Oligonucleotide preparations

Abstract

This disclosure relates to oligonucleotide formulations. In particular, this disclosure relates to oligonucleotide formulations containing oligonucleotides (such as ASOs, siRNAs, and saRNAs) and calcium, methods for preparing such formulations, and their use. The oligonucleotide formulations of this disclosure have excellent applicability because they reduce acute toxicity in the body (particularly to the central nervous system) and extend the safety margin in the body, especially compared to lipid-conjugated oligonucleotides.

Description

Technical Field

[0001] Relates to a preparation comprising a nucleotide substance and a calcium-containing solution having a specific calcium concentration, and a specific oligonucleotide preparation containing calcium and other components.

[0002] Cross-reference to related applications This application claims priority to the filing date of provisional patent application No. PCT / CN2023 / 100743 filed on June 16, 2023, the disclosure of which is incorporated herein by reference.

[0003] Sequence listing This application includes a sequence listing submitted electronically in a computer-readable format, which is incorporated herein by reference in its entirety.

Background Art

[0004] Oligonucleotide-based therapeutic agents are emerging as a promising class of therapeutic agents for neurological diseases. By administering oligonucleotides to patients, organs, tissues or cells, various biochemical reactions can be induced or affected, and functions such as gene expression silencing, inhibition, activation and / or regulation can be achieved.

[0005] Single-stranded ASOs in the form of "gapmers" can be used to suppress gene expression by degrading target mRNA via the RNase H mechanism. Another category of ASOs are steric blockers (i.e., splicing regulators), which are usually composed uniformly of ribonucleotides, bind to pre-mRNA in the nucleus, and alter mRNA splicing by inhibiting the binding of specific splicing factors to mRNA.

[0006] Double-stranded oligonucleotides or double-stranded RNAs (dsRNAs) include small interfering RNAs (siRNAs), small activating RNAs (saRNAs), and microRNAs (miRNAs), all of which are loaded onto argonaute (AGO) proteins in the cytoplasm as an initial step before specifically acting on their targets. siRNAs mainly bind to target mRNAs in the cytoplasm and downregulate gene expression post-transcriptionally via the RNA interference (RNAi) mechanism. saRNAs target gene regulatory sequences such as gene promoters in the nucleus and upregulate gene expression at the transcriptional level via the RNA activation (RNAa) mechanism. miRNAs are also double-stranded RNAs and, after being loaded onto AGO proteins, usually bind with incomplete complementarity to the 3' untranslated region (3'UTR) of mRNAs and partially inhibit expression via translational repression and / or mRNA degradation.

[0007] Oligonucleotides (such as ASOs, siRNAs or saRNAs) having 2'-position modified (such as 2'-fluoro-2'-deoxynucleoside, 2'-O-methyl, or 2'-O-(2-methoxyethyl) modification), backbone modified (such as phosphorothioate (PS) modification), and / or conjugated to hydrophilic moieties (such as fatty acid moieties, cholesterol or lipids, etc.) are thought to have improved nuclease stability and reduced hydrophilicity. However, chemically modified (such as fluorinated, PS-modified) and / or conjugated (such as conjugated to hydrophilic moieties, etc.) oligonucleotide / oligonucleotide substances induce dose-limiting acute toxicity, and as a result, the therapeutic window may be narrowed or clinical trials may fail. Summary of the Invention Problems to be Solved by the Invention

[0008] Therefore, there is a need for an improved formulation for oligonucleotide substances to address the above problems.

[0009] To address the aforementioned problems and challenges, this application provides a novel oligonucleotide formulation, a method for preparing the same, and its use.

[0010] Aspects of the present disclosure include oligonucleotide formulations. The formulation comprises (a) a calcium-containing solution comprising calcium at a concentration of at least 15 mM, and (b) an oligonucleotide substance in the solution.

[0011] In some embodiments, when the oligonucleotide substance is a non-conjugated oligonucleotide, the calcium concentration in the formulation is between 15 mM and less than 25 mM. For example, when the oligonucleotide substance is a non-conjugated oligonucleotide, the calcium concentration in the formulation is in the range of 15 mM to 24, 23, 22, 21, 20, 19, 18, 17, or 16 mM, or any lower range or numerical value within that range.

[0012] In some embodiments, when the oligonucleotide substance is a conjugated oligonucleotide, the calcium concentration in the formulation is 15 mM to 150 mM. For example, the calcium concentration in the formulation is in the range of 15 mM to 150 mM, e.g., 20 mM to 150 mM, 20 mM to 130 mM, 20 mM to 120 mM, 30 mM to 110 mM, or 35 mM to 100 mM, for example, the calcium concentrations in the formulation are approximately 20, 35, 47.5, 60, 82.5, 100, and 120 mM. In some embodiments, when the oligonucleotide substance is a conjugated oligonucleotide, the calcium concentration in the formulation is from 15 mM to less than 25 mM, e.g., in the range of 15 mM to 24, 23, 22, 21, 20, 19, 18, 17, or 16 mM, or any sub-range or numerical value within that range.

[0013] In some embodiments, the oligonucleotide formulation has an osmotic pressure in the range of 250 mOsmol / kg to 350 mOsmol / kg.

[0014] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide (e.g., an antisense RNA (ASO) selected from gapmers, steric blockers, or mixed mers), or a double-stranded oligonucleotide (e.g., a double-stranded RNA selected from siRNA, saRNA, or miRNA, a combination of siRNA and saRNA, or a conjugate of siRNA and / or saRNA), or a combination thereof (e.g., a combination of a single-stranded oligonucleotide and a double-stranded oligonucleotide, e.g., an oligonucleotide agent comprising a target double-stranded RNA and a single-stranded non-target accessory oligonucleotide (ACO), which are linked to each other with or without a linker). In some embodiments, the oligonucleotide substance is an oligonucleotide conjugate in which a double-stranded RNA is conjugated to an ACO. In some embodiments, the oligonucleotide is conjugated to one or more conjugation sites, such as a lipid (e.g., fatty acid) site. In some embodiments, the oligonucleotide is conjugated to a C5×5 derived site.

[0015] In some embodiments, one or more nucleotides in the oligonucleotide material are modified. In some embodiments, one or more nucleoside bonds in the oligonucleotide or ACO are selected from the group consisting of phosphodiesters, phosphotriesters, methylphosphonates, phosphoamidates, and phosphorothioates.

[0016] In some aspects of this disclosure, a method for preparing an oligonucleotide formulation is provided. The method includes combining an oligonucleotide substance with a calcium-containing substance in solution to form an oligonucleotide formulation.

[0017] In some embodiments, calcium is incorporated into the oligonucleotide material by replacing sodium with calcium in a solution containing the oligonucleotide material. In some embodiments, the formulation is prepared by adding 35 mM CaCl2 to an artificial cerebrospinal fluid (aCSF) solution and dissolving the oligonucleotide material in the CaCl2-containing aCSF solution.

[0018] In some embodiments of this disclosure, products comprising oligonucleotide formulations are provided. These products may be selected from pharmaceuticals, vaccines, diagnostic products, and imaging products.

[0019] In some embodiments of this disclosure, the use of oligonucleotide formulations in the manufacture of products for the treatment, prevention, or detection of diseases or disorders of a subject is provided. In some embodiments, the products act within the central nervous system (CNS).

[0020] In some aspects of this disclosure, a method is provided for treating, preventing or detecting a disease or disorder in a subject, comprising administering an oligonucleotide formulation to the subject in need thereof.

[0021] In some embodiments of this disclosure, oligonucleotide formulations of the present invention are provided for use in the treatment, prevention, or detection of a disease or disorder in a subject requiring the present invention.

[0022] In some embodiments, the oligonucleotides target genes selected from the group consisting of SOD1, FUS, C9orf72, MAPT(Tau), APP, SMN2, SCN9A, SCN10A, HTT, p21, UTRN, DUX4, SNCA, ATXN1, ATXN2, ATXN3, SCA1, SCA7, SCA8, UCP1, VEGFA, MeCP2, PRNP, DMPK, TARDBP, and TTR.

[0023] In some embodiments, the disease or disorder is a disease or disorder of the central nervous system (CNS). In some embodiments, the action of the oligonucleotide in the CNS is selected from the treatment, prevention and / or diagnosis of a disease or disorder in the CNS, or imaging of the site(s) of the disease or disorder in the CNS. In some embodiments, the disease or disorder in the CNS is selected from brain diseases, myelopathy, and peripheral neuropathy, for example, spinal muscular atrophy (SMA), Duchenne muscular dystrophy (DMD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD) and Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), brain tumors, frontotemporal dementia, spinocerebellar degeneration, prion diseases, Lafora disease, migraines, schizophrenia, depression, pain, and stroke.

[0024] Oligonucleotide formulations and products containing them can be used to reduce acute toxicity to oligonucleotides (e.g., acute toxicity in the CNS) and extend the safety margin, and therefore have extremely promising application potential.

[0025] Additional aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description, which illustrates and describes only exemplary embodiments of the present application. As will be understood, the present application may take other and different embodiments, and some of its details may be modified in various obvious ways without departing from the present disclosure. Accordingly, the drawings and description should be considered essentially illustrative and not restrictive. Novel features of the present invention are specifically described in the appended claims. The features and advantages of the present invention will be better understood by referring to the following detailed description illustrating exemplary embodiments in which the principles of the present invention are used, and by referring to the accompanying drawings (hereinafter also referred to as "figure" and "FIG."). [Brief explanation of the drawing]

[0026] [Figure 1]This document demonstrates the purification of calcium-exchange oligonucleotide (OS) of TA2 (described in Example 1) from a free CaCl2 salt solution desalted using a size exclusion column, by UV and conductivity signaling. The second fraction was recovered and freeze-dried to produce calcium OS of TA2. [Figure 2] This document demonstrates the purification of calcium-substituted OS of TA5 (described in Example 3) from a free CaCl2 salt solution desalted using a size exclusion column, by UV and conductivity signaling. The second fraction was recovered and freeze-dried to produce calcium OS of TA5. [Figure 3] This document demonstrates the purification of calcium-substituted OS of TA15 (described in Example 4) from a free CaCl2 salt solution desalted using a size exclusion column, by UV and conductivity signaling. The second fraction was recovered and freeze-dried to produce calcium OS of TA15. [Figure 4] This document demonstrates the purification of calcium-substituted OS of TA6 (described in Example 5) from a free CaCl2 salt solution desalted using a size exclusion column, by UV and conductivity signaling. The second fraction was recovered and freeze-dried to produce calcium OS of TA6. [Figure 5] This shows the purification of calcium-substituted OS of TA16 (described in Example 6) from a free CaCl2 salt solution desalted using a size exclusion column, by UV and conductivity signaling. The calcium OS of TA16 was prepared by recovering fraction 2 and freeze-drying it. In the figure, "------10 Conductivity Signal" shows the conductivity signal curve, "---10 Fraction" shows the fraction portion indicated as fraction numbers 1, 2, 3..., and "---10 UV1 260 nm" indicates that the UV measurement wavelength for oligonucleotide detection is 260 nm. [Modes for carrying out the invention]

[0027] Although various embodiments of the present invention are illustrated and described herein, it will be apparent to those skilled in the art that such embodiments are provided merely as examples. Without departing from the present invention, various modifications, changes, and substitutions can be made, as will be apparent to those skilled in the art. It should be understood that various substitutes can be used in the embodiments of the present invention described herein.

[0028] Prior to describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, and that such embodiments are naturally subject to various modifications. Furthermore, it should be understood that the terms used herein are for the purpose of describing specific embodiments and are not intended to limit them, and that the scope of the present invention is limited only by the appended claims.

[0029] Where a numerical range is provided, unless the context clearly indicates otherwise, each intermediate value between the upper and lower limits of that range, in units of 1 / 10 of the lower limit, should also be understood to be specifically disclosed. Each smaller range between any value or intermediate value within the stated range and any other value or intermediate value within that stated range should also be understood to be included in the present invention. The upper and lower limits of these smaller ranges may or may not be independently included in the range, while ranges that do not include either or both should also be understood to be included in the present invention unless specifically excluded within the stated range. If one or both limits are included in the stated range, the range excluding one or both of those included limits should also be understood to be included in the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but exemplary methods and materials are described below. All references herein are incorporated herein by reference to disclose and illustrate the cited methods and / or materials. In the event of any conflict between the disclosures of any incorporated reference and this disclosure, this disclosure shall prevail.

[0031] In this specification and the attached claims, the singular forms "a," "an," and "the" should be understood to include multiple subjects unless the context clearly indicates otherwise. For example, a reference to "a sample" should be understood to include multiple such samples, and a reference to "the molecule" should be understood to include one or more molecules and their equivalents known to those skilled in the art.

[0032] The documents referenced herein are provided only to the extent disclosed prior to the filing date of this application. Nothing in this specification should be construed as acknowledging that the present invention does not have prior rights to such documents based on prior inventions. Furthermore, the publication dates provided may differ from the actual publication dates, and such dates should be verified separately.

[0033] As used herein, the term "calcium" refers to any form of calcium that may be present in an oligonucleotide formulation and that can reduce acute toxicity in the body (e.g., in the central nervous system) and extend the body's safety margin. In some embodiments, calcium is a form selected from molecular calcium, ionized calcium, and complex calcium, or any combination thereof. Calcium (e.g., in CaCl2) can be incorporated into an oligonucleotide formulation by adding it to a solution containing oligonucleotides (e.g., aCSF solution) or by replacing sodium in a solution containing oligonucleotides (e.g., synthetic oligonucleotide material).

[0034] As used herein, the term “calcium-containing solution” in a nucleotide formulation refers to a solution containing calcium at a predetermined concentration for supporting the nucleotides of the formulation, or the supporting solution thereof. The calcium in the formulation may be provided by one or more calcium sources selected from, for example, calcium chloride, calcium gluconate, calcium lactate, calcium bicarbonate, calcium dihydrogen phosphate, calcium hydrogen phosphate, and any combination thereof.

[0035] In some embodiments, the calcium concentration in the formulation is varied. In some embodiments, when the oligonucleotide substance is a non-conjugate oligonucleotide, the calcium concentration in the formulation is between 15 mM and less than 25 mM. For example, when the oligonucleotide substance is a non-conjugate oligonucleotide, the calcium concentration in the formulation is in the range of 15 mM to 24, 23, 22, 21, 20, 19, 18, 17, or 16 mM, or any sub-range or numerical value within that range.

[0036] In some embodiments, when the oligonucleotide substance is a conjugated oligonucleotide, the calcium concentration in the formulation is 15 mM to 150 mM. For example, the calcium concentration in the formulation is in the range of 15 mM to 150 mM, e.g., 20 mM to 150 mM, 20 mM to 130 mM, 20 mM to 120 mM, 30 mM to 110 mM, or 35 mM to 100 mM, for example, the calcium concentrations in the formulation are approximately 20, 35, 47.5, 60, 82.5, 100, and 120 mM. In some embodiments, when the oligonucleotide substance is a conjugated oligonucleotide, the calcium concentration in the formulation is from 15 mM to less than 25 mM, e.g., in the range of 15 mM to 24, 23, 22, 21, 20, 19, 18, 17, or 16 mM, or any sub-range or numerical value within that range. Those skilled in the art can adjust the calcium concentration in the oligonucleotide formulation as needed based on the disclosures of this application.

[0037] As used herein, the term “oligonucleotide substance (OS)” means a substance composed of oligonucleotides, or a substance containing an oligonucleotide moiety (e.g., an oligonucleotide moiety conjugated with a non-target site), preferably such oligonucleotides or oligonucleotide moieties can target a specific gene and modulate gene expression and / or function.

[0038] In some embodiments, the oligonucleotide substance in an oligonucleotide formulation is one or more selected from single-stranded oligonucleotides, double-stranded oligonucleotides, or combinations thereof. The oligonucleotide substance may be in the form of isolated oligonucleotides or conjugated oligonucleotides. Single-stranded oligonucleotides may be antisense RNA (ASO) selected from gapmers, steric blockers, or mixmers. Double-stranded oligonucleotides may be double-stranded RNA selected from siRNA, saRNA, combinations of siRNA and saRNA, conjugates of siRNA and / or saRNA, or miRNA. Single-stranded oligonucleotides, double-stranded oligonucleotides, or combinations or conjugates of single-stranded and double-stranded oligonucleotides may also be used in the formulations of the present application. For example, the present disclosure also includes oligonucleotide conjugates in which a target double helix and an auxiliary oligonucleotide (ACO) are conjugated to each other with or without a linker.

[0039] As used herein, the term “gapmer” refers to a short DNA antisense oligonucleotide (ASO) structure having modified RNA segments on both sides of a central DNA structure. In some embodiments, at least one of the modified RNA segments may contain one or more modified nucleotides selected from locked nucleic acids (LNAs), 2'-OMe modified nucleotides, or 2'-F modified nucleotides to increase affinity to a target, enhance nuclease resistance, reduce immunogenicity, and / or decrease toxicity. In some embodiments, the gapmer contains a nucleotide modified with at least one phosphorothioate (PS) group. In some embodiments, the gapmer is designed to silence a gene transcript by hybridizing with a target RNA fragment and inducing cleavage by RNase H.

[0040] As used herein, the term "LNA" refers to locked nucleic acids in which a 2'-oxygen atom and a 4'-carbon atom are linked by additional crosslinks. As used herein, the term "BNA" refers to nucleic acids crosslinked by 2'-O and 4'-aminoethylene, and may include five-membered or six-membered ring crosslinking structures having NO bonds. As used herein, the term "PNA" refers to nucleic acid mimes having a pseudopeptide skeleton composed of N-(2-aminoethyl)glycine units, in which nucleic acid bases are linked to glycine nitrogen via carbonylmethylene linkers.

[0041] As used herein, the term “mixmer” refers to an antisense oligonucleotide (ASO) characterized structurally as a mixture of DNA and a chemically modified nucleic acid analog. Mixmers may, as needed, consist of fully modified nucleotides or nucleic acid analogs. In some embodiments, mixmers are designed to bind to and mask a complementary RNA sequence, thereby sterically blocking interaction between a protein, factor, or other RNA and the target RNA. In some embodiments, mixmers are designed to alter the splicing of pre-mRNA by displacing a spliceosome. In some embodiments, mixmers are designed to bind to and sequester microRNAs (miRNAs), some of which are also called “antagomyls” or “anti-miRs.”

[0042] As used herein, the term “splicing modifier” typically refers to antisense oligonucleotides (ASOs) that are uniformly composed of ribonucleotides and alter mRNA splicing by binding to pre-mRNA in the nucleus and blocking the binding of specific splicing factors to mRNA (steric blockers).

[0043] As used herein, the term “sense strand” of a dsRNA (e.g., siRNA, saRNA) double helix refers to a strand that has sequence homology or sequence identity with a fragment of the coding strand of the target gene sequence.

[0044] As used herein, the term “antisense strand” of a dsRNA (e.g., siRNA, saRNA) double helix refers to the strand having a sequence complementary to the sense strand.

[0045] As used herein, the term "overhang" refers to a nucleotide in a double-stranded oligonucleotide that does not form a base pair at the end (5' or 3') of the oligonucleotide chain, formed by the extension of one strand from the other. As used herein, a single-stranded region extending from the 3' and / or 5' ends of a double helix is ​​called an overhang.

[0046] As used herein, the term “natural overhang” refers to an overhang consisting of one or more nucleotides that are identical or complementary to the corresponding position in the target sequence.

[0047] As used herein, the terms “gene activation,” “gene expression activation,” “gene upregulation,” and “gene expression upregulation” are interchangeable and mean an increase or upregulation of the transcription, translation, expression, or activity of a particular nucleic acid sequence, determined by measuring the transcriptional level, mRNA level, protein level, enzyme activity, methylation status, chromatin state or structure, translational level, or the activity or state of a gene in a cell or biological system. These activities or states can be determined directly or indirectly. Also, “gene activation” or “gene expression activation” refers to an increase in activity related to a nucleic acid sequence, regardless of the mechanism of such activation. For example, gene activation can occur at the transcriptional level, increasing transcription to RNA, and increasing protein expression as the RNA is translated into protein.

[0048] As used herein, the terms “gene silencing,” “knockdown of gene expression,” “gene downregulation,” and “downregulation of gene expression” are interchangeable and mean a reduction or downregulation of the transcription, translation, expression, or activity of a particular nucleic acid sequence, determined by measuring the transcriptional level, mRNA level, protein level, enzyme activity, methylation status, chromatin state or structure, translational level, or the activity or state of the gene in a cell or biological system. These activities or states can be determined directly or indirectly. Furthermore, “gene downregulation” or “downregulation of gene expression” refers to a reduction in activity related to a nucleic acid sequence, regardless of the mechanism of such downregulation. For example, gene downregulation can occur at the transcriptional level, reducing transcription to RNA, and thereby reducing or silencing protein expression by preventing the RNA from being translated into protein.

[0049] As used herein, the terms “short interfering RNA,” “siRNA,” and “silencing RNA” are interchangeable and refer to ribonucleic acid molecules that can downregulate, knock down, or silence the expression of a target gene. These can be double-stranded nucleic acid molecules. siRNA inhibits the expression of specific genes with complementary nucleotide sequences by degrading mRNA post-transcriptionally and inhibiting translation. siRNA primarily binds to target mRNA in the cytoplasm and downregulates gene expression post-transcriptionally via RNA interference (RNAi) mechanisms. siRNA may be designed to target the mRNA sequence of a gene, for example, to silence it via an RNAi mechanism such as SOD1, in order to maximize therapeutic efficacy in patients with ALS. siRNA is a molecule having endogenous RNA bases or chemically modified nucleotides. The modifications do not cause loss of intracellular activity, but rather enhance stability and / or intracellular efficacy. Examples of chemical modifications include phosphorothioate groups, 2'-deoxyribonucleotides, 2'-OCH3-containing ribonucleotides, 2'-F-ribonucleotides, 2'-methoxyethylribonucleotides, and combinations thereof. siRNAs vary in length (e.g., 10-200 bp) and structure (e.g., hairpin, single-stranded / double-stranded, bulge, nick / gap, mismatch) and are processed in cells to provide active gene silencing. Double-stranded siRNAs may have an equal number of nucleotides on each strand (blunt ends) or asymmetric ends (overhangs). For example, a 1-2 nucleotide overhang may be present on the sense strand and / or antisense strand, as well as at the 5' and / or 3' ends of a given strand. The lengths of siRNA molecules are typically about 10–60, 10–50, 15–30, 17–29, 18–28, 19–27, 20–26, 21–25, 22–24 base pairs, and also typically about 15, 16, 17, 18, 19, 20, 21, 23, 25, 30, 40, or 50 base pairs.Furthermore, the terms "small interfering RNA," "silencing RNA," and "siRNA" include nucleic acids other than ribonucleotides, including but not limited to modified nucleotides or analogues.

[0050] As used herein, the terms “small activated RNA,” “saRNA,” and “small activated ribonucleic acid” are interchangeable and refer to ribonucleic acid molecules capable of upregulating the expression of a target gene. It may be a double-stranded nucleic acid molecule comprising a first nucleic acid strand containing a ribonucleotide sequence having sequence homology to the non-coding nucleic acid sequence (such as a promoter and enhancer) of the target gene, and a second nucleic acid strand containing a nucleotide sequence complementary to the first strand. saRNA may also consist of a synthetic or vector-expressed single-stranded RNA molecule that readily forms a hairpin structure by two complementary regions within the molecule, the first region containing a ribonucleotide sequence having sequence homology to the target sequence of the gene's promoter, and the ribonucleotide sequence contained in the second region being complementary to the first region. The length of the double-stranded region of a saRNA molecule is typically about 10–60, 10–50, 10–40, 12–30, 14–28, 16–26, 18–24, and 20–22 base pairs, and also typically about 10, 13, 15, 17, 18, 19, 20, 21, 22, 25, 30, 40, 50, or 60 base pairs. The terms “small activated RNA,” “saRNA,” and “small activated ribonucleic acid” also include nucleic acids other than ribonucleotides, including but not limited to modified nucleotides or analogues.

[0051] As used herein, the term “complementary” refers to the ability to form base pairs between two oligonucleotide chains. Base pairs are typically formed by hydrogen bonds between nucleotides in antiparallel oligonucleotide chains. The bases of complementary oligonucleotide chains can form pairs in the Watson-Crick type (e.g., A and T, A and U, C and G) or in any other form that allows for the formation of double helix (e.g., Hoogsteen type or inverse Hoogsteen type base pairing).

[0052] The terms “conjugated oligonucleotide” or “oligonucleotide conjugate” can be used interchangeably and refer to a chimeric oligonucleotide molecule comprising a target oligonucleotide and a non-target site that can facilitate the delivery of the target oligonucleotide, for example. The target oligonucleotide includes, but is not limited to, double-stranded nucleic acid molecules of DNA, RNA, or DNA / RNA hybrids, oligonucleotide chains in which deoxyribose and / or ribose moieties are regularly or irregularly alternating, and modified and naturally occurring or unnaturally occurring skeletons for such oligonucleotides. The target oligonucleotides disclosed herein may be small inhibitory nucleic acid molecules (siRNA), small activating nucleic acid molecules (saRNA), or antisense oligonucleotide molecules (ASO). Specifically, the oligonucleotide conjugate for inhibiting mRNA transcription levels of a target gene described herein is an siRNA molecule with a conjugated non-target site, and the oligonucleotide agent for activating transcription of a target gene described herein is an saRNA molecule with a conjugated non-target site.

[0053] As used herein, the term “non-target” means that a reference auxiliary oligonucleotide (ACO) conjugated with a target oligonucleotide (e.g., siRNA, saRNA, etc.) is not specifically complementary to the target sequence on which the target oligonucleotide functions, and / or the reference oligonucleotide (i.e., the ACO) does not share the same target sequence on which the target oligonucleotide (e.g., siRNA, saRNA, etc.) specifically acts. The target oligonucleotides disclosed herein are nucleic acid sequences specifically complementary to the target sequence or region thereof. In some embodiments, the term “non-target oligonucleotide” may include any reference oligonucleotide other than the “target sequence.” In some cases, “specifically complementary” may mean that the complementarity between the target oligonucleotide and the target sequence or region thereof is at least about 95%. Non-target oligonucleotides (i.e., accessory oligonucleotides, or interchangeably used "ACOs") do not induce biological activity through any known mechanism, nor are they intended to induce activity that exhibits ASO (i.e., "mixmer" or "gapmer") function to a specific subject, the subject's organs, the subject's tissues, or complementary nucleic acid sequences (i.e., mRNA) within the subject's cells when the oligonucleotide is administered. Non-target oligonucleotides (i.e., ACOs) are intended to facilitate the introduction of the target oligonucleotide (e.g., siRNA, saRNA, etc.) that the oligonucleotide conjugate is conjugated into a specific subject, the subject's organs, the subject's tissues, the subject's cells, or the subject's cell nucleus when the oligonucleotide conjugate is administered.

[0054] The oligonucleotide may further contain one or more conjugation sites to facilitate its introduction into cells. In certain embodiments, the conjugation sites modify one or more properties of the conjugated oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, intracellular distribution, intracellular uptake, charge, and clearance. In certain embodiments, the conjugation sites impart novel properties to the conjugated oligonucleotide, such as a fluorescent labeling group or reporter group that enables the detection of the oligonucleotide.

[0055] In some embodiments, the oligonucleotide conjugates with one or more conjugation sites selected from intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycol, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, fluorophores, and pigments.

[0056] In some embodiments, the conjugation site contains an active drug component, such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, holinic acid, benzothiadiazide, chlorothiazide, diazepine, indomethacin, barbiturates, cephalosporins, sulfonamides, antidiabetic drugs, antibacterial agents or antibiotics. In some embodiments, the oligonucleotide is conjugated with one or more conjugation sites selected from lipids, fatty acids, fluorophores, ligands, sugars, peptides and antibodies. For example, the lipid or fatty acid conjugation site is a saturated or unsaturated carbon chain having 4 to 30 carbon atoms, such as saturated or unsaturated C4, C6, C8, C 10 , C12 , C 14 , C 16 , C 18 , C 20 , C 22 , or C 24 may contain a carbon chain.

[0057] In certain embodiments, one or more conjugation sites are derived from C5×5 as shown herein. [Chemical formula]

[0058] According to some embodiments, the oligonucleotide may further comprise one or more linking sites or linkers. As used herein, the term "linking site" or "linker" refers to a molecule for covalently linking two molecules, such as a non-target site and a dsRNA (e.g., siRNA or saRNA), two dsRNAs, etc. As described in more detail below, the above term includes, for example, nucleic acid linkers, peptide linkers, etc., and also includes disulfide linkers. When present, the linking site or linker is -O-, -S-, -C(O)-, -NH-, -N((C1-C 12 )alkyl)-, -N((C1-C 12 )alkyl)-C(O)-O-, -O-C(O)-, -C(O)-O-, -O-C(O)-O-, -C(O)-NH-, -OP(O)2O-, -P(O)(O - )O-, -OP(O)O-, -P(O)-O-, -OP(O)(S)O-, -O-S(O)2-O-, -S(O)2-O-, -S(O)-O-, -(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-NH-, -NH-(C1-C 22 )alkylene-, -(C1-C 22 )alkylene-NH-C(O)-, -(C1-C 22 )alkylene-C(O)-, -(C1-C 22 )alkylene-C(O)-O-, -C(O)-(C1-C 22 )alkylene-, -NH-C(O)-(C1-C22 )Alkylene-,-C(O)-NH-(C1-C 22 )Alkylene-,-C(O)-(C1-C 22 ) Alkylene-NH-,-NH-(C1-C 22 ) Alkylene-C(O)-, -C(O)-(C1-C 22 )Alkylene-C(O)-,-NH-(C1-C 22 )Alkylene-NH-,-C(O)-(C1-C 22 )Alkylene-C(O)O-,-OC(O)-(C1-C 22 ) Alkylene-C(O)-O-, -C(O)-O-(C1-C 22 ) Alkylene-OC(O)-, -C(O)-(C1-C 22 )Alkylene-NH-C(O)-,-NH-C(O)-(C1-C 22 )Alkylene-C(O)-,-NH-C(O)-(C1-C 22 ) Alkylene-C(O)-NH-,-C(O)-NH-(C1-C 22 )Alkylene-NH-C(O)-,-(C1-C 22 )Alkylene-OP(O)2O-,-(C1-C 22 )Alkilen-OP(O)(O - )O-,-(C1-C 22 )Alkilen-OP(O)(O - )O-(C1-C 22 )Alkylene-,-(C1-C 22 )Alkilen-OP(O)O-,-(C1-C 22 )Alkilen-OP(O)(S)O-,-(C1-C 22 )Alkylene-OS(O)2-O-,-(C1-C 22 )Alkylene-S(O)2-O-,-(C1-C 22 )Alkylene-S(O)-O-,-OP(O)2-O-(C1-C 22 )Alkylene-OP(O)2O-,-OP(O)-O-(C1-C 22 )Alkilen-OP(O)O-,-OP(O)(S)O-(C1-C 22 )Alkylene-OP(O)(S)O-,-OS(O)2-O-(C1-C 22)Alkylene-OS(O)2-O-,-S(O)2-O-(C1-C 22 )Alkylene-S(O)2-O- and -OS(O)-(C1-C 22 )Can be selected from the group consisting of alkylene-S(O)-O-, in the formula, the linking site is -(C1-C 22 The alkylene group can be an alkylene group having 1 to 22 carbon atoms, for example, an alkylene group having 2 to 20 carbon atoms, or 3 to 18 carbon atoms, or 4 to 16 carbon atoms, or 5 to 12 carbon atoms, or 6 to 10 carbon atoms. In one embodiment, when the linking site is a direct bond, the conjugation site directly bonds to the oligonucleotide. For example, a conjugation site derived from C5×5 may be conjugated to the 5' end of the nucleotide chain via -OP(O)2O- or -P(O)-O-.

[0059] All nucleotides of the oligonucleotides described herein may be natural, i.e., unchemically modified nucleotides, or at least one nucleotide may be chemically modified. Non-limiting examples of chemical modifications include modification of phosphodiester bonds of nucleotides in the nucleotide sequence of functional oligonucleotides, modification of the 2'-OH group of ribose in the nucleotide sequence of functional oligonucleotides, and modification of bases in the nucleotide sequence of functional oligonucleotides, or a combination thereof. These modifications may enhance the bioavailability of oligonucleotides, improve affinity to target sequences, and strengthen resistance to intracellular nuclease hydrolysis.

[0060] The chemical modifications of nucleotides or oligonucleotides in this disclosure are well known to those skilled in the art, and phosphodiester bond modifications may refer to modifications of oxygen in the phosphodiester bond, including phosphorothioate modifications and boronated phosphate modifications. Any modification stabilizes the structure of the oligonucleotide and maintains high specificity and affinity for base pairing. Ribose modifications refer to modifications of the 2'-OH in the pentose of a nucleotide, i.e., the introduction of specific substituents to the hydroxyl position of ribose, such as 2'-fluoro modifications, 2'-oxomethyl modifications, 2'-oxyethylene methoxy modifications, 2,4'-dinitrophenol modifications, locked nucleic acid (LNA), 2'-amino modifications, and 2'-deoxy modifications. Base modifications refer to modifications of the bases of a nucleotide, such as 5'-bromouracil modifications, 5'-iodouracil modifications, N-methyluracil modifications, and 2,6-diaminopurine modifications.

[0061] As used herein, the term “synthetic” refers to a method of synthesis of oligonucleotides and includes any means by which RNA can be synthesized or chemically modified, such as chemical synthesis, in vitro transcription, and vector expression.

[0062] As used herein, the terms “subject” and “individual” may be used interchangeably and mean any living organism that can be treated with the agent of this application. The term “patient” means a human subject or individual, including infants, children, and adults.

[0063] As used herein, the terms “amyotrophic lateral sclerosis” or “ALS” include, but are not limited to, familial ALS (fALS), sporadic ALS (sALS), Lou Gehrig's disease, and diseases associated with mutant genes, namely, the chromosome 9 open reading frame 72 gene (C9orf72; 40%), superoxide dismutase 1 (SOD1; 20%), trans-active responsive DNA-binding protein 43 (TDP43; 4%), and fused-in-sarcoma / translocated-in-liposarcoma (FUS / TLS; 4%).

[0064] In certain embodiments of the present application, the target gene is SOD1. “Target sequence” refers to a sequence fragment in which the sense strand or antisense oligonucleotide of an siRNA or saRNA has sequence homology or complementarity. For example, in certain embodiments, the SOD1 siRNA has homology or complementarity to a target selection sequence in a human SOD1 transcript. In certain embodiments, the sequence of the sense strand of the SOD1 siRNA is shown in SEQ ID NO: 1, and the sequence of the antisense strand of the SOD1 siRNA is shown in SEQ ID NO: 2.

[0065] In certain embodiments of the present application, the target gene is FUS. For example, in certain embodiments, the FUS siRNA is homologous or complementary to a target selection sequence in a human FUS transcript. In certain embodiments, the FUS siRNA strand is fully chemically modified. In certain embodiments, the sense strand of the FUS siRNA contains a single-stranded auxiliary oligonucleotide ACO or a C5×5 lipid conjugate. In certain embodiments, the sequence of the sense strand of the FUS siRNA is shown in SEQ ID NOs: 3, 5, 6, 8, 10, 11, 13, or 15, and the sequence of the antisense strand of the FUS siRNA is shown in SEQ ID NOs: 4, 7, 9, 12, or 14. In some embodiments, the sense strand and antisense strand sequences of the FUS siRNA are selected from (a) SEQ ID NOs. 3 and 4, (b) SEQ ID NOs. 5 and 4, (c) SEQ ID NOs. 6 and 7, (d) SEQ ID NOs. 8 and 9, (e) SEQ ID NOs. 10 and 9, (f) SEQ ID NOs. 11 and 12, (g) SEQ ID NOs. 13 and 14, and (h) SEQ ID NOs. 15 and 14.

[0066] An effective dose refers to an amount sufficient to produce the desired effect. For example, a "therapeutic effective dose" or "effective dose for treatment" refers to an amount sufficient to achieve the desired therapeutic effect, and does not necessarily require a cure or complete remission. In the embodiments of this application, therapeutic effectiveness means improvement of any disease indicator, and a therapeutic effective dose refers to an amount sufficient to cause a clinically significant improvement in the disease state / symptoms of the treated individual.

[0067] The effective dose may vary depending on factors such as the size and weight of the subject, the type of disease, or the specific drug being administered. For example, the components of the "effective dose" may be affected by the choice of drug to be administered. Those skilled in the art will be able to consider the factors included herein and determine the effective dose of the drug to be administered without requiring excessive experimentation.

[0068] The administration regimen may influence the determination of the effective dose. The drug of this application can be administered to the subject before or after the diagnosis of the disease or condition. Furthermore, it can be administered daily or continuously in divided doses or with time staggers, or the dose can be administered by continuous infusion or bolus injection. Moreover, the dosage of the drug(s) of this application can be relatively increased or decreased as required depending on the treatment or prevention situation.

[0069] As used herein, the terms “to treat,” “to be treated,” “to treat,” or “treatment” have the meanings commonly understood in the medical field and include beneficial or desirable clinical outcomes, not necessarily requiring a cure or complete remission. Non-limiting examples of such beneficial or desirable clinical outcomes include extending survival compared to the expected survival without treatment, and alleviating symptoms including one or more of the following: weakness and atrophy of proximal skeletal muscles, inability to sit or walk independently, and difficulty swallowing and / or breathing. As used herein, “prevention” or “delay” means suppressing the complete onset of a disease.

[0070] Specific Embodiments Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art in the field relating to this application.

[0071] This invention is based on the remarkable discovery that the presence of calcium in a formulation containing oligonucleotides reduces the acute toxicity induced by oligonucleotide substances in the body, thereby expanding the safety margin of the oligonucleotide formulation.

[0072] In this application, further screening of the calcium concentration range in oligonucleotide formulations revealed that calcium concentrations of 15 mM to less than 25 mM are effective for non-conjugate oligonucleotides, and calcium concentrations of 10 mM to 210 mM (preferably 35 mM to 100 mM) are effective for conjugate oligonucleotides in reducing acute toxicity in the body, particularly in the CNS. Furthermore, calcium can be easily incorporated into oligonucleotides by conventional technical means, such as adding CaCl2 to an aCSF solution and directly dissolving the oligonucleotide sample in this solution, or by replacing sodium in the original oligonucleotide solution with calcium. Furthermore, the oligonucleotide formulation preferably has an osmotic pressure of 250 to 350 mOsmol / kg, and more preferably an isotonic osmotic pressure of 280 to 320 mOsmol / kg.

[0073] Oligonucleotide formulations are suitable for carrying various oligonucleotide substances, including ASOs, double-stranded RNAs (e.g., siRNAs, saRNAs, or combinations thereof targeting various genes), and related conjugates such as lipid conjugates and ACO conjugates. Preferably, the oligonucleotides target genes associated with diseases or disorders in the CNS, such as spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), and Duchenne muscular dystrophy (DMD) (e.g., SOD1, SMN2, UTRN, FUS).

[0074] The present invention provides the following specific embodiments. 1. Oligonucleotide preparations, (a) A calcium-containing solution having a calcium concentration of at least 15 mM in the above preparation, (b) An oligonucleotide preparation comprising the oligonucleotide substance in the above solution. 2. If the above oligonucleotide substance is a non-conjugate oligonucleotide, the calcium concentration in the above preparation is between 15 mM and less than 25 mM, or If the above oligonucleotide substance is a conjugated oligonucleotide, the calcium concentration in the above formulation is 15 mM to 150 mM, and / or The above oligonucleotide preparation is the oligonucleotide preparation according to Embodiment 1, having an osmotic pressure in the range of 250 mOsmol / kg to 350 mOsmol / kg.

[0075] 3. The oligonucleotide preparation according to Embodiment 1, wherein the oligonucleotide substance includes a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a combination thereof. 4. The single-stranded oligonucleotide described above is one or more antisense oligonucleotides (ASOs) selected from gapmers, mixmers, steric blockers, or splicing modifiers, and / or The oligonucleotide formulation according to Embodiment 1, wherein the double-stranded oligonucleotide is one or more double-stranded RNAs selected from siRNA, saRNA, a combination of siRNA and / or saRNA, a conjugate of siRNA and / or saRNA, or miRNA. 5. The above conjugated oligonucleotide is conjugated to one or more conjugation sites, and / or One or more of the above conjugation sites are selected from lipids, luminescent foci, ligands, sugars, peptides, and antibodies, and / or The above conjugation site is an accessory oligonucleotide (ACO) conjugated to the above oligonucleotide, and / or The oligonucleotide formulation according to any one of Embodiments 2 to 4, wherein one or more of the above-mentioned conjugation sites conjugate to the oligonucleotide at one or more terminals of the oligonucleotide chain or at an internal position of the oligonucleotide. 6. One or more of the above conjugation sites are selected from fatty acids, cell-penetrating peptides, fluorophores, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolones, amino acids, cholesterol, glucose, and N-acetylgalactosamine, and / or The oligonucleotide preparation according to Embodiment 5, wherein the above-mentioned oligonucleotide substance is an oligonucleotide conjugated to ACO or an oligonucleotide conjugated to a lipid moiety.

[0076] 7. The above lipid region is C4-C 30 An oligonucleotide formulation according to Embodiment 5, wherein the fatty acid portion is the fatty acid moiety. 8. The above lipid regions are saturated or unsaturated C6, C8, C 10 , C 12 , C 14 , C 16 , C 18 , C 20 , C 22 , or C 24 An oligonucleotide formulation according to Embodiment 5, comprising a carbon chain. 9. The above conjugation site is derived from C5×5, the oligonucleotide formulation according to Embodiment 4. [ka]

[0077] 10. The oligonucleotide formulation according to Embodiment 9, wherein C5×5 is conjugated to the 5' end of the sense or antisense strand in the oligonucleotide via -OP(O)2O-, -OP(O)(S)O-, and / or -P(O)-O-. 11. The above auxiliary oligonucleotide (ACO) is a single-stranded oligonucleotide having a length of at least 6 nucleotides, and / or The above auxiliary oligonucleotide (ACO) is a non-target oligonucleotide, and / or The oligonucleotide formulation according to Embodiment 5, wherein the auxiliary oligonucleotide (ACO) has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 phosphorothioate modifications on its skeleton, or all nucleotides in the skeleton are phosphorothioate modified.

[0078] 12. The oligonucleotide formulation according to Embodiment 1, wherein the calcium is in a form selected from molecular calcium, ionized calcium, and complex calcium, or any combination thereof. 13. The oligonucleotide formulation according to Embodiment 1, wherein the calcium is provided as calcium chloride, calcium gluconate, calcium lactate, calcium bicarbonate, calcium dihydrogen phosphate, calcium hydrogen phosphate, and any combination thereof. 14. If the oligonucleotide substance is a non-conjugate oligonucleotide, the calcium concentration in the preparation is in the range of 15 mM to 24, 23, 22, 21, 20, 19, 18, 17, or 16 mM, or any lower range or numerical value within the above range, and / or The oligonucleotide formulation according to Embodiment 2, wherein, when the above-mentioned oligonucleotide substance is a non-conjugated oligonucleotide, the osmotic pressure of the formulation is in the range of 280 mOsmol / kg to 320 mOsmol / kg. 15. If the above oligonucleotide substance is a conjugated oligonucleotide, The calcium concentration in the above preparations is in the range of 15 mM to 140 mM, 20 mM to 130 mM, 20 mM to 120 mM, 30 mM to 110 mM, or 35 mM to 100 mM, and / or the calcium concentration in the above preparations is approximately 20, 35, 47.5, 60, 82.5, 100 mM, 120 mM, and / or The calcium concentrations in the above preparations are approximately 35, 47.4, 60, and 100 mM, and / or The calcium concentration in the above preparation is between 15 mM and less than 25 mM, and / or The calcium concentration in the above preparation is in the range of 15 mM to 24, 23, 22, 21, 20, 19, 18, 17, or 16 mM, or any lower range or numerical value within the above range, and / or The oligonucleotide formulation according to Embodiment 2, wherein, when the above oligonucleotide substance is a conjugated oligonucleotide, the osmotic pressure of the formulation is in the range of 280 mOsmol / kg to 320 mOsmol / kg.

[0079] 16. The oligonucleotide preparation according to any one of Embodiments 1 to 15, wherein at least one nucleotide of the oligonucleotide substance is a chemically modified nucleotide. 17. The above chemically modified nucleotides are located within the antisense strand of the ASO, or within the sense strand, antisense strand, or both strands of the double-stranded RNA, or within the ACO, and / or The above chemically modified nucleotides are nucleotides modified at the 5' end, 3' end, both ends, or the internal portion of the above chain(s), and / or The oligonucleotide formulation according to Embodiment 16, wherein at least 50% of the nucleotides in the above oligonucleotide substance are chemically modified.

[0080] 18. The oligonucleotide formulation according to Embodiment 16, wherein the chemically modified nucleotide is one or more selected from 2' sugar modification, base modification, phosphorothioate (PS) skeleton modification, and addition of a 5'-phosphate moiety or 5-methylcytosine moiety to the 5' end of the nucleotide chain. 19. The above 2' sugar modification is one or more selected from 2'-fluoro-2'-deoxynucleoside (2'-F) modification, 2'-O-methyl (2'-O-Me) modification, and / or 2'-O-(2-methoxyethyl) (2'-O-MOE) modification. The oligonucleotide formulation according to Embodiment 18, wherein the addition of the 5'-phosphate moiety is one or more selected from the addition of an (E)-vinylphosphonate moiety to the 5' end of the nucleotide chain.

[0081] 20. The oligonucleotide formulation according to any one of Embodiments 1 to 19, further comprising one or more other excipients selected from preservatives, humectants, emulsifiers, dispersants, isotonic agents, and antioxidants. 21. The above preparation comprises one or more selected from salts, polyols, sugars, and / or alcohols, and / or The carrier in the above-mentioned preparation is artificial cerebrospinal fluid (aCSF) or water, and / or The calcium-containing solution mentioned above is aCSF or an aqueous solution, and / or The above formulation is an oligonucleotide formulation according to Embodiment 1, comprising one or more components selected from NaCl, KCl, MgSO4, KH2PO4, NaHCO3, glucose, and sucrose.

[0082] 22. The oligonucleotide formulation according to Embodiment 1, wherein the formulation comprises 50-150 mM NaCl, 0.5-5.0 mM KCl, 0.5-5.0 mM MgSO4, 0.4-3.0 mM KH2PO4, 10-50 mM NaHCO3, 1-20 mM glucose, and 1-10 mM sucrose.

[0083] 23. The oligonucleotide formulation according to Embodiment 1, wherein the formulation comprises 124 mM NaCl, 2.5 mM KCl, 2.0 mM MgSO4, 1.25 mM KH2PO4, 26 mM NaHCO3, 10 mM glucose, and 4 mM sucrose.

[0084] 24. The oligonucleotide formulation according to Embodiment 1, wherein the formulation contains a component selected from the solution groups A1, B1, A2, B2, A3, B3, A4, B4, A5, and B5 shown in Tables 17, 18, 19, 20, and 21. 25. The oligonucleotides in the above oligonucleotide preparations target the SOD1 gene and / or The oligonucleotides in the above oligonucleotide formulations target the FUS gene and / or The oligonucleotide preparation described above targets a gene selected from the group consisting of C9orf72, MAPT(Tau), APP, SMN2, SCN9A, SCN10A, HTT, p21, UTRN, DUX4, SNCA, ATXN1, ATXN2, ATXN3, SCA1, SCA7, SCA8, UCP1, VEGFA, MeCP2, PRNP, DMPK, TARDBP, and TTR, as described in Embodiment 1. 26. The oligonucleotides in the above oligonucleotide preparations include one or more oligonucleotide chains or chain pairs from the oligonucleotide substances in Table 1 and / or Table 12, and / or The oligonucleotide preparation according to Embodiment 1, wherein the oligonucleotide in the above-mentioned oligonucleotide preparation comprises one or more oligonucleotide chains or chain pairs having the sequence shown in one or more of Sequence IDs 1 to 15. 27. The above oligonucleotide preparation has a calcium concentration in the range of 20 mM to 100 mM, and / or The above oligonucleotide preparations have an osmotic pressure in the range of 280 mOsmol / kg to 320 mOsmol / kg, and / or The oligonucleotide preparation described above is an isotonic solution, as described in Embodiment 25.

[0085] 28. The oligonucleotide preparation according to any one of Embodiments 1 to 27, wherein the preparation is in a form suitable for an administration route selected from subcutaneous injection, intravenous injection, intraocular injection, intradermal injection, intramuscular injection, intraperitoneal injection, intratracheal administration, intrafat administration, intra-articular administration, intrathecal administration, epidural administration, inhalation, intranasal administration, oral administration, sublingual administration, buccal administration, rectal administration, vaginal administration, intracisional administration, transdermal administration and local administration, or administration by local delivery.

[0086] 29. A method for preparing an oligonucleotide preparation according to any one of Embodiments 1 to 28, comprising combining the above oligonucleotide substance with a calcium-containing substance in solution to form the above oligonucleotide preparation.

[0087] 30. The method according to Embodiment 29, wherein the calcium is provided as a calcium salt such as calcium chloride (CaCl2), calcium gluconate, calcium lactate, calcium bicarbonate, calcium dihydrogen phosphate, or calcium hydrogen phosphate. 31. In order to dissolve the above oligonucleotide substance, calcium is contained in aCSF, and / or By exchanging the sodium in the solution containing the above oligonucleotide substance for calcium, calcium is incorporated into the above solution, and / or If the above oligonucleotide substance is a non-conjugate oligonucleotide, the calcium concentration in the above formulation is between 15 mM and less than 25 mM, or The method according to Embodiment 29, wherein, when the above-mentioned oligonucleotide substance is a conjugated oligonucleotide, the calcium concentration in the above-mentioned oligonucleotide preparation is in the range of 15 mM to 150 mM.

[0088] 32. The above formulation is prepared by adding CaCl2 to an aCSF solution and dissolving an oligonucleotide substance in the aCSF solution containing CaCl2, as described in Embodiment 29.

[0089] 33. A product comprising an oligonucleotide preparation according to any one of Embodiments 1 to 28.

[0090] 34. The above product is the product according to Embodiment 33, selected from pharmaceuticals, vaccines, diagnostic products, and imaging products.

[0091] 35. Use of an oligonucleotide preparation according to any one of Embodiments 1 to 28 in the manufacture of a product for the treatment, prevention, or detection of a disease or disorder of a subject.

[0092] 36. A method for treating, preventing, or detecting a disease or disorder in a subject, comprising administering an oligonucleotide preparation described in any one of Embodiments 1 to 28 to the subject in need thereof in an effective amount.

[0093] 37. An oligonucleotide preparation according to any one of Embodiments 1 to 28 for the treatment, prevention, or detection of a disease or disorder in a subject.

[0094] 38. The oligonucleotide preparation described above exhibits reduced acute toxicity in the body compared to a reference oligonucleotide preparation that does not contain calcium within a predetermined concentration range, and is an oligonucleotide preparation for use as described in Embodiment 35, the method described in Embodiment 36, or the use described in Embodiment 37. 39. The above oligonucleotide preparation exhibits reduced acute toxicity in the body compared to a reference oligonucleotide preparation that does not contain calcium within a specified concentration range, and the above acute toxicity in the body is toxicity to the somatic motor nervous system, and / or The above oligonucleotide preparations act on the somatic motor nervous system, and / or The above oligonucleotide preparations act on the central nervous system (CNS), and / or Oligonucleotide formulations for use according to Embodiment 35, the method according to Embodiment 36, or the use according to Embodiment 37, wherein the acute toxicity described above is CNS acute toxicity.

[0095] 40. An oligonucleotide formulation for use as described in Embodiment 35, as described in Embodiment 36, or as described in Embodiment 37, wherein the subject requires a pharmaceutical for the treatment of the disease or disorder, a vaccine for the prevention of the disease or disorder, a diagnostic product for the diagnosis of the disease or disorder, and an imaging product for imaging of the lesion site(s) of the disease or disorder.

[0096] 41. Oligonucleotide formulations for use according to Embodiment 35, the method described in Embodiment 36, or the use described in Embodiment 37, selected from the above diseases or disorders, including brain diseases, spinal cord disorders, and peripheral nerve disorders.

[0097] 42. The above oligonucleotide is an oligonucleotide formulation for use as described in Embodiment 35, as described in Embodiment 36, or as described in Embodiment 37, targeting a gene selected from the group consisting of SOD1, FUS, C9orf72, MAPT(Tau), APP, SMN2, SCN9A, SCN10A, HTT, p21, UTRN, DUX4, SNCA, ATXN1, ATXN2, ATXN3, SCA1, SCA7, SCA8, UCP1, VEGFA, MeCP2, PRNP, DMPK, TARDBP, and TTR.

[0098] 43. The above-mentioned diseases or disorders are selected from spinal muscular atrophy (SMA), Duchenne-Becker muscular dystrophy (DMD and BMD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), brain tumors, frontotemporal dementia, spinocerebellar degeneration, prion diseases, Lafora disease, migraines, schizophrenia, depression, pain, and stroke, and are oligonucleotide formulations for use according to Embodiment 35, the method according to Embodiment 36, or the use according to Embodiment 37. [Examples]

[0099] The following examples illustrate some embodiments of the present invention, where all parts and percentages are by mass unless otherwise specified. However, the scope of this disclosure is not limited to the formulations described in these examples. Rather, these examples are merely illustrative of the present disclosure.

[0100] Unless otherwise specified, all starting materials, reagents, and solvents used below were purchased from commercially available suppliers and used as is. Reaction products were purified by column chromatography using silica gel (200-300 mesh), with hexane / ethyl acetate and DCM / MeOH used as eluents. Thin-layer chromatography (TLC) was performed using plates pre-coated with silica gel GF, and visualization was performed by KMnO4 staining. 1 H-NMR spectra were recorded at 400 or 500 MHz (Varian) using CDCl3 in conjunction with TMS. High-resolution mass spectrometry (HRMS) was recorded using LC / MS (Agilent Technologies 1260 Infinity II / 6120 Quadrupole) and time-of-flight mass spectrometers with ESI or matrix-assisted laser desorption / ionization (MALDI).

[0101] Materials and methods oligonucleotide synthesis (1) Synthesis of single-stranded oligonucleotides Single-stranded oligonucleotides were synthesized by solid-phase synthesis using the K&A DNA synthesizer (K&A Laboratories GbR, Schaafheim, Germany). The starting materials were either commercially available universal solid supports or special solid supports, or the synthesis disclosed in the above description. Generally, phosphoramidite monomers (0.1 M in acetonitrile or dichloromethane) containing various linkers and conjugates were sequentially added to the solid support in a DNA synthesizer to produce the desired full-length oligonucleotide.

[0102] Amidite addition: Each cycle of amidite addition consisted of four chemical reactions: detritylation, coupling, oxidation / thiolation, and capping. In the first step, detritylation was performed for 45 seconds using 3% dichloroacetic acid (DCA) in DCM. In the second step, phosphoramidite coupling was performed on all amidites at 12 eq for 6 minutes. In the third step, as an oxidation reaction, treatment was performed for 1 minute with a 0.02 M iodine solution in THF:pyridine:water (70:20:10, v / v / v). If phosphorothioate modification was required, thiolation was performed instead of oxidation, and treatment was performed for 3 minutes with a 0.1 M xanthan hydride solution in pyridine:ACN (50:50, v / v). In the fourth step, capping was performed for 20 seconds using THF:acetic anhydride:pyridine (80:10:10, v / v / v) (CAP A) and N-methylimidazole:THF (10:90, v / v) (CAP B). The number of cycles for the four chemical reactions depended on the length of the single-stranded oligonucleotide.

[0103] Deprotection I (nucleoside base deprotection): After synthesis was complete, the solid support was transferred to a microcentrifuge tube with a screw cap. For synthesis on a 1 μmol scale, 1 mL of a mixture of methylamine and ammonium hydroxide was added. The tube containing the solid support was then heated in an oven at 60°C to 65°C for 15 minutes, and then cooled to room temperature. The cleavage solution was collected and evaporated to dryness by speed vacuum to obtain crude single-stranded oligonucleotides.

[0104] Deprotection II (Removal of 2'-TBDMS group): If the crude RNA oligonucleotide retained a 2'-TBDMS group, it was dissolved in 0.1 mL of DMSO. After adding 1 mL of triethylamine hydrofluoric acid, the tube was capped, and the mixture was shaken vigorously to completely dissolve it. Then, it was heated in a 65°C oven for 15 minutes. The tube was removed from the oven and allowed to cool to room temperature. The solution containing the completely desilylated oligonucleotide was cooled on dry ice. To precipitate the oligonucleotide, 2 mL of ice-cold n-butanol (-20°C) was carefully added in 0.5 mL increments. The precipitate was filtered, washed with 1 mL of ice-cold n-butanol, and then dissolved in 0.01 M tris(hydroxymethyl)aminomethane hydrochloride buffer.

[0105] (2) Purification of single-strand oligonucleotides Purification of single-stranded oligonucleotides was performed using an AKTA explorer 10 equipped with a Source 15Q 4.6 / 100 PE column, under the following conditions: Buffer A: (10 mM Tris-HCl, 1 mM EDTA, pH 7.5), Buffer B: (10 mM Tris-HCl, 1 mM EDTA, 2 M NaCl, pH 7.5), gradient: 10%B to 60%B for 25 minutes, flow rate: 1 mL / min or less. The purified oligonucleotides were collected and desalted using a HiPrep 26 / 10 Desalting column.

[0106] (3) Formation of double-strand oligonucleotides by annealing To form double chains, a desalted, purified single-chain solution was prepared, and then the sense and antisense chains were mixed in equimolar concentrations and volumes in a tube. The tube was placed on a 95°C heat block for 5 minutes, and then cooled to room temperature. The resulting double chains were then freeze-dried to produce a powder. General method A: Exchange of sodium and calcium in oligonucleotide substances (OS) CaCl2 (Bidepharm, lot number: BD119632-500g) was weighed and dissolved in injection medium [e.g., commercially available artificial cerebrospinal fluid (aCSF, TOCRIS, batch number: 57A) or water] to obtain calcium solutions of 5 mM, 10 mM, 20 mM, or 100 mM, respectively. Oligonucleotide material RD-12500 (WuXi STA, COA: P220505001-C-MC00759-5-C-Lyo-V04, containing 6.3% w / w sodium, calcium-free) was dissolved in a predetermined concentration of CaCl2 solution to obtain a 20 mg / mL oligonucleotide sample. RD-12500 is an siRNA double helix conjugated to ACO via an S9 spacer (Table 1). The obtained sample solution was allowed to stand for 30 minutes. Subsequently, to remove free CaCl2, the sample was loaded onto a desalting column (AKTA, HiPrep 26 / 10), and the oligonucleotide fraction was recovered based on the conductivity signal (only the fraction before the trough of the conductivity signal curve was recovered, i.e., the fraction with the least salt or free salt). The oligonucleotides recovered together with the salt were freeze-dried, and the sodium (sodium salt for oligopharmaceutical substances) and calcium content was measured by inductively coupled plasma mass spectrometry (NSF LABORTORY or Medinoah Company, China). The calcium-exchange oligonucleotides were dissolved in aCSF (TOCRIS, batch number: 57A) to prepare a test substance (TA) for intrathecal (IT) injection at a predetermined concentration. The oligonucleotide sequence of RD-12500 is shown in Table 1. [Table 1]

[0107] General method B: Introduction of calcium into oligonucleotide preparations (OFs) CaCl2 (Bidepharm, lot number: BD119632-500g) was weighed and dissolved in solvent medium (e.g., aCSF or water) to obtain calcium solutions of 5 mM, 10 mM, 20 mM, 35 mM, and 100 mM for intrathecal (IT) injection. Oligonucleotides of predetermined concentrations were dissolved in calcium (Ca)-containing solutions to obtain oligonucleotide preparations.

[0108] Animal experiments Sprague-Dawley female rats (A102, SPF, China) were purchased from SPF Biotechnology Co., LTD (Suzhou, Jiangsu, China). All animal experiment procedures were approved by the Animal Experiment Committee (IACUC) of Ractigen Therapeutics. The freshly prepared formulations used for treating animals were prepared immediately before use by dissolving lyophilized oligonucleotide aliquots in aCSF to create a stock solution for dilution, thereby achieving the desired dosage concentration. aCSF alone was used as a vehicle control. Animals were randomly assigned to test groups (4-6 animals per group) based on body weight (140 g-260 g).

[0109] Intrathecal (IT) injection Anesthesia was induced by administering 3.0% isoflurane continuously for 10 minutes in an induction chamber. The fur around the injection site at the base of the tail was shaved, and then the injection site was washed with 75% ethanol. The space between the L5 and L6 spinous processes was identified, and a 30-gauge needle attached to a microliter syringe filled with the appropriate drug formulation was slowly inserted into the intradural space and advanced until tail flick was observed. The needle was then fixed in place, and a total volume of 30 μL of solution was injected over 1 minute.

[0110] Functional Observation and Assessment (FOB) Sprague Dawley rats were administered the test substance as a single intra-articular (IT) dose. Within 3 hours post-administration, the rats were evaluated for signs of motor dysfunction using the Focus on Body (FOB) scale. The FOB was developed to provide an unbiased assessment of the effects of drugs on the central and peripheral nervous systems based on motor dysfunction. The FOB was evaluated based on seven different rat body parts: (1) tail, (2) posterior position, (3) hind limbs, (4) hind feet, (5) forelimbs, (6) anterior position, and (7) head. Each rat was assigned a subscore based on these seven different body parts, with a score of 0 indicating no signs of motor dysfunction and a score of 1 indicating paralysis in the evaluated area. After evaluation of each body part, the subscore and its mean value were calculated and compiled for each rat.

[0111] Example 1. Preparation of TA2 In this embodiment, TA2 was prepared according to the following procedure in accordance with general method A. Oligonucleotide material RD-12500 (WuXi STA, COA: P220505001-C-MC00759-5-C-Lyo-V04) was dissolved in 5 mL of 5 mM CaCl2 solution to obtain a 20 mg / mL oligonucleotide sample. The resulting TA solution was allowed to stand for 30 minutes. The sample was then loaded onto a desalting column to remove free CaCl2, and the oligonucleotide fraction was recovered based on the conductivity signal (Figure 1, Fraction 2). The recovered oligonucleotides, along with the salt, were freeze-dried, and the sodium and calcium content was then characterized by inductively coupled plasma mass spectrometry (NSF Laboratory, China). The results are summarized in Table 2. Na: 3.9 × 10⁻⁶ 4 (mg / kg) (3.9%, percentage); Ca: 1.3 × 10 4 (mg / kg) (1.3%, percentage). Calcium-exchange oligonucleotides were dissolved in aCSF to obtain a 100 mg / mL TA2 solution.

[0112] Example 2. Preparation of TA3 In this embodiment, TA3 was prepared by the following procedure. Oligonucleotide substance RD-12500 (WuXi STA, COA: P220505001-C-MC00759-5-C-Lyo-V04) was dissolved in lactated Ringer's solution (Phygene, lot number: 20221129) to obtain TA3 at a concentration of 100 mg / mL for IT injection.

[0113] Example 3. Preparation of TA5 In this embodiment, TA5 was prepared according to the following procedure in accordance with general method A. Oligonucleotide material RD-12500 (WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01, containing 5.6% w / w sodium and no calcium) was dissolved in 5 mL of 10 mM CaCl2 solution to obtain a 20 mg / mL oligonucleotide sample. The resulting TA solution was allowed to stand for 30 minutes. The sample was then loaded onto a desalting column to remove free CaCl2, and the oligonucleotide fraction was recovered based on the conductivity signal (Figure 2, fraction 2). The recovered oligonucleotides, along with the salt, were freeze-dried, and the sodium and calcium content was then characterized by inductively coupled plasma mass spectrometry (Medinoah Company, China). The results are summarized in Table 2. Na: 5.3%, Ca: 0.9%. Calcium-exchange oligonucleotides were dissolved in aCSF to obtain TA5 at a concentration of 200 mg / mL.

[0114] Example 4. Preparation of TA15 In this embodiment, TA15 was prepared according to the following procedure in accordance with general method A. Oligonucleotide material RD-12500 was dissolved in 5 mL of 20 mM CaCl2 solution to obtain a 50 mg / mL oligonucleotide sample. The resulting TA solution was allowed to stand for 30 minutes. The sample was then loaded onto a desalting column to remove free CaCl2, and the oligonucleotide fraction was recovered based on the conductivity signal (Figure 3, fraction 2). The recovered oligonucleotides, along with the salt, were freeze-dried, and the sodium and calcium content was then characterized by inductively coupled plasma mass spectrometry (Medinoah Company, China). The results are summarized in Table 2. Na: 4.5%, Ca: 1.6%. Calcium-exchange oligonucleotides were dissolved in aCSF to obtain TA15 at a concentration of 200 mg / mL.

[0115] Example 5. Preparation of TA6 In this embodiment, TA6 was prepared according to the following procedure in accordance with general method A. Oligonucleotide material RD-12500 was dissolved in 5 mL of 100 mM CaCl2 solution to obtain a 20 mg / mL oligonucleotide sample. The resulting TA solution was allowed to stand for 30 minutes. The sample was then loaded onto a desalting column to remove free CaCl2, and the oligonucleotide fraction was recovered based on the conductivity signal (Figure 4, fraction 2). The recovered oligonucleotides, along with the salt, were freeze-dried, and the sodium and calcium content was then characterized by inductively coupled plasma mass spectrometry (Medinoah Company, China). The results are summarized in Table 2. Na: 1.8%, Ca: 4.0%. Calcium-exchange oligonucleotides were dissolved in aCSF to obtain TA6 at a concentration of 200 mg / mL.

[0116] Example 6. Preparation of TA16 In this embodiment, TA16 was prepared according to the following procedure in accordance with general method A. The oligonucleotide substance RD-12500 was dissolved in water and loaded onto a size exclusion column. Elution was performed using PBS (pH=7.4) buffer and a 2 M CaCl2 aqueous solution, and the oligonucleotide fraction was recovered. Subsequently, the sample was desalted by loading it onto a desalting column to remove free CaCl2, and the oligonucleotide fraction was recovered based on the conductivity signal (Figure 5, fraction 2). The oligonucleotides recovered together with the salt were freeze-dried, and then the sodium and calcium content was characterized by inductively coupled plasma mass spectrometry (Medinoah Company, China). The results are summarized in Table 2. Na: 0.8%, Ca: 5.3%. Calcium-exchange oligonucleotides were dissolved in aCSF to obtain TA16 at a concentration of 200 mg / mL.

[0117] Example 7. Preparation of TA7 In this embodiment, TA7 was prepared according to the following procedure in accordance with general method B. 3.6 mg of CaCl2 was weighed out and dissolved in 1 mL of aCSF to obtain a 35 mM calcium-containing aCSF solution. The oligonucleotide substance sodium RD-12500 was dissolved in a 35 mM calcium-containing aCSF solution to obtain TA7 at a concentration of 200 mg / mL.

[0118] Example 8. Preparation of TA8 In this embodiment, TA8 was prepared according to the following procedure in accordance with general method B. 6.4 mg of CaCl2 was weighed and dissolved in 1 mL of aCSF to obtain a 60 mM calcium-containing aCSF solution. The oligonucleotide substance sodium RD-12500 was dissolved in a 60 mM calcium-containing aCSF solution to obtain TA8 at a concentration of 200 mg / mL.

[0119] Example 9. Preparation of TA9 In this embodiment, TA9 was prepared according to the following procedure in accordance with general method B. 10.8 mg of CaCl2 was weighed and dissolved in 1 mL of aCSF to obtain a 100 mM calcium-containing aCSF solution. The oligonucleotide substance sodium RD-12500 was dissolved in a 100 mM calcium-containing aCSF solution to obtain TA9 at a concentration of 200 mg / mL.

[0120] Example 10. Preparation of TA11 In this embodiment, TA11 was prepared according to the following procedure in accordance with general method B. 0.3 mg of CaCl2 was weighed and dissolved in 1 mL of aCSF to obtain a 5 mM calcium-containing aCSF solution. The oligonucleotide substance sodium RD-12500 was dissolved in a 5 mM calcium-containing aCSF solution to obtain TA8 at a concentration of 200 mg / mL for IT injection. The mean osmotic pressure of TA11 was 703 mOsmol / kg (WuXi AppTec, China).

[0121] Example 11. Preparation of TA12 In this embodiment, TA12 was prepared according to the following procedure in accordance with general method B. 0.8 mg of CaCl2 was weighed and dissolved in 1 mL of aCSF solvent to obtain a 10 mM calcium-containing aCSF solution. The oligonucleotide substance sodium RD-12500 was dissolved in a 10 mM calcium-containing aCSF solution to obtain TA12 at a concentration of 200 mg / mL for IT injection. The mean osmotic pressure of TA12 was 716 mOsmol / kg (WuXi AppTec, China).

[0122] Example 12. Preparation of TA13 In this embodiment, TA13 was prepared according to the following procedure in accordance with general method B. 2.0 mg of CaCl2 was weighed and dissolved in 1 mL of aCSF to obtain a 20 mM calcium-containing aCSF solution. The oligonucleotide substance sodium RD-12500 was dissolved in a 20 mM calcium-containing aCSF solution to obtain TA13 at a concentration of 200 mg / mL for IT injection. The mean osmotic pressure of TA13 was 739 mOsmol / kg (WuXi AppTec, China).

[0123] Example 13. Preparation of TA14 In this embodiment, TA14 was prepared according to the following procedure in accordance with general method B. 3.6 mg of CaCl2 was weighed out and dissolved in 1 mL of aCSF to obtain a 35 mM calcium-containing aCSF solution. The oligonucleotide substance sodium RD-12500 was dissolved in a 35 mM calcium-containing aCSF solution to obtain TA14 at a concentration of 200 mg / mL for IT injection. The mean osmotic pressure of TA14 was 794 mOsmol / kg (WuXi AppTec, China).

[0124] Example 14. Calcium introduction into oligonucleotide substances by cation exchange (Method A) As summarized in Table 2, the difference in CaCl2 concentration in Method A and the use of a size exclusion column in Method B increased the calcium concentration of oligonucleotide substances TA5, TA15, TA6, and TA16 from 0.9% to 4%, and the calcium exchange rate increased from 16% to 71%. In Example 6, sodium was removed as much as possible from TA16 using a size exclusion column, and then the oligonucleotide sample was diluted on the column with a 2 M calcium solution. As a result, the OS calcium content was 5.3% and the calcium exchange rate was 84%. When TA16 OS was dissolved in aCSF to make a 200 mg / mL oligonucleotide preparation (OF) solution, the calculated calcium concentration reached 267.5 mM, which is an extremely high concentration. The sodium-calcium exchange rate was calculated using formula I (OS RD-12500 contains 6.3% sodium).

number

[0125] The calculated calcium concentration (mM) was determined using Equation II (aCSF contains 2.5 mM calcium).

number

[0126] In Equation II, the oligonucleotide concentration refers to the concentration of the oligonucleotide sodium substrate dissolved in CaCl2 solution in mg / mL, and OS Ca% (i.e., calcium %) in the oligonucleotide substrate refers to the value detected by inductively coupled plasma mass spectrometry in Table 2.

[0127] In summary, Method A is effective for preparing oligonucleotide solutions containing relatively low concentrations of calcium, while Method B is more suitable for preparing oligonucleotide solutions with moderate and controllable calcium concentrations. [Table 2]

[0128] Example 15. Acute CNS toxicity of oligonucleotides via calcium exchange in IT-injected SD rats To test the acute toxicity of oligonucleotide formulations, the specified test substances (i.e., TA1, TA2, and TA3, see Table 3) were prepared and administered via IT injection to adult female SD rats (body weight range: 140 g to 260 g) at a dose of 3 mg / dose (100 mg / mL, 30 μL). aCSF was used as a vehicle control to establish baseline FOB. Cage-side clinical symptoms and FOB were recorded within 3 hours after IT injection.

[0129] The results are shown in Tables 4 and 5. In the TA1 and TA3 groups, acute neurotoxicity characterized by convulsions, abnormal vocalizations, abnormal gait, or hind limb weakness was observed in 3 out of 4 rats. No abnormal behavior was observed in any of the rats in the TA2 group. These results suggest that calcium exchange reduced the acute CNS toxicity of oligonucleotides. [Table 3]

[0130] [Table 4]

[0131] [Table 5]

[0132] Example 16. CNS acute toxicity of different calcium exchange-mediated oligonucleotides in IT-injected SD rats To further test the acute toxicity of oligonucleotide formulations, the specified test substances (i.e., TA4, TA5, TA6, TA7, TA8, and TA9; see Table 6) were prepared and administered via IT injection to adult female SD rats (body weight range: 140 g to 260 g) at a dose of 6 mg / dose (200 mg / mL, 30 μL). TA4 was prepared by directly dissolving RD-12500 in aCSF and used as a positive control for the Functional Observational Balance (FOB) assessment. Cage-side clinical symptoms and FOB were recorded within 3 hours after IT injection.

[0133] The results are shown in Tables 7 and 8. In the TA4 control group, acute neurotoxicity characterized by severe convulsions, abnormal vocalizations, pallor of the ears and limbs, hind limb weakness, abnormal gait, or sensitivity to touch was observed in 3 rats, and 1 rat died approximately 1 hour after IT administration. In the TA5, TA7, TA8, and TA9 groups, no abnormal clinical symptoms were observed in cage-side clinical observations or FOB records. In the TA6 group, when the calcium concentration in the oligonucleotide preparation reached 203 mM, 3 out of 6 rats showed paralysis and did not recover within 3 hours. Table 8 shows the results for TA5, TA7, and TA9.

[0134] This study also demonstrated that both Method A and Method B are effective in preparing oligonucleotide formulations with the desired calcium concentration, while Method B was shown to be more reproducible and easier to perform than Method A. Furthermore, including more than 10 mM of calcium, particularly 20 mM or more (e.g., 35 mM), in the oligonucleotide formulation makes it possible to eliminate most or all of the acute toxicity caused by the oligonucleotide substrate injected into the CNS using IT. [Table 6]

[0135] [Table 7] JPEG2026523055000012.jpg252170

[0136] [Table 8]

[0137] Example 17. Acute CNS toxicity of oligonucleotides accompanied by increased calcium concentration in IT-injected SD rats To further investigate the optimal calcium concentration of oligonucleotide formulations to minimize acute toxicity and to verify the reproducibility of the protocol, the indicated test substances (i.e., TA10, TA11, TA12, TA13, and TA14, see Table 9) were prepared and administered via IT injection at a dose of 6 mg / dose (200 mg / mL, 30 μL) to adult female SD rats (body weight range: 140 g to 260 g). TA10 was prepared by directly dissolving the oligonucleotide substance RD-12500 (WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01, sodium 5.6% w / w, calcium-free) in aCSF and used as a positive control for neurotoxicity. Cage-side clinical symptoms and FOB were recorded within 3 hours after IT injection. The results are shown in Tables 10 and 11. In the TA10 group, acute neurotoxicity, including convulsions, abnormal vocalizations, and abnormal gait, was observed in 5 out of 6 rats. TA10 was identical to TA4, the same formulation, except for a different oligonucleotide batch (the difference in sodium content was within 1% w / w).

[0138] In the TA11 group, all rats exhibited abnormal clinical symptoms. In the TA12 group, 4 out of 6 rats exhibited abnormal clinical symptoms such as convulsions, abnormal vocalizations, and / or abnormal gait. In the TA13 group, 2 out of 6 rats exhibited abnormal clinical symptoms. Surprisingly, in the TA14 group, no abnormal clinical symptoms were observed despite an increase in calcium concentration to 35 mM. TA14 was consistent with TA7, and both were prepared using the same formulation by Method B. TA14 and TA7 demonstrated reproducibility in reducing acute toxicity in two independent studies. In conclusion, the results indicate that oligonucleotide formulations with a calcium concentration of 10 mM or higher, preferably at least 20 mM, and more preferably 35 mM or higher, can effectively reduce the acute toxicity of oligonucleotide substances and / or their formulations.

[0139] [Table 9]

[0140] [Table 10] JPEG2026523055000016.jpg250170

[0141] [Table 11]

[0142] Example 18: Preparation of compound C5×5 In this example, compound C5×5 was prepared using the following procedure. [ka]

[0143] (1) Preparation of compound 42 [ka]

[0144] Under a nitrogen atmosphere, compound 40 (19.56 g, 81 mmol, 1.0 eq) was added to a solution of methylmethyl 2-(4-fluoro-3-nitrophenyl) acetate compound 41 (17.3 g, 81 mmol, 1.0 eq) and K2CO3 (11.2 g, 81 mmol) in anhydrous DMF (200 mL) under a nitrogen atmosphere. The reaction mixture was stirred at 55°C for 6 hours, and then cold water (100 mL) was added. The mixture was extracted three times with ethyl acetate, the organic phase was washed three times with saturated LiCl solution, followed by one wash with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain a yellow oily compound 42, which was used directly in the next step without further purification.

[0145] (2) Preparation of compound 43 [ka]

[0146] Under ice bath conditions, compound 42 (35.18 g, 81 mmol, 1.0 eq) was dissolved in THF / H2O (9:1, 280 mL) and HCOONH4 (30.67 g, 486 mmol, 6.0 eq) and Zn powder (31.78 g, 486 mmol, 6.0 eq) were added. After 10 minutes, the reaction mixture was transferred to room temperature and stirred overnight. The reaction mixture was then filtered and concentrated under reduced pressure. Water (200 mL) was then added to the mixture, and after extraction three times with ethyl acetate, the organic phase was washed once with brine. After drying over anhydrous Na2SO4 and concentration under reduced pressure, compound 43 was obtained and used directly in the next step without further purification. Compound 43 was characterized by mass spectrometry. Calculated MW: 404.34; MW. Measured: 405.3 [M+H] + .

[0147] (3) Preparation of compound 45 [ka]

[0148] Under a nitrogen atmosphere, compound 43 (19.36 g, 48 mmol, 1.0 eq) was dissolved in EtOH (200 mL) and 3-((tert-butyldimethylsilyl)oxy)propanal compound 44 (9.0 g, 48 mmol, 1.0 eq) and AcOH (11 mL, 192 mmol, 4.0 eq) were added. The reaction mixture was stirred overnight at 80°C and then concentrated under reduced pressure. Next, saturated NaHCO3 solution (100 mL) was added, and the mixture was extracted three times with ethyl acetate. The organic phases were then combined, washed with brine, dried over Na2SO4, and concentrated. The resulting residue, compound 45, was used directly in the next step without further purification.

[0149] (4) Preparation of compound 46 [ka]

[0150] Under a nitrogen atmosphere, compound 45 (10 g, 17.5 mmol, 1.0 eq) was dissolved in anhydrous THF (50 mL) to which 1 M TBAF THF solution (26.3 mL, 26.3 mmol, 1.5 eq) was added. The reaction mixture was stirred at room temperature for 1 hour and then concentrated under reduced pressure. Next, water (100 mL) was added, and the mixture was extracted three times with DCM. The organic phases were then combined, washed with brine, dried over Na2SO4, and concentrated. The resulting residue was dissolved in 50 mL of pyridine and DMTrCl (7.12 g, 21 mmol, 1.2 eq) was added. The reaction mixture was stirred at room temperature for 6 hours and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, gradient elution: 1-3% MeOH / DCM) to obtain compound 46 (8.1 g, yield 61%) as a yellow solid. The product was analyzed by mass spectrometry and 1 Characterized by 1H NMR. Calculated MW value: 760.48; MW. Measured value: 761.8 [M + H] + . 1 H NMR (400 MHz, CDCl3) δ 7.59 - 7.53 (m, 1H), 7.38 - 7.31 (m, 2H), 7.23 - 7.19 (m, 6H), 7.18 - 7.14 (m, 3H), 6.76 (dd, J = 7.8, 5.6 Hz, 4H), 4.17 - 4.02 (m, 2H), 3.76 (s, 6H), 3.73 (s, 2H), 3.67 (s, 3H), 3.59 (t, J = 7.0 Hz, 2H), 3.19 - 3.05 (m, 2H), 1.29 - 1.25 (m, 28H), 0.88 (t, J = 6.5 Hz (3H).

[0151] (5) Preparation of compound C5×5 [ka]

[0152] Under a nitrogen atmosphere and in an ice bath, LiAlH4 (202 mg, 5.33 mmol, 1.5 eq) was added to a solution of compound 46 (2.7 g, 3.55 mmol, 1.0 eq) in anhydrous THF (20 mL). After 10 minutes, the mixture was transferred to room temperature and stirred for 1 hour. The reaction mixture was then transferred to an ice bath, and saturated potassium sodium tartrate solution (20 mL) was slowly added to the mixture. After 30 minutes, the reaction mixture was extracted three times with Et2O, then the organic phases were combined, washed with brine, dried over Na2SO4, and concentrated. Under a nitrogen atmosphere, the crude product (300 mg, 0.41 mmol, 1.0 eq) was dissolved in anhydrous DCM (5 mL), and then DIPEA (204 μL, 1.23 mmol, 3.0 eq) and 3-((chloro(diisopropylamino)phosphanyl)oxy)propanenitrile compound 47 (274 μL, 1.23 mmol, 3.0 eq) were added at 25°C. The reaction mixture was stirred for 1 hour. The mixture was extracted twice with DCM, washed with brine, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient elution: 1-5% MeOH / DCM, 1% Et3N) to obtain compound C5×5 (299 mg, yield 78%) as a colorless oil. The product was analyzed by mass spectrometry and 1 Characterized by 1H NMR. 1 H NMR (400 MHz, CDCl3) δ 7.62 (s, 1H), 7.32 (dd, J= 7.6, 4.1 Hz, 2H), 7.27 - 7.11 (m, 9H), 6.73 (dd, J = 7.9, 5.6 Hz, 4H), 4.34 - 4.31 (m, 2H), 4.10 - 4.06 (m, 2H), 3.75 (s, 6H), 3.64 - 3.61 (m, 2H), 3.58 (dd, J = 11.8, 5.1 Hz, 2H), 3.24 - 3.18 (m, 2H), 3.09 - 2.88 (m, 4H), 2.65 -2.55 (m, 4H), 1.37 - 1.28 (m, 28H), 1.22 (dd, J = 6.8, 3.2 Hz, 12H), 0.88 (t, J = 6.5 Hz, 3H).

[0153] Example 19: Preparation of a conjugate oligonucleotide linked to a conjugation group derived from compound C5×5 Following the above method, which is a common method for synthesizing oligonucleotides, an oligonucleotide conjugate was prepared using a C5×5 compound with a conjugation group derived from the C5×5 compound, by using C5×5 as the terminal amidite.

[0154] An exemplary structure of a lipid-conjugated oligonucleotide is shown in O1 below. [ka]

[0155] In the structure of O1, it is confirmed that the conjugation derived from the delivery-promoting compound C5×5 is linked to a double helix of double-stranded RNA (dsRNA) (including, but not limited to, saRNA or siRNA) via linker sites such as -OP(O)2O-, -OP(O)(S)O-, or -P(O)-O- at the 5' end of the sense strand (S) (wherein (S) represents the sense strand and (AS) represents the antisense strand).

[0156] Example 20. Design and Synthesis of Oligonucleotides To further evaluate the toxicity of oligonucleotide formulations, the oligonucleotides shown in Table 12 were designed and synthesized with complete chemical modification (i.e., 2'-O-methyl, 2'-O-methoxyethyl, 5'-(E)-vinylphosphonate, or phosphorothioate (PS) skeleton modification) and conjugated with ACO or lipids (i.e., C5×5), respectively. The oligonucleotide formulations were prepared according to general method B. [Table 12]

[0157] Example 21: Acute CNS toxicity in SD rats injected with an oligonucleotide dissolved in 1.35 mM calcium-containing aCSF using IT. Lipid-conjugated oligonucleotides or double-chain ACOs (e.g., siRNA-ACOs) typically exhibit severe acute toxicity within 3 hours after moderate dose IT injection (see, for example, Tables 13 and 14). However, our observations have confirmed that an appropriate (preferably optimized) calcium concentration in the oligonucleotide formulation can significantly reduce the acute toxicity derived from oligonucleotide substances such as oligonucleotide lipid conjugates (RD-16234 and RD-16237) or siRNA-ACOs (RD-16223, RD-16224, RD-16226, RD-16229, RD-16230 and RD-16236).

[0158] To evaluate the acute toxicity of oligonucleotide preparations, siRNA-ACOs (RD-16223, RD-16224, RD-16226, RD-16229, RD-16230, and RD-16236, see Table 12) and lipid-conjugated oligonucleotides (RD-16234 and RD-16237, see Table 12) were dissolved in aCSF and 35 mM calcium-containing aCSF, respectively. The oligonucleotides were administered to adult female SD rats at a dose of 6 mg / dose (200 mg / mL, 30 μL) by IT injection. aCSF was used as the solvent and served as a negative control. Cage-side clinical findings and FOB were recorded within 3 hours after IT injection for oligonucleotides dissolved in commercially available aCSF (TOCRIS, batch number: 57A) or 35 mM calcium-containing aCSF, and the results are summarized in Tables 13, 15, 14, and 16. For comparison, oligonucleotide preparations dissolved in aCSF containing 35 mM calcium showed reduced acute CNS toxicity, particularly with lipid-conjugated oligonucleotides (i.e., RD-16234 and RD-16237).

[0159] [Table 13] JPEG2026523055000027.jpg243170

[0160] [Table 14] JPEG2026523055000029.jpg244170

[0161] [Table 15]

[0162] [Table 16]

[0163] Example 22. Preparation of DP-10 In this embodiment, DP-10 was prepared by the following procedure. 1) Solutions A1 and B1 were prepared according to Table 17 below. [Table 17]

[0164] 2) The prescribed amount of oligonucleotide substance RD-12500 (500 mg, WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01) was dissolved in 2.5 mL of solution A1 in a 15 mL tube, and then 1.0 mL of solution B1 was added. The mixed solution was transferred to a 5 mL volumetric flask and the volume was adjusted to 5 mL using sterile water for injection. The osmotic pressure was measured and a result of 278 mOsmol / kg was obtained. Subsequently, 3.21 mg of sodium chloride was added to bring the final concentration of DP-10 to 100 mg / mL and the osmotic pressure to 305 mOsmol / kg.

[0165] Example 23. Preparation of DP-15 In this embodiment, DP-15 was prepared by the following procedure. 1) Solutions A2 and B2 were prepared according to Table 18 below. [Table 18]

[0166] 2) The prescribed amount of oligonucleotide substance RD-12500 (500 mg, WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01) was dissolved in 2.5 mL of solution A2 in a 15 mL tube, and then 1.0 mL of solution B2 was added. The mixed solution was transferred to a 5 mL volumetric flask and the volume was adjusted to 5 mL using sterile water for injection. The osmotic pressure was measured and a result of 263 mOsm / kg was obtained. Subsequently, 5.12 mg of sodium chloride was added to bring the final concentration of DP-15 to 100 mg / mL and the osmotic pressure to 298 mOsmol / kg.

[0167] Example 24. Preparation of DP-20 In this embodiment, DP-20 was prepared by the following procedure. 1) Solutions A3 and B3 were prepared according to Table 19 below. [Table 19]

[0168] 2) The prescribed amount of oligonucleotide substance RD-12500 (500 mg, WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01) was dissolved in 2.5 mL of solution A3 in a 15 mL tube, and then 1.0 mL of solution B3 was added. The mixed solution was transferred to a 5 mL volumetric flask and the volume was adjusted to 5 mL using sterile water for injection. The osmotic pressure was measured and a result of 267 mOsmol / kg was obtained. Subsequently, 4.12 mg of sodium chloride was added to bring the final concentration of DP-20 to 100 mg / mL and the osmotic pressure to 297 mOsmol / kg.

[0169] Example 25. Preparation of DP-25 In this embodiment, DP-25 was prepared by the following procedure. 1) Solutions A4 and B4 were prepared according to Table 20 below.

Table 20

[0170] 2) The formulated amount of the oligonucleotide substance RD-12500 (500 mg, WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01) was dissolved in 2.5 mL of Solution A4 in a 15 mL tube, and then 1.0 mL of Solution B4 was added. The mixed solution was transferred to a 5 mL volumetric flask and the volume was adjusted to 5 mL using water for injection. When the osmotic pressure was measured, a result of 249 mOsmol / kg was obtained. Thereafter, 6.58 mg of sodium chloride was added to set the final concentration of DP-25 to 100 mg / mL and the osmotic pressure to 297 mOsmol / kg.

[0171] Example 26. Preparation of DP-35 In this example, DP-35 was prepared by the following procedure. 1) According to Table 21 below, Solution A5 and Solution B5 were prepared.

Table 21

[0172] 2) The formulated amount of the oligonucleotide substance RD-12500 (500 mg, WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01) was dissolved in 2.5 mL of Solution A5 in a 15 mL tube, and then 1.0 mL of Solution B5 was added. The mixed solution was transferred to a 5 mL volumetric flask and the volume was adjusted to 5 mL using water for injection. When the osmotic pressure was measured, a result of 268 mOsmol / kg was obtained. Thereafter, 4.37 mg of sodium chloride was added to set the final concentration of DP-35 to 100 mg / mL and the osmotic pressure to 305 mOsmol / kg.

[0173] Example 27. Acute CNS toxicity of oligonucleotides using isotonic formulations in IT-injected SD rats To further evaluate the acute toxicity of oligonucleotide formulations, the specified test substances (i.e., DP-10, DP-15, DP-20, DP-25, and DP-35, see Table 22) were prepared and administered via IT injection to adult female SD rats (body weight range: 140 g to 260 g) at a dose of 3 mg / dose (100 mg / mL, 30 μL). The preparations of DP-10, DP-15, DP-20, DP-25, and DP-35 are described in Examples 22, 23, 24, 25, and 26, respectively. Cage-side clinical symptoms and FOB were recorded within 3 hours after IT injection, and the results are shown in Tables 22 and 23. No significant acute toxicity was observed in any group. The results suggest that isotonic oligonucleotide formulations with an osmotic pressure of 280–320 mOsmol / kg can significantly reduce acute toxicity.

[0174] While the present invention has been shown and described in particular in terms of preferred embodiments and various alternative embodiments, those skilled in the art will understand that various modifications can be made to the form and details without departing from the spirit and scope of the invention.

[0175] [Table 22] JPEG2026523055000038.jpg252170

[0176] [Table 23]

Claims

1. Oligonucleotide preparations, (a) A calcium-containing solution having a calcium concentration of at least 15 mM in the preparation, (b) comprising the oligonucleotide substance in the solution, Oligonucleotide preparations.

2. If the oligonucleotide substance is a non-conjugate oligonucleotide, the calcium concentration in the formulation is between 15 mM and less than 25 mM, or If the oligonucleotide substance is a conjugated oligonucleotide, the calcium concentration in the formulation is 15 mM to 150 mM, and / or The oligonucleotide preparation has an osmotic pressure in the range of 250 mOsmol / kg to 350 mOsmol / kg. The oligonucleotide formulation according to claim 1.

3. The oligonucleotide material includes single-stranded oligonucleotides, double-stranded oligonucleotides, or combinations thereof. The oligonucleotide formulation according to claim 1.

4. The single-stranded oligonucleotide is one or more antisense oligonucleotides (ASOs) selected from gapmers, mixmers, steric blockers, or splicing modifiers, and / or The double-stranded oligonucleotide is one or more double-stranded RNAs selected from siRNA, saRNA, a combination of siRNA and / or saRNA, a conjugate of siRNA and / or saRNA, or miRNA. The oligonucleotide formulation according to claim 1.

5. The conjugated oligonucleotide is conjugated to one or more conjugation sites, and / or The one or more conjugation sites are selected from lipids, luminescent foci, ligands, sugars, peptides, and antibodies, and / or The conjugation site is an accessory oligonucleotide (ACO) conjugated to the oligonucleotide, and / or The one or more conjugation sites conjugate to the oligonucleotide at one or more ends of the oligonucleotide chain or at an internal position of the oligonucleotide. An oligonucleotide preparation according to any one of claims 2 to 4.

6. The one or more conjugation sites are selected from fatty acids, cell-penetrating peptides, fluorophores, polyethylene glycol, alkaloids, tryptamine, benzimidazole, quinolone, amino acids, cholesterol, glucose, and N-acetylgalactosamine, and / or The oligonucleotide substance is either an oligonucleotide conjugated to ACO, or an oligonucleotide conjugated to a lipid moiety. The oligonucleotide formulation according to claim 5.

7. The aforementioned lipid portion is C 4 -C 30 The fatty acid portion, The oligonucleotide formulation according to claim 5.

8. The lipid moiety is saturated or unsaturated C 6 - 8 - 10 - 12 - 14 - 16 - 18 - 20 - 22 - 24 carbon chain-containing, The oligonucleotide formulation according to claim 5.

9. The oligonucleotide formulation according to claim 4, wherein the conjugation site is derived from C5×5. 【Chemistry 10】

10. C5×5 is -OP(O) 2 Conjugate to the 5' end of the sense or antisense strand in oligonucleotides via O-, -OP(O)(S)O-, and / or -P(O)-O-. The oligonucleotide formulation according to claim 9.

11. The auxiliary oligonucleotide (ACO) is a single-stranded oligonucleotide having a length of at least 6 nucleotides, and / or The auxiliary oligonucleotide (ACO) is a non-target oligonucleotide, and / or The auxiliary oligonucleotide (ACO) has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 phosphorothioate modifications on its backbone, or all nucleotides in the backbone are phosphorothioate modified. The oligonucleotide formulation according to claim 5.

12. The calcium is in a form selected from molecular calcium, ionized calcium, and complex calcium, or any combination thereof. The oligonucleotide formulation according to claim 1.

13. The calcium is provided as calcium chloride, calcium gluconate, calcium lactate, calcium bicarbonate, calcium dihydrogen phosphate, calcium hydrogen phosphate, and any combination thereof. The oligonucleotide formulation according to claim 1.

14. If the oligonucleotide substance is a non-conjugate oligonucleotide, the calcium concentration in the formulation is in the range of 15 mM to 24, 23, 22, 21, 20, 19, 18, 17, or 16 mM, or any lower range or numerical value within the said range, and / or If the oligonucleotide substance is a non-conjugated oligonucleotide, the osmotic pressure of the formulation is in the range of 280 mOsmol / kg to 320 mOsmol / kg. The oligonucleotide formulation according to claim 2.

15. If the oligonucleotide substance is a conjugate oligonucleotide, The calcium concentration in the preparation is in the range of 15 mM to 140 mM, 20 mM to 130 mM, 20 mM to 120 mM, 30 mM to 110 mM, or 35 mM to 100 mM, and / or the calcium concentration in the preparation is approximately 20, 35, 47.5, 60, 82.5, 100 mM, 120 mM, and / or The calcium concentrations in the aforementioned preparations are approximately 35, 47.4, 60, and 100 mM, and / or The calcium concentration in the aforementioned preparation is between 15 mM and less than 25 mM, and / or The calcium concentration in the preparation is in the range of 15 mM to 24, 23, 22, 21, 20, 19, 18, 17, or 16 mM, or any lower range or numerical value within the said range, and / or If the oligonucleotide substance is a conjugated oligonucleotide, the osmotic pressure of the formulation is in the range of 280 mOsmol / kg to 320 mOsmol / kg. The oligonucleotide formulation according to claim 2.

16. At least one nucleotide of the oligonucleotide substance is a chemically modified nucleotide. An oligonucleotide preparation according to any one of claims 1 to 15.

17. The chemically modified nucleotide is located within the antisense strand of the ASO, or within the sense strand, antisense strand, or both strands of the double-stranded RNA, or within the ACO, and / or The chemically modified nucleotide is a nucleotide modified at the 5' end, 3' end, both ends, or the internal portion of the chain(s), and / or At least 50% of the nucleotides in the oligonucleotide substance are chemically modified. The oligonucleotide formulation according to claim 16.

18. The chemically modified nucleotide is one or more selected from 2' sugar modification, base modification, phosphorothioate (PS) skeleton modification, and addition of a 5'-phosphate moiety or 5-methylcytosine moiety to the 5' end of the nucleotide chain. The oligonucleotide formulation according to claim 16.

19. The 2' sugar modification is one or more selected from 2'-fluoro-2'-deoxynucleoside (2'-F) modification, 2'-O-methyl (2'-O-Me) modification, and / or 2'-O-(2-methoxyethyl) (2'-O-MOE) modification, and / or The addition of the 5'-phosphate moiety is one or more selected from the addition of (E)-vinylphosphonate moieties to the 5' end of the nucleotide chain. The oligonucleotide formulation according to claim 18.

20. The formulation further comprises one or more other excipients selected from preservatives, humectants, emulsifiers, dispersants, isotonic agents, and antioxidants. An oligonucleotide formulation according to any one of claims 1 to 19.

21. The preparation comprises one or more selected from salts, polyols, sugars, and / or alcohols, and / or The carrier in the aforementioned formulation is artificial cerebrospinal fluid (aCSF) or water, and / or The calcium-containing solution is aCSF or an aqueous solution, and / or The aforementioned preparation consists of NaCl, KCl, and MgSO4. 4 , KH 2 PO 4 NaHCO 3 , comprising one or more components selected from glucose and sucrose, The oligonucleotide formulation according to claim 1.

22. The aforementioned formulation contains 50–150 mM NaCl, 0.5–5.0 mM KCl, and 0.5–5.0 mM MgSO4. 4 , 0.4–3.0 mM KH 2 PO 4 , 10-50 mM NaHCO 3 , comprising 1-20 mM glucose and 1-10 mM sucrose, The oligonucleotide formulation according to claim 1.

23. The aforementioned formulation contains 124 mM NaCl, 2.5 mM KCl, and 2.0 mM MgSO4. 4 , 1.25 mM KH 2 PO 4 , 26 mM NaHCO 3 , containing 10 mM glucose and 4 mM sucrose, The oligonucleotide formulation according to claim 1.

24. The aforementioned formulation contains components selected from the solution groups A1, B1, A2, B2, A3, B3, A4, B4, A5, and B5 shown in Tables 17, 18, 19, 20, and 21. The oligonucleotide formulation according to claim 1.

25. The oligonucleotides in the oligonucleotide formulation target the SOD1 gene, and / or The oligonucleotides in the oligonucleotide formulation target the FUS gene, and / or The oligonucleotides in the oligonucleotide preparation target genes selected from the group consisting of C9orf72, MAPT (Tau), APP, SMN2, SCN9A, SCN10A, HTT, p21, UTRN, DUX4, SNCA, ATXN1, ATXN2, ATXN3, SCA1, SCA7, SCA8, UCP1, VEGFA, MeCP2, PRNP, DMPK, TARDBP, and TTR. The oligonucleotide formulation according to claim 1.

26. The oligonucleotide in the oligonucleotide preparation comprises one or more oligonucleotide chains or chain pairs from the oligonucleotide substances in Table 1 and / or Table 12, and / or The oligonucleotide in the oligonucleotide preparation includes one or more oligonucleotide chains or chain pairs having the sequence shown in one or more of Sequence IDs 1 to 15. The oligonucleotide formulation according to claim 1.

27. The oligonucleotide preparation has a calcium concentration in the range of 20 mM to 100 mM, and / or The oligonucleotide formulation has an osmotic pressure in the range of 280 mOsmol / kg to 320 mOsmol / kg, and / or The oligonucleotide preparation is an isotonic solution. The oligonucleotide formulation according to claim 25.

28. The aforementioned preparation is in a form suitable for a route of administration selected from subcutaneous administration, intravenous administration, intraocular injection, intradermal injection, intramuscular injection, intraperitoneal injection, intratracheal administration, intrafat administration, intra-articular administration, intrathecal administration, epidural administration, inhalation, intranasal administration, oral administration, sublingual administration, buccal administration, rectal administration, vaginal administration, intracisional administration, transdermal administration, and local administration, or local delivery. An oligonucleotide formulation according to any one of claims 1 to 27.

29. The process includes combining the oligonucleotide substance with a calcium-containing substance in solution to form the oligonucleotide preparation. A method for preparing an oligonucleotide preparation according to any one of claims 1 to 28.

30. The aforementioned calcium is calcium chloride (CaCl 2 ), provided as calcium salts such as calcium gluconate, calcium lactate, calcium bicarbonate, calcium dihydrogen phosphate, and calcium hydrogen phosphate. The method according to claim 29.

31. In order to dissolve the oligonucleotide substance, calcium is contained in aCSF, and / or By exchanging the sodium in the solution containing the oligonucleotide substance for calcium, calcium is incorporated into the solution, and / or If the oligonucleotide substance is a non-conjugate oligonucleotide, the calcium concentration in the formulation is between 15 mM and less than 25 mM, or When the oligonucleotide substance is a conjugated oligonucleotide, the calcium concentration in the oligonucleotide preparation is in the range of 15 mM to 150 mM. The method according to claim 29.

32. The aforementioned formulation is made by adding CaCl to aCSF solution. 2 Add CaCl 2 It is prepared by dissolving an oligonucleotide substance in an aCSF solution containing the following: The method according to claim 29.

33. A product comprising an oligonucleotide preparation according to any one of claims 1 to 28.

34. The aforementioned products are selected from pharmaceuticals, vaccines, diagnostic products, and imaging products. The product according to claim 33.

35. Use of an oligonucleotide formulation according to any one of claims 1 to 28 in the preparation of a product for the treatment, prevention, or detection of a disease or disorder of a subject.

36. A method for treating, preventing, or detecting a disease or disorder in a subject, comprising administering an oligonucleotide formulation according to any one of claims 1 to 28 to the subject in need in an effective amount.

37. An oligonucleotide formulation according to any one of claims 1 to 28 for the treatment, prevention, or detection of a disease or disorder in a subject.

38. The oligonucleotide formulation for use according to claim 35, the method according to claim 36, or the use according to claim 37, wherein the oligonucleotide formulation exhibits reduced acute toxicity in the body compared to a reference oligonucleotide formulation that does not contain calcium within a predetermined concentration range.

39. The oligonucleotide formulation exhibits reduced acute toxicity in the body compared to a reference oligonucleotide formulation that does not contain calcium within a predetermined concentration range, and the acute toxicity in the body is toxicity to the somatic motor nervous system, and / or The oligonucleotide preparation acts on the somatic motor nervous system, and / or The oligonucleotide formulation acts on the central nervous system (CNS), and / or Oligonucleotide formulations for use according to claim 35, the method according to claim 36, or the use according to claim 37, wherein the acute toxicity is CNS acute toxicity.

40. An oligonucleotide formulation for use according to claim 35, the method according to claim 36, or the use according to claim 37, wherein the subject requires a pharmaceutical for the treatment of the disease or disorder, a vaccine for the prevention of the disease or disorder, a diagnostic product for the diagnosis of the disease or disorder, and an imaging product for imaging of the lesion site(s) of the disease or disorder.

41. The disease or disorder is selected from brain diseases, spinal cord disorders, and peripheral nerve disorders, and is an oligonucleotide formulation for use according to claim 35, the method according to claim 36, or the use according to claim 37.

42. The oligonucleotide formulation for use according to claim 35, the method according to claim 36, or the use according to claim 37, wherein the oligonucleotide targets a gene selected from the group consisting of SOD1, FUS, C9orf72, MAPT (Tau), APP, SMN2, SCN9A, SCN10A, HTT, p21, UTRN, DUX4, SNCA, ATXN1, ATXN2, ATXN3, SCA1, SCA7, SCA8, UCP1, VEGFA, MeCP2, PRNP, DMPK, TARDBP, and TTR.

43. The disease or disorder is selected from spinal muscular atrophy (SMA), Duchenne and Becker muscular dystrophy (DMD and BMD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), brain tumor, frontotemporal dementia, spinocerebellar degeneration, prion disease, Lafora disease, migraine, schizophrenia, depression, pain, and stroke, and is an oligonucleotide formulation for use according to claim 35, the method according to claim 36, or the use according to claim 37.

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