.09 11.17 58.90 6.99 0016 80.36 9.88 104.85 4.26 0017 44.48 12.79 83.96 0.85 0018 33.94 10.13 78.06 4.09 0019 43.71 11.39 61.61 2.09 0020 23.26 15.79 53.17 0.42 0021 22.24 2.98 29.68 8.17 0022 32.88 6.91 93.91 8.64 0023 46.22 12.89 80.16 2.63 0024 23.64 10.04 59.94 3.64 0025 17.05 9.43 52.10 5.90 0026 36.77 6.77 56.93 3.44 0027 25.91 9.49 53.57 6.36 0028 60.29 8.12 82.76 2.68 0029 44.92 9.71 84.62 5.99 0030 41.74 4.47 91.29 1.28 0031 31.59 9.87 60.76 2.98 0032 29.14 2.63 58.25 6.94 0033 39.85 9.26 77.69 1.71 0034 50.86 7.49 84.93 2.09 0035 41.31 4.91 58.04 6.45 0036 34.48 7.07 40.03 15.21 0037 34.20 13.39 29.98 9.43 0038 42.94 11.11 56.44 2.02 0039 36.21 11.30 99.22 3.30 0040 64.98 3.56 91.81 5.36 0041 37.97 9.89 91.51 8.21 0042 38.68 3.47 53.20 3.76 0043 43.70 9.52 93.30 5.82 0044 57.75 9.94 106.44 3.05 0045 36.07 5.86 107.03 1.96 0046 18.66 9.16 101.40 6.51 0047 29.00 18.03 80.36 7.28 0048 83.05 9.36 74.41 4.55 0049 30.08 8.09 58.53 3.63 0050 42.45 7.27 40.41 8.10 0051 39.46 16.67 64.33 3.90 0052 38.22 10.52 75.78 2.90 0053 28.70 9.16 66.52 8.88 0054 44.29 2.67 64.42 2.04 0055 33.45 13.48 46.89 5.63 0056 22.72 3.49 36.35 5.49 0057 57.37 8.08 63.42 7.31 0058 26.19 8.24 91.50 1.71 0059 50.98 13.59 61.36 3.03 0060 47.31 7.97 90.03 3.75 0061 83.55 7.14 131.18 6.05 0062 50.90 13.25 89.57 5.10 0063 51.20 10.40 101.18 2.53 0064 42.75 13.00 120.94 6.21 0065 56.24 20.12 84.44 12.70 0066 223.57 9.41 177.95 14.37 0067 44.42 10.49 84.05 26.54 0068 35.61 12.37 81.16 1.23 0069 41.63 9.36 105.26 10.01 0070 59.80 4.60 150.31 6.62 0071 173.97 12.68 113.21 3.89 0072 40.45 9.12 80.80 7.72 0073 77.78 10.43 77.19 18.71 0074 112.56 5.18 119.47 10.21 0075 86.26 4.81 74.01 9.72 0076 195.11 2.52 103.22 8.38 0077 117.52 7.92 101.63 4.39 0078 140.89 4.01 133.88 6.34 0079 169.53 5.55 120.17 9.68 0080 125.95 8.96 99.66 5.04 0081 32.41 7.68 63.79 4.44 0082 40.28 5.67 95.08 5.36 0083 47.27 2.93 66.52 1.37 0084 58.04 2.54 107.06 1.62 0085 84.74 1.64 114.08 8.73 0086 84.00 4.88 177.14 2.28 0087 56.57 9.32 92.42 10.63 0088 59.06 4.79 47.74 1.70 0089 91.16 2.05 91.44 3.45 0090 62.50 6.08 86.70 10.68 0091 87.42 4.71 96.43 2.80 0092 44.33 3.93 63.12 8.71 0093 83.42 3.41 136.73 9.43 0094 63.65 1.81 180.51 2.75 0095 58.37 2.54 44.78 4.36 0096 69.27 7.17 97.09 0.80 0097 64.00 11.68 73.65 5.74 0098 90.51 5.99 96.34 7.55 0099 41.11 6.52 78.35 4.59 0100 38.63 8.78 99.41 7.26

[0218] A2: Screening of Modified 19 / 21mer siRNAs

[0219] Based on the above results, 20 19 / 21mer siRNAs with better performance (comprehensive evaluation of 2 pmol and 0.02 pmol results, selecting 20 siRNAs with lower mRNA expression levels) were selected. For these 20 preferred 19 / 21mer siRNAs, two chemical modification methods were used to synthesize the modified siRNAs. Among them, siRNA ID# 0101-0120 were synthesized using Modification Pattern A, with the modified sequences shown in Table 8 below. The other modification pattern is Pattern B, described later. Seq ID NOs: 201-220 are the sense strands, and Seq ID NOs: 221-240 are the antisense strands. The first modified 19 / 21mer siRNA is siRNA ID# 0101, with its sense strand being Seq ID NO: 201 and its antisense strand being Seq ID NO: 221, and so on. Sequence information is shown in Table 8, where the numbers in parentheses in the first column indicate the corresponding naked sequence numbers prior to modification.

[0220] Using the in vitro evaluation method 2 for siRNA from Example 2, predetermined amounts of siRNA (see Table 9) were transfected into SJCRH30 cells. DMPK mRNA levels were calculated as described in the method, and the results are shown in Table 8. The values in Table 8 represent the mRNA levels. KDmax refers to the maximum mRNA knockdown level calculated from the knockdown curve; IC50 refers to the half-maximal inhibitory concentration calculated from the knockdown curve; and absolute IC50 refers to the intersection point of the horizontal line at 50% mRNA level and the knockdown curve.

[0221] Table 8. Modified 19 / 21mer siRNAs siRNA ID# Unmodi fied siRNA ID# Modified sense strand sequence (5'^3') - 19 nt Seq ID NO: Modified antisense strand sequence (5'^3') - 21 nt Seq ID NO: 0101 0009 5'- / mC* / mU* / mG / mA / i2FC / mA / i2FC / i2FU / i2FG / mC / mU / mG / mA / mG / mC / m A / mA* / mG* / mA / -3' 201 5'- / mU* / i2FC* / mU / mU / mG / i2 FC / mU / i2FC / i2FA / mG / mC / mA / mG / i2FU / mG / i2FU / mC / mA / mG* / mU* / mU / -3' 221 0102 0010 5'- / mG* / mG* / mC / mA / i2FA / mG / i2FA / i2FU / i2FC / mG / mU / mC / mC / mA / mC / m U / mA* / mC* / mA / -3' 202 5'- / mU* / i2FG* / mU / mA / mG / i2 FU / mG / i2FG / i2FA / mC / mG / mA / mU / i2FC / mU / i2FU / mG / mC / mC* / mU* / mU / -3' 222 0103 0011 5'- / mG* / mU* / mG / mA / i2FG / mG / i2FA / i2FG / i2FA / mG / mG / mG / mA / mC / mG / m U / mG* / mU* / mA / -3' 203 5'- / mU* / i2FA* / mC / mA / mC / i2 FG / mU / i2FC / i2FC / mC / mU / mC / mU / i2FC / mC / i2FU / mC / mA / mC* / mU* / mU / -3' 223 0104 0012 5'- / mC* / mU* / mU / mC / i2FU / mA / i2FC / i2FC / i2FU / mG / mG / mC / mG / mG / mA / m G / mA* / mU* / mA / -3' 204 5'- / mU* / i2FA* / mU / mC / mU / i2 FC / mC / i2FG / i2FC / mC / mA / mG / mG / i2FU / mA / i2FG / mA / mA / mG* / mU* / mU / -3' 224 0105 0020 5'- / mG* / mU* / mG / mU / i2FA / mU / i2FG / i2FC / i2FC / mA / mU / mG / mA / mA / mG / m A / mU* / mC* / mA / -3' 205 5'- / mU* / i2FG* / mA / mU / mC / i2 FU / mU / i2FC / i2FA / mU / mG / mG / mC / i2FA / mU / i2FA / mC / mA / mC* / mU* / mU / -3' 225 0106 0021 5'- / mC* / mG* / mG / mU / i2FA / mG / i2FU / i2FG / i2FA / m A / mG / mA / mU / mG / mA / mA / mG* / mC* / mA / -3' 206 5'- / mU* / i2FG* / mC / mU / mU / i2 FC / mA / i2FU / i2FC / mU / mU / mC / mA / i2FC / mU / i2FA / mC / mC / mG* / mU* / mU / -3' 226 0107 0025 5'- / mC* / mC* / mG / mA / i2FC / mA / i2FC / i2FA / i2FU / mG / mC / mA / mA / mC / mU / m U / mC* / mG* / mA / -3' 207 5'- / mU* / i2FC* / mG / mA / mA / i2 FG / mU / i2FU / i2FG / mC / mA / mU / mG / i2FU / mG / i2FU / mC / mG / mG* / mU* / mU / -3' 227 0108 0026 5'- / mC* / mA* / mG / mG / i2FU / mG / i2FU / i2FA / i2FU / m G / mC / mC / mA / mU / mG / mA / mA* / mG* / mA / -3' 208 5'- / mU* / i2FC* / mU / mU / mC / i2 FA / mU / i2FG / i2FG / mC / mA / mU / mA / i2FC / mA / i2FC / mC / mU / mG* / mU* / mU / -3' 228 0109 0027 5'- / mG* / mA* / mC / mG / i2FA / mC / i2FU / i2FU / i2FC / mG / mA / mG / mA / mU / mU / m C / mU* / mG* / mA / -3' 209 5'- / mU* / i2FC* / mA / mG / mA / i2 FA / mU / i2FC / i2FU / mC / mG / mA / mA / i2FG / mU / i2FC / mG / mU / mC* / mU* / mU / -3' 229 0110 0031 5'- / mG* / mG* / mG / mU / i2FG / mU / i2FA / i2FU / i2FU / mC / mG / mC / mC / mU / mA / m U / mG* / mA* / mA / -3' 210 5'- / mU* / i2FU* / mC / mA / mU / i2 FA / mG / i2FG / i2FC / mG / mA / mA / mU / i2FA / mC / i2FA / mC / mC / mC* / mU* / mU / -3' 230 0111 0032 5'- / mG* / mA* / mC / mA / i2FU / mU / i2FC / i2FC / i2FU / mC / mG / mG / mU / mA / mU / m U / mU* / mA* / mA / -3' 211 5'- / mU* / i2FU* / mA / mA / mA / i2 FU / mA / i2FC / i2FC / mG / mA / mG / mG / i2FA / mA / i2FU / mG / mU / mC* / mU* / mU / -3' 231 0112 0035 5'- / mC* / mC* / mG / mA / i2FG / mA / i2FU / i2FG / i2FG / m C / mG / mC / mG / mC / mU / mU / mC* / mU* / mA / -3' 212 5'- / mU* / i2FA* / mG / mA / mA / i2 FG / mC / i2FG / i2FC / mG / mC / mC / mA / i2FU / mC / i2FU / mC / mG / mG* / mU* / mU / -3' 232 0113 0038 5'- / mG* / mA* / mC / mA / i2FC / mU / i2FG / i2FC / i2FU / m G / mA / mG / mC / mA / mA / mG / mU* / mU* / mA / -3' 213 5'- / mU* / i2FA* / mA / mC / mU / i2 FU / mG / i2FC / i2FU / mC / mA / mG / mC / i2FA / mG / i2FU / mG / mU / mC* / mU* / mU / -3' 233 0114 0042 5'- / mC* / mC* / mC / mG / i2FA / mC / i2FA / i2FU / i2FU / mC / mC / mU / mC / mG / mG / m U / mA* / mU* / mA / -3' 214 5'- / mU* / i2FA* / mU / mA / mC / i2 FC / mG / i2FA / i2FG / mG / mA / mA / mU / i2FG / mU / i2FC / mG / mG / mG* / mU* / mU / -3' 234 0115 0046 5'- / mG* / mA* / mC / mU / i2FG / mC / i2FA / i2FG / i2FA / mG / mG / mG / mA / mC / mG / m A / mC* / mU* / mA / -3' 215 5'- / mU* / i2FA* / mG / mU / mC / i2 FG / mU / i2FC / i2FC / mC / mU / mC / mU / i2FG / mC / i2FA / mG / mU / mC* / mU* / mU / -3' 235 0116 0047 5'- / mC* / mG* / mU / mG / i2FC / mA / i2FC / i2FA / i2FG / mG / mG / mA / mC / mA / mU / m C / mA* / mA* / mA / -3' 216 5'- / mU* / i2FU* / mU / mG / mA / i2 FU / mG / i2FU / i2FC / mC / mC / mU / mG / i2FU / mG / i2FC / mA / mC / mG* / mU* / mU / -3' 236 0117 0049 5'- / mC* / mC* / mC / mC / i2FA / mA / i2FC / i2FA / i2FA / mC / mC / mC / mC / mA / mA / mU / mC* / mC* / mA / -3' 217 5'- / mU* / i2FG* / mG / mA / mU / i2 FU / mG / i2FG / i2FG / mG / mU / mU / mG / i2FU / mU / i2FG / m G / mG / mG* / mU* / mU / -3' 237 0118 0051 5'- / mG* / mC* / mA / mG / i2FA / mC / i2FG / i2FG / i2FG / mC / mC / mA / mG / mG / mU / m G / mU* / mA* / mA / -3' 218 5'- / mU* / i2FU* / mA / mC / mA / i2 FC / mC / i2FU / i2FG / mG / mC / mC / mC / i2FG / mU / i2FC / mU / mG / mC* / mU* / mU / -3' 238 0119 0056 5'- / mG* / mC* / mA / mC / i2FU / mG / i2FA / i2FG / i2FA / mC / mC / mC / mC / mG / mA / mC / mA* / mU* / mA / -3' 219 5'- / mU* / i2FA* / mU / mG / mU / i2 FC / mG / i2FG / i2FG / mG / mU / mC / mU / i2FC / mA / i2FG / mU / mG / mC* / mU* / mU / -3' 239 0120 0058 5'- / mC* / mC* / mG / mC / i2FU / mU / i2FC / i2FG / i2FG / mC / mG / mG / mU / mU / mU / m G / mG* / mA* / mA / -3' 220 5'- / mU* / i2FU* / mC / mC / mA / i2 FA / mA / i2FC / i2FC / mG / mC / mC / mG / i2FA / mA / i2FG / mC / mG / mG* / mU* / mU / -3' 240

[0222] Modification description: m = 2'-O-methyl; i2F = 2'-fluoro; gn = ethylene glycol nucleic acid; * / = phosphorothioate linkage; / = phosphodiester linkage.

[0223] Table 9. Screening results of modified 19 / 21mer siRNAs siR NA ID# 200p mol 20p mol 2pm ol 0.2p mol 0.02p mol 0.002p mol KDm ax IC50(p mol) Absolut e IC50(p mol) Modifica tion type 010 1 12.97 23.2 6 21.6 7 76.40 107.1 2 128.14 16.2 4 0.2213 0.4824 A 010 2 21.46 32.3 3 36.8 5 103.1 9 119.1 7 126.08 23.1 9 0.5147 1.4634 A 010 3 33.47 35.4 1 39.8 7 90.05 109.1 6 122.79 32.1 4 0.3247 1.2842 A 010 4 31.55 31.1 4 50.0 8 96.68 114.4 7 128.39 30.5 1 0.4797 1.8165 A 010 5 25.19 21.2 9 27.7 2 41.32 78.63 129.84 26.4 5 0.0180 0.0691 A 010 6 26.67 18.6 4 25.3 9 68.67 127.0 6 137.35 20.9 2 0.1329 0.4146 A 010 7 13.37 17.2 9 18.7 4 76.66 97.71 111.40 10.5 3 0.3377 0.5011 A 010 8 15.54 21.2 7 25.1 8 66.98 114.4 3 147.15 19.9 0 0.1000 0.3118 A 010 9 25.97 17.8 4 32.4 5 59.44 109.5 0 119.40 23.8 2 0.1206 0.3316 A 011 0 18.24 13.6 2 21.5 4 53.20 101.3 9 117.79 16.3 0 0.1089 0.2220 A 011 1 26.20 24.7 9 46.0 8 55.05 103.6 8 114.50 30.0 6 0.0960 0.3229 A 011 2 18.68 16.9 8 15.6 0 66.38 105.4 5 116.13 15.2 6 0.1873 0.3588 A 011 3 23.13 19.6 8 28.1 5 77.45 95.52 135.62 20.8 6 0.1945 0.4878 A 011 4 62.63 59.7 5 47.4 5 78.57 169.2 4 185.40 53.2 9 0.0601 1.0000 A 011 5 48.65 45.4 5 59.9 6 79.40 108.4 1 110.17 49.0 1 0.2288 14.2198 A 011 6 26.31 26.5 4 42.1 2 111.9 0 113.1 5 130.92 22.8 0 0.6815 1.9138 A 011 7 31.47 48.0 1 41.0 6 90.32 100.5 4 112.90 35.3 2 0.4252 1.7348 A 011 8 39.07 37.8 6 45.6 9 124.0 3 127.0 9 141.13 33.6 5 0.5793 3.1640 A 011 9 33.35 45.9 6 33.1 9 81.18 121.0 9 134.12 34.3 5 0.1572 0.8470 A 012 0 20.45 27.8 7 34.7 0 67.53 124.1 5 133.90 24.7 7 0.1342 0.4599 A

[0224] siRNA ID# 0121-0140 were synthesized using Modification Pattern B (see Figure 4 for details), and the modified sequences are shown in Table 10. Seq ID NOs: 241-260 are the sense strands, and Seq ID NOs: 261-280 are the antisense strands. The first modified 19 / 21mer siRNA is siRNA ID# 0121, with its sense strand being Seq ID NO: 241 and its antisense strand being Seq ID NO: 261, and so on. Sequence information is shown in Table 10.

[0225] Using the in vitro evaluation method 2 for siRNA from Example 2, predetermined amounts of siRNA were transfected into SJCRH30 cells. DMPK mRNA levels were calculated according to this evaluation method, and the results are shown in Table 11. The values in the table represent the mRNA levels. KDmax refers to the maximum mRNA knockdown level calculated from the knockdown curve; curve IC50 refers to the half-maximal inhibitory concentration calculated from the knockdown curve; and absolute IC50 refers to the intersection point of the horizontal line at 50% mRNA level and the knockdown curve.

[0226] Table 10. Modified 19 / 21mer siRNAs siRNA ID# Unmod ified siRNA ID# Modified sense strand sequence (5'^3') - 19 nt Seq ID NO. Modified antisense strand sequence (5'^3‘) - 21 nt Seq ID NO. 0121 0009 5'- ZmC* / mU* / mG / mA / mC / m A / i2FCZi2FU / i2FG / mC / mU ZmGZmAZmG / mC / mA / mA* / mG* / mA / -3' 241 5'- / mU* / i2FC* / mU / mU / mGZi 2FC / mU / mC / mA / mG / mC / mAZmGZi2FUZmGZi2FU / m CZmAZmG*ZmU*ZmUZ-3‘ 261 0122 0010 5'- / mG* / mG* / mC / mA / mA / m GZi2FA / i2FU / i2FC / mG / mU / mC / mC / mA / mC / mU / mA* ZmC* / mA / -3' 242 5'- / mU* / i2FG* / mU / mA / mGZi 2FUZmGZmGZmAZmCZmGZ mAZmUZi2FCZmUZi2FUZm GZmC / mC*ZmU* / mU / -3' 262 0123 0011 5'- ZmG* / mU* / mG / mA / mG / m G / i2FA / i2FG / i2FA / mG / mG ZmGZmAZmC / mGZmU / mG* / mU* / mA / -3' 243 5'- / mU* / i2FA* / mCZmAZmC / i2 FGZmUZmC / mC / mC / mUZm C / mU / i2FC / mC / i2FU / mC / mA / mC*ZmU*ZmUZ-3' 263 0124 0012 5'- / mC* / mU* / mU / mC / mU / m A / i2FC / i2FC / i2FU / mG / mG / mC / mG / mG / mA / mG / mA* / mU* / mA / -3' 244 5'- / mU* / i2FA* / mU / mC / mU / i2 F C / mC / mG / mC / mC / mA / m G / mG / i2FU / mA / i2FG / mA / mA / mG* / mU* / mU / -3' 264 0125 0020 5'- / mG* / mU* / mG / mU / mA / m U / i2FG / i2FC / i2FC / mA / mU / mG / mA / mA / mG / mA / mU* / mC* / mA / -3' 245 5'- / mU* / i2FG* / mA / mU / mC / i 2FU / mU / mC / mA / mU / mG / mG / mC / i2FA / mU / i2FA / mC / mA / mC* / mU* / mU / -3' 265 0126 0021 5'- / mC* / mG* / mG / mU / mA / m G / i2FU / i2FG / i2FA / mA / m G / mA / mU / mG / mA / mA / m G* / mC* / mA / -3' 246 5'- / mU* / i2FG* / mC / mU / mU / i 2FC / mA / mU / mC / mU / mU / mC / mA / i2FC / mU / i2FA / mC / mC / mG* / mU* / mU / -3' 266 0127 0025 5'- / mC* / mC* / mG / mA / mC / m A / i2FC / i2FA / i2FU / mG / mC / mA / mA / mC / mU / mU / mC* / mG* / mA / -3' 247 5'- / mU* / i2FC* / mG / mA / mA / i 2FG / mU / mU / mG / mC / mA / mU / mG / i2FU / mG / i2FU / m C / mG / mG* / mU* / mU / -3' 267 0128 0026 5'- / mC* / mA* / mG / mG / mU / m G / i2FU / i2FA / i2FU / mG / mC / mC / mA / mU / mG / mA / mA* / mG* / mA / -3' 248 5'- / mU* / i2FC* / mU / mU / mC / i 2FA / mU / mG / mG / mC / mA / mU / mA / i2FC / mA / i2FC / mC / mU / mG* / mU* / mU / -3' 268 0129 0027 5'- / mG* / mA* / mC / mG / mA / m C / i2FU / i2FU / i2FC / mG / mA / mG / mA / mU / mU / mC / mU* / mG* / mA / -3' 249 5'- / mU* / i2FC* / mA / mG / mA / i 2FA / mU / mC / mU / mC / mG / mA / mA / i2FG / mU / i2FC / m G / mU / mC* / mU* / mU / -3' 269 0130 0031 5'- / mG* / mG* / mG / mU / mG / m U / i2FA / i2FU / i2FU / mC / mG / mC / mC / mU / mA / mU / mG* / mA* / mA / -3' 250 5'- / mU* / i2FU* / mC / mA / mU / i 2FA / mG / mG / mC / mG / mA / mA / mU / i2FA / mC / i2FA / mC / mC / mC* / mU* / mU / -3' 270 0131 0032 5'- / mG* / mA* / mC / mA / mU / m U / i2FC / i2FC / i2FU / mC / mG / mG / mU / mA / mU / mU / mU* / mA* / mA / -3' 251 5'- / mU* / i2FU* / mA / mA / mA / i 2FU / mA / mC / mC / mG / mA / mG / mG / i2FA / mA / i2FU / m G / mU / mC* / mU* / mU / -3' 271 0132 0035 5'- / mC* / mC* / mG / mA / mG / m A / i2FU / i2FG / i2FG / mC / m G / mC / mG / mC / mU / mU / m C* / mU* / mA / -3' 252 5'- / mU* / i2FA* / mG / mA / mA / i 2FG / mC / mG / mC / mG / mC / mC / mA / i2FU / mC / i2FU / mC / mG / mG* / mU* / mU / -3' 272 0133 0038 5'- / mG* / mA* / mC / mA / mC / m U / i2FG / i2FC / i2FU / mG / m A / mG / mC / mA / mA / mG / m U* / mU* / mA / -3' 253 5'- / mU* / i2FA* / mA / mC / mU / i2 FU / mG / mC / mU / mC / mA / m G / mC / i2FA / mG / i2FU / mG / mU / mC* / mU* / mU / -3' 273 0134 0042 5'- / mC* / mC* / mC / mG / mA / m C / i2FA / i2FU / i2FU / mC / mC / mU / mC / mG / mG / mU / mA* / mU* / mA / -3' 254 5'- / mU* / i2FA* / mU / mA / mC / i2 FC / mG / mA / mG / mG / mA / m A / mU / i2FG / mU / i2FC / mG / mG / mG* / mU* / mU / -3' 274 0135 0046 5'- / mG* / mA* / mC / mU / mG / m C / i2FA / i2FG / i2FA / mG / mG / mG / mA / mC / mG / mA / mC * / mU* / mA / -3' 255 5'- / mU* / i2FA* / mG / mU / mC / i2 FG / mU / mC / mC / mC / mU / m C / mU / i2FG / mC / i2FA / mG / mU / mC* / mU* / mU / -3' 275 0136 0047 5'- / mC* / mG* / mU / mG / mC / m A / i2FC / i2FA / i2FG / mG / mG / mA / mC / mA / mU / mC / mA* / mA* / mA / -3' 256 5'- / mU* / i2FU* / mU / mG / mA / i 2FU / mG / mU / mC / mC / mC / mU / mG / i2FU / mG / i2FC / m A / mC / mG* / mU* / mU / -3' 276 0137 0049 5'- / mC* / mC* / mC / mC / mA / m A / i2FC / i2FA / i2FA / mC / mC / mC / mC / mA / mA / mU / mC * / mC* / mA / -3' 257 5'- / mU* / i2FG* / mG / mA / mU / i 2FU / mG / mG / mG / mG / mU / mU / mG / i2FU / mU / i2FG / m G / mG / mG* / mU* / mU / -3' 277 0138 0051 5'- / mG* / mC* / mA / mG / mA / m C / i2FG / i2FG / i2FG / mC / mC / mA / mG / mG / mU / mG / mU* / mA* / mA / -3' 258 5'- / mU* / i2FU* / mA / mC / mA / i 2FC / mC / mU / mG / mG / mC / mC / mC / i2FG / mU / i2FC / mU / mG / mC* / mU* / mU / -3' 278 0139 0056 5'- / mG* / mC* / mA / mC / mU / m G / i2FA / i2FG / i2FA / mC / mC / mC / mC / mG / mA / mC / mA* / mU* / mA / -3' 259 5'- / mU* / i2FA* / mU / mG / mU / i 2FC / mG / mG / mG / mG / mU / mC / mU / i2FC / mA / i2FG / m U / mG / mC* / mU* / mU / -3' 279 0140 0058 5'- / mC* / mC* / mG / mC / mU / m U / i2FC / i2FG / i2FG / mC / mG / mG / mU / mU / mU / mG / mG* / mA* / mA / -3' 260 5'- / mU* / i2FU* / mC / mC / mA / i 2FA / mA / mC / mC / mG / mC / mC / mG / i2FA / mA / i2FG / mC / mG / mG* / mU* / mU / -3' 280

[0227] Modification description: m = 2'-O-methyl; i2F = 2'-fluoro; gn = ethylene glycol nucleic acid; * / = phosphorothioate linkage; / = phosphodiester linkage.

[0228] Table 11. Screening results of modified 19 / 21mer siRNAs siRN A ID# Seq ID NO: 20p mol 2pm ol 0.2p mol 0.02p mol 0.002p mol KDm ax IC50(p mol) Absolute IC50(pm ol) Modificat ion type 0121 026 1 17.3 3 26.2 2 53.69 115.59 120.82 17.82 0.1130 0.2683 B 0122 026 2 33.9 1 39.6 8 70.91 114.92 115.38 32.85 0.1680 0.6829 B 0123 026 3 26.0 8 21.0 0 38.15 107.29 127.20 20.48 0.0513 0.1459 B 0124 026 4 18.4 7 21.2 1 53.48 76.94 104.68 18.47 0.1160 0.1805 B 0125 026 5 23.5 3 21.5 1 45.03 91.92 115.78 21.57 0.0582 0.1390 B 0126 026 6 16.4 1 15.8 6 43.54 76.20 90.73 14.28 0.1138 0.1297 B 0127 026 7 9.71 13.0 0 48.64 82.03 93.50 7.861 0.1740 0.1774 B 0128 026 8 28.5 8 22.5 5 54.73 86.61 80.89 22.87 0.2001 0.2689 B 0129 026 9 20.7 2 11.2 6 45.37 84.35 92.53 14.04 0.1202 0.1497 B 0130 027 0 38.7 9 32.2 6 72.35 90.42 92.35 32.26 0.2940 0.7303 B 0131 027 1 21.5 8 25.7 1 41.63 51.45 96.61 27.7 0.0089 0.0246 B 0132 027 2 15.1 6 20.3 5 31.29 62.20 80.05 17.19 0.0498 0.0485 B 0133 027 3 33.9 3 32.6 7 50.43 91.59 111.36 32.36 0.0540 0.1962 B 0134 027 4 32.4 7 29.4 0 36.70 80.84 108.81 29.24 0.0280 0.0877 B 0135 027 5 51.4 9 42.5 2 47.06 75.20 102.27 45.57 0.0155 0.2094 B 0136 027 6 26.8 6 22.3 6 69.78 87.70 98.21 20.39 0.2921 0.4593 B 0137 027 7 54.5 2 56.0 2 75.70 111.94 98.60 52.41 0.1859 1.0000 B 0138 027 8 22.2 0 21.4 6 69.25 107.35 111.07 17.07 0.2227 0.4305 B 0139 027 9 24.2 2 21.4 8 42.49 95.50 130.29 22.38 0.0381 0.1180 B 0140 028 0 21.9 5 22.0 0 76.16 80.16 109.49 17.18 0.3550 0.5584 B

[0229] A3: Validation of Modified 19 / 21mer siRNAs

[0230] Based on the above results, 10 modified 19 / 21mer siRNAs were further selected. For these 10 modified 19 / 21mer siRNAs, the in vitro evaluation method 3 for siRNA from Example 2 was used for activity validation. Predetermined amounts of siRNA were transfected into SJCRH30 cells. DMPK mRNA levels were calculated according to this evaluation method, and the results are shown in Table 12. The values represent the mRNA levels. KDmax refers to the maximum mRNA knockdown level calculated from the knockdown curve; curve IC50 refers to the half-maximal inhibitory concentration calculated from the knockdown curve; and absolute IC50 refers to the intersection point of the horizontal line at 50% mRNA level and the knockdown curve.

[0231] Table 12. Validation of modified 19 / 21mer siRNAs siRNA ID# 100nM 10nM 1nM 0.1nM 0.01nM KDmax IC50(pmol) Absolute IC50(pmol) 0105 23.60 20.26 29.26 86.96 145.32 20.33 0.0896 0.3306 0110 12.99 14.26 41.74 114.57 153.33 12.16 0.2388 0.6864 0112 15.21 14.86 24.32 37.28 117.44 17.06 0.0136 0.0583 0124 29.11 27.47 65.11 113.40 116.78 24.8 0.7387 2.0815 0126 27.77 28.69 31.67 110.01 141.25 23.54 0.1725 0.6561 0127 8.15 9.42 25.72 98.99 149.33 7.127 0.1503 0.3823 0129 39.47 31.31 45.85 103.49 127.73 33.11 0.2004 0.9959 0131 14.79 17.83 18.97 40.42 117.88 16.15 0.0182 0.0666 0132 15.66 22.25 20.68 73.48 136.75 16.57 0.0677 0.2122 0139 20.01 22.07 28.12 54.75 108.53 21.53 0.0468 0.1266

[0232] For the 10 preferred modified 19 / 21mer siRNAs, the in vitro evaluation method 4 for siRNA from Example 2 was used for activity validation. Predetermined amounts of siRNA were transfected into SJCRH30 cells. DMPK mRNA levels were calculated as described in the method, and the results are shown in Table 13. The values represent the mRNA levels. KDmax refers to the maximum mRNA knockdown level calculated from the knockdown curve; curve IC50 refers to the half-maximal inhibitory concentration calculated from the knockdown curve; and absolute IC50 refers to the intersection point of the horizontal line at 50% mRNA level and the knockdown curve.

[0233] Table 13. Validation of modified 19 / 21mer siRNAs siRNA ID# 100nM 10nM 1nM 0.1nM 0.01nM 0.001nM KDmax Absolute IC50(pmol) 0105 14.14 22.51 34.71 77.36 126.68 101.03 17.54 0.39 0110 13.14 14.25 31.74 77.79 98.60 107.46 12.84 0.36 0112 14.60 16.72 23.13 50.53 91.15 86.64 15.41 0.12 0124 31.68 34.00 44.85 79.05 91.59 103.17 32.19 0.62 0126 15.28 20.30 31.17 75.98 94.53 100.24 16.32 0.35 0127 15.73 18.89 24.05 66.70 100.78 99.36 15.73 0.22 0129 26.27 28.94 36.76 73.80 98.88 102.35 26.76 0.36 0131 16.65 19.09 24.50 41.76 67.35 81.19 18.96 0.05 0132 19.19 18.16 24.29 54.11 88.69 97.98 18.38 0.12 0139 24.39 26.68 38.07 69.00 98.35 99.56 25.80 0.31

[0234] B1. Primary Screening of 21 / 23mer siRNA Naked Sequences

[0235] Using the in vitro evaluation method 1 for siRNA from Example 2, predetermined amounts of siRNA were transfected into SJCRH30 cells. DMPK mRNA levels were calculated as described in the method, and the results are shown in Tables 14-15. Values are expressed as the percentage of mRNA levels relative to the control group, along with the coefficient of variation (CV).

[0236] Table 14. Primary screening results of 21 / 23mer siRNA naked sequences siRNA ID# 1nM CV% 0.01nM CV% 0176 49.67 22.90 83.61 10.07 0177 23.00 34.51 70.33 9.99 0181 32.33 34.21 58.67 25.53 0183 34.50 14.35 64.00 13.26 0184 24.33 9.49 59.01 42.61 0185 45.67 13.91 81.29 32.66 0186 74.00 15.23 77.00 16.53 0203 108.00 7.41 83.24 27.21 0187 49.00 16.70 76.81 25.83 0188 29.33 7.87 60.00 30.05 0189 33.00 0.00 78.67 31.38 0190 49.67 20.27 50.00 25.46 0191 37.67 8.11 54.61 16.29 0192 34.33 30.59 56.96 36.76 0196 17.33 43.68 36.67 23.51 0197 36.33 25.57 37.32 22.51 0198 23.67 20.84 67.00 27.88 0201 33.67 13.39 46.33 21.62 0202 32.00 19.01 45.50 7.77 0214 50.19 2.28 55.76 1.07 0215 58.96 3.50 61.75 16.33 0217 67.00 21.16 82.49 4.57 0218 54.47 16.03 65.67 12.77 0219 54.20 14.46 66.05 10.89 0220 49.67 14.57 75.08 4.35 0221 53.67 3.88 61.21 32.99 0153 44.64 40.60 76.00 16.80 0154 38.25 20.65 59.50 13.07 0161 36.67 41.03 44.33 36.46 0166 88.00 14.46 99.00 14.67 0168 68.33 2.24 96.00 12.80 0171 26.67 14.20 56.50 1.25 0174 28.77 17.27 43.91 19.43 0175 27.33 26.47 40.67 2.84 0208 35.19 23.97 73.14 2.71 0213 38.50 1.84 81.07 4.54 0223 27.67 28.99 43.55 3.94 0224 31.00 11.63 71.54 16.08 0225 37.25 22.85 72.86 19.13 0226 35.66 40.64 58.00 11.35 0228 35.17 2.16 39.67 6.34 0230 43.75 32.87 102.41 23.99 0234 50.98 25.44 82.90 28.36 0237 71.67 27.95 84.94 8.52 0238 73.64 12.04 84.52 9.26 0239 21.00 21.82 54.73 14.75 0240 11.33 18.37 37.49 7.97 0242 50.94 20.90 80.18 27.85 0244 43.00 11.63 56.33 7.39 0245 49.49 15.85 72.85 12.08 0143 31.00 28.12 59.96 7.67 0144 52.33 8.83 84.50 2.51 0146 70.33 22.67 87.79 8.63 0148 34.33 35.11 65.09 14.55 0150 30.67 10.48 73.26 27.35 0151 62.80 15.13 98.16 18.63 0152 41.44 11.30 80.91 7.97 0155 57.74 8.88 63.14 2.37 0156 33.67 4.54 63.33 14.92 0158 26.67 17.32 70.83 14.54 0159 15.99 10.77 52.33 9.03 0160 21.67 9.61 60.00 25.66 0205 24.67 4.68 41.12 19.27 0207 50.86 3.24 40.95 11.08 0222 25.34 15.31 35.28 2.93 0227 45.56 13.14 78.12 7.49 0232 43.43 11.83 63.00 11.36 0235 63.92 3.64 63.40 10.19 0169 43.33 21.26 77.41 33.38 0170 56.57 28.24 81.66 18.39 0172 33.55 26.20 62.08 28.23 0173 23.58 37.90 39.23 21.51 0179 46.68 21.05 56.09 6.41 0180 45.12 3.13 63.97 14.08 0182 34.74 9.88 75.90 16.90 0209 28.18 34.53 48.64 18.66 0210 27.69 7.86 43.96 19.98 0211 14.09 6.61 46.71 9.72 0212 20.41 16.06 36.39 2.96 0216 31.22 17.75 65.14 22.41 0229 36.29 4.98 57.77 30.56 0231 56.87 35.44 67.27 18.93 0233 43.80 3.64 99.70 28.82 0241 42.25 12.47 102.63 20.89 0243 81.90 18.37 68.47 25.17 0141 117.16 4.25 132.94 38.30 0142 51.61 6.70 109.48 5.59 0145 59.08 16.20 55.70 3.21 0147 57.74 12.71 80.22 7.36 0149 41.18 10.70 45.27 8.67 0157 37.32 8.68 72.92 16.18 0162 31.39 33.30 45.49 15.19 0163 25.50 41.76 46.77 3.33 0164 33.87 40.03 38.16 30.32 0165 25.16 9.47 38.40 13.82 0167 30.29 20.22 44.07 13.51 0178 24.77 10.96 34.71 0.73 0195 49.91 8.33 89.17 4.34 0199 35.86 19.45 42.13 14.04 0200 26.03 20.89 43.95 17.83 0204 33.11 24.76 53.18 24.15 0206 29.79 7.33 39.43 20.05

[0237] Table 15. Primary screening results of 21 / 23mer siRNA naked sequences siRNA ID# 1nM CV% 0.01nM CV% 0211 18.19 14.11 86.91 27.43 0212 32.26 3.09 72.87 13.71 0216 63.03 12.11 74.22 6.03 0222 45.67 20.13 48.78 26.30 0223 39.22 23.57 70.49 23.16 0239 23.47 35.21 56.44 24.72 0240 11.13 17.61 56.13 10.67 0143 30.21 11.83 85.16 21.11 0159 29.56 20.48 74.87 3.94 0160 55.14 2.92 67.64 11.10 0163 41.53 16.79 72.63 14.24 0164 44.66 14.70 58.86 9.93 0165 31.88 35.94 52.07 14.34 0174 49.73 12.56 98.18 2.90 0175 25.62 17.77 45.75 15.01 0177 36.08 13.27 51.29 25.02 0178 23.93 8.67 56.02 22.09 0181 31.16 16.86 35.99 11.62 0196 32.37 36.57 61.72 14.70 0198 36.32 20.49 68.37 6.11 0236 38.11 23.00 73.96 15.14 0193 43.67 17.15 73.74 7.84 0194 39.79 8.65 71.20 29.61

[0238] B2: Validation of 21 / 23mer siRNA Naked Sequences

[0239] Based on the above results, 20 21 / 23mer siRNA naked sequences were selected. For these 20 preferred 21 / 23mer siRNA naked sequences, the in vitro evaluation method 1 for siRNA from Example 2 was used for activity validation. Predetermined amounts of siRNA were transfected into SJCRH30 cells. DMPK mRNA levels were calculated according to this method, and the results are shown in Table 16. The values represent the mRNA levels. KDmax refers to the maximum mRNA knockdown level calculated from the knockdown curve; curve IC50 refers to the half-maximal inhibitory concentration calculated from the knockdown curve; and absolute IC50 refers to the intersection point of the horizontal line at 50% mRNA level and the knockdown curve.

[0240] Table 16. Validation of 21 / 23mer siRNA naked sequences siRNA ID# 100n M 14.29n M 2.04n M 0.2915 nM 0.042n M 0.0059 nM 0.0008 5nM 0.0001 21nM KDma x IC50( nM) 0175 15.73 13.67 21.08 18.60 51.17 103.56 100.99 120.01 16.43 0.0286 0177 26.66 23.15 23.21 23.18 43.12 84.26 93.91 nd 23.74 0.0210 0178 19.37 11.52 18.05 18.65 32.02 51.98 79.27 101.34 15.06 0.0022 0181 14.78 22.64 16.75 30.05 67.65 nd 82.46 nd 17.79 0.1003 0196 12.67 14.44 12.14 20.96 56.07 73.06 62.86 nd 13.05 0.1010 0165 17.20 22.43 25.83 23.71 33.44 58.77 114.73 95.77 24.08 0.0047 0174 38.12 21.19 25.73 41.01 68.40 90.16 nd 108.09 27.50 0.0385 0223 42.40 33.47 33.02 72.71 89.91 105.95 108.39 115.01 34.61 0.2077 0239 20.43 14.39 14.48 29.54 46.82 56.79 71.72 nd 15.19 0.0303 0240 nd 13.20 17.72 42.55 60.90 85.19 nd 98.07 8.11 0.0801 0211 26.66 23.15 23.21 32.35 43.12 84.26 93.91 nd 25.06 0.0198 0212 15.73 13.67 21.08 18.60 51.17 103.56 100.99 120.01 16.43 0.0286 0216 nd 27.44 28.76 30.27 58.05 76.44 89.68 nd 26.57 0.0386 0222 23.52 24.22 27.59 18.43 42.50 75.58 84.75 94.28 23.13 0.0172 0143 17.75 13.29 15.47 21.91 39.16 111.75 nd 111.33 17.09 0.0292 0159 25.23 19.33 30.17 32.62 60.85 82.45 nd 105.01 22.35 0.0264 0160 14.28 14.51 19.03 23.17 44.06 83.09 nd 108.80 15.38 0.0166 0164 22.20 21.94 21.75 67.62 104.34 117.06 nd nd 20.28 0.2780 0198 17.51 14.19 13.61 16.91 40.41 80.87 78.37 91.80 15.08 0.0290 0163 14.19 22.61 21.64 39.58 56.44 100.60 100.36 119.29 16.98 0.0307 nd: not measured

[0241] B3: Screening of Modified 21 / 23mer siRNAs

[0242] Based on the above results, 9 21 / 23mer siRNA naked sequences were selected. For these 9 preferred 21 / 23mer siRNAs, modified siRNAs were synthesized using 5 chemical modification methods, resulting in a total of 45 sequences. The modified sequences are shown in Table 17, and the modification types are shown in Table 18. Seq ID NOs: 491-535 are the sense strands, and Seq ID NOs: 536-580 are the antisense strands. The first modified 21 / 23mer siRNA is siRNA ID# 0246, with its sense strand being Seq ID NO: 491 and its antisense strand being Seq ID NO: 536, and so on. Sequence information is shown in Table 17.

[0243] Using the in vitro evaluation method 1 for siRNA from Example 2, predetermined amounts of siRNA were transfected into SJCRH30 cells. DMPK mRNA levels were calculated as described in the method, and the results are shown in Tables 18-19. Values are expressed as the percentage of mRNA levels relative to the control group, along with the coefficient of variation (CV).

[0244] Table 17. Modified 21 / 23mer siRNAs siRNA ID # Unmodi fied siRNA ID # Modified sense strand sequence (5'^-3') - 21 nt Seq ID NO. Modified antisense strand sequence (5'^3') - 23 nt Seq ID NO. 0246 0143 5'- / mG* / mG* / mC / mA / mA / m G / i2FA / mU / i2FC / i2FG / i2 FU / mC / mC / mA / mC / mU / mA / mC / mA* / mA* / mG / -3' 491 5'- / mC* / i2FU* / mU / mG / mU / i2 FA / mG / i2FU / i2FG / mG / mA / mC / mG / i2FA / mU / i2FC / mU / mU / mG / mC / mC* / mA* / mU / -3' 536 0247 0143 5'- / mG* / mG* / mC / mA / mA / m G / i2FA / mU / i2FC / i2FG / i2 FU / mC / mC / mA / mC / mU / mA / mC / mA* / mA* / mG / -3' 492 5'- / mC* / i2FU* / mU / mG / mU / i2 FA / mG / mU / mG / mG / mA / mC / mG / i2FA / mU / i2FC / mU / mU / mG / mC / mC* / mA* / mU / -3' 537 0248 0143 5'- / i2FG* / mG* / i2FC / mA / i2F A / mG / i2FA / mU / i2FC / i2F G / i2FU / mC / i2FC / mA / i2F C / mU / i2FA / mC / i2FA* / m A* / i2FG / -3' 493 5'- / mC* / i2FU* / mU / i2FG / mU / i 2FA / mG / i2FU / mG / i2FG / mA / mC / mG / i2FA / mU / i2FC / mU / i2FU / mG / i2FC / mC* / mA* / m U / -3' 538 0249 0143 5'- / mG* / mG* / mC / mA / mA / m G / mA / mU / i2FC / i2FG / i2F U / mC / mC / mA / mC / mU / m A / mC / mA* / mA* / mG / -3' 494 5'- / mC* / i2FU* / mU / mG / mU / i2 FA / gnG / i2FU / i2FG / mG / mA / mC / mG / i2FA / mU / i2FC / mU / mU / mG / mC / mC* / mA* / mU / -3' 539 0250 0143 5'- / mG* / mG* / mC / mA / mA / m G / mA / mU / i2FC / i2FG / i2F U / mC / mC / mA / mC / mU / m A / mC / mA* / mA* / mG / -3' 495 5'- / mC* / i2FU* / mU / mG / mU / i2 FA / mG / mU / mG / mG / mA / mC / mG / i2FA / mU / i2FC / mU / mU / mG / mC / mC* / mA* / mU / -3' 540 0251 0159 5'- / mU* / mC* / mC / mC / mU / m G / i2FA / mA / i2FC / i2FC / i2 F C / mU / mA / mG / mA / mA / mC / mU / mG* / mU* / mC / -3' 496 5'- / mG* / i2FA* / mC / mA / mG / i2F U / mU / i2FC / i2FU / mA / mG / m G / mG / i2FU / mU / i2FC / mA / m G / mG / mG / mA* / mG* / mC / -3' 541 0252 0159 5'- / mU* / mC* / mC / mC / mU / m G / i2FA / mA / i2FC / i2FC / i2 F C / mU / mA / mG / mA / mA / mC / mU / mG* / mU* / mC / -3' 497 5'- / mG* / i2FA* / mC / mA / mG / i2F U / mU / mC / mU / mA / mG / mG / mG / i2FU / mU / i2FC / mA / mG / mG / mG / mA* / mG* / mC / -3' 542 0253 0159 5'- / i2FU* / mC* / i2FC / mC / i2F U / mG / i2FA / mA / i2FC / i2F C / i2FC / mU / i2FA / mG / i2F A / mA / i2FC / mU / i2FG* / m U* / i2FC / -3' 498 5'- / mG* / i2FA* / mC / i2FA / mG / i2 FU / mU / i2FC / mU / i2FA / mG / mG / mG / i2FU / mU / i2FC / mA / i2FG / mG / i2FG / mA* / mG* / m C / -3' 543 0254 0159 5'- / mU* / mC* / mC / mC / mU / m G / mA / mA / i2FC / i2FC / i2F C / mU / mA / mG / mA / mA / m C / mU / mG* / mU* / mC / -3' 499 5'- / mG* / i2FA* / mC / mA / mG / i2F U / gnT / i2FC / i2FU / mA / mG / m G / mG / i2FU / mU / i2FC / mA / m G / mG / mG / mA* / mG* / mC / -3' 544 0255 0159 5'- / mU* / mC* / mC / mC / mU / m G / mA / mA / i2FC / i2FC / i2F C / mU / mA / mG / mA / mA / m C / mU / mG* / mU* / mC / -3' 500 5'- / mG* / i2FA* / mC / mA / mG / i2F U / mU / mC / mU / mA / mG / mG / mG / i2FU / mU / i2FC / mA / mG / mG / mG / mA* / mG* / mC / -3' 545 0256 0175 5'- / mA* / mC* / mC / mU / mC / m G / i2FU / mC / i2FC / i2FU / i2 FC / mC / mG / mA / mC / mU / mC / mG / mC* / mU* / mG / -3' 501 5'- / mC* / i2FA* / mG / mC / mG / i2F A / mG / i2FU / i2FC / mG / mG / m A / mG / i2FG / mA / i2FC / mG / m A / mG / mG / mU* / mC* / mA / -3' 546 0257 0175 5'- / mA* / mC* / mC / mU / mC / m G / i2FU / mC / i2FC / i2FU / i2 502 5'- / mC* / i2FA* / mG / mC / mG / i2F A / mG / mU / mC / mG / mG / mA / 547 FC / mC / mG / mA / mC / mU / mC / mG / mC* / mU* / mG / -3' mG / i2FG / mA / i2FC / mG / mA / mG / mG / mU* / mC* / mA / -3' 0258 0175 5'- / i2FA* / mC* / i2FC / mU / i2F C / mG / i2FU / mC / i2FC / i2F U / i2FC / mC / i2FG / mA / i2F C / mU / i2FC / mG / i2FC* / m U* / i2FG / -3' 503 5'- / mC* / i2FA* / mG / i2FC / mG / i2 FA / mG / i2FU / mC / i2FG / mG / mA / mG / i2FG / mA / i2FC / mG / i2FA / mG / i2FG / mU* / mC* / m A / -3' 548 0259 0175 5'- / mA* / mC* / mC / mU / mC / m G / mU / mC / i2FC / i2FU / i2F C / mC / mG / mA / mC / mU / m C / mG / mC* / mU* / mG / -3' 504 5'- / mC* / i2FA* / mG / mC / mG / i2F A / gnG / i2FU / i2FC / mG / mG / m A / mG / i2FG / mA / i2FC / mG / m A / mG / mG / mU* / mC* / mA / -3' 549 0260 0175 5'- / mA* / mC* / mC / mU / mC / m G / mU / mC / i2FC / i2FU / i2F C / mC / mG / mA / mC / mU / m C / mG / mC* / mU* / mG / -3‘ 505 5'- / mC* / i2FA* / mG / mC / mG / i2F A / mG / mU / mC / mG / mG / mA / mG / i2FG / mA / i2FC / mG / mA / mG / mG / mU* / mC* / mA / -3' 550 0261 0178 5'- / mG* / mU* / mC / mC / mU / m C / i2FC / mG / i2FA / i2FC / i2F U / mC / mG / mC / mU / mG / m A / mC / mA* / mG* / mG / -3' 506 5'- / mC* / i2FC* / mU / mG / mU / i2F C / mA / i2FG / i2FC / mG / mA / m G / mU / i2FC / mG / i2FG / mA / m G / mG / mA / mC* / mG* / mA / -3' 551 0262 0178 5'- / mG* / mU* / mC / mC / mU / m C / i2FC / mG / i2FA / i2FC / i2F U / mC / mG / mC / mU / mG / m A / mC / mA* / mG* / mG / -3' 507 5'- / mC* / i2FC* / mU / mG / mU / i2F C / mA / mG / mC / mG / mA / mG / mU / i2FC / mG / i2FG / mA / mG / mG / mA / mC* / mG* / mA / -3' 552 0263 0178 5'- / i2FG* / mU* / i2FC / mC / i2F U / mC / i2FC / mG / i2FA / i2F C / i2FU / mC / i2FG / mC / i2F U / mG / i2FA / mC / i2FA* / m G* / i2FG / -3' 508 5'- / mC* / i2FC* / mU / i2FG / mU / i2 FC / mA / i2FG / mC / i2FG / mA / mG / mU / i2FC / mG / i2FG / mA / i2FG / mG / i2FA / mC* / mG* / m A / -3' 553 0264 0178 5'- / mG* / mU* / mC / mC / mU / m C / mC / mG / i2FA / i2FC / i2F U / mC / mG / mC / mU / mG / m A / mC / mA* / mG* / mG / -3' 509 5'- / mC* / i2FC* / mU / mG / mU / i2F C / gnA / i2FG / i2FC / mG / mA / m G / mU / i2FC / mG / i2FG / mA / m G / mG / mA / mC* / mG* / mA / -3' 554 0265 0178 5'- / mG* / mU* / mC / mC / mU / m C / mC / mG / i2FA / i2FC / i2F U / mC / mG / mC / mU / mG / m A / mC / mA* / mG* / mG / -3' 510 5'- / mC* / i2FC* / mU / mG / mU / i2F C / mA / mG / mC / mG / mA / mG / mU / i2FC / mG / i2FG / mA / mG / mG / mA / mC* / mG* / mA / -3' 555 0266 0196 5'- / mC* / mA* / mC / mC / mC / m C / i2FC / mG / i2FA / i2FC / i2F C / mC / mU / mC / mG / mC / m G / mA / mA* / mU* / mA / -3' 511 5'- / mU* / i2FA* / mU / mU / mC / i2F G / mC / i2FG / i2FA / mG / mG / m G / mU / i2FC / mG / i2FG / mG / m G / mG / mU / mG* / mG* / mG / -3' 556 0267 0196 5'- / mC* / mA* / mC / mC / mC / m C / i2FC / mG / i2FA / i2FC / i2F C / mC / mU / mC / mG / mC / m G / mA / mA* / mU* / mA / -3' 512 5'- / mU* / i2FA* / mU / mU / mC / i2F G / mC / mG / mA / mG / mG / mG / mU / i2FC / mG / i2FG / mG / mG / mG / mU / mG* / mG* / mG / -3' 557 0268 0196 5'- / i2FC* / mA* / i2FC / mC / i2F C / mC / i2FC / mG / i2FA / i2F C / i2FC / mC / i2FU / mC / i2F G / mC / i2FG / mA / i2FA* / m U* / i2FA / -3' 513 5'- / mU* / i2FA* / mU / i2FU / mC / i2 FG / mC / i2FG / mA / i2FG / mG / mG / mU / i2FC / mG / i2FG / mG / i2FG / mG / i2FU / mG* / mG* / m G / -3' 558 0269 0196 5'- / mC* / mA* / mC / mC / mC / m C / mC / mG / i2FA / i2FC / i2F C / mC / mU / mC / mG / mC / m G / mA / mA* / mU* / mA / -3' 514 5'- / mU* / i2FA* / mU / mU / mC / i2F G / gnC / i2FG / i2FA / mG / mG / m G / mU / i2FC / mG / i2FG / mG / m G / mG / mU / mG* / mG* / mG / -3' 559 0270 0196 5'- / mC * / mA* / mC / mC / mC / m C / mC / mG / i2FA / i2FC / i2F C / mC / mU / mC / mG / mC / m G / mA / mA* / mU* / mA / -3' 515 5'- / mU* / i2FA* / mU / mU / mC / i2F G / mC / mG / mA / mG / mG / mG / mU / i2FC / mG / i2FG / mG / mG / mG / mU / mG* / mG* / mG / -3' 560 0271 0211 5'- / mG* / mC* / mC / mA / mU / m G / i2FA / mA / i2FG / i2FA / i2 FU / mC / mA / mU / mG / mA / mA / mC / mA* / mA* / mG / -3' 516 5'- / mC* / i2FU* / mU / mG / mU / i2 FU / mC / i2FA / i2FU / mG / mA / mU / mC / i2FU / mU / i2FC / mA / mU / mG / mG / mC* / mA* / mU / -3' 561 0272 0211 5'- / mG* / mC* / mC / mA / mU / m G / i2FA / mA / i2FG / i2FA / i2 FU / mC / mA / mU / mG / mA / mA / mC / mA* / mA* / mG / -3' 517 5'- / mC* / i2FU* / mU / mG / mU / i2 FU / mC / mA / mU / mG / mA / mU / mC / i2FU / mU / i2FC / mA / mU / mG / mG / mC* / mA* / mU / -3' 562 0273 0211 5'- / i2FG* / mC* / i2FC / mA / i2F U / mG / i2FA / mA / i2FG / i2F A / i2FU / mC / i2FA / mU / i2F G / mA / i2FA / mC / i2FA* / m A* / i2FG / -3' 518 5'- / mC* / i2FU* / mU / i2FG / mU / i 2FU / mC / i2FA / mU / i2FG / mA / mU / mC / i2FU / mU / i2FC / mA / i 2FU / mG / i2FG / mC* / mA* / m U / -3' 563 0274 0211 5'- / mG* / mC* / mC / mA / mU / m 519 5'- / mC* / i2FU* / mU / mG / mU / i2 564 G / mA / mA / i2FG / i2FA / i2F U / mC / mA / mU / mG / mA / m A / mC / mA* / mA* / mG / -3' FU / gnC / i2FA / i2FU / mG / mA / mU / mC / i2FU / mU / i2FC / mA / mU / mG / mG / mC* / mA* / mU / -3' 0275 0211 5'- / mG* / mC* / mC / mA / mU / m G / mA / mA / i2FG / i2FA / i2F U / mC / mA / mU / mG / mA / m A / mC / mA* / mA* / mG / -3' 520 5'- / mC* / i2FU* / mU / mG / mU / i2 FU / mC / mA / mU / mG / mA / mU / mC / i2FU / mU / i2F C / mA / mU / mG / mG / mC* / mA* / mU / -3' 565 0276 0216 5'- / mG* / mA* / mA / mC / mA / m A / i2FG / mU / i2FG / i2FG / i2 FG / mA / mC / mA / mU / mG / mC / mU / mG* / mA* / mA / -3' 521 5'- / mU* / i2FU* / mC / mA / mG / i2 FC / mA / i2FU / i2FG / mU / mC / mC / mC / i2FA / mC / i2FU / mU / mG / mU / mU / mC* / mA* / mU / -3' 566 0277 0216 5'- / mG* / mA* / mA / mC / mA / m A / i2FG / mU / i2FG / i2FG / i2 F G / mA / mC / mA / mU / mG / mC / mU / mG* / mA* / mA / -3' 522 5'- / mU* / i2FU* / mC / mA / mG / i2 FC / mA / mU / mG / mU / mC / mC / mC / i2FA / mC / i2FU / mU / mG / mU / mU / mC* / mA* / mU / -3' 567 0278 0216 5'- / i2FG* / mA* / i2FA / mC / i2F A / mA / i2FG / mU / i2FG / i2F G / i2FG / mA / i2FC / mA / i2F U / mG / i2FC / mU / i2FG* / m A* / i2FA / -3' 523 5'- / mU* / i2FU* / mC / i2FA / mG / i2 FC / mA / i2FU / mG / i2FU / mC / mC / mC / i2FA / mC / i2FU / mU / i 2FG / mU / i2FU / mC* / mA* / m U / -3' 568 0279 0216 5'- / mG* / mA* / mA / mC / mA / m A / mG / mU / i2FG / i2FG / i2F G / mA / mC / mA / mU / mG / m C / mU / mG* / mA* / mA / -3' 524 5'- / mU* / i2FU* / mC / mA / mG / i2 FC / gnA / i2FU / i2FG / mU / mC / mC / mC / i2FA / mC / i2FU / mU / mG / mU / mU / mC* / mA* / mU / -3' 569 0280 0216 5'- / mG* / mA* / mA / mC / mA / m A / mG / mU / i2FG / i2FG / i2F G / mA / mC / mA / mU / mG / m C / mU / mG* / mA* / mA / -3' 525 5'- / mU* / i2FU* / mC / mA / mG / i2 FC / mA / mU / mG / mU / mC / mC / mC / i2FA / mC / i2FU / mU / mG / mU / mU / mC* / mA* / mU / -3' 570 0281 0222 5'- / mG* / mG* / mC / mG / mA / m G / i2FG / mU / i2FG / i2FU / i2 FC / mG / mU / mG / mC / mU / mU / mC / mC* / mG* / mU / -3' 526 5'- / mA* / i2FC* / mG / mG / mA / i2 FA / mG / i2FC / i2FA / mC / mG / mA / mC / i2FA / mC / i2FC / mU / mC / mG / mC / mC* / mC* / mC / -3' 571 0282 0222 5'- / mG* / mG* / mC / mG / mA / m 527 5'- / mA* / i2FC* / mG / mG / mA / i2 572 G / i2FG / mU / i2FG / i2FU / i2 FC / mG / mU / mG / mC / mU / mU / mC / mC* / mG* / mU / -3' FA / mG / mC / mA / mC / mG / mA / mC / i2FA / mC / i2FC / mU / mC / mG / mC / mC* / mC* / mC / -3' 0283 0222 5'- / i2FG* / mG* / i2FC / mG / i2F A / mG / i2FG / mU / i2FG / i2F U / i2FC / mG / i2FU / mG / i2F C / mU / i2FU / mC / i2FC* / m G* / i2FU / -3' 528 5'- / mA* / i2FC* / mG / i2FG / mA / i 2FA / mG / i2FC / mA / i2FC / mG / mA / mC / i2FA / mC / i2FC / mU / i 2FC / mG / i2FC / mC* / mC* / mC / -3' 573 0284 0222 5'- / mG* / mG* / mC / mG / mA / m G / mG / mU / i2FG / i2FU / i2F C / mG / mU / mG / mC / mU / m U / mC / mC* / mG* / mU / -3' 529 5'- / mA* / i2FC* / mG / mG / mA / i2 FA / gnG / i2FC / i2FA / mC / mG / mA / mC / i2FA / mC / i2FC / mU / mC / mG / mC / mC* / mC* / mC / -3' 574 0285 0222 5'- / mG* / mG* / mC / mG / mA / m G / mG / mU / i2FG / i2FU / i2F C / mG / mU / mG / mC / mU / m U / mC / mC* / mG* / mU / -3' 530 5'- / mA* / i2FC* / mG / mG / mA / i2 FA / mG / mC / mA / mC / mG / mA / mC / i2FA / mC / i2FC / mU / mC / mG / mC / mC* / mC* / mC / -3' 575 0286 0240 5'- / mA* / mU* / mU / mC / mG / m C / i2FC / mU / i2FA / i2FU / i2 F G / mA / mA / mA / mU / mG / mU / mU / mC* / mU* / mA / -3' 531 5'- / mU* / i2FA* / mG / mA / mA / i2 FC / mA / i2FU / i2FU / mU / mC / mA / mU / i2FA / mG / i2FG / mC / mG / mA / mA / mU* / mA* / mC / -3' 576 0287 0240 5'- / mA* / mU* / mU / mC / mG / m C / i2FC / mU / i2FA / i2FU / i2 FG / mA / mA / mA / mU / mG / mU / mU / mC* / mU* / mA / -3' 532 5'- / mU* / i2FA* / mG / mA / mA / i2 FC / mA / mU / mU / mU / mC / mA / mU / i2FA / mG / i2FG / mC / mG / mA / mA / mU* / mA* / mC / -3' 577 0288 0240 5'- / i2FA* / mU* / i2FU / mC / i2F G / mC / i2FC / mU / i2FA / i2F U / i2FG / mA / i2FA / mA / i2F U / mG / i2FU / mU / i2FC* / m U* / i2FA / -3' 533 5'- / mU* / i2FA* / mG / i2FA / mA / i2 FC / mA / i2FU / mU / i2FU / mC / mA / mU / i2FA / mG / i2FG / mC / i 2FG / mA / i2FA / mU* / mA* / m C / -3' 578 0289 0240 5'- / mA* / mU* / mU / mC / mG / m C / mC / mU / i2FA / i2FU / i2F G / mA / mA / mA / mU / mG / m U / mU / mC* / mU* / mA / -3' 534 5'- / mU* / i2FA* / mG / mA / mA / i2 FC / gnA / i2FU / i2FU / mU / mC / mA / mU / i2FA / mG / i2FG / mC / mG / mA / mA / mU* / mA* / mC / -3' 579 0290 0240 5'- / mA* / mU* / mU / mC / mG / m 535 5'- / mU* / i2FA* / mG / mA / mA / i2 580 C / mC / mU / i2FA / i2FU / i2F G / mA / mA / mA / mU / mG / m U / mU / mC* / mU* / mA / -3' FC / mA / mU / mU / mU / mC / mA / mU / i2FA / mG / i2FG / mC / mG / mA / mA / mU* / mA* / mC / -3'

[0245] Modification description: m = 2'-O-methyl; i2F = 2'-fluoro; gn = ethylene glycol nucleic acid; * / = phosphorothioate linkage; / = phosphodiester linkage.

[0246] Table 18. Screening of modified 21 / 23mer siRNAs siRNA ID# 1nM CV% 0.01nM CV% Modification Type 0246 57.80 35.76 121.87 29.52 C 0247 47.13 10.62 107.34 16.81 D 0248 65.09 26.53 80.81 19.25 E 0249 43.90 12.61 64.73 2.64 F 0250 60.59 17.71 118.71 39.15 G 0251 47.48 17.52 85.97 21.18 C 0252 43.34 0.19 91.41 23.39 D 0253 37.15 17.12 84.59 2.47 E 0254 40.99 6.99 70.43 3.55 F 0255 45.39 12.47 76.90 4.39 G 0256 71.45 13.88 103.49 21.74 C 0257 51.86 17.73 126.87 5.00 D 0258 46.90 30.65 84.46 5.79 E 0259 65.29 14.72 130.76 17.31 F 0260 39.36 20.61 86.85 14.85 G 0261 43.42 10.71 73.96 21.73 C 0262 37.78 16.75 66.73 3.49 D 0263 30.35 17.11 57.24 10.18 E 0264 35.57 7.29 89.19 9.15 F 0265 56.16 24.00 72.93 29.30 G 0266 49.77 17.56 131.98 33.85 C 0267 76.85 15.25 122.82 10.02 D 0268 29.03 23.08 77.17 15.04 E 0269 40.57 20.17 88.34 19.68 F 0270 30.34 23.59 65.67 12.74 G 0271 35.70 4.21 90.71 5.81 C 0272 41.76 13.25 87.60 14.33 D 0273 22.45 14.21 90.04 4.41 E 0274 75.94 21.83 112.25 3.84 F 0275 23.58 13.73 101.91 16.04 G 0276 39.56 7.41 91.76 12.43 C 0277 30.84 23.51 61.14 24.63 D 0278 46.78 14.51 83.34 28.41 E 0279 51.94 18.97 78.27 37.90 F 0280 34.90 10.59 85.23 4.35 G 0281 77.58 13.85 127.03 16.89 C 0282 51.66 5.59 96.31 6.34 D 0283 51.20 13.22 106.07 13.74 E 0284 76.88 33.97 104.06 9.33 F 0285 56.40 31.96 114.61 14.99 G 0286 36.14 10.66 97.07 2.35 C 0287 30.77 14.04 100.24 8.93 D 0288 28.02 11.35 103.29 16.27 E 0289 52.70 12.31 102.25 5.90 F 0290 27.83 18.98 98.70 24.83 G

[0247] Table 19. Screening of modified 21 / 23mer siRNAs siRNA ID# 1nM CV% 0.01nM CV% 253 24.17 27.73 81.01 24.51 263 50.37 18.55 87.46 16.29 268 46.74 15.11 85.82 20.87 270 34.44 16.01 98.89 23.62 273 32.2 46.6 79.39 4.22 275 41.13 38.87 84.06 21.08 277 41.42 31.97 67.83 22.06 280 48.36 2.8 97.04 17.01 287 27.61 6.91 72.14 27.4 288 30.58 46.41 96.92 11.18 290 27.58 10.56 88.08 18.71

[0248] B4: Validation of Modified 21 / 23mer siRNAs

[0249] Based on the above results, 6 modified 21 / 23mer siRNAs were selected. For these 6 preferred modified 21 / 23mer siRNAs, the in vitro evaluation method 1 for siRNA from Example 2 was used for activity validation. The predetermined amounts of siRNA were transfected into SJCRH30 cells. DMPK mRNA levels were calculated as described in the method, and the results are shown in Table 20. The values represent the mRNA levels. KDmax refers to the maximum mRNA knockdown level calculated from the knockdown curve; curve IC50 refers to the half-maximal inhibitory concentration calculated from the knockdown curve; and absolute IC50 refers to the intersection point of the horizontal line at 50% mRNA level and the knockdown curve.

[0250] Table 20. Validation of modified 21 / 23mer siRNAs siRN 100n 14.29 2.04n 0.291 54519 nM 0.042 0.005 0.000 0.000 121n M KDm IC50( A ID# M nM M nM 9nM 85nM ax nM) 0273 18.64 20.58 19.90 29.25 78.78 68.69 119.7 6 98.77 78.34 0.041 9 0277 37.79 31.28 42.23 31.75 66.57 105.7 3 97.21 nd 64.24 0.040 8 0287 35.46 12.74 12.22 43.31 60.48 88.68 100.6 0 96.68 79.32 0.051 1 0290 22.35 16.20 14.53 58.73 108.0 8 100.0 6 nd 121.0 4 82.31 0.284 4 0253 18.98 16.31 20.05 40.24 104.2 2 110.1 3 101.9 5 107.9 7 81.65 0.184 6 0270 34.37 27.75 29.83 39.06 51.61 104.2 7 133.4 8 110.6 2 67.75 0.018 6

[0251] Example 4. Further Validation and Screening of Modified siRNAs

[0252] A1. The preferred modified 19 / 21mer siRNAs from Example 3 and the preferred modified 21 / 23mer siRNAs from Example 10 were mixed. For the 9 modified siRNAs with the best activity, the in vitro evaluation method 2 for siRNA from Example 2 was used for activity validation. Predetermined amounts of siRNA were transfected into SJCRH30 cells. DMPK mRNA levels were calculated according to the above method, and the results are shown in Table 21. The values represent the mRNA levels. KDmax refers to the maximum mRNA knockdown level calculated from the knockdown curve; curve IC50 refers to the half-maximal inhibitory concentration calculated from the knockdown curve; and absolute IC50 refers to the intersection point of the horizontal line at 50% mRNA level and the knockdown curve.

[0253] Table 21. Validation of modified siRNAs siRNA ID# 200pm ol 20pm ol 2pm ol 0.2pm ol 0.02pm ol 0.002pm ol KDma x IC50(pm ol) Absolute IC50(pm ol) 0112 18.81 23.66 19.5 0 46.26 127.78 149.41 18.41 0.0611 0.2063 0131 17.44 22.12 20.4 8 30.41 97.87 142.74 18.38 0.0274 0.0889 0132 34.18 30.38 31.7 6 67.75 159.81 152.68 28.78 0.0985 0.5542 0253 14.15 17.58 11.8 7 32.67 121.36 140.37 11.65 0.0547 0.1423 0270 34.52 29.69 39.5 1 68.82 124.87 134.97 32.24 0.1120 0.5578 0273 16.15 12.45 13.2 9 69.92 135.93 130.52 10.39 0.1762 0.3983 0277 36.50 38.48 39.3 8 46.15 113.89 142.49 35.8 0.0362 0.2558 0287 13.01 7.83 5.90 27.03 69.59 114.69 9.32 0.0245 0.0437

[0254] A2. The preferred modified 19 / 21mer siRNAs and the preferred modified 21 / 23mer siRNAs from Example 3 were mixed. For the 9 modified siRNAs with the best activity, the in vitro evaluation method 3 for siRNA from Example 2 was used for activity validation. Predetermined amounts of siRNA were transfected into SJCRH30 cells. DMPK mRNA levels were calculated according to this method, and the results are shown in Table 22. The values represent the mRNA levels. KDmax refers to the maximum mRNA knockdown level calculated from the knockdown curve; curve IC50 refers to the half-maximal inhibitory concentration calculated from the knockdown curve; and absolute IC50 refers to the intersection point of the horizontal line at 50% mRNA level and the knockdown curve.

[0255] Table 22. Validation of modified siRNAs siRNA ID# 100nM 10nM 1nM 0. 1nM 0.01nM 0.001nM KDmax Absolute IC50(pmol) 112 14.31 15.17 16.59 37.25 75.64 94.52 14.65 0.0449 131 12.26 10.9 11.56 19.98 47.58 86.62 11.9 0.0092 132 15.25 14.69 15.21 34.57 71.33 91.84 14.65 0.0357 253 14.42 13.45 16.28 44.66 76.31 85.65 13.03 0.0712 270 27.35 23.12 25.92 51.43 114.43 111.64 23.11 0.1362 273 17.46 11.3 10.73 35.11 94.57 101.43 11.36 0.0594 277 18.55 21.03 19.08 38.71 79.56 110.12 19.7 0.0409 287 7.64 4.08 2.74 27.42 75.51 117.6 5.422 0.0266

[0256] B. Human-Mouse Cross-Reactivity of Modified siRNAs siRNAs from Example 3 were mixed. Since all the aforementioned screening and validation were performed using human cells, but the human DMPK transcript differs significantly from that of the mouse, 8 modified siRNAs with predicted human-mouse cross-reactivity based on transcript analysis are shown in Table 23 (some sequences predicted to be non-reactive were not selected from Table 22; therefore, the sequences in Table 23 are not entirely identical to those in Table 22). The in vitro evaluation method 5 for siRNA from Example 2 was used for activity validation. Predetermined amounts of siRNA were transfected into SJCRH30 cells. DMPK mRNA levels were calculated as described in the method, and the results are shown in Table 23. The values represent the mRNA levels. KDmax refers to the maximum mRNA knockdown level calculated from the knockdown curve; curve IC50 refers to the half-maximal inhibitory concentration calculated from the knockdown curve; and absolute IC50 refers to the intersection point of the horizontal line at 50% mRNA level and the knockdown curve.

[0258] Table 23. Human-mouse cross-reactivity of modified siRNAs siRNA ID# 100nM 10nM 1nM 0. 1nM 0.01nM 0.001nM KDmax Absolute IC50(pmol) 101 108.26 99.39 107.99 107.21 107.15 102.71 100 > 100 111 28.19 33.51 29.12 71.67 86.62 89.61 27.61 0.3147 116 77.25 59.23 59.13 98.33 94.24 101.41 64.41 > 100 121 111.31 111.78 132.94 109.17 106.68 112.67 117.7 > 100 131 30.71 22.86 32.3 68.99 82.78 102.32 25.88 0.2537 136 44.18 55.57 73.87 93.74 98.44 105.21 46.74 15.9605 253 94.92 99.36 90.77 95.38 94.05 87.71 97.05 > 100 287 70.26 61.81 79.7 88.64 90.15 102.18 66.01 > 100

[0259] C: Serum Stability Test of Modified siRNAs

[0260] Based on activity, 6 modified siRNAs were selected (to cover different modification types, not for the purpose of screening for active sequences) to evaluate their stability in serum. The experimental method included: mixing each test siRNA in 50% mouse serum, incubating at 37°C for 0 h, 24 h, and 48 h. Three biological replicates were set up for the experiment. After serum incubation, both 10% nondenaturing PAGE nucleic acid electrophoresis and LCMS analysis were performed to determine the stability of the test siRNAs. The following sequences were selected as controls: PCa was an unmodified siRNA, and PCb was a modified siRNA (positive control) derived from SEQ ID NOs: 114-115 in U.S. Patent Application Publication No. US 2021 / 0299266 A1. Sequence information is shown in Table 24.

[0261] Table 24. Positive control sequences for serum stability test Name siRNA ID# Unmodified sense strand sequence (5'^3') - 19 nt Seq ID NO: Unmodified antisense strand sequence (5'^3') -21 nt Seq ID NO: Mod ificat ion Type PCa 0291 CCCUAGAACUGUCU UCGAA 581 UUCGAAGACAGUUCU AGGGUU 583 - PCb 0292 5'- / mC* / mC* / mC / mU / mA / mG / i2FA / i2FA / i2FC / mU / mG / mU / mC / mU / mU / m C / mG* / mA* / mA / -3' 582 5'- / mU* / i2FU* / mC / mG / mA / i 2FA / mG / mA / mC / mA / mG / mU / mU / i2FC / mU / i2FA / m G / mG / mG* / mU* / mU / -3' 584 B

[0262] Modification description: m = 2'-O-methyl; i2F = 2'-fluoro; gn = ethylene glycol nucleic acid; * / = phosphorothioate linkage; / = phosphodiester linkage.

[0263] PAGE electrophoresis results showed that the unmodified siRNA had poor stability in 50% serum, with the target band being completely degraded after 24 hours of incubation. The modified siRNAs exhibited similar stability to the positive control, with no significant degradation of the target band after 48 hours of incubation in 50% serum, indicating good stability (results shown in Figure 5). LCMS results showed that after 48 hours of serum incubation, the remaining amounts of both the sense and antisense strands of the modified siRNAs were greater than 85%, demonstrating good stability and no significant difference compared to the positive control (Table 25).

[0264] Table 25. Serum stability test results of modified siRNAs (LC-MS) siRNA ID# 0h 24h 48h antisense strand sense strand antisense strand sense strand antisense strand sense strand 0291 100.00%±16. 55% 100.00%±18. 82% / / / / 0292 100.00%±4.4 100.00%±4.6 92.01%±4.86 91.46%±1.6 96.69%±0.4 91.34% 8% 8% % 2% 4% ±4.06% 0273 100.00%±2.8 6% 100.00%±2.0 0% 93.90%±0.23 % 99.77%±2.1 9% 89.89%±1.2 9% 100.34 %±3.27 % 0287 100.00%±0.9 100.00%±1.6 95.71%±2.30 106.23%±3. 99.84%±0.4 113.65% 6% 2% % 18% 5% ±2.65% 0290 100.00%±2.7 2% 100.00%±3.7 9% 96.09%±2.33 % 104.56%±1. 87% 91.68%±2.0 4% 104.61 %±1.02 % 0253 100.00%±12. 100.00%±5.4 84.96%±0.96 93.44%±3.3 86.50%±2.1 96.91% 80% 2% % 2% 8% ±1.95% 0277 100.00%±3.8 100.00%±2.6 100.33%±1.4 99.60%±1.2 87.96%±4.0 88.54% 2% 2% 7% 5% 3% ±4.78% 0270 100.00%±25. 40% 100.00%±26. 34% 109.92%±4.0 0% 117.00%±5. 18% 94.35%±3.8 2% 106.27 %±2.22 %

[0265] D. Cytotoxicity of Modified siRNAs

[0266] The 8 sequences from Table 21 (siRNA ID: #0112, #0131, #0132, #0253, #0270, #0273, #0277, #0287) were further evaluated for cytotoxicity in SJCRH30 cells. The specific method was as follows: 5 x 104 cells per well were seeded in 24-well plates (or 8 x 103 cells per well in 96-well plates). The test siRNAs at final concentrations of 1 nM and 20 nM were transfected using Lipofectamine RNAiMAX according to the manufacturer's instructions. Blank (untransfected), luciferase siRNA (siLUC, 20 nM, commercially purchased), a known cytotoxic siRNA TX (1 nM and 20 nM, targeting PLK, commercially purchased), and PCb used in the serum stability test described above were used as controls. Sequence information is shown in Table 26. After 48 hours of incubation, 10% CCK-8 solution was added, and cell viability was determined by measuring OD450.

[0267] The experimental results were normalized for cell viability using the Blank result as the baseline. Figures 6-7 show the results from two replicate experiments. The results demonstrated that TX significantly reduced cell viability in all three experiments (independent experiments using the 24-well plate system), whereas none of the candidate siRNAs significantly reduced cell viability compared to siLUC.

[0268] Table 26. Control and positive control sequences for cytotoxicity test Name siRNA ID# sense strand (5'-3') - 19nt Seq ID NO: antisense strand (5'-3') -21nt Seq ID NO: siLU C 0293 AAGACGACUCGAAAUC CACAU 585 GUGGAUUUCGAGUCGU CUUAA 587 TX 0294 GUGAUCUUCUUCAUCA AGGTT 586 CCUUGAUGAAGAAGAU CACTT 588

[0269] E. Immunogenicity of Modified siRNAs

[0270] The eight sequences listed in Table 21 were further evaluated for immunogenicity following transfection of the siRNAs into peripheral blood mononuclear cells (PBMCs). The commonly used transfection reagent lipofectamine is known to stimulate PBMC immunogenicity; therefore, GenePORTER 2 was used for transfection in this experiment. Specifically, using in vitro evaluation method 1 for siRNA, 106 cells per well were seeded in 24-well plates, and the test siRNAs were transfected at a final concentration of 100 nM according to the manufacturer's instructions. The following controls were included: Blank (untransfected), Mock (transfection reagent GenePORTER 2 only, commercially purchased), luciferase siRNA (siLUC, 100 nM), the known immunogenic poly I:C (2 pg / mL, commercially purchased), and PCb used in the serum stability test. After 6 hours of incubation, cell supernatants were collected and IFN-a and IL-6 expression were measured by ELISA. After 24 hours of incubation, total mRNA was extracted from PBMCs, and the expression levels of OAS1 and P56 mRNAs were quantified by qRT-PCR using the primer sequences shown in Table 27.

[0271] The experimental results were normalized to the Blank baseline for cellular activity. Figures 8 and 9 show the ELISA results for IFN-a and IL-6 expression, while Figures 10 and 11 present the qRT-PCR results for OAS1 and P56 mRNA levels. The results demonstrated that poly I:C significantly increased the supernatant levels of IFN-a and IL-6 from PBMCs and also activated OAS1 and P56 gene transcription in PBMCs. In contrast, the candidate siRNAs did not exhibit significant immunogenicity. Human OAS1-F CGAGGGAGCATGAAAACACATTT ( Seq ID NO: 631 ) Human OAS1-R GCAGAGTTGCTGGTAGTTTATGAC ( Seq ID NO: 632 ) Human P56 -F GCCTCCTTGGGTTCGTCTATAA ( Seq ID NO: 633 ) Human P56 -R CTCAGGGCCCGCTCATAGTA ( Seq ID NO: 634 )

[0273] F. Off-Target Risk Assessment and Mitigation Strategy for Modified siRNAs

[0274] The eight sequences listed in Table 21 were further evaluated for off- target potential using next-generation sequencing (RNA-seq). To reduce off-target gene downregulation mediated by the modified siRNAs, an ethylene glycol nucleic acid (GNA) modification was introduced at positions 5-8 of the antisense strand seed region (numbering revised). The eight modified siRNAs used in the experiment, along with their respective GNA-modified counterparts, are shown in Table 28.

[0275] Table 28 siRNAs and GNA modifications used for off-target risk assessment siRNA ID# Modified sense strand sequence (5'-3') - 21 nt Seq ID NO: Modified antisense strand sequence (5'-3‘) - 23 nt Seq ID NO: GN A Mo difi ed 0277 5'- / mG* / mA* / mA / mC / mA / m A / i2FG / mU / i2FG / i2FG / i2F G / mA / mC / mA / mU / mG / mC / mU / mG* / mA* / mA / -3' 522 5'- / mU* / i2FU* / mC / mA / mG / i2F C / mA / mU / mG / mU / mC / mC / m C / i2FA / mC / i2FU / mU / mG / m U / mU / mC* / mA* / mU / -3' 567 / 0270 5'- / mC* / mA* / mC / mC / mC / mC / mC / mG / i2FA / i2FC / i2FC / m C / mU / mC / mG / mC / mG / mA / mA* / mU* / mA / -3' 515 5'- / mU* / i2FA* / mU / mU / mC / i2F G / mC / mG / mA / mG / mG / mG / mU / i2FC / mG / i2FG / mG / mG / mG / mU / mG* / mG* / mG / -3' 560 / 0287 5'- / mA* / mU* / mU / mC / mG / m C / i2FC / mU / i2FA / i2FU / i2F G / mA / mA / mA / mU / mG / mU 532 5'- / mU* / i2FA* / mG / mA / mA / i2F C / mA / mU / mU / mU / mC / mA / mU / i2FA / mG / i2FG / mC / mG / 577 / / mU / mC* / mU* / mA / -3' mA / mA / mU* / mA* / mC / -3' 0273 5'- / i2FG* / mC* / i2FC / mA / i2FU / mG / i2FA / mA / i2FG / i2FA / i2 FU / mC / i2FA / mU / i2FG / mA / i2FA / mC / i2FA* / mA* / i2FA / -3' 518 5'- / mU* / i2FU* / mU / i2FG / mU / i2 FU / mC / i2FA / mU / i2FG / mA / m U / mC / i2FU / mU / i2FC / mA / i2F U / mG / i2FG / mC* / mA* / mU / -3' 563 / 0253 5'- / i2FU* / mC* / i2FC / mC / i2FU / mG / i2FA / mA / i2FC / i2FC / i2 FC / mU / i2FA / mG / i2FA / mA / i2FC / mU / i2FG* / mU* / i2FA / -3' 498 5'- / mU* / i2FA* / mC / i2FA / mG / i2 FU / mU / i2FC / mU / i2FA / mG / m G / mG / i2FU / mU / i2FC / mA / i2F G / mG / i2FG / mA* / mG* / mC / -3' 543 / 0131 5'- / mG* / mA* / mC / mA / mU / m U / i2FC / i2FC / i2FU / mC / mG / mG / mU / mA / mU / mU / mU* / mA* / mA / -3' 251 5'- / mU* / i2FU* / mA / mA / mA / i2F U / mA / mC / mC / mG / mA / mG / mG / i2FA / mA / i2FU / mG / mU / mC* / mU* / mU / -3' 271 / 0112 5'- / mC* / mC* / mG / mA / i2FG / m A / i2FU / i2FG / i2FG / mC / mG / mC / mG / mC / mU / mU / mC* / mU* / mA / -3' 212 5'- / mU* / i2FA* / mG / mA / mA / i2F G / mC / i2FG / i2FC / mG / mC / mC / mA / i2FU / mC / i2FU / mC / mG / mG* / mU* / mU / -3' 232 / 0132 5'- / mC* / mC* / mG / mA / mG / m A / i2FU / i2FG / i2FG / mC / mG / mC / mG / mC / mU / mU / mC* / mU* / mA / -3' 252 5'- / mU* / i2FA* / mG / mA / mA / i2F G / mC / mG / mC / mG / mC / mC / m A / i2FU / mC / i2FU / mC / mG / m G* / mU* / mU / -3' 272 / 0295 5'- / mG* / mA* / mA / mC / mA / m A / i2FG / mU / i2FG / i2FG / i2F G / mA / mC / mA / mU / mG / mC / mU / mG* / mA* / mA / -3' 522 5'- / mU* / i2FU* / mC / mA / gnG / i2F C / mA / mU / mG / mU / mC / mC / m C / i2FA / mC / i2FU / mU / mG / m U / mU / mC* / mA* / mU / -3' 589 GN A5 0296 5'- / mG* / mA* / mA / mC / mA / m A / i2FG / mU / i2FG / i2FG / i2F G / mA / mC / mA / mU / mG / mC / mU / mG* / mA* / mA / -3' 522 5'- / mU* / i2FU* / mC / mA / mG / gn C / mA / mU / mG / mU / mC / mC / m C / i2FA / mC / i2FU / mU / mG / m U / mU / mC* / mA* / mU / -3' 590 GN A6 0297 5'- / mG* / mA* / mA / mC / mA / m A / i2FG / mU / i2FG / i2FG / i2F G / mA / mC / mA / mU / mG / mC / mU / mG* / mA* / mA / -3' 522 5'- / mU* / i2FU* / mC / mA / mG / i2F C / gnA / mU / mG / mU / mC / mC / mC / i2FA / mC / i2FU / mU / mG / mU / mU / mC* / mA* / mU / -3' 591 GN A7 0298 5'- / mG* / mA* / mA / mC / mA / m A / i2FG / mU / i2FG / i2FG / i2F G / mA / mC / mA / mU / mG / mC / mU / mG* / mA* / mA / -3' 522 5'- / mU* / i2FU* / mC / mA / mG / i2F C / mA / gnU / mG / mU / mC / mC / mC / i2FA / mC / i2FU / mU / mG / mU / mU / mC* / mA* / mU / -3' 592 GN A8 0299 5'- / mC* / mA* / mC / mC / mC / mC / mC / mG / i2FA / i2FC / i2FC / m C / mU / mC / mG / mC / mG / mA / mA* / mU* / mA / -3' 515 5'- / mU* / i2FA* / mU / mU / gnC / i2F G / mC / mG / mA / mG / mG / mG / mU / i2FC / mG / i2FG / mG / mG / mG / mU / mG* / mG* / mG / -3' 593 GN A5 0300 5'- / mC* / mA* / mC / mC / mC / mC / mC / mG / i2FA / i2FC / i2FC / m C / mU / mC / mG / mC / mG / mA / mA* / mU* / mA / -3‘ 515 5'- / mU* / i2FA* / mU / mU / mC / gn G / mC / mG / mA / mG / mG / mG / mU / i2FC / mG / i2FG / mG / mG / mG / mU / mG* / mG* / mG / -3' 594 GN A6 0301 5'- / mC* / mA* / mC / mC / mC / mC / mC / mG / i2FA / i2FC / i2FC / m C / mU / mC / mG / mC / mG / mA / mA* / mU* / mA / -3' 515 5'- / mU* / i2FA* / mU / mU / mC / i2F G / gnC / mG / mA / mG / mG / mG / mU / i2FC / mG / i2FG / mG / mG / mG / mU / mG* / mG* / mG / -3' 595 GN A7 0302 5'- / mC* / mA* / mC / mC / mC / mC / mC / mG / i2FA / i2FC / i2FC / m C / mU / mC / mG / mC / mG / mA / mA* / mU* / mA / -3' 515 5'- / mU* / i2FA* / mU / mU / mC / i2F G / mC / gnG / mA / mG / mG / mG / mU / i2FC / mG / i2FG / mG / mG / mG / mU / mG* / mG* / mG / -3' 596 GN A8 0303 5'- / mA* / mU* / mU / mC / mG / m C / i2FC / mU / i2FA / i2FU / i2F G / mA / mA / mA / mU / mG / mU / mU / mC* / mU* / mA / -3' 532 5'- / mU* / i2FA* / mG / mA / gnA / i2F C / mA / mU / mU / mU / mC / mA / mU / i2FA / mG / i2FG / mC / mG / mA / mA / mU* / mA* / mC / -3' 597 GN A5 0304 5'- / mA* / mU* / mU / mC / mG / m C / i2FC / mU / i2FA / i2FU / i2F G / mA / mA / mA / mU / mG / mU / mU / mC* / mU* / mA / -3' 532 5'- / mU* / i2FA* / mG / mA / mA / gn C / mA / mU / mU / mU / mC / mA / mU / i2FA / mG / i2FG / mC / mG / mA / mA / mU* / mA* / mC / -3' 598 GN A6 0305 5'- / mA* / mU* / mU / mC / mG / m C / i2FC / mU / i2FA / i2FU / i2F G / mA / mA / mA / mU / mG / mU / mU / mC* / mU* / mA / -3' 532 5'- / mU* / i2FA* / mG / mA / mA / i2F C / gnA / mU / mU / mU / mC / mA / mU / i2FA / mG / i2FG / mC / mG / mA / mA / mU* / mA* / mC / -3' 599 GN A7 0306 5'- / mA* / mU* / mU / mC / mG / m C / i2FC / mU / i2FA / i2FU / i2F G / mA / mA / mA / mU / mG / mU / mU / mC* / mU* / mA / -3' 532 5'- / mU* / i2FA* / mG / mA / mA / i2F C / mA / gnU / mU / mU / mC / mA / mU / i2FA / mG / i2FG / mC / mG / mA / mA / mU* / mA* / mC / -3' 600 GN A8 0307 5'- / i2FG* / mC* / i2FC / mA / i2FU / mG / i2FA / mA / i2FG / i2FA / i2 FU / mC / i2FA / mU / i2FG / mA / i2FA / mC / i2FA* / mA* / i2FA / -3' 518 5'- / mU* / i2FU* / mU / i2FG / gnU / i2 FU / mC / i2FA / mU / i2FG / mA / m U / mC / i2FU / mU / i2FC / mA / i2F U / mG / i2FG / mC* / mA* / mU / -3' 601 GN A5 0308 5'- / i2FG* / mC* / i2FC / mA / i2FU / mG / i2FA / mA / i2FG / i2FA / i2 FU / mC / i2FA / mU / i2FG / mA / i2FA / mC / i2FA* / mA* / i2FA / -3' 518 5'- / mU* / i2FU* / mU / i2FG / mU / gn U / mC / i2FA / mU / i2FG / mA / m U / mC / i2FU / mU / i2FC / mA / i2F U / mG / i2FG / mC* / mA* / mU / -3' 602 GN A6 0309 5'- / i2FG* / mC* / i2FC / mA / i2FU / mG / i2FA / mA / i2FG / i2FA / i2 FU / mC / i2FA / mU / i2FG / mA / i2FA / mC / i2FA* / mA* / i2FA / -3' 518 5'- / mU* / i2FU* / mU / i2FG / mU / i2 FU / gnC / i2FA / mU / i2FG / mA / mU / mC / i2FU / mU / i2FC / mA / i 2FU / mG / i2FG / mC* / mA* / mU / -3' 603 GN A7 0310 5'- / i2FG* / mC* / i2FC / mA / i2FU / mG / i2FA / mA / i2FG / i2FA / i2 FU / mC / i2FA / mU / i2FG / mA / i2FA / mC / i2FA* / mA* / i2FA / -3' 518 5'- / mU* / i2FU* / mU / i2FG / mU / i2 FU / mC / gnA / mU / i2FG / mA / m U / mC / i2FU / mU / i2FC / mA / i2F U / mG / i2FG / mC* / mA* / mU / -3' 604 GN A8 0311 5'- / i2FU* / mC* / i2FC / mC / i2FU / mG / i2FA / mA / i2FC / i2FC / i2 FC / mU / i2FA / mG / i2FA / mA / i2FC / mU / i2FG* / mU* / i2FA / -3' 498 5'- / mU* / i2FA* / mC / i2FA / gnG / i2 FU / mU / i2FC / mU / i2FA / mG / m G / mG / i2FU / mU / i2FC / mA / i2F G / mG / i2FG / mA* / mG* / mC / -3' 605 GN A5 0312 5'- / i2FU* / mC* / i2FC / mC / i2FU / mG / i2FA / mA / i2FC / i2FC / i2 FC / mU / i2FA / mG / i2FA / mA / i2FC / mU / i2FG* / mU* / i2FA / -3' 498 5'- / mU* / i2FA* / mC / i2FA / mG / gn U / mU / i2FC / mU / i2FA / mG / m G / mG / i2FU / mU / i2FC / mA / i2F G / mG / i2FG / mA* / mG* / mC / -3' 606 GN A6 0313 5'- / i2FU* / mC* / i2FC / mC / i2FU / mG / i2FA / mA / i2FC / i2FC / i2 FC / mU / i2FA / mG / i2FA / mA / i2FC / mU / i2FG* / mU* / i2FA / -3' 498 5'- / mU* / i2FA* / mC / i2FA / mG / i2 FU / gnU / i2FC / mU / i2FA / mG / mG / mG / i2FU / mU / i2FC / mA / i 2FG / mG / i2FG / mA* / mG* / mC / -3' 607 GN A7 0314 5'- / i2FU* / mC* / i2FC / mC / i2FU / mG / i2FA / mA / i2FC / i2FC / i2 498 5'- / mU* / i2FA* / mC / i2FA / mG / i2 FU / mU / gnC / mU / i2FA / mG / m 608 GN A8 FC / mU / i2FA / mG / i2FA / mA / i2FC / mU / i2FG* / mU* / i2FA / -3' G / mG / i2FU / mU / i2FC / mA / i2F G / mG / i2FG / mA* / mG* / mC / -3' 0315 5'- / mG* / mA* / mC / mA / mU / m U / i2FC / i2FC / i2FU / mC / mG / mG / mU / mA / mU / mU / mU* / mA* / mA / -3' 251 5'- / mU* / i2FU* / mA / mA / gnA / i2F U / mA / mC / mC / mG / mA / mG / mG / i2FA / mA / i2FU / mG / mU / mC* / mU* / mU / -3' 609 GN A5 0316 5'- / mG* / mA* / mC / mA / mU / m U / i2FC / i2FC / i2FU / mC / mG / mG / mU / mA / mU / mU / mU* / mA* / mA / -3' 251 5'- / mU* / i2FU* / mA / mA / mA / gn U / mA / mC / mC / mG / mA / mG / mG / i2FA / mA / i2FU / mG / mU / mC* / mU* / mU / -3' 610 GN A6 0317 5'- / mG* / mA* / mC / mA / mU / m U / i2FC / i2FC / i2FU / mC / mG / mG / mU / mA / mU / mU / mU* / mA* / mA / -3' 251 5'- / mU* / i2FU* / mA / mA / mA / i2F U / gnA / mC / mC / mG / mA / mG / mG / i2FA / mA / i2FU / mG / mU / mC* / mU* / mU / -3' 611 GN A7 0318 5'- / mG* / mA* / mC / mA / mU / m U / i2FC / i2FC / i2FU / mC / mG / mG / mU / mA / mU / mU / mU* / mA* / mA / -3' 251 5'- / mU* / i2FU* / mA / mA / mA / i2F U / mA / gnC / mC / mG / mA / mG / mG / i2FA / mA / i2FU / mG / mU / mC* / mU* / mU / -3' 612 GN A8 0319 5'- / mC* / mC* / mG / mA / i2FG / m A / i2FU / i2FG / i2FG / mC / mG / mC / mG / mC / mU / mU / mC* / mU* / mA / -3' 212 5'- / mU* / i2FA* / mG / mA / gnA / i2F G / mC / i2FG / i2FC / mG / mC / mC / mA / i2FU / mC / i2FU / mC / mG / mG* / mU* / mU / -3' 613 GN A5 0320 5'- ZmC* / mC* / mG / mA / i2FG / m A / i2FU / i2FG / i2FG / mC / mG / mC / mG / mC / mU / mU / mC* / mU* / mA / -3' 212 5'- / mU* / i2FA* / mG / mA / mA / gn G / mC / i2FG / i2FC / mG / mC / mC / mA / i2FU / mC / i2FU / mC / mG / mG* / mU* / mU / -3' 614 GN A6 0321 5'- / mC* / mC* / mG / mA / i2FG / m A / i2FU / i2FG / i2FG / mC / mG / mC / mG / mC / mU / mU / mC* / mU* / mA / -3' 212 5'- / mU* / i2FA* / mG / mA / mA / i2F G / gnC / i2FG / i2FC / mG / mC / m C / mA / i2FU / mC / i2FU / mC / m G / mG* / mU* / mU / -3' 615 GN A7 0322 5'- / mC* / mC* / mG / mA / i2FG / m A / i2FU / i2FG / i2FG / mC / mG / mC / mG / mC / mU / mU / mC* / mU* / mA / -3' 212 5'- / mU* / i2FA* / mG / mA / mA / i2F G / mC / gnG / i2FC / mG / mC / mC / mA / i2FU / mC / i2FU / mC / mG / mG* / mU* / mU / -3' 616 GN A8 0323 5'- / mC* / mC* / mG / mA / mG / m A / i2FU / i2FG / i2FG / mC / mG / mC / mG / mC / mU / mU / mC* / mU* / mA / -3' 252 5'- / mU* / i2FA* / mG / mA / gnA / i2F G / mC / mG / mC / mG / mC / mC / m A / i2FU / mC / i2FU / mC / mG / m G* / mU* / mU / -3' 617 GN A5 0324 5'- / mC* / mC* / mG / mA / mG / m A / i2FU / i2FG / i2FG / mC / mG / mC / mG / mC / mU / mU / mC* / mU* / mA / -3' 252 5'- / mU* / i2FA* / mG / mA / mA / gn G / mC / mG / mC / mG / mC / mC / m A / i2FU / mC / i2FU / mC / mG / m G* / mU* / mU / -3' 618 GN A6 0325 5'- / mC* / mC* / mG / mA / mG / m A / i2FU / i2FG / i2FG / mC / mG / mC / mG / mC / mU / mU / mC* / mU* / mA / -3‘ 252 5'- / mU* / i2FA* / mG / mA / mA / i2F G / gnC / mG / mC / mG / mC / mC / mA / i2FU / mC / i2FU / mC / mG / mG* / mU* / mU / -3' 619 GN A7 0326 5'- / mC* / mC* / mG / mA / mG / m A / i2FU / i2FG / i2FG / mC / mG / mC / mG / mC / mU / mU / mC* / mU* / mA / -3' 252 5'- / mU* / i2FA* / mG / mA / mA / i2F G / mC / gnG / mC / mG / mC / mC / mA / i2FU / mC / i2FU / mC / mG / mG* / mU* / mU / -3' 620 GN A8

[0276] Modification descriptions: m = 2'-O-methyl; i2F = 2'-fluoro; gn = ethylene glycol nucleic acid; * / = phosphorothioate linkage; / = phosphodiester linkage.

[0277] The detailed procedure for the off-target risk assessment is as follows: Using in vitro siRNA evaluation method 1 from Example 2, SJCRH30 cells were transfected with siRNAs at final concentrations of 1 nM and 20 nM. After 48 hours, changes in on-target and off-target genes were assessed by next-generation sequencing (RNA-seq). The analysis was limited to mRNA off-targets. The criteria for identification were: log2(target / blank) < -1 at 20 nM; padj < 10-5 at both concentrations; better knockdown efficiency at 20 nM than at 1 nM; and some sequence similarity between the siRNA and the target gene. Two batches from two different suppliers of siRNA ID #253 were used in these experiments. The RNA-seq results are shown in Figure 12, where recurrently identified genes were selected for subsequent off-target validation. For off-target validation, primers were designed for each candidate off-target gene. qPCR validation results identified COL4A1, RPL38, and HIGD2A as off-target genes (Figure 13). The primer design for these genes is shown in Table 29.

[0278] Table 29 Primer information for off-target genes Human COL4A1-F CAGGCACCCCATCTGTTGAT (Seq ID NO: 635) Human COL4A1-R CATTGCCTTGCACGTAGAGC (Seq ID NO: 636) Human RPL38-F CAAGGACTTCCTGCTCACAGC (Seq ID NO: 637 ) Human RPL38-R ACCAGGGTGTAAAGGTATCTGC (Seq ID NO: 638 ) Human HIGD2A-F AGAACCCGGTGGTACCCATA (Seq ID NO: 639) Human HIGD2A-R TGCGCATCATGAGCTGAGAG ( Seq ID NO: 640 )

[0279] By introducing an ethylene glycol nucleic acid (GNA) modification at positions 5-8 of the antisense strand seed region, siRNA ID #253 was modified to generate siRNA ID #311 through siRNA ID #314. Specifically, siRNA ID #311 contains a GNA modification at position 5 of the antisense strand seed region, siRNA ID #314 contains a GNA modification at position 8 of the antisense strand seed region, and so forth (Table 28). The in vitro siRNA evaluation method 1 from Example 2 was repeated. The results demonstrated that all four GNA modifications at different positions effectively reduced the knockdown efficiency of the off-target genes COL4A1, RPL38, and HIGD2A, without significantly affecting the knockdown efficiency of the on-target gene DMPK (Figures 14-17).

[0280] Example 5: Expression and Affinity Validation of Anti-TfR1 Antibody

[0281] Stable cell pools were selected and expanded, and seeded into shake flasks or cell culture bags using Serum-free Expression medium (GenScript) at 37°C in a 5% CO2 atmosphere. On the day of transfection, the culture medium was warmed to 25-37°C. One hour prior to transfection, 50 pM swainsonine (MedChemExpress) was added to the medium. Appropriate amounts of plasmid and transfection reagent were added to the transfection mix, thoroughly mixed, and incubated at room temperature. Depending on cell growth, appropriate feed medium was added to the cell culture. On day 7, the culture supernatant was collected for purification. The supernatant was loaded onto a MabSelect™ PrismA Crude column at an appropriate flow rate. After washing and elution with suitable buffers, the buffer of the eluted fraction was exchanged into the final formulation buffer. The eluted protein buffer was exchanged into PBS (pH 7.2). The purified protein was analyzed by SDS-PAGE and SEC-HPLC (column: TSKgel G3000SWxl, Tosoh) to determine molecular weight and purity. The final protein purity was >98%.

[0282] The anti-human TfR1 antibody used in the experiment is shown in Table 30. Its Fab region is derived from 13E4-Variant 2iii described in US 10,913,800 B2, and its Fc region is derived from a human IgG1 subclass monoclonal antibody. In some embodiments, the Fc region was engineered, for example, with LALA-LR mutations to reduce Fc immunogenicity, or with an S238C mutation to create a site suitable for site-specific nucleic acid conjugation. The affinity between the anti-human TfR1 antibody and human TfR1 was confirmed by Octet BLI, and the results are shown in Table 31.

[0283] Table 30 Antibody sequences for anti-human TfR1 Name Sequence Var2iii >VH, 445aa, LALA-LR QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLE WIGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSRLRSDDTAVYYCA RGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKARPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG (Seq ID NO: 641) >VL, 214aa DIQMTQSPSSLSASVGDRVTITCRTSENIYNNLAWYQQKPGKSPKLLIYAA TNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGGGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC (Seq ID NO: 642) Var2iii-S238C >VH, 445aa, LALA-LR + S238C QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLE WIGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSRLRSDDTAVYYCA RGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPCVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKARPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG (Seq ID NO: 643) >VL, 214aa DIQMTQSPSSLSASVGDRVTITCRTSENIYNNLAWYQQKPGKSPKLLIYAA TNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGGGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC (Seq ID NO: 644)

[0284] Table 31 Binding affinity of anti-human TfR1 antibody to human and cynomolgus monkey TfR1 Antibody Antigen Chi2 (RU2) ka (1 / Ms) kd (1 / s) KD (M) Rmax (RU) hIgGI TfR1-Var2iii Human TfR1 5.88E-01 4.36E+05 7.42E-05 1.70E-10 96.3 hIgG1 TfR1-Var2iii Cyno TfR1 6.30E-01 3.31E+05 1.33E-03 4.02E-09 69.1 “Chi2” refers to the chi-square test; “ka” refers to the association rate constant; “kd” refers to the dissociation rate constant; “KD” refers to the affinity, calculated as kd / ka; and “Rmax” refers to the maximum signal intensity at saturation

[0285] The anti-mouse TfR1 antibodies used in the experiments are shown in Table 32. The variable regions were derived from the rat anti-mouse TfR1 antibody TIB-219 (light chain GenBank: ABV48920.1; heavy chain GenBank: ABV48917.1), and the Fc region was derived from a human IgG1 subclass monoclonal antibody. In some embodiments, the Fc region was engineered, for example, with LALA-LR mutations to reduce Fc immunogenicity, or with an S245C mutation to create a site suitable for site-specific nucleic acid conjugation. The affinity between the antimouse TfR1 antibody and mouse TfR1 was confirmed by Octet BLI, and the results are shown in Table 33.

[0286] Table 32. Antibody Sequences for Anti-mouse TfR1 Name Sequence TIB-219 >VH, 452aa, LALA-LR EVQLVESGGGLVQSGRSLKLSCVASGFTFSSYGMNWIRQAPGKGLEWVA YISSAGKYIYYAETMKGRFTISRDNARNTLYLQMTSLRSENTALYYCARL GTGSEGHYWYFDFWGPGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKARPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG (Seq ID NO: 645) >VL, 214aa DVQMTQSPYNLAASPGESISISCKASKSISKYLAWYQQKPGKANKLLIYD GSTLQSGIPSRFSGSGSGTNFTLTIRSLEPEDFGLYYCQQHDESPPTFGAGT KLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC (Seq ID NO: 646) TIB-219- S245C >VH, 452aa, LALA-LR + S245C EVQLVESGGGLVQSGRSLKLSCVASGFTFSSYGMNWIRQAPGKGLEWVA YISSAGKYIYYAETMKGRFTISRDNARNTLYLQMTSLRSENTALYYCARL GTGSEGHYWYFDFWGPGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPCVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKARPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG (Seq ID NO: 647) >VL, 214aa DVQMTQSPYNLAASPGESISISCKASKSISKYLAWYQQKPGKANKLLIYD GSTLQSGIPSRFSGSGSGTNFTLTIRSLEPEDFGLYYCQQHDESPPTFGAGT KLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC (Seq ID NO: 648)

[0287] Table 33. Binding affinity of anti-mouse TfR1 antibody to mouse and cynomolgus monkey TfR1 Antibody Antigen Chi2 (RU 2) ka (1 / Ms) kd (1 / s) KD (M) Rmax (RU) TIB-219 Mouse TfR1 3.73E-02 1.34E+05 1.06E-04 7.87E-10 67.2

[0288] Example 6: Conjugation of Modified siRNA and Antibody

[0289] Conjugation of wild-type antibody and siRNA

[0290] The conjugation of wild-type antibody and siRNA was performed using a random conjugation process. Specifically, the interchain disulfide bonds of the antibody were first reduced, and the resulting thiol groups were conjugated to a linker-siRNA activated with a maleimide (SMCC) linker at the 5' end of the siRNA sense strand, ultimately forming an antibody-siRNA conjugate. For the linkersiRNA used in this experiment, the single strand was fully assembled on a solid phase using standard phosphoramidite chemistry and purified by HPLC. Base, sugar, and phosphate modifications well described in the RNAi field were used to optimize duplex stability. The 5' end of the siRNA sense strand contained a C6-NH2 conjugation handle, which was attached to the siRNA sense strand via a phosphodiester bond on the terminal base (see Figure 18). In some embodiments, this conjugation process is generally applicable to different antibodies and sequences; differences in antibody targets or sequences and nucleic acid sequences typically do not cause significant changes in the conjugation process.

[0291] Step 1: Antibody reduction with TCEP

[0292] The antibody was buffer-exchanged into 25 mM Tris buffer (pH 8) and adjusted to a concentration of 10 mg / mL. To this solution, 0.8 equivalents of TCEP in the same buffer were added, and the mixture was incubated at room temperature for 2 hours. The reaction solution was then buffer-exchanged by ultrafiltration centrifugation into 25 mM Tris buffer (pH 8) containing 2 mM EDTA. SMCC-siRNA (3 equivalents) was added, and the reaction proceeded at 22°C for 3 hours. The reaction mixture was analyzed by analytical SAX column chromatography to resolve the antibody-siRNA conjugate, unreacted antibody, and unreacted siRNA.

[0293] Step 2: Purification and analysis

[0294] The crude reaction mixture was purified by AKTA Pure FPLC using an anion exchange chromatography (SAX) method #1 (Table 34). Fractions containing the antibody-siRNA conjugate were isolated, concentrated, and buffer-exchanged into PBS (pH 7.4). The purity of the conjugate was assessed by analytical HPLC using SAX method #2 (Table 35).

[0295] Table 34 Anion exchange chromatography method (SAX) #1 Column: CytivaTM HiTrap Capto Q 5 ml Solvent A: 10 mM TRIS buffer, pH 8.0; Solvent B: 10 mM TRIS, 1.5 M NaCl, pH 8.0; Flow rate: 6.0 ml / min Gradient Time (min) A(%) B(%) 0 100 0 20 70 30 28 70 30 48 34 66 56 34 66 76 0 100 77 100 0 87 100 0

[0296] Table 35 Anion exchange chromatography method (SAX) #2 Column: Thermo Scientific, ProPacTM SAX-10, Bio LCTM, 4X 250mm SolventA: 80% 10 mM TRIS pH 8, 20% Ethanol; Solvent B: 80% 10 mM TRIS pH 8, 20%Ethanol, 1.5 M NaCl;  Flow rate: 0.6 ml / min Gradient Time (min) A(%) B(%) 0 100 0 1 100 0 3 92 8 12 0 100 15 0 100 17 100 0 20 100 0

[0297] Conjugation of cysteine-mutant antibody and siRNA

[0298] The conjugation of cysteine-mutant antibody and siRNA was performed using a site-specific conjugation process. Specifically, the cysteine site- directed conjugation positions on the antibody (depending on the specific antibody sequence, e.g., S239C, S238C, S245C, etc.) and interchain disulfide bonds were first reduced. The interchain disulfide bonds were then re-oxidized while maintaining the reduced state of the cysteine conjugation sites. Finally, the antibody was conjugated to a linker-siRNA activated with a maleimide (SMCC) linker at the 5' end of the siRNA sense strand to form the antibody-siRNA conjugate. For the linker-siRNA used in this experiment, the single strand was fully assembled on a solid phase using standard phosphoramidite chemistry and purified by HPLC. Base, sugar, and phosphate modifications well described in the RNAi field were used to optimize duplex stability. The 5' end of the siRNA sense strand contained a C6-NH2 conjugation handle, which was attached to the siRNA sense strand via a phosphodiester bond on the terminal base (see Figure 18). In some embodiments, this conjugation process is generally applicable to different antibodies and sequences; differences in antibody targets or sequences and nucleic acid sequences typically do not cause significant changes in the conjugation process.

[0299] Step 1: Antibody reduction with TCEP

[0300] The antibody was buffer-exchanged into 25 mM Tris buffer (pH 8) and adjusted to a concentration of 10 mg / mL. To this solution, 100 equivalents of DTT in the same buffer were added, and the mixture was incubated at room temperature for 16 hours. The reaction solution was then buffer-exchanged by ultrafiltration centrifugation into 25 mM Tris buffer (pH 8) containing 2 mM EDTA. Subsequently, 20 equivalents of DHAA were added, and the reaction was allowed to proceed at room temperature (RT) for 2 hours. The resulting reaction mixture was passed through a desalting column to remove excess DHAA, then combined with SMCC-siRNA (0.8 equivalents) and reacted at 22°C for 2 hours. Analysis of the reaction mixture by analytical SAX column chromatography revealed the antibody-siRNA conjugate along with unreacted antibody and siRNA.

[0301] Step 2 : Purification and analysis

[0302] The crude reaction mixture was purified by AKTA Pure FPLC using anion exchange chromatography (SAX) method #1 (Table 34). Fractions containing the antibody-siRNA conjugate were isolated, concentrated, and buffer-exchanged into PBS (pH 7.4). The purity of the conjugate was assessed by analytical HPLC using SAX method #2 (Table 35).

[0303] The analytical data for the conjugates synthesized in this example, including HPLC retention time (RT) in minutes and percent purity determined by chromatographic peak area, are shown in Table 36 for representative purified antibody-siRNA conjugate molecules. All purities were above 97%, and the corresponding spectra are shown in Figures 19-26. The antibody used was TIB-219-S245C. This example is a validation example, and therefore the specific sequence details are omitted.

[0304] Table 36 Design and analytical results of antibody-siRNA conjugates AOC DAR SAX Retention Time (min) %Purity (by peak area) CGBC-1001 2 8.338 99 CGBC-1002 2 8.230 98 CGBC-1004-1 1 7.903 99 CGBC-1004-2 2 8.333 97 CGBC-1005-1 1 7.931 99 CGBC-1005-2 2 8.341 98 CGBC-1012-1 1 7.982 100 CGBC-1012-2 1 8.413 99

[0305] Example 7: Study of HPRT Knockdown in Wild-Type CD-1 Mouse Muscle by Antibody-siRNA Conjugates (Validation)

[0306] The experimental design and dosing are shown in Table 37. The HPRT siRNAs used for synthesizing the antibody-siRNA conjugates in the experiment are shown in Table 38. CGBC-1001 was synthesized using the antibody TIB-219 from Example 5 and the wild-type antibody-siRNA conjugation process from Example 6. CGBC-1002 was synthesized using the antibody TIB-219-S245C from Example 5 and the cysteine-mutant antibody-siRNA conjugation process from Example 6.

[0307] PBS control and antibody-siRNA conjugates were administered to mice via intravenous injection. Animals were euthanized at 7 days and 14 days postinjection. Tissue samples including cardiac muscle, diaphragm, gastrocnemius (gastro), liver, kidney, and brain were collected, treated with RNALater overnight, and stored at -80°C. mRNA knockdown in target tissues was determined using a comparative qPCR assay. Total RNA was extracted from tissues, reverse transcribed, and mRNA levels were quantified by TaqMan qPCR using appropriately designed primers and probes. GAPDH was used as an internal RNA loading control. Results were calculated using the comparative Ct method, where the difference between the target gene Ct value and the GAPDH Ct value (ACt) was calculated, and then further normalized to the PBS control group by taking the double difference (AACt).

[0308] Table 37 Study of HPRT knockdown in wild-type CD-1 mouse muscle by antibody-siRNA conjugates Group Test article N siRNA equivalent dose (mg / kg) Injection volume (mL / kg) Time (Days ) 1 PBS 4 / 5 0 2 CGBC-1001 4 0.5 5 7, 14 3 CGBC-1001 4 1.5 5 7, 14 4 CGBC-1001 4 5 5 7, 14 5 CGBC-1002 4 0.5 5 7, 14 6 CGBC-1002 4 1.5 5 7, 14 7 CGBC-1002 4 5 5 7, 14

[0309] Table 38 HPRT siRNA sequences design siRNA ID# Modified sense strand sequence (5'-3') Seq ID NO: Modified antisense strand sequence (5'-3') Seq ID NO: 0327 SMCC-NH-C6- 5'- / i2FU / mC / i2FC / mU / i2FA / mU / i2FG / mA / i2FC / mU / i2F G / mU / i2FA / mG / i2FA / mU / i 2FU / mU / i2FU / mA / i2FU / -3' 649 5'- / mA / i2FU / mA / i2FA / mA / i2FA / mU / i2FC / mU / i2FA / mC / i2FA / m G / i2FU / mC / i2FA / mU / i2FA / mG / i2FG / mA* / i2FA* / mU / -3' 650

[0310] Modification descriptions: m = 2'-O-methyl; i2F = 2'-fluoro; gn = ethylene glycol nucleic acid; * / = phosphorothioate linkage; / = phosphodiester linkage.

[0311] The experimental results demonstrated that the antibody-siRNA conjugates showed dose-dependent knockdown of the target gene in cardiac muscle, diaphragm, and gastrocnemius muscle, while no significant target gene knockdown was observed in liver, kidney, or brain tissues. Furthermore, compared to the randomly conjugated CGBC-1001, the site-specifically conjugated CGBC-1002 exhibited advantages in both onset time and maximum knockdown efficiency (Figures 27-30).

[0312] Example 8: Study of DMPK Knockdown in Wild-Type CD-1 Mouse Muscle by Antibody-siRNA Conjugates

[0313] The experimental design and dosing are shown in Table 39. All antibody-siRNA conjugates were synthesized using the antibody TIB-219-S245C from Example 5 and the cysteine-mutant antibody-siRNA conjugation process from Example 6. The DMPK siRNAs used for synthesizing the antibody-siRNA conjugates in the experiment are shown in Table 40. siRNA ID #0296 was used to synthesize CGBC-1004-1 and CGBC-1004-2, and siRNA ID #0297 was used to synthesize CGBC-1005-1 and CGBC-1005-2.

[0314] PBS control and antibody-siRNA conjugates were administered to mice via intravenous injection. Animals were euthanized at 7 days and 14 days postinjection. Plasma and various tissue samples including cardiac muscle, diaphragm, and gastrocnemius (gastro) were collected, treated with RNALater overnight, and stored at -80°C. mRNA knockdown in target tissues was determined using a comparative qPCR assay. Total RNA was extracted from tissues, reverse transcribed, and mRNA levels were quantified by TaqMan qPCR using appropriately designed primers and probes. GAPDH was used as an internal RNA loading control. Results were calculated using the comparative Ct method, where the difference between the target gene Ct value and the GAPDH Ct value (ACt) was calculated, and then further normalized to the PBS control group by taking the double difference (AACt). Bioinformatics analysis was performed according to siRNA in vitro evaluation method 5.

[0315] Table 39 Study of DMPK knockdown in wild-type CD-1 mouse muscle by antibody-siRNA conjugates Group Test article N siRNA equivalent dose (mg / kg) Injection volume (mL / kg) Time (Days ) 1 4 0 2 CGBC-1004-1 4 5 5 7, 14 3 CGBC-1004-2 4 5 5 7, 14 4 CGBC-1005-1 4 5 5 7, 14 5 CGBC-1005-2 4 5 5 7, 14

[0316] Table 40 DMPK siRNA sequences design siRNA ID# Modified sense strand sequence (5'-3') Seq ID NO: Modified antisense strand sequence (5'-3') Seq ID NO: 0328 SMCC-NH-C6-5'- / mG* / mA* / mC / mA / i2FU / mU / i2FC / i2FC / i2FU / mC / mG / mG / mU / mA / mU / mU / mU* / mA* / mA / -3' 211 5'- / mU* / i2FU* / mA / mA / mA / i2FU / mA / i2FC / i2FC / mG / mA / mG / mG / i2FA / mA / i2FU / mG / mU / mC* / m U* / mU / -3' 231 0329 SMCC-NH-C6-5'- / mG* / mA* / mC / mA / i2FU / mU / i2FC / i2FC / i2FU / mC / mG / mG / mU / mA / mU / mU / mU* / mA* / mA / -3' 211 5'- / VPmU* / i2FU* / mA / mA / mA / i2F U / mA / i2FC / i2FC / mG / mA / mG / mG / i2FA / mA / i2FU / mG / mU / mC * / mU* / mU / -3' 659

[0317] Modification descriptions: m = 2'-O-methyl; i2F = 2'-fluoro; gn = ethylene glycol nucleic acid; * / = phosphorothioate linkage; / = phosphodiester linkage; VP = 5-(E)-VP vinylphosphonate modified nucleic acid.

[0318] The pharmacokinetic experimental method primarily used SL-PCR to measure siRNA concentrations in plasma and tissues. Plasma and various tissue samples, including quadriceps muscle and liver, were collected and snap-frozen in liquid nitrogen for storage. Tissues were homogenized and digested with proteinase K, and the supernatant was collected, diluted, and subjected to SL-PCR analysis. SL-PCR mainly consisted of a first step of reverse transcription using a stem-loop primer sequence and a second step of quantification by qPCR. The reaction systems are shown in Tables 41-42, and the primer sequences are shown in Table 43.

[0319] Table 41 SL-PCR reverse transcription reaction system PCR reaction solution component Volume required per reaction (pL) 100mM dNTPs (with dTTP) 0.15 MultiScribe Reverse Transcriptase, 50 U / pL 1 10 X Reverse Transcription Buffer 1.5 RNase Inhibitor, 20 U / pL 0.19 Nuclease-free Water 7.085 5 X RT primer (4pM) 0.1875 siRNA / miRNA 5 Total 15

[0320] Table 42 qPCR reaction system for SL-PCR PCR reaction solution component Volume required per reaction (pL) Universal PCR Master Mix 2 x 5 Forward primer (10pM) 1.5 Reverse primer (10pM) 0.7 Probe (10pM) 0.2 cDNA 2 H2O 0.6 Total 10

[0321] Table 43 Primer sequences for SL-PCR in mouse experiments Primer Sequence (5’-3‘) Stem-loop primer GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAG ACAT (Seq ID NO: 651) Forward GCGCTTAAATACCGAGGAA (Seq ID NO: 652) Reverse GTGCAGGGTCCGAGGT (Seq ID NO: 653 ) Probe    |    FAM-CGCACTGGATACGACAAGAC-MGB (Seq ID NO: 654)

[0322] In both the mRNA knockdown in target tissues and the pharmacokinetic experimental methods, a standard curve of Ct versus log[concentration] was required. The standard curve for mRNA knockdown in target tissues was prepared using serially diluted freshly extracted cDNA from tissues. The standard curve for pharmacokinetics was prepared by diluting the antibody-siRNA conjugate compound and then spiking it into blank mouse tissue homogenate. Except for the steps described above, the actual sample processing steps were kept consistent with those of the standard curve samples. The amplification efficiency of the standard curve was required to be within the range of 90-110%.

[0323] The experimental results demonstrated that the antibody-siRNA conjugates showed target gene knockdown in cardiac muscle, diaphragm, and gastrocnemius muscle, while no significant target gene knockdown was observed in liver, kidney, or brain tissues. Compared to the DAR2 antibody-siRNA conjugates, the DAR1 molecules exhibited stronger target gene knockdown (Figures 31-32). Additionally, the 5-(E)-VP design significantly improved the target gene knockdown efficiency of the antibody-siRNA conjugates in cardiac muscle, diaphragm, and gastrocnemius muscle (Figure 32).

[0324] The antibody-siRNA conjugates exhibited a much slower plasma clearance rate and muscle-specific siRNA delivery efficiency compared to conventional siRNA drugs. At 14 days post-administration, siRNA in plasma was still detectable by the SL-PCR method (Figures 33-34), which is significantly different from the plasma half-life of several hours for conventional siRNA drugs, indicating that the antibody-siRNA conjugates prolonged the plasma half-life of siRNA. In terms of tissue distribution, the antibody-siRNA conjugates achieved greater siRNA distribution in muscle, with muscle siRNA levels higher than or close to liver siRNA levels, demonstrating the muscle-targeting effect of the TfR1 antibody. In terms of molecular design, the DAR1 molecule exhibited higher plasma concentration and greater muscle enrichment than the DAR2 molecule, and the 5-(E)-VP design also significantly increased muscle enrichment of the antibodysiRNA conjugates (Figure 35).

[0325] Example 9: In Vivo Pharmacokinetic study of antibody-siRNA conjugates in cynomolgus monkeys

[0326] The experimental design and dosing are shown in Table 44. The DMPK siRNAs used for synthesizing the antibody-siRNA conjugates in the experiment are shown in Table 45. The antibody used was Var2iii-S238C from Example 18, and the conjugation process from Example 19 (cysteine-mutant antibody and siRNA conjugation) was employed.

[0327] The antibody-siRNA conjugates were administered to cynomolgus monkeys via intravenous injection. Plasma samples were collected before dosing and at 3, 6, 12 hours and 1, 3, 7, 14, 28, 42 days post-dosing according to the study protocol. Simultaneously, gastrocnemius muscle samples were collected by muscle biopsy before dosing and at 3, 7, 14, 28, 56, 84 days post-dosing for pharmacokinetic analysis. The standard curve generation method was the same as described in Example 20 and will not be repeated here. The primer sequences are shown in Table 46.

[0328] Table 44 In vivo study of antibody-siRNA conjugates in cynomolgus monkeys Group Test article N siRNA equivalent dose (mg / kg) Injection volume (mL / kg) Time (Days ) 1 CGBC-1012-1 2 0.6 5 84 2 CGBC-1012-1 2 6 5 84

[0329] Table 45 siRNA sequence design for DMPK siRNA ID# Modified sense strand sequence (5'-3') Seq ID NO: Modified antisense strand sequence (5'-3') Seq ID NO: 0330 SMCC-NH-C6-5'- / mG* / mA* / mC / mA / mU / mU / i2 FC / i2FC / i2FU / mC / mG / mG / m U / mA / mU / mU / mU* / mA* / mA / -3' 251 5'- / mU* / i2FU* / mA / mA / mA / i2FU / mA / mC / mC / mG / mA / mG / mG / i2F A / mA / i2FU / mG / mU / mC* / mU* / mU / -3' 271

[0330] Modification descriptions: m = 2'-O-methyl; i2FN = 2'-fluoro; gn = ethylene glycol nucleic acid; * / = phosphorothioate linkage; / = phosphodiester linkage; VpN = 5-(E)-VP vinylphosphonate modified nucleic acid.

[0331] Table 46 Primer sequences for SL-PCR in cynomolgus monkey experiments Primer Sequence (5’-3’) Stem-loop primer 5’- GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACATT CCTA-3’ (Seq ID NO: 655) Forward 5’-GCGCATAAAATCTACAGTCA-3’ (Seq ID NO: 656 ) Reverse 5’-GTGCAGGGTCCGAGGT-3’ (Seq ID NO: 657) Probe 5’-FAM-CGCACTGGATACGACATTCC-MGB-3’ ( Seq ID NO: 658 )

[0332] The results showed that the antibody-siRNA conjugates exhibited a typical two-compartment model in cynomolgus monkey plasma, showing linear pharmacokinetics at both doses. The distribution half-life at the high dose was 27.6 hours, and the elimination half-life was 279 hours. For the low-dose group, due to the lower limit of detection, only the distribution phase curve was accurately recorded (Figure 36). In terms of tissue distribution, the antibody-siRNA conjugates accumulated in the gastrocnemius muscle of cynomolgus monkeys, reaching a Cmax exceeding 300 pmol / g on day 3 post-dose. As the active pharmaceutical ingredient, the clinically effective dose range is 0.1-20 mg / kg, with a more preferred range of 1-10 mg / kg. The elimination half-life of the antibody-siRNA conjugates in cynomolgus monkey gastrocnemius muscle was 239 hours, and the siRNA concentration in the gastrocnemius muscle remained above 1 pmol / g throughout the 84-day period prior to the end of the study (Figure 37).

[0333] The above experiments fully demonstrate that the antibody-siRNA conjugates can exert their effects in mice.

[0334] Example 10: In vitro activity of antibody-siRNA conjugates

[0335] The antibody-siRNA conjugates used in this experiment are the same as those in Example 23. The objective of the experiment was to demonstrate that the antibody-siRNA conjugates can be internalized by human muscle cells in vitro without the aid of lipofectamine and produce significant target gene knockdown. The specific experimental method is as follows: RD human rhabdomyosarcoma cells (4201HUM-CCTCC00295) were cultured in DMEM (Gibco) containing 10% fetal bovine serum (Gibco). The antibody-siRNA conjugates were diluted to a maximum dose of 10 mM. The conjugates were transfected at final siRNA concentrations of 200, 100, 10, 1, and 0.1 nM. Cells were seeded into 48-well plates at 10,000 cells per well 24 hours prior to dosing. The conjugates were added to the wells. PBS was added to some wells as an additional negative control. The cells were placed at 37 ° C in 5% CO2 for 72 hours. The medium was removed from the wells, and 150 pL of Trizol (Life) was added. The plates were frozen at -80°C overnight or longer before analysis. RNA was isolated using the Direct-zol-96 RNA Kit (Zymo Research) according to the manufacturer's instructions. RNA was reverse transcribed into cDNA using ReverTra Ace™ qPCR RT Master Mix (TOYOBO) according to the manufacturer's instructions, and cDNA samples were evaluated by qPCR using TaqMan human gene expression probes (Thermo Fisher). The percentage of mRNA remaining was calculated using the standard 2 AACT method, with PBS-treated cells set at 100% expression. All experiments were performed in triplicate. Primer information is the same as in siRNA in vitro evaluation method 1.

[0336] The experimental results showed that CGBC-1012, without the aid of lipofectamine, entered RD cells under in vitro co-incubation conditions and exerted dose-dependent target gene knockdown. The knockdown effect of the antibodysiRNA conjugates reached saturation at a concentration of 1 nM. The maximum knockdown efficiency of the DAR2 antibody-siRNA conjugate was approximately 40%, while that of the DAR1 molecule was approximately 50% (Figure 38).

[0337] The above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A polynucleotide molecule that binds to DMPK gene mRNA, characterized in that the polynucleotide molecule comprises at least 12 consecutive nucleotides of any one of nucleotide sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 200 and SEQ ID NO: 281 to SEQ ID NO: 490, or a sequence complementary thereto, or a sequence that differs from at least 12 consecutive nucleotides by no more than 3 nucleotides, or a sequence complementary thereto, wherein the nucleotides are in a modified or unmodified state.

2. The polynucleotide molecule of claim 1, wherein the polynucleotide molecule is complementary to at least 8 consecutive nucleotides of a target sequence of DMPK.

3. The polynucleotide molecule according to claim 1, characterized in that the polynucleotide molecule mediates RNA interference against DMPK, thereby modulating muscle atrophy or myotonic dystrophy in a subject.

4. The polynucleotide molecule according to claim 1, characterized in that the polynucleotide molecule has a length of 8 to 50 nucleotides, preferably 10 to 30 nucleotides, more preferably 15 to 25 nucleotides.

5. The polynucleotide molecule according to claim 1, characterized in that the polynucleotide molecule is a single-stranded oligonucleotide or a double-strandedoligonucleotide.

6. The polynucleotide molecule according to claim 1, characterized in that the polynucleotide molecule comprises one or more modified nucleotides, one or more modified internucleoside linkages, or one or more inverted abasic moieties.

7. The polynucleotide molecule according to claim 6, characterized in that the modified nucleotides are 2'-modified nucleotides.

8. The polynucleotide molecule according to claim 7, characterized in that the 2'-modified nucleotides comprise 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE),     2'-O-dimethylaminopropyl     (2'-O-DMAP),     2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA) modified nucleotides.

9. The polynucleotide molecule according to claim 7, characterized in that the 2'-modified nucleotides comprise locked nucleic acid (LNA), ethylene nucleic acid (ENA), or glycol nucleic acid (GNA).

10. The polynucleotide molecule according to claim 6, characterized in that the modified internucleoside linkages comprise phosphorothioate linkages, phosphorodithioate linkages, methylphosphonate linkages, phosphotriester linkages,or amide linkages.

11. The polynucleotide molecule according to claim 10, characterized in that the modified internucleoside linkages comprise phosphorothioate linkages or phosphorodithioate linkages.

12. The polynucleotide molecule according to claim 6, characterized in that the inverted abasic moiety is present on at least one terminus.

13. The polynucleotide molecule according to claim 6, characterized in that the polynucleotide molecule comprises at least 12 consecutive nucleotides of any one of the nucleotide sequences set forth in SEQ ID NO: 201 to SEQ ID NO: 280, SEQ ID NO: 491 to SEQ ID NO: 580, and SEQ ID NO: 589 to SEQ ID NO: 620, or a sequence that differs from said at least 12 consecutive nucleotides by no more than 3 nucleotides, or a sequence complementary thereto.

14. The polynucleotide molecule according to claim 6, characterized in that the polynucleotide molecule comprises a double-stranded structure having an antisense strand and a sense strand.

15. The polynucleotide molecule according to any one of claims 1-14, characterized in that the antisense strand comprises any one of SEQ ID NO: 101200, SEQ ID NO: 221-240, SEQ ID NO: 261-280, SEQ ID NO: 386-490, SEQ IDNO: 536-580, or SEQ ID NO: 589-620, or a sequence that differs therefrom by no more than 3 nucleotides.

16. The polynucleotide molecule according to claim 1, characterized in that the sense strand comprises any one of SEQ ID NO: 1-100, SEQ ID NO: 201-220, SEQ ID NO: 241-260, SEQ ID NO: 281-385, or SEQ ID NO: 491-535, or a sequence that differs therefrom by no more than 3 nucleotides.

17. A conjugate, characterized in that the conjugate comprises the polynucleotide molecule according to any one of claims 1-16, and further comprises an antibody, an antibody fragment, or an antigen-binding sequence.

18. The conjugate according to claim 17, characterized in that the polynucleotide molecule and the antibody or antigen-binding sequence are conjugated to each other via random conjugation or site-specific conjugation.

19. The conjugate according to claim 18, characterized in that the antibody, antibody fragment, or antigen-binding sequence is used to bind to a transferrin receptor.

20. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the polynucleotide molecule according to any one of claims 1-16 or the conjugate according to any one of claims 17-19, wherein thepharmaceutical composition is used for treating muscle atrophy or myotonic dystrophy.

21. The pharmaceutical composition according to claim 20, characterized in that the pharmaceutical composition is formulated for intravenous, subcutaneous, parenteral, oral, intranasal, buccal, rectal, or transdermal administration.

22. The pharmaceutical composition according to claim 20, characterized in that the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

23. The pharmaceutical composition according to claim 20, characterized in that the pharmaceutical composition further comprises a therapeutic agent, wherein the therapeutic agent comprises a small molecule drug, an antibody, an antibody fragment, and / or a vaccine.

24. The pharmaceutical composition according to claim 23, characterized in that the antibody comprises an anti-transferrin receptor antibody.

25. A use of the polynucleotide molecule according to any one of claims 1-16 or the conjugate according to any one of claims 17-19 for the preparation of a medicament, characterized in that the medicament is used for reducing DMPK mRNA expression, or for preventing and / or treating muscle atrophy or myotonicdystrophy.

26. The use according to claim 25, characterized in that the muscle atrophy comprises muscle atrophy associated with insulin deficiency, associated with chronic renal failure, associated with congestive heart failure, associated with chronic respiratory disease, associated with chronic infection, associated with fasting, associated with denervation, associated with sarcopenia, or associated with glucocorticoid therapy.

27. The use according to claim 26, characterized in that the muscle atrophy comprises muscle atrophy associated with myotonic dystrophy type 1 or myotonic dystrophy.

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