Oligonucleotide formulations

Abstract

The present disclosure relates to oligonucleotide formulations. In particular, the disclosure relates to oligonucleotide formulations comprising oligonucleotides (such as ASO, siRNA, saRNA) and calcium, methods of making the formulations, and uses thereof. According to the oligonucleotide preparation, the in-vivo acute toxicity (especially toxicity to a central nervous system) of oligonucleotide, especially lipid conjugates, is reduced, and the in-vivo safety window of the oligonucleotide is expanded, so that the oligonucleotide preparation has a great application prospect.

Description

TECHNICAL FIELD

[0001] The present disclosure relates generally to the fields of biopharmaceuticals and oligonucleotide therapeutics. Specifically, the present disclosure relates to the formulation of oligonucleotide substances with calcium-containing solutions having specific calcium concentrations, and specific oligonucleotide formulations with calcium and other components.

[0002] Cross Reference to Related Applications

[0003] This application claims priority to the filing date of Provisional Patent Application Serial No. PCT / CN2023 / 100743 filed June 16, 2023, the disclosure of which is hereby incorporated by reference in its entirety.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted electronically in computer readable format and is hereby incorporated by reference in its entirety. BACKGROUND

[0006] Oligonucleotide-based therapeutics have emerged as a promising class of therapeutic agents for neurological diseases. Administration of oligonucleotides to a patient, organ, tissue, or cell can initiate or influence various biochemical reactions to achieve functions such as silencing, inhibiting, activating, and / or modulating gene expression.

[0007] Single-stranded ASOs in the form of gapmers can be used to inhibit gene expression via the RNase H mechanism of degrading target mRNA. Another class of ASOs are steric blockers (i.e., splice modulators), which are typically composed of ribonucleotides uniformly and bind to pre-mRNA in the nucleus to alter mRNA splicing by blocking the binding of certain splicing factors to the mRNA.

[0008] Duplex oligonucleotides or double-stranded RNA (dsRNA) include small interfering RNA (siRNA) and small activating RNA (saRNA) as well as microRNA (miRNA), all of which are loaded onto Argonaute (AGO) proteins in the cytoplasm as an initial step before acting specifically on their targets. siRNAs primarily bind to target mRNAs in the cytoplasm to downregulate gene expression post-transcriptionally through the RNA interference (RNAi) mechanism. saRNAs target gene regulatory sequences in the nucleus, such as gene promoters, to upregulate gene expression at the transcriptional level through the RNA activation (RNAa) mechanism. miRNAs are also duplex RNAs that typically bind to the 3' untranslated region (3' UTR) of mRNAs in an imperfectly complementary manner after being loaded onto AGO proteins and partially inhibit expression through translational repression or / and mRNA degradation.

[0009] Oligonucleotides (e.g., ASOs, siRNAs, or saRNAs) with 2'-substitution modifications (such as 2'-fluoro-2'-deoxynucleosides, 2'-O-methyl, or 2'-O-(2-methoxyethyl) modifications), backbone modifications (such as phosphorothioate (PS)), and / or conjugated to moieties such as hydrophilic moieties (such as fatty acid moieties, cholesterol, or lipids) are believed 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) oligonucleotides / oligonucleotide substances induce dose-limiting acute toxicities, resulting in a narrow therapeutic window or even clinical trial failure.

[0010] Accordingly, there is a need for improved oligonucleotide substance formulations to address the aforementioned challenges. SUMMARY

[0011] To address the aforementioned problems and challenges, the present application provides novel oligonucleotide formulations, methods of making the same, and uses thereof.

[0012] One aspect of the present disclosure includes an oligonucleotide formulation. The formulation includes (a) a calcium-containing solution including calcium at a concentration of at least 15 mM; and (b) an oligonucleotide substance in the solution.

[0013] In some embodiments, when the oligonucleotide substance is a non-conjugated oligonucleotide, the concentration of calcium in the formulation is 15 mM to less than 25 mM. For example, when the oligonucleotide substance is a non-conjugated oligonucleotide, the concentration of calcium in the formulation is in the range of 15 mM to 24 mM, 23 mM, 22 mM, 21 mM, 20 mM, 19 mM, 18 mM, 17 mM, or 16 mM, or any sub-range or value within the range.

[0014] In some embodiments, when the oligonucleotide substance is a conjugated oligonucleotide, the concentration of calcium in the formulation is 15 mM to 150 mM. For example, the concentration of calcium in the formulation is in the range of 15 mM to 150 mM, such as 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 concentration of calcium in the formulation is about 20 mM, 35 mM, 47.5 mM, 60 mM, 82.5 mM, 100 mM, 120 mM. In some embodiments, when the oligonucleotide substance is a conjugated oligonucleotide, the concentration of calcium in the formulation is 15 mM to less than 25 mM, such as in the range of 15 mM to 24 mM, 23 mM, 22 mM, 21 mM, 20 mM, 19 mM, 18 mM, 17 mM, or 16 mM, or any sub-range or value within the range.

[0015] In some embodiments, the osmolarity of the oligonucleotide formulation is in the range of 250 mOsmol / kg to 350 mOsmol / kg.

[0016] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide (such as an antisense RNA (ASO) selected from gapmer, steric blocker, or mixmer) or a double-stranded oligonucleotide (such as 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 (such as a combination of single-stranded and double-stranded oligonucleotides, for example, an oligonucleotide agent comprising a targeting duplex and a single-stranded non-targeting accessory oligonucleotide (ACO) conjugated to each other with or without a linker). In some embodiments, the oligonucleotide agent is an oligonucleotide conjugate comprising a duplex RNA conjugated to an ACO. In some embodiments, the oligonucleotide is conjugated to one or more conjugating moieties, such as a lipid (e.g., fatty acid) moiety. In some embodiments, the oligonucleotide is conjugated to a moiety derived from C5x5.

[0017] In some embodiments, one or more of the nucleotides in the oligonucleotide agent is modified. In some embodiments, one or more of the internucleoside linkages in the oligonucleotide or ACO is selected from the group consisting of phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate.

[0018] In some aspects of the disclosure, a method for preparing an oligonucleotide formulation is provided. The method comprises combining an oligonucleotide agent with a calcium-containing agent in solution to form the oligonucleotide formulation.

[0019] In some embodiments, calcium is included in the oligonucleotide agent by exchanging sodium for calcium in a solution comprising the oligonucleotide agent. In some embodiments, the formulation is prepared by adding 35 mM CaCl2 in an artificial cerebrospinal fluid (aCSF) solution and dissolving the oligonucleotide agent in the aCSF solution containing CaCl2.

[0020] In some aspects of the disclosure, a product comprising the oligonucleotide formulation is provided. The product can be selected from the group consisting of a drug, a vaccine, a diagnostic product, and an imaging product.

[0021] In some aspects of the disclosure, use of the oligonucleotide formulation in the preparation of a product for treating, preventing, or detecting a disease or condition in a subject is provided. In some embodiments, the product acts in the central nervous system (CNS).

[0022] In some aspects of the disclosure, a method for treating, preventing, or detecting a disease or disorder in a subject in need thereof is provided, comprising administering to the subject an oligonucleotide formulation.

[0023] In some aspects of the disclosure, the oligonucleotide formulation of the invention is provided for use in treating, preventing, or detecting a disease or disorder in a subject in need thereof.

[0024] In some embodiments, 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.

[0025] In some embodiments, the disease or disorder is a disease or disorder in the central nervous system (CNS). In some embodiments, the effect of the oligonucleotide on the CNS is selected from treating, preventing, and / or diagnosing a disease or disorder in the CNS, or imaging a site of a disease or disorder in the CNS. In some embodiments, the disease or disorder in the CNS is selected from brain diseases, spinal cord diseases, and peripheral neuropathies, such as spinal muscular atrophy (SMA), Duchenne muscular dystrophy (DMD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), multiple sclerosis (MS), brain tumors, frontotemporal dementia, spinocerebellar diseases, prion diseases, lafora, migraines, schizophrenia, depression, pain, and stroke.

[0026] The oligonucleotide formulations of the invention and products comprising the oligonucleotide formulations can be used to reduce acute toxicity (e.g., in the CNS) and expand the safety window of the oligonucleotide, thus having great application prospects.

[0027] Other aspects and advantages of the application will become apparent to those skilled in the art from a consideration of the following detailed description taken in conjunction with the accompanying drawings. As will be realized, the application is capable of other and different embodiments and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0028] The novel features of the application are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present application will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the application are utilized, and the accompanying drawings (also "Figure" and "FIG." herein), of which: Figure 1 Purification of calcium exchanged oligonucleotide material (OS) of TA2 from free CaCl2 salt solution (described in Example 1) is shown, where size exclusion column was used to desalt by UV and conductivity signals. Fraction 2 was collected and lyophilized to produce calcium OS of TA2.

[0029] Figure 2 Purification of calcium exchanged OS of TA5 from free CaCl2 salt solution (described in Example 3) is shown, where size exclusion column was used to desalt by UV and conductivity signals. Fraction 2 was collected and lyophilized to produce calcium OS of TA5.

[0030] Figure 3 Purification of calcium exchanged OS of TA15 from free CaCl2 salt solution (described in Example 4) is shown, where size exclusion column was used to desalt by UV and conductivity signals. Fraction 2 was collected and lyophilized to produce calcium OS of TA15.

[0031] Figure 4 Purification of calcium exchanged OS of TA6 from free CaCl2 salt solution (described in Example 5) is shown, where size exclusion column was used to desalt by UV and conductivity signals. Fraction 2 was collected and lyophilized to produce calcium OS of TA6.

[0032] Figure 5 Purification of calcium exchanged OS of TA16 from free CaCl2 salt solution (described in Example 6) is shown, where size exclusion column was used to desalt by UV and conductivity signals. Fraction 2 was collected and lyophilized to produce calcium OS of TA16.

[0033] In the Figures, "— " represents the conductivity signal curve; "— 10 Cond " represents the fraction parts numbered 1, 2, 3,...; and "— 10 fraction " indicates that the UV measurement wavelength for oligonucleotide detection was 260 nm. 10 UV1 260 nm DETAILED DESCRIPTION

[0034] While various embodiments of the application have been shown and described herein, it will be apparent to those skilled in the art that many changes, modifications, and substitutions can be made therein without departing from the application. It should be understood that various alternatives to the embodiments of the application described herein can be employed in practicing the application.

[0035] ​Before the present application is described, it is to be understood that this application is not limited to the particular implementation described, as such will of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present application will be limited only by the appended claims.

[0036] It should be understood that, in providing a range of values, every intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range is also specifically disclosed. Every smaller range between any stated or intervening value, or intervening values, in a stated range, is encompassed within the application. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and each range where either, both or neither limit of the range is included is also encompassed within the application, subject to any explicitly excluded limit(s) in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the application.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although exemplary methods and materials are described herein, any method and material similar or equivalent to those described herein can be used in the practice and testing of the present application. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It will be understood that the disclosures in this application supersede any disclosure of an incorporated publication to the extent that there is a contradiction.

[0038] It should be noted that, as used in this application and the appended claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a sample" includes a plurality of such samples, and a reference to "the molecule" includes reference to one or more molecules known to one of skill in the art, and equivalents thereof, and the like.

[0039] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present application is not entitled to antedate such publication by virtue of prior application. Further, the dates of publication provided can be different from the actual publication dates, which can need to be independently confirmed.

[0040] As used herein, the term "calcium" refers to calcium present in any form in an oligonucleotide formulation that is capable of reducing the in vivo acute toxicity of the oligonucleotide (such as in the central nervous system) and expanding the in vivo safety window of the oligonucleotide. In some embodiments, the calcium is in a form selected from the group consisting of molecular calcium, ionic calcium, complexed calcium, or any combination thereof. Calcium (such as in CaCl2) can be incorporated into an oligonucleotide formulation by adding it to a solution comprising the oligonucleotide (such as in an aCSF solution) or exchanging sodium in a solution comprising the oligonucleotide (such as a synthetic oligonucleotide material).

[0041] As used herein, the term "calcium-containing solution" in a nucleotide formulation refers to a solution comprising a defined concentration of calcium and used to carry or has carried the nucleotide of the formulation. The calcium in the formulation can be provided by a calcium source, for example, one or more selected from the group consisting of calcium chloride, calcium gluconate, calcium lactate, calcium bicarbonate, calcium dihydrogen phosphate, calcium hydrogen phosphate, and any combination thereof.

[0042] In some embodiments, the concentration of calcium in the formulation is varied. In some embodiments, when the oligonucleotide material is a non-conjugated oligonucleotide, the concentration of calcium in the formulation is 15 mM to less than 25 mM. For example, when the oligonucleotide material is a non-conjugated oligonucleotide, the concentration of calcium in the formulation is in the range of 15 mM to 24 mM, 23 mM, 22 mM, 21 mM, 20 mM, 19 mM, 18 mM, 17 mM, or 16 mM, or any sub-range or value within this range.

[0043] In some embodiments, when the oligonucleotide material is a conjugated oligonucleotide, the concentration of calcium in the formulation is 15 mM to 150 mM. For example, the concentration of calcium in the formulation is in the range of 15 mM to 150 mM, for example, 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 concentration of calcium in the formulation is about 20 mM, 35 mM, 47.5 mM, 60 mM, 82.5 mM, 100 mM, 120 mM. In some embodiments, when the oligonucleotide material is a conjugated oligonucleotide, the concentration of calcium in the formulation is 15 mM to less than 25 mM, such as in the range of 15 mM to 24 mM, 23 mM, 22 mM, 21 mM, 20 mM, 19 mM, 18 mM, 17 mM, or 16 mM, or any sub-range or value within this range. Based on the disclosure herein, one of skill in the art can adjust the concentration of calcium in an oligonucleotide formulation as needed for practice.

[0044] As used herein, the term "oligonucleotide substance (OS)" refers to a substance consisting of or comprising an oligonucleotide moiety, such as an oligonucleotide moiety conjugated to other non-targeting moieties, preferably, the oligonucleotide or oligonucleotide moiety is capable of targeting a specific gene and modulating the expression and / or function of that gene.

[0045] In some embodiments, the oligonucleotide substance in the oligonucleotide formulation is one or more selected from a single-stranded oligonucleotide or a double-stranded oligonucleotide or a combination thereof. The oligonucleotide substance can be in the form of an isolated oligonucleotide or a conjugated oligonucleotide. The single-stranded oligonucleotide can be an antisense RNA (ASO) selected from a gapmer, a steric blocker, or a mixmer. The double-stranded oligonucleotide can be a duplex RNA selected from a siRNA or a saRNA, a combination of siRNA and saRNA, or a conjugate of siRNA and / or saRNA, or a miRNA. A combination or conjugation of single-stranded oligonucleotides, double-stranded oligonucleotides, or a combination of single- and double-stranded oligonucleotides can also be used in the formulations of the present application. For example, the present disclosure also includes oligonucleotide conjugates comprising a targeting duplex and an accessory oligonucleotide (ACO) conjugated to each other with or without a linker.

[0046] As used herein, the term "gapmer" refers to a short DNA antisense oligonucleotide (ASO) structure with modified RNA segments on both sides of a central DNA structure. In some embodiments, at least one of the modified RNA segments can include one or more selected from a locked nucleic acid (LNA) and a 2'-OMe or 2'-F modified nucleotide to increase affinity to the target, increase nuclease resistance, decrease immunogenicity, and / or decrease toxicity. In some embodiments, the gapmer comprises at least one nucleotide modified with a phosphorothioate (PS) group. In some embodiments, the gapmer is designed to hybridize to a target segment of RNA and silence the gene transcript by inducing RNase H cleavage.

[0047] As used herein, the term "LNA" refers to a locked nucleic acid, in which the 2'-oxygen and 4'-carbon atom are connected by an extra bridge. As used herein, the term "BNA" refers to a 2'-O and 4'-aminoethylene bridged nucleic acid that can comprise a five- or six-membered bridged structure with a N-O bond. As used herein, the term "PNA" refers to a nucleic acid mimic with a pseudopeptide backbone, which is composed of N-(2-aminoethyl)glycine units, in which the nucleobase is connected to the glycine nitrogen via a carbonylmethylene linker.

[0048] As used herein, the term "mixed-polymer" refers to an antisense oligonucleotide (ASO) characterized as a mixture of DNA and structurally chemically modified nucleic acid analogs. Optionally, the mixed-polymer is composed of fully modified nucleotides or nucleic acid analogs. In some embodiments, the mixed-polymer is designed to bind and mask a complementary RNA sequence, thereby sterically blocking protein, factor or other RNA interaction with the targeted RNA. In some embodiments, the mixed-polymer is designed to alter pre-mRNA splicing by displacing a spliceosome. In some embodiments, the mixed-polymer is designed to bind and sequester microRNA (miRNA), in the process it adopts another name, called "miR antagomir" or "anti-miR".

[0049] As used herein, the term "splicing modulator" refers to an antisense oligonucleotide (ASO) that typically uniformly consists of ribonucleotides and binds to a pre-mRNA in the nucleus to alter mRNA splicing, such as by blocking the binding of certain splicing factors to the mRNA (steric blocker).

[0050] As used herein, the term "sense strand" of a dsRNA (e.g., siRNA, saRNA) duplex refers to the strand that has sequence homology or sequence identity to a segment of the coding strand of a target gene sequence.

[0051] As used herein, the term "antisense strand" of a dsRNA (e.g., siRNA, saRNA) duplex refers to the strand that has sequence complementarity to the sense strand.

[0052] As used herein, the term "overhang" refers to unpaired nucleotides at the end (5' or 3') of an oligonucleotide strand that are formed by one strand of a double-stranded oligonucleotide extending beyond the other strand. Single-stranded regions that extend beyond the 3' end and / or 5' end of a duplex are referred to as overhangs.

[0053] As used herein, the term "natural overhang" refers to an overhang composed of one or more nucleotides identical or complementary to the corresponding position on the target sequence.

[0054] As used herein, the terms "gene activation," "activate gene expression," "gene upregulation," and "upregulate gene expression" are used interchangeably and mean an increase or upregulation in the transcription, translation, expression, or activity of a certain nucleic acid sequence as determined by measuring the level of transcription, mRNA level, protein level, enzyme activity, methylation state, chromatin state or conformation, level of translation, or activity or state of a gene in a cellular or biological system. These activities or states can be determined directly or indirectly. Further, "gene activation" or "activate gene expression" refers to an increase in the activity associated with a nucleic acid sequence regardless of the mechanism of such activation. For example, gene activation occurs at the level of transcription to increase transcription into RNA, and the RNA is translated into a protein, thereby increasing expression of the protein.

[0055] As used herein, the terms "gene silencing," "knock down gene expression," "gene downregulation," and "downregulate gene expression" are used interchangeably and mean a decrease or downregulation in the transcription, translation, expression, or activity of a certain nucleic acid sequence as determined by measuring the level of transcription, mRNA level, protein level, enzyme activity, methylation state, chromatin state or conformation, level of translation, or activity or state of a gene in a cellular or biological system. These activities or states can be determined directly or indirectly. Further, "gene downregulation" or "downregulate gene expression" refers to a decrease in the activity associated with a nucleic acid sequence regardless of the mechanism of such downregulation. For example, gene downregulation occurs at the level of transcription to decrease or silence transcription into RNA, and the RNA is not translated into a protein, thereby decreasing or silencing expression of the protein.

[0056] As used herein, the terms "short interfering RNA," "siRNA," and "silencing RNA" are used interchangeably and refer to a ribonucleic acid molecule that can downregulate, knock down, or silence the expression of a target gene. It can be a double-stranded nucleic acid molecule. It interferes with the expression of a specific gene with complementary nucleotide sequences by degrading the mRNA after transcription, thereby preventing translation. siRNA binds to the target mRNA primarily in the cytoplasm to downregulate gene expression post-transcriptionally via the mechanism of RNA interference (RNAi). For example, for ALS patients, siRNA can be designed to target genes such as SOD1, TDP-43, FUS, C9ORF72, and so on. SOD1mRNA sequence of the target gene to silence its expression via an RNAi mechanism, thereby maximizing the therapeutic outcome. siRNAs are molecules with endogenous RNA bases or chemically modified nucleotides. These modifications do not eliminate cellular activity, but rather impart increased stability and / or increased cellular potency. Examples of chemical modifications include phosphorothioate groups, 2'-deoxynucleotides, 2'-OCH3-containing ribonucleotides, 2'-F-ribonucleotides, 2'-methoxyethyl ribonucleotides, combinations thereof, and the like. siRNAs can have different lengths (e.g., 10-200 bp) and structures (e.g., hairpins, single / double stranded, bulges, nicks / gaps, mismatches), and are processed in the cell to provide active gene silencing. Double stranded siRNAs can have the same number of nucleotides on each strand (blunt ends) or asymmetric ends (overhangs). For example, 1-2 nucleotide overhangs can be present on the sense and / or antisense strand, as well as on the 5' and / or 3' end of a given strand. The length of the siRNA molecule is typically about 10 to about 60 base pairs, about 10 to about 50 base pairs, about 15 to about 30 base pairs, about 17 to about 29 base pairs, about 18 to about 28 base pairs, about 19 to about 27 base pairs, about 20 to about 26 base pairs, about 21 to about 25 base pairs, and about 22 to about 24 base pairs, and is typically about 15 base pairs, about 16 base pairs, about 17 base pairs, about 18 base pairs, about 19 base pairs, about 20 base pairs, about 21 base pairs, about 23 base pairs, about 25 base pairs, about 30 base pairs, about 40 base pairs, or about 50 base pairs. Furthermore, the terms "small interfering RNA," "silencing RNA," and "siRNA" also encompass nucleic acids other than ribonucleotides, including but not limited to modified nucleotides or analogs.

[0057] As used herein, the terms "small activating RNA," "saRNA," and "small activating ribonucleic acid" are used interchangeably and refer to a ribonucleic acid molecule that can upregulate the expression of a target gene. It can be a double-stranded nucleic acid molecule composed of a first nucleic acid strand containing a ribonucleotide sequence having sequence homology to a non-coding nucleic acid sequence of a target gene, such as a promoter and enhancer, and a second nucleic acid strand containing a nucleotide sequence complementary to the first strand. A saRNA can also be composed of a synthetic or vector-expressed single-stranded RNA molecule that is prone to form a hairpin structure through two complementary regions within the molecule, where the first region contains a ribonucleotide sequence having sequence homology to a target sequence of a promoter of a gene, and the ribonucleotide sequence contained in the second region is complementary to the first region. The duplex region of a saRNA molecule is typically about 10 to about 60 base pairs, about 10 to about 50 base pairs, about 10 to about 40 base pairs, about 12 to about 30 base pairs, about 14 to about 28 base pairs, about 16 to about 26 base pairs, about 18 to about 24 base pairs, and about 20 to about 22 base pairs in length, and is typically about 10 base pairs, about 13 base pairs, about 15 base pairs, about 17 base pairs, about 18 base pairs, about 19 base pairs, about 20 base pairs, about 21 base pairs, about 22 base pairs, about 25 base pairs, about 30 base pairs, about 40 base pairs, about 50 base pairs, or about 60 base pairs in length. Furthermore, the terms "small activating RNA," "saRNA," and "small activating ribonucleic acid" also encompass nucleic acids other than ribonucleotides, including but not limited to modified nucleotides or analogs.

[0058] As used herein, the term "complementary" refers to the ability of two oligonucleotide strands to form base pairs between them. These base pairs are typically formed through hydrogen bonds between nucleotides in anti-parallel oligonucleotide strands. The bases of complementary oligonucleotide strands can pair in a Watson-Crick fashion (such as A with T, A with U, and C with G) or in any other way that allows a duplex to form (such as Hoogsteen or anti-Hoogsteen base pairing).

[0059] The terms "conjugated oligonucleotide" or "oligonucleotide conjugate" are used interchangeably and refer to a chimeric oligonucleotide molecule that includes a targeting oligonucleotide and a non-targeting moiety (e.g., capable of facilitating delivery of the targeting oligonucleotide). Targeting oligonucleotides include, but are not limited to, double-stranded nucleic acid molecules of DNA, RNA, or DNA / RNA hybrids, oligonucleotide chains containing regularly and irregularly alternating deoxyribosyl moieties and / or ribosyl moieties, and modified and naturally or non-naturally occurring backbones of such oligonucleotides. The targeting oligonucleotides as disclosed herein can be small inhibitory nucleic acid molecules (siRNAs), small activating nucleic acid molecules (saRNAs), or antisense oligonucleotide molecules (ASOs). In particular, the oligonucleotide conjugates described herein for inhibiting mRNA transcript levels of a target gene are non-targeting moiety-conjugated siRNA molecules, and the oligonucleotide agents described herein for activating transcription of a target gene are non-targeting moiety-conjugated saRNA molecules.

[0060] As used herein, the term "non-targeting" means that the referenced ancillary oligonucleotide (ACO) conjugated to a targeting oligonucleotide (e.g., siRNA, saRNA, etc.) is not specifically complementary to the target sequence at which the targeting oligonucleotide functions, and / or the referenced oligonucleotide (i.e., ACO) does not share the same target sequence at which the targeting oligonucleotide (e.g., siRNA, saRNA, etc.) specifically functions. The targeting oligonucleotides disclosed herein are nucleic acid sequences that are specifically complementary to a target sequence or region thereof. In some embodiments, the term "non-targeting oligonucleotide" can include any referenced oligonucleotide other than a "targeting sequence." In some cases, "specifically complementary" can mean that the complementarity between the targeting oligonucleotide and the target sequence or region thereof is at least about 95%. When the oligonucleotide is administered, the non-targeting oligonucleotide (i.e., ancillary oligonucleotide or "ACO" used interchangeably) is not intended to elicit biological activity via any known mechanism, nor is it intended to elicit activity indicative of ASO (i.e., "mixmer" or "gapmer") function on a complementary nucleic acid sequence (i.e., mRNA) in a particular subject, organ of a subject, tissue of a subject, or cell of a subject. When the oligonucleotide conjugate is administered, the non-targeting oligonucleotide (i.e., ACO) is intended to facilitate introduction of its conjugated targeting oligonucleotide (e.g., siRNA, saRNA, etc.) into a particular subject, organ of a subject, tissue of a subject, cell of a subject, or nucleus of a cell of a subject.

[0061] Oligonucleotides can also include one or more conjugate moieties, such as to facilitate cellular uptake of the oligonucleotide. In certain embodiments, the conjugate moiety alters one or more properties of the attached oligonucleotide, including but not limited to pharmacokinetics, pharmacodynamics, stability, binding, absorption, tissue distribution, cellular uptake, charge, and clearance. In certain embodiments, the conjugate moiety imparts a new property to the attached oligonucleotide, such as a fluorophore or reporter group that enables detection of the oligonucleotide.

[0062] In some embodiments, the oligonucleotide agent is conjugated to one or more conjugate moieties selected from the group consisting of an intercalator, a reporter molecule, a polyamine, a polyamide, a peptide, a carbohydrate, a vitamin moiety, a polyethylene glycol, a sulfide, a polyether, a cholesterol, a thiocholesterol, a cholic acid moiety, a folate, a lipid, a phospholipid, a biotin, a phenazine, a phenanthridine, an anthraquinone, an adamantane, an acridine, a fluorescein, a rhodamine, a coumarin, a fluorophore, and a dye.

[0063] In some embodiments, the conjugate moiety comprises an active pharmaceutical agent, such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, daniscofalamine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, benzothiadiazide, chlorothiazide, diazepine, indomethacin, barbiturate, cephalosporin, sulfonamide, anti-diabetic drug, anti-bacterial agent, or antibiotic. In some embodiments, the oligonucleotide is conjugated to one or more conjugate moieties selected from the group consisting of a lipid, a fatty acid, a fluorophore, a ligand, a sugar, a peptide, and an antibody. For example, a lipid or fatty acid conjugate moiety can comprise a saturated or unsaturated carbon chain having 4 to 30 carbon atoms, such as a saturated or unsaturated C4, C6, C8, C 10 , C 12 , C 14 , C 16 , C 18 , C 20 , C 22 or C 24 carbon chain.

[0064] In certain embodiments, the one or more conjugate moieties are derived from C5x5: (C5x5) shown in this application.

[0065] According to some embodiments, the oligonucleotide may further include one or more linker moieties or adapters. As used herein, the terms "linker moieties" or "adapters" refer to molecules used for covalently linking two molecules (e.g., a non-targeted portion and dsRNA (e.g., siRNA or saRNA), two dsRNAs, etc.). As described in more detail below, the term may include, for example, nucleic acid adapters, peptide adapters, etc., and also includes disulfide adapters. When a linker moieties or adapters are present, the linker moieties or adapters may be selected from the group consisting of: -O-, -S-, -C(O)-, -NH-, -N((Cl-C) ...C((Cl-C)-C)-, -N((Cl-C)-C)-C((Cl-C)-C)-C((Cl-C)-C)-C((Cl-C)-C)- 12 )alkyl)-、-N((C1-C 12 )alkyl)-C(O)-O-, -OC(O)-, -C(O)-O-, -OC(O)-O-, -C(O)-NH-, -OP(O)2O-, -P(O)(O - )O-, -OP(O)O-, -P(O)-O-, -OP(O)(S)O-, -OS(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-C 22 )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 22alkylene-C(O)-, -NH-C(O)-(Ci-C 22 alkylene-C(O)-NH-, -C(O)-NH-(Ci-C 22 alkylene-NH-C(O)-, -(Ci-C 22 alkylene-OP(O)2O-, -(Ci-C 22 alkylene-OP(O)(O - )-O-, -(Ci-C 22 alkylene-OP(O)(O - )-O-(Ci-C 22 alkylene-, -(Ci-C 22 alkylene-OP(O)O-, -(Ci-C 22 alkylene-OP(O)(S)O-, -(Ci-C 22 alkylene-O-S(O)2-O-, -(Ci-C 22 alkylene-S(O)2-O-, -(Ci-C 22 alkylene-S(O)-O-, -O-P(O)2-O-(Ci-C 22 alkylene-OP(O)2O-, -O-P(O)-O-(Ci-C 22 alkylene-OP(O)O-, -OP(O)(S)O-(Ci-C 22 alkylene-OP(O)(S)O-, -O-S(O)2-O-(Ci-C 22 alkylene-O-S(O)2-O-, -S(O)2-O-(Ci-C 22 alkylene-S(O)2-O-, and -O-S(O)-(Ci-C 22 alkylene-S(O)-O-; wherein the -(Ci-C 22 alkylene- comprised in the linking moiety can be an alkylene comprising 1 to 22 carbon atoms, such as 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 moiety is a direct bond, the conjugation moiety is directly linked to the oligonucleotide. For example, a conjugation moiety derived from C5x5 can be conjugated to the 5'-end of the nucleotide chain via -OP(O)2O- or -P(O)-O-.

[0066] All nucleotides of the oligonucleotides described herein can be natural nucleotides, i.e., non-chemically modified nucleotides, or at least one nucleotide can be chemically modified. Non-limiting examples of chemical modifications can include one or a combination of modification to the phosphodiester bond of the nucleotides in the nucleotide sequence of the functional oligonucleotide, modification to the 2’-OH of the ribose in the nucleotide sequence of the functional oligonucleotide, and modification to the base in the nucleotide sequence of the functional oligonucleotide. These modifications can increase the bioavailability of the oligonucleotide, increase the affinity to the target sequence, and enhance the resistance to nuclease hydrolysis in the cell.

[0067] Chemical modifications of the nucleotides or oligonucleotides in the present disclosure are well known to those skilled in the art, and modification of the phosphodiester bond can refer to modification of the oxygen in the phosphodiester bond, including phosphorothioate modification and boronated phosphate modification. Both modifications stabilize the oligonucleotide structure, thereby maintaining high specificity and high affinity to base pairing. Modification of the ribose refers to modification to the 2’-OH in the pentose sugar of the nucleotide, i.e., introducing certain substituents at the hydroxyl position of the ribose, such as 2’-fluoro modification, 2’-oxymethyl modification, 2’-oxyethylmethyl modification, 2,4’-dinitrophenol modification, locked nucleic acid (LNA), 2’-amino modification, 2’-deoxy modification. Modification of the base refers to modification of the base of the nucleotide, such as 5’-bromouracil modification, 5’-iodouracil modification, N-methyluracil modification, 2,6-diaminopurine modification.

[0068] As used herein, the term “synthetic” refers to the manner in which the oligonucleotide is synthesized, including any manner that enables synthesis or chemical modification of RNA, such as chemical synthesis, in vitro transcription, vector expression, and the like.

[0069] As used herein, the terms “subject” and “individual” are used interchangeably herein to mean any living organism that can be treated with the agents of the present application. The term “patient” means a human subject or individual, including infants, children, and adults.

[0070] The term “amyotrophic lateral sclerosis” or “ALS” includes, but is not limited to, familial ALS (fALS), sporadic ALS (sALS), Lou Gehrig’s disease, diseases associated with the following mutant genes: chromosome 9 open reading frame 72 gene (C9orf72; 40%), superoxide dismutase 1 (SOD1; 20%), transactive DNA-binding protein 43 (TDP43; 4%), and fused in sarcoma / translocated in liposarcoma mutant gene (FUS / TLS; 4%).

[0071] In certain embodiments of the present application, a target gene is SOD1“Target sequence” means a segment of sequence that is homologous or complementary to the sense strand or antisense oligonucleotide of an siRNA or saRNA. For example, in certain embodiments, the SOD1 siRNA is homologous or complementary to a target selection sequence in a SOD1 transcript. In certain embodiments, the sequence of the sense strand of the SOD1 siRNA is set forth in SEQ ID NO: 1, and the sequence of the antisense strand of the SOD1 siRNA is set forth in SEQ ID NO: 2. SOD1 “Target sequence” means a segment of sequence that is homologous or complementary to the sense strand or antisense oligonucleotide of an siRNA or saRNA. For example, in certain embodiments, the SOD1 siRNA is homologous or complementary to a target selection sequence in a SOD1 transcript. In certain embodiments, the sequence of the sense strand of the SOD1 siRNA is set forth in SEQ ID NO: 1, and the sequence of the antisense strand of the SOD1 siRNA is set forth in SEQ ID NO: 2. “Target sequence” means a segment of sequence that is homologous or complementary to the sense strand or antisense oligonucleotide of an siRNA or saRNA. For example, in certain embodiments, the SOD1 siRNA is homologous or complementary to a target selection sequence in a SOD1 transcript. In certain embodiments, the sequence of the sense strand of the SOD1 siRNA is set forth in SEQ ID NO: 1, and the sequence of the antisense strand of the SOD1 siRNA is set forth in SEQ ID NO: 2.

[0072] “Target sequence” means a segment of sequence that is homologous or complementary to the sense strand or antisense oligonucleotide of an siRNA or saRNA. For example, in certain embodiments, the SOD1 siRNA is homologous or complementary to a target selection sequence in a SOD1 transcript. In certain embodiments, the sequence of the sense strand of the SOD1 siRNA is set forth in SEQ ID NO: 1, and the sequence of the antisense strand of the SOD1 siRNA is set forth in SEQ ID NO: 2. FUS “Target sequence” means a segment of sequence that is homologous or complementary to the sense strand or antisense oligonucleotide of an siRNA or saRNA. For example, in certain embodiments, the SOD1 siRNA is homologous or complementary to a target selection sequence in a SOD1 transcript. In certain embodiments, the sequence of the sense strand of the SOD1 siRNA is set forth in SEQ ID NO: 1, and the sequence of the antisense strand of the SOD1 siRNA is set forth in SEQ ID NO: 2. FUS “Target sequence” means a segment of sequence that is homologous or complementary to the sense strand or antisense oligonucleotide of an siRNA or saRNA. For example, in certain embodiments, the SOD1 siRNA is homologous or complementary to a target selection sequence in a SOD1 transcript. In certain embodiments, the sequence of the sense strand of the SOD1 siRNA is set forth in SEQ ID NO: 1, and the sequence of the antisense strand of the SOD1 siRNA is set forth in SEQ ID NO: 2. “Target sequence” means a segment of sequence that is homologous or complementary to the sense strand or antisense oligonucleotide of an siRNA or saRNA. For example, in certain embodiments, the SOD1 siRNA is homologous or complementary to a target selection sequence in a SOD1 transcript. In certain embodiments, the sequence of the sense strand of the SOD1 siRNA is set forth in SEQ ID NO: 1, and the sequence of the antisense strand of the SOD1 siRNA is set forth in SEQ ID NO: 2.

[0073] “Target sequence” means a segment of sequence that is homologous or complementary to the sense strand or antisense oligonucleotide of an siRNA or saRNA. For example, in certain embodiments, the SOD1 siRNA is homologous or complementary to a target selection sequence in a SOD1 transcript. In certain embodiments, the sequence of the sense strand of the SOD1 siRNA is set forth in SEQ ID NO: 1, and the sequence of the antisense strand of the SOD1 siRNA is set forth in SEQ ID NO: 2. “Target sequence” means a segment of sequence that is homologous or complementary to the sense strand or antisense oligonucleotide of an siRNA or saRNA. For example, in certain embodiments, the SOD1 siRNA is homologous or complementary to a target selection sequence in a SOD1 transcript. In certain embodiments, the sequence of the sense strand of the SOD1 siRNA is set forth in SEQ ID NO: 1, and the sequence of the antisense strand of the SOD1 siRNA is set forth in SEQ ID NO: 2.

[0074] The effective amount can vary according to factors such as the size and weight of the subject, the type of disease, or the particular agent being applied. For example, the choice of agent being applied can influence the constitution of the "effective amount". One of ordinary skill in the art will be able to study the factors contained herein and determine an effective amount of an agent of the application without undue experimentation.

[0075] The administration regimen can influence the constitution of the effective amount. An agent of the application can be administered to a subject before or after diagnosis of a disease or condition. Furthermore, several separate doses and staggered doses can be administered daily or in succession, or the dose can be continuously infused or can be bolus injected. Furthermore, the dose of an agent of the application can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0076] As used herein, the term "treatment" has the meaning generally understood in the medical arts, and thus does not require a cure or complete remission, and includes any beneficial or desirable clinical outcome. Non-limiting examples of such beneficial or desirable clinical outcomes are prolonged survival as compared to untreated expected survival, alleviation of symptoms including one or more of proximal skeletal muscle weakness and atrophy, inability to sit or walk independently, difficulty swallowing and / or breathing, and the like. As used herein, "preventing" or "delaying" a disease means inhibiting full development of the disease.

[0077] Particular embodiments

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application encompasses.

[0079] The present application is based on the surprising finding that the presence of calcium in formulations comprising oligonucleotides can attenuate acute toxicity induced by oligonucleotide substances in vivo, thereby broadening the safety window of oligonucleotide formulations.

[0080] In the present application, we further screened the range of calcium concentrations in oligonucleotide formulations and found that for non-conjugated oligonucleotides, a calcium concentration in the range of 15 mM to less than 25 mM in the oligonucleotide formulation, and for conjugated oligonucleotides, a calcium concentration in the range of 10 mM to 210 mM (preferably 35 mM to 100 mM) effectively attenuates acute toxicity in vivo, especially in the CNS. Furthermore, calcium can be easily incorporated into oligonucleotides by routine technical means, such as by adding CaCl2 in aCSF solution and dissolving the oligonucleotide sample directly in this solution; or by exchanging sodium with calcium in the original oligonucleotide solution. Furthermore, it is also preferred that the oligonucleotide formulation has an osmolarity in the range of 250 to 350 mOsmol / kg, more preferably an isotonic osmolarity in the range of 280 to 320 mOsmol / kg.

[0081] The oligonucleotide formulation is suitable for carrying various oligonucleotide agents, such as ASOs, duplex RNAs (e.g., siRNAs, saRNAs, or combinations thereof targeting various genes), and related conjugates (such as lipid conjugates or ACO conjugates). Preferably, the oligonucleotide targets a gene associated with a disease or disorder in the CNS, such as spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy (DMD), and the like. SOD1 、 SMN2 、 UTRN, FUS ).

[0082] The present application provides the following specific embodiments: 1. An oligonucleotide formulation, comprising: (a) a calcium-containing solution having a calcium concentration of at least 15 mM in the formulation; and (b) an oligonucleotide agent in the solution.

[0083] 2. The oligonucleotide formulation according to embodiment 1, wherein the calcium concentration in the formulation is from 15 mM to less than 25 mM when the oligonucleotide agent is a non-conjugated oligonucleotide; or

[0084] wherein the calcium concentration in the formulation is from 15 mM to 150 mM when the oligonucleotide agent is a conjugated oligonucleotide; and / or

[0085] wherein the osmolality of the oligonucleotide formulation is in the range of 250 mOsmol / kg to 350 mOsmol / kg.

[0086] 3. The oligonucleotide formulation according to embodiment 1, wherein the oligonucleotide agent comprises a single-stranded oligonucleotide or a double-stranded oligonucleotide or a combination thereof.

[0087] 4. The oligonucleotide formulation according to embodiment 1, wherein the single-stranded oligonucleotide is one or more antisense oligonucleotides (ASOs) selected from gapmers, mixmers, steric blockers, or splice modulators; and / or

[0088] the double-stranded oligonucleotide is one or more duplex RNAs selected from siRNAs or saRNAs, or a combination of siRNAs and / or saRNAs, or conjugates of siRNAs and / or saRNAs, or miRNAs.

[0089] 5. The oligonucleotide formulation according to any one of embodiments 2-4, wherein the conjugated oligonucleotide is conjugated to one or more conjugating moieties; and / or

[0090] wherein the one or more conjugating moieties are selected from the group consisting of lipids, luminophores, ligands, sugars, peptides, and antibodies; and / or

[0091] wherein the conjugation moiety is an auxiliary oligonucleotide (ACO) conjugated to the oligonucleotide; and / or

[0092] wherein the one or more conjugation moieties are conjugated to the oligonucleotide at one or more terminal ends of the oligonucleotide chain or at an internal position of the oligonucleotide.

[0093] 6. The oligonucleotide formulation according to embodiment 5, wherein the one or more conjugation moieties are selected from the group consisting of: a fatty acid, a cell penetrating peptide, a fluorophore, a polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, a glucose and an N-acetylgalactosamine; and / or

[0094] wherein the oligonucleotide species is an oligonucleotide conjugated to an ACO or an oligonucleotide conjugated to a lipid moiety.

[0095] 7. The oligonucleotide formulation according to embodiment 5, wherein the lipid moiety is a C4-C 30 fatty acid moiety.

[0096] 8. The oligonucleotide formulation according to embodiment 5, wherein the lipid moiety comprises a saturated or unsaturated C6, C8, C 10 , C 12 , C 14 , C 16 , C 18 , C 20 , C 22 or C 24 carbon chain.

[0097] 9. The oligonucleotide formulation according to embodiment 4, wherein the conjugation moiety is derived from C5x5: (C5x5).

[0098] 10. The oligonucleotide formulation according to embodiment 9, wherein C5x5 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-.

[0099] 11. The oligonucleotide formulation according to embodiment 5, wherein the auxiliary oligonucleotide (ACO) is a single-stranded oligonucleotide having a length of at least 6 nucleotides; and / or

[0100] wherein the auxiliary oligonucleotide (ACO) is a non-targeting oligonucleotide; and / or

[0101] wherein the helper oligonucleotide (ACO) comprises 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 the backbone, or all of the nucleotides in the backbone are phosphorothioate modified.

[0102] 12. The oligonucleotide formulation according to embodiment 1, wherein the calcium is in a form selected from the group consisting of molecular calcium, ionic calcium, complexed calcium, or any combination thereof.

[0103] 13. The oligonucleotide formulation according to embodiment 1, wherein the calcium is provided by calcium chloride, calcium gluconate, calcium lactate, calcium bicarbonate, calcium dihydrogen phosphate, calcium hydrogen phosphate, and any combination thereof.

[0104] 14. The oligonucleotide formulation according to embodiment 2, wherein when the oligonucleotide species is a non-conjugated oligonucleotide, the concentration of calcium in the formulation is in the range of 15 mM to 24 mM, 23 mM, 22 mM, 21 mM, 20 mM, 19 mM, 18 mM, 17 mM, or 16 mM, or any sub-range or value within the range; and / or

[0105] wherein when the oligonucleotide species is a non-conjugated oligonucleotide, the osmolality of the formulation is in the range of 280 mOsmol / kg to 320 mOsmol / kg.

[0106] 15. The oligonucleotide formulation according to embodiment 2, wherein, when the oligonucleotide species is a conjugated oligonucleotide, the concentration of calcium in the formulation 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 concentration of calcium in the formulation is about 20 mM, 35 mM, 47.5 mM, 60 mM, 82.5 mM, 100 mM, 120 mM; and / or wherein the concentration of calcium in the formulation is about 35 mM, 47.4 mM, 60 mM, 100 mM; and / or wherein the concentration of calcium in the formulation is 15 mM to less than 25 mM; and / or wherein the concentration of calcium in the formulation is in the range of 15 mM to 24 mM, 23 mM, 22 mM, 21 mM, 20 mM, 19 mM, 18 mM, 17 mM, or 16 mM, or any sub-range or value within the range; and / or wherein the osmolarity of the formulation is in the range of 280 mOsmol / kg to 320 mOsmol / kg when the oligonucleotide agent is a conjugated oligonucleotide.

[0107] 16. The oligonucleotide formulation according to any one of embodiments 1-15, wherein at least one nucleotide of the oligonucleotide agent is a chemically modified nucleotide.

[0108] 17. The oligonucleotide formulation according to embodiment 16, wherein the chemically modified nucleotide is located in the antisense strand of the ASO, or in the sense strand, the antisense strand, or both strands of the duplex RNA, or in the ACO; and / or

[0109] wherein the chemically modified nucleotide is a 5’-end, 3’-end, both ends, or internal modified nucleotide of the strand; and / or

[0110] wherein at least 50% of the nucleotides in the oligonucleotide agent are chemically modified.

[0111] 18. The oligonucleotide formulation according to embodiment 16, wherein the chemically modified nucleotide is one or more selected from: a 2’ sugar modification; a base modification; a phosphorothioate (PS) backbone modification; an addition of a 5’-phosphate moiety or a 5-methylcytosine moiety at the 5’-end of the nucleotide strand.

[0112] 19. The oligonucleotide formulation according to embodiment 18, wherein the 2’ sugar modification is one or more selected from: a 2’-fluoro-2’-deoxynucleoside (2’-F) modification, a 2’-O-methyl (2’-O-Me) modification, and a 2’-O-(2-methoxyethyl) (2’-O-MOE) modification; and / or

[0113] wherein the addition of the 5’-phosphate moiety is selected from one or more additions of a (E)-vinylphosphonate moiety at the 5’-end of the nucleotide strand.

[0114] 20. The oligonucleotide formulation according to any one of embodiments 1-19, wherein the formulation further comprises one or more additional excipients selected from a preservative, a wetting agent, an emulsifying agent, a dispersing agent, an isotonic agent, and an antioxidant.

[0115] 21. The oligonucleotide formulation according to embodiment 1, wherein the formulation comprises one or more selected from a salt, a polyol, a sugar, and / or an alcohol; and / or

[0116] wherein the carrier in the formulation is artificial cerebrospinal fluid (aCSF) or water; and / or

[0117] wherein the calcium-containing solution is aCSF or an aqueous solution; and / or

[0118] wherein the formulation comprises one or more components selected from the group consisting of NaCl, KCl, MgS04, KH2P04, NaHC03, glucose, and sucrose.

[0119] 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 MgS04, 0.4-3.0 mM KH2P04, 10-50 mM NaHC03, 1-20 mM glucose, and 1-10 mM sucrose.

[0120] 23. The oligonucleotide formulation according to embodiment 1, wherein the formulation comprises 124 mM NaCl, 2.5 mM KCl, 2.0 mM MgS04, 1.25 mM KH2P04, 26 mM NaHC03, 10 mM glucose, and 4 mM sucrose.

[0121] 24. The oligonucleotide formulation according to embodiment 1, wherein the formulation comprises components selected from the group consisting of Solution Sets Al, Bl, A2, B2, A3, B3, A4, B4, A5, and B5 as listed in Table 17, Table 18, Table 19, Table 20, and Table 21.

[0122] 25. The oligonucleotide formulation according to embodiment 1, wherein the oligonucleotide in the oligonucleotide formulation targets SOD1 gene; and / or

[0123] wherein the oligonucleotide in the oligonucleotide formulation targets FUS gene; and / or

[0124] wherein the oligonucleotide in the oligonucleotide formulation targets one 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 .

[0125] 26. The oligonucleotide formulation according to embodiment 1, wherein the oligonucleotide in the oligonucleotide formulation comprises one or more oligonucleotide strands or strand pairs of the oligonucleotide species of Table 1 and / or Table 12; and / or

[0126] wherein the oligonucleotide in the oligonucleotide formulation comprises one or more oligonucleotide strands or strand pairs having a sequence as set forth in one or more of SEQ ID NOS: 1-15.

[0127] 27. The oligonucleotide formulation of embodiment 25, wherein the oligonucleotide formulation has a calcium concentration in the range of 20 mM to 100 mM; and / or

[0128] wherein the oligonucleotide formulation has an osmolarity in the range of 280 mOsmol / kg to 320 mOsmol / kg; and / or

[0129] wherein the oligonucleotide formulation is an isotonic solution.

[0130] 28. The oligonucleotide formulation of any one of embodiments 1-27, wherein the formulation is in a form suitable for an administration route selected from the group consisting of subcutaneous injection, intravenous injection, intraocular injection, intradermal injection, intramuscular injection, intraperitoneal injection, intratracheal administration, intraadipose administration, intraarticular administration, intrathecal administration, epidural administration, inhalation, intranasal administration, oral administration, sublingual administration, buccal administration, rectal administration, vaginal administration, intracisternal administration, transdermal administration, and topical administration or administration by local delivery.

[0131] 29. A method of making the oligonucleotide formulation of any one of embodiments 1-28, comprising: combining the oligonucleotide material with a calcium-containing material in solution to form the oligonucleotide formulation.

[0132] 30. The method of embodiment 29, wherein the calcium is provided by a calcium salt such as calcium chloride (CaCl2), calcium gluconate, calcium lactate, calcium bicarbonate, calcium dihydrogen phosphate, calcium hydrogen phosphate.

[0133] 31. The method of embodiment 29, wherein the calcium is included in aCSF used to dissolve the oligonucleotide material; and / or

[0134] by exchanging sodium for calcium in a solution comprising the oligonucleotide material; and / or

[0135] wherein when the oligonucleotide material is a non-conjugated oligonucleotide, the concentration of calcium in the formulation is 15 mM to less than 25 mM; or

[0136] wherein when the oligonucleotide material is a conjugated oligonucleotide, the concentration of the calcium in the oligonucleotide formulation is in the range of 15 mM to 150 mM.

[0137] 32. The method of embodiment 29, wherein the formulation is made by adding CaCl2 to aCSF solution and dissolving the oligonucleotide material in the aCSF solution containing CaCl2.

[0138] 33. A product comprising the oligonucleotide formulation of any one of embodiments 1-28.

[0139] 34. The product of embodiment 33, wherein the product is a drug, a vaccine, a diagnostic product, an imaging product, or a kit.

[0140] 35. Use of the oligonucleotide formulation of any one of embodiments 1-28 in the manufacture of a product for treating, preventing, or detecting a disease or condition in a subject.

[0141] 36. A method for treating, preventing, or detecting a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically, preventively, or detectively effective amount of the oligonucleotide formulation of any one of embodiments 1-28.

[0142] 37. The oligonucleotide formulation of any one of embodiments 1-28 for use in treating, preventing, or detecting a disease or condition in a subject.

[0143] 38. The use of embodiment 35, the method of embodiment 36, or the oligonucleotide formulation for use of embodiment 37, wherein the oligonucleotide formulation has reduced acute toxicity in vivo as compared to a reference oligonucleotide formulation in the absence of calcium within a defined concentration range.

[0144] 39. The use of embodiment 35, the method of embodiment 36, or the oligonucleotide formulation for use of embodiment 37, wherein the oligonucleotide formulation has reduced acute toxicity in vivo as compared to a reference oligonucleotide formulation in the absence of calcium within a defined concentration range, and wherein the acute toxicity in vivo is toxicity to the somatic motor nervous system; and / or

[0145] wherein the oligonucleotide formulation is for use in acting on the somatic motor nervous system; and / or

[0146] wherein the oligonucleotide formulation is for use in acting on the central nervous system (CNS); and / or

[0147] the acute toxicity is CNS acute toxicity.

[0148] 40. The use of embodiment 35, the method of embodiment 36, or the oligonucleotide formulation for use of embodiment 37, wherein the subject is in need of a drug for treating the disease or condition, a vaccine for preventing the disease or condition, a diagnostic product for diagnosing the disease or condition, and an imaging product for imaging one or more sites of the disease or condition.

[0149] 41. The use according to embodiment 35, the method according to embodiment 36 or the preparation of oligonucleotides for use according to embodiment 37, wherein the disease or disorder is selected from brain diseases, spinal cord diseases and peripheral neuropathies.

[0150] 42. The use according to embodiment 35, the method according to embodiment 36 or the preparation of oligonucleotides for use according to embodiment 37, wherein the oligonucleotide targets a sequence 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 .

[0151] 43. The use according to embodiment 35, the method according to embodiment 36 or the preparation of oligonucleotides for use according to embodiment 37, wherein 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 tumors, frontotemporal dementia, spinocerebellar diseases, prion diseases, Lafora disease, migraine, schizophrenia, depression, pain and stroke.

[0152] Example

[0153] Some embodiments of the present application will now be described in the following examples, in which all parts and percentages are by weight unless otherwise stated. The scope of the present disclosure is of course not limited to the formulations set out in these examples. Rather, the examples are merely illustrative of the inventive concept.

[0154] All starting materials, reagents and solvents used hereinafter were purchased from commercial sources and used as received unless otherwise stated. Purification of reaction products was carried out by column chromatography using silica gel (200-300 mesh) and hexane / ethyl acetate, DCM / MeOH eluents. Thin layer chromatography (TLC) was performed using pre-coated silica gel GF plates and visualized using KMnO4staining. 1 H NMR spectra were recorded using CDC13with TMS at 400 or 500 MHz (Varian). High resolution mass spectrometry (HRMS) was recorded by ESI or matrix assisted laser desorption / ionization (MALDI) on LC / MS (Agilent Technologies 1260 Infinity II / 6120 Quadrupole) and time-of-flight mass spectrometers.

[0155] Materials and Methods

[0156] Oligonucleotide synthesis

[0157] (1) Synthesis of single-stranded oligonucleotides

[0158] Single-stranded oligonucleotides were synthesized by solid-phase synthesis techniques on a K&A DNA synthesizer (K&A Laborgeraete GbR, Schaffhausen, Germany).

[0159] The starting material is a general solid support or a special solid support, which is commercially available or synthesized as disclosed in the foregoing. Typically, phosphoramidite monomers (0.1 M in acetonitrile or dichloromethane) including various linkers and conjugates are added sequentially to the solid support in a DNA synthesizer to generate the desired full-length oligonucleotide.

[0160] Phosphoramidite addition: Each phosphoramidite addition cycle consists of four chemical reactions, including detritylation, coupling, oxidation / thiolation, and capping. In the first step, a detritylation reaction is performed using 3% dichloroacetic acid (DCA) in DCM for 45 seconds. In the second step, phosphoramidite coupling is performed on all amidites at 12 eq for 6 minutes. In the third step, oxidation is performed by using 0.02 M iodine in THF:pyridine:water (70:20:10, v / v / v) for 1 minute; if a phosphorothioate modification is required, thiolation is performed by using 0.1 M hydriodum in pyridine:ACN (50:50, v / v) for 3 minutes instead of oxidation. In the fourth step, capping is performed using THF:acetic anhydride:pyridine (80:10:10, v / v / v) (CAP A) and N-methylimidazole:THF (10:90, v / v) (CAP B) for 20 seconds. The cycle of four chemical reactions depends on the length of the single-stranded oligonucleotide.

[0161] Deprotection I (nucleobase deprotection): After completion of synthesis, the solid support is transferred to a microcentrifuge tube with a screw cap. For a synthesis scale of 1 pmol, a mixture of 1 mL methylamine and ammonium hydroxide is added. The tube containing the solid support is then heated in an oven at 60-65 °C for 15 minutes, and then allowed to cool to room temperature. The cleavage solution is collected and evaporated to dryness in a Speed-Vac to obtain the oligonucleotide single-stranded crude product.

[0162] Deprotection II (removal of 2'-TBDMS groups): If the crude RNA oligonucleotide still carries 2'-TBDMS groups, it is dissolved in 0.1 mL DMSO. After addition of 1 mL triethylamine trifluoride, the tube is capped and the mixture is shaken vigorously to ensure complete dissolution, then heated in an oven at 65°C for 15 min. The tube is removed from the oven and cooled to room temperature. The solution containing the completely desilylated oligonucleotide is cooled on dry ice. 2 mL ice-cold n-butanol (-20°C) is carefully added in 0.5 mL portions to precipitate the oligonucleotide. The precipitate is filtered, washed with 1 mL ice-cold n-butanol and subsequently dissolved in 0.01 M tris(hydroxymethyl)aminomethanol hydrochloride buffer.

[0163] (2) Purification of single-stranded oligonucleotides

[0164] Purification of single-stranded oligonucleotides was performed on an AKTA explorer 10 equipped with a Source 15Q 4.6 / 100 PE column using 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 in 25 min, Flow rate: 1 mL / min. Purified oligonucleotides were collected and desalted by a HiPrep 26 / 10 desalting column.

[0165] (3) Annealing to form duplex oligonucleotides

[0166] To form duplexes, after generating the desalted purified single-stranded solution, the sense and antisense strands were mixed in equal molar concentrations in equal volumes in a tube. The tube was placed in a heating block at 95°C for 5 min, then cooled to room temperature. The duplex thus obtained was subsequently lyophilized to a powder.

[0167] General Method A: Oligonucleotide substance (OS) calcium to sodium exchange.

[0168] CaCl2(Biochemica, batch number: BD119632-500 g) was weighed out and dissolved in injection medium [e.g. commercially available artificial cerebrospinal fluid (aCSF, TOCRIS, batch number: 57A) or water] to obtain 5 mM, 10 mM, 20 mM, 100 mM calcium solutions, respectively.

[0169] Oligonucleotide material RD-12500 (WuXi Biologies, COA: P220505001-C-MC00759-5-C-Lyo-V04, 6.3% w / w sodium, no calcium) was dissolved in CaCl2solution at the indicated concentration to obtain 20 mg / mL oligonucleotide samples. RD-12500 is a siRNA duplex conjugated to ACO through S9 spacer (Table 1). The resulting sample solution was left for 30 minutes. After that, the sample was loaded onto a desalting column (AKTA, Hiprep 26 / 10) to remove free CaCl2, and the oligonucleotide fraction was collected based on the conductivity signal (only the fraction before the valley point of the conductivity signal curve was collected, which means the lowest or no salt content). The collected oligonucleotide with combined salt was lyophilized and the percentage of sodium (oligonucleotide drug substance is sodium salt) and calcium was measured by inductively coupled plasma mass spectrometry (NSF LABORATORY or Medinoah Company, China). The calcium exchanged oligonucleotide was dissolved in aCSF (TOCRIS, batch number: 57A) to produce the test article (TA) at the indicated concentration for intrathecal (IT) injection. The oligonucleotide sequence of RD-12500 is shown in Table 1.

[0170] Table 1. Oligonucleotide chain sequence and duplex composition

[0171] Note: Capital letters represent RNA; indicates phosphorothioate (PS) backbone modification; f indicates 2'-fluoro; m indicates 2'-O-methyl (2'-OMe); me indicates 2'-O-methoxyethyl (2'MOE); Vp indicates 5'-( E )-vinyl phosphonate; meC indicates 2'-O-methoxyethyl-5-methylcytosine; meU indicates 2'-O-methoxyethyl-5-methyluracil; S9 indicates a 9-atom long triethylene glycol.

[0172] General Method B: Introduction of calcium into an oligonucleotide formulation (OF)

[0173] CaCl2(Bide Pharmatech, batch number: BD119632-500g) was weighed and dissolved in solvent media (e.g., aCSF or water) to obtain 5 mM, 10 mM, 20 mM, 35 mM, 100 mM calcium solutions for intrathecal (IT) injection. Oligonucleotide was dissolved in calcium (Ca) containing solution at the indicated concentration to obtain oligonucleotide formulations.

[0174] Animal experiments

[0175] Female Sprague-Dawley rats (A102, SPF, China) were purchased from Sibeifu Biotechnology Co., Ltd. (Suzhou, Jiangsu, China). All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Charles River Laboratories. Fresh preparations for animal treatment were prepared prior to use by dissolving an aliquot of lyophilized oligonucleotide in aCSF to generate a stock solution for dilution to the intended treatment concentration. Separate aCSF was used as a vehicle control. Animals were randomly assigned to study groups (4-6 rats per group) based on body weight (140-260 g).

[0176] Intrathecal (IT) injection

[0177] Anesthesia was induced by administration of 3.0% isoflurane for 10 minutes continuously in an induction chamber. The hair around the base of the tail injection site was shaved, and the injection site was cleaned with 75% ethanol. The gap between the L5-L6 spinous processes was found, and a 30-gauge needle attached to a microliter syringe containing the appropriate drug preparation was slowly inserted into the intradural space until a tail flick was observed. The needle position was subsequently fixed, and a 30 μL total volume of solution was injected over 1 minute.

[0178] Functional observation battery test (FOB)

[0179] Sprague Dawley rats were administered test articles as a single IT dose. Within 3 hours post-injection, rats were evaluated for motor deficit indicators using the FOB, which 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 assigned based on the following 7 different locations of the rat: (1) tail; (2) hind posture; (3) hind limb; (4) hind paw; (5) forepaw; (6) fore posture; (7) head. Based on each of the 7 different locations, each rat was given a subscore: 0 if there were no motor dysfunction indicators, and 1 if the assessed location was paralyzed. After each location was evaluated, the subscores and mean values for each rat were calculated and summarized.

[0180] Example 1. Preparation of TA2

[0181] In this example, TA2 was prepared by using the following method, as shown in General Method A.

[0182] Oligonucleotide material RD-12500 (WuXi Biologies, COA: P220505001-C-MC00759-5-C-Lyo-V04) was dissolved in 5 mL of 5 mM CaCl2solution to give a 20 mg / mL oligonucleotide sample. The resulting TA solution was allowed to stand for 30 minutes. After that, the sample was loaded onto a desalting column to remove free CaCl2, and the oligonucleotide fraction was collected based on the conductivity signal.Figure 1 (Section 2). The collected oligonucleotides with combined salts were lyophilized and the sodium and calcium percentages were measured by inductively coupled plasma mass spectrometry (NSF LABORTORY, 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).

[0183] Calcium-exchange oligonucleotides were dissolved in aCSF to obtain a TA2 solution with a concentration of 100 mg / mL.

[0184] Example 2. Preparation of TA3

[0185] In this embodiment, TA3 is prepared using the following method.

[0186] Oligonucleotide RD-12500 (WuXi AppTec, COA: P220505001-C-MC00759-5-C-Lyo-V04) was dissolved in lactated Ringer's solution (Phygene, batch number 20221129) to obtain TA3 with a concentration of 100 mg / mL, which was used for IT injection.

[0187] Example 3. Preparation of TA5

[0188] In this embodiment, TA5 is prepared using the following method, as shown in General Method A.

[0189] Oligonucleotide RD-12500 (WuXi AppTec, COA: P220505001-C-MC00655-16-C-Lyo-V01, 5.6% w / w sodium, calcium-free) was dissolved in 5 mL of 10 mM CaCl2 solution to obtain an oligonucleotide sample of 20 mg / mL. The resulting TA solution was allowed to stand for 30 minutes. Then, the sample was loaded onto a desalting column to remove free CaCl2, and the oligonucleotide fractions were collected based on the conductivity signal. Figure 2 (Section 2). The collected oligonucleotides with combined salts were lyophilized and the sodium and calcium percentages were measured by inductively coupled plasma mass spectrometry (Medinoah, China). The results are summarized in Table 2. Na: 5.3%, Ca: 0.9%.

[0190] Calcium-exchange oligonucleotides were dissolved in aCSF to obtain a TA5 solution with a concentration of 200 mg / mL.

[0191] Example 4. Preparation of TA15

[0192] In this embodiment, TA15 is prepared by using the following method, as shown in General Method A.

[0193] Oligonucleotide material RD-12500 was dissolved in 5 mL of 20 mM CaCl2solution to obtain a 50 mg / mL oligonucleotide sample. The resulting TA solution was left to stand for 30 minutes. Afterwards, the sample was loaded onto a desalting column to remove free CaCl2and oligonucleotide fractions were collected based on conductivity signals (Fraction 1), (Fraction 2). The collected oligonucleotide with combined salts was lyophilized and the percentage of sodium and calcium was measured by inductively coupled plasma mass spectrometry (Medinoah, China). The results are summarized in Table 2. Na: 4.5%, Ca: 1.6%. Figure 3

[0194] The calcium exchanged oligonucleotide was dissolved in aCSF to obtain a TA15 solution with a concentration of 200 mg / mL.

[0195] Example 5. Preparation of TA6

[0196] In this example, TA6 was prepared by using the following method, as indicated in General Method A.

[0197] Oligonucleotide material RD-12500 was dissolved in 5 mL of 20 mM CaCl2solution to obtain a 50 mg / mL oligonucleotide sample. The resulting TA solution was left to stand for 30 minutes. Afterwards, the sample was loaded onto a desalting column to remove free CaCl2and oligonucleotide fractions were collected based on conductivity signals (Fraction 1), (Fraction 2). The collected oligonucleotide with combined salts was lyophilized and the percentage of sodium and calcium was measured by inductively coupled plasma mass spectrometry (Medinoah, China). The results are summarized in Table 2. Na: 4.5%, Ca: 1.6%. Figure 4

[0198] The calcium exchanged oligonucleotide was dissolved in aCSF to obtain a TA6 solution with a concentration of 200 mg / mL.

[0199] Example 6. Preparation of TA16

[0200] In this example, TA16 was prepared by using the following method, as indicated in General Method A.

[0201] Oligonucleotide material RD-12500 was dissolved in 5 mL of 20 mM CaCl2solution to obtain a 50 mg / mL oligonucleotide sample. The resulting TA solution was left to stand for 30 minutes. Afterwards, the sample was loaded onto a desalting column to remove free CaCl2and oligonucleotide fractions were collected based on conductivity signals (Fraction 1), (Fraction 2). The collected oligonucleotide with combined salts was lyophilized and the percentage of sodium and calcium was measured by inductively coupled plasma mass spectrometry (Medinoah, China). The results are summarized in Table 2. Na: 4.5%, Ca: 1.6%. Figure 5 ​​, fraction 2). The collected oligonucleotide with combined salts was lyophilized and the percentage of sodium and calcium was measured by inductively coupled plasma mass spectrometry (Medinoah, China). The results are summarized in Table 2. Na: 0.8%, Ca: 5.3%.

[0202] The calcium exchanged oligonucleotide was dissolved in aCSF to give a TA16 solution at a concentration of 200 mg / mL.

[0203] Example 7. Preparation of TA7

[0204] In this example, TA7 was prepared using the following method, as indicated in General Method B.

[0205] A 3.6 mg of CaCl2was weighed into 1 mL of aCSF to give a 35 mM calcium containing aCSF solution.

[0206] The oligonucleotide material sodium RD-12500 was dissolved in the 35 mM calcium containing aCSF solution to give a TA7 solution at a concentration of 200 mg / mL.

[0207] Example 8. Preparation of TA8

[0208] In this example, TA8 was prepared using the following method, as indicated in General Method B.

[0209] A 6.4 mg of CaCl2was weighed into 1 mL of aCSF to give a 60 mM calcium containing aCSF solution.

[0210] The oligonucleotide material sodium RD-12500 was dissolved in the 60 mM calcium containing aCSF solution to give a TA8 solution at a concentration of 200 mg / mL.

[0211] Example 9. Preparation of TA9

[0212] In this example, TA9 was prepared using the following method, as indicated in General Method B.

[0213] A 10.8 mg of CaCl2was weighed into 1 mL of aCSF to give a 100 mM calcium containing aCSF solution.

[0214] The oligonucleotide material sodium RD-12500 was dissolved in the 100 mM calcium containing aCSF solution to give a TA9 solution at a concentration of 200 mg / mL.

[0215] Example 10. Preparation of TA11

[0216] In this example, TA11 was prepared using the following method, as indicated in General Method B.

[0217] Weigh 0.3 mg CaCl2into 1 mL aCSF to obtain a 5 mM calcium-containing aCSF solution.

[0218] Dissolve oligonucleotide substance sodium RD-12500 in the 5 mM calcium-containing aCSF solution to obtain a TA8 solution with a concentration of 200 mg / mL for IT injection. The average osmotic pressure of TA11 is 703 mOsmol / kg (Pharmaceuticals, China).

[0219] Example 11. Preparation of TA12

[0220] In this example, TA12 is prepared by using the following method, as shown in General Method B.

[0221] Weigh 0.8 mg CaCl2into 1 mL solvent aCSF to obtain a 10 mM calcium-containing aCSF solution.

[0222] Dissolve oligonucleotide substance sodium RD-12500 in the 10 mM calcium-containing aCSF solution to obtain a TA12 solution with a concentration of 200 mg / mL for IT injection. The average osmotic pressure of TA12 is 716 mOsmol / kg (Pharmaceuticals, China).

[0223] Example 12. Preparation of TA13

[0224] In this example, TA13 is prepared by using the following method, as shown in General Method B.

[0225] Weigh 2.0 mg CaCl2into 1 mL aCSF to obtain a 20 mM calcium-containing aCSF solution.

[0226] Dissolve oligonucleotide substance sodium RD-12500 in the 20 mM calcium-containing aCSF solution to obtain a TA13 solution with a concentration of 200 mg / mL for IT injection. The average osmotic pressure of TA13 is 739 mOsmol / kg (Pharmaceuticals, China).

[0227] Example 13. Preparation of TA14

[0228] In this example, TA14 is prepared by using the following method, as shown in General Method B.

[0229] Weigh 3.6 mg CaCl2into 1 mL aCSF to obtain a 35 mM calcium-containing aCSF solution.

[0230] Oligonucleotide material Sodium RD-12500 was dissolved in 35 mM calcium containing aCSF solution to get a TA14 solution with a concentration of 200 mg / mL for IT injection. The average osmolality of TA14 was 794 mOsmol / kg (WuXi Biotechnology, China).

[0231] Example 14. Introduction of calcium into oligonucleotide substances by cation exchange (Method A)

[0232] As summarized in Table 2, the calcium concentration in oligonucleotide materials TA5, TA15, TA6 and TA16 was increased from 0.9% to 4% or the calcium exchange percentage was increased from 16% to 71% by using different CaCl2concentrations in Method A or different size exclusion chromatography columns in Method B. In Example 6, TA16 was passed through a size exclusion chromatography column to remove as much sodium as possible, then the oligonucleotide sample was diluted on the column using a 2 M calcium solution to get a 5.3% OS calcium percentage and 84% calcium exchange percentage. When TA16 OS was dissolved in aCSF at 200 mg / mL as an oligonucleotide formulation (OF) solution, the calculated calcium concentration reached an extremely high value of 267.5 mM.

[0233] The calcium to sodium exchange percentage was calculated by Formula I (OS RD-12500 contains 6.3% sodium):

[0234] (Formula I)

[0235] The calculated calcium concentration (mM) was calculated by Formula II (aCSF includes 2.5 mM calcium):

[0236] (Formula II)

[0237] In Formula II, the oligonucleotide concentration refers to the concentration of the oligonucleotide sodium substrate dissolved in the CaCl2solution in mg / mL; OS Ca% (i.e., oligonucleotide substrate calcium%) refers to the value detected by inductively coupled plasma mass spectrometry in Table 2.

[0238] In summary, Method A is effective for preparing oligonucleotide solutions with relatively low calcium concentrations, while Method B is more suitable for preparing oligonucleotide solutions with moderate and controllable calcium concentrations.

[0239] Table 2. Summary of calcium exchange data by Method A

[0240] Note: “-” means not applicable.

[0241] a: The calcium to sodium exchange percentage was calculated by Formula I.

[0242] b: Calcium exchanged OS was dissolved in aCSF to form a 200 mg / mL OF solution. The theoretical calcium concentration of the OF solution was calculated by Equation II.

[0243] Example 15. CNS acute toxicity of calcium exchanged oligonucleotides in IT injected SD rats

[0244] To test the acute toxicity of the oligonucleotide formulations, the indicated test articles (i.e., TA1, TA2, and TA3, see Table 3) were prepared and administered to adult female SD rats (body weight range: 140-260 g) via IT injection at 3 mg / dose (100 mg / mL, 30 μL). aCSF was administered as a vehicle control to establish a baseline FOB. Cage-side clinical signs and FOB were recorded within 3 hours post-IT injection.

[0245] The results are shown in Tables 4 and 5. In the TA1 and TA3 groups, 3 out of 4 rats exhibited acute neurotoxicity characterized by convulsions, abnormal vocalization, abnormal gait, or hind limb weakness. None of the rats in the TA2 group exhibited behavioral abnormalities. The results indicate that calcium exchange reduced the CNS acute toxicity of the oligonucleotide.

[0246] Table 3. Oligonucleotide sample information

[0247] Note: "-" means not applicable.

[0248] n represents the number of rats.

[0249] a: represents the oligonucleotide material (sodium salt) that was dissolved directly in aCSF solution.

[0250] b: represents the oligonucleotide material that was dissolved in aCSF solution by calcium exchange in Example 1.

[0251] c: Lac represents the oligonucleotide material (sodium salt) that was dissolved in lactated Ringer's solution in Example 2.

[0252] d: aCSF contained 2.5 mM calcium.

[0253] e: 35 mM calcium was calculated by Equation II.

[0254] Table 4. Abnormal clinical signs recorded within 3 hours post TA single IT administration

[0255] Note: n represents the number of rats. N means not observed, i.e., no symptoms were observed. Y means yes, i.e., symptoms were observed. "min" represents the duration of the observed symptoms in minutes.

[0256] Table 5. Functional observation battery (FOB) recording within 3 hours after a single IT administration of TA

[0257] Note: n represents the number of rats, 0 represents no motor dysfunction signs, and 1 represents paralysis of the assessed area.

[0258] Example 16. CNS acute toxicity of different calcium exchanged oligonucleotides in IT injected SD rats

[0259] To further test the acute toxicity of the oligonucleotide formulations, the indicated test articles (i.e., TA4, TA5, TA6, TA7, TA8, and TA9, see Table 6) were prepared and administered to adult female SD rats (body weight range: 140-260 g) via IT injection at 6 mg / dose (200 mg / mL, 30 μL). TA4 was prepared by directly dissolving RD-12500 in aCSF and served as a functional observation battery (FOB) positive control. Cage-side clinical signs and FOB were recorded within 3 hours after IT injection.

[0260] The results are shown in Tables 7 and 8. In the TA4 control group, 3 out of 6 rats showed acute neurotoxicity, manifested as severe convulsions, abnormal vocalization, ear and limb pallor, hind limb weakness, abnormal gait, or touch sensitivity, and 1 out of 6 rats died about 1 hour after IT administration. No abnormal clinical signs were observed in the cage-side clinical observation and FOB recording for each of the TA5, TA7, TA8, and TA9 groups. In the TA6 group, when the calcium concentration in the oligonucleotide formulation reached 203 mM, 3 out of 6 rats showed paralysis and did not recover within 3 hours. Table 8 presents the results for TA5, TA7, and TA9.

[0261] This study also showed that both Method A and Method B were effective in preparing oligonucleotide formulations with the desired calcium concentration, and Method B was more reproducible and simpler in process than Method A. Calcium in excess of 10 mM, particularly 20 mM or more, e.g., 35 mM, in the oligonucleotide formulation was able to eliminate most or all of the acute toxicity of the oligonucleotide substrate administered into the CNS via IT injection.

[0262] Table 6. Oligonucleotide sample information

[0263] Note: “-” means not applicable.

[0264] n represents the number of rats.

[0265] a: MA-10 means TA was prepared by Method A and the final calcium concentration was 10 mM.

[0266] b: MB-35 indicates that the TA was prepared by Method B and the final calcium concentration was 35 mM.

[0267] c: 47.5 mM calcium for TA5 and 203 mM calcium for TA6 were calculated by Formula II.

[0268] Table 7. Abnormal clinical symptom recording within 3 hours post single IT administration of TA

[0269] Note: n represents the number of rats. N means no, i.e. no symptoms were observed. Y means yes, i.e. symptoms were observed. “min” means the duration of the observed symptoms. Indicates the time of death.

[0270] Table 8. FOB recording within 3 hours post single IT administration of TA

[0271] Note: n represents the number of rats, 0 means no motor dysfunction signs, 1 means paralysis of the assessed area.

[0272] Example 17. CNS acute toxicity of high calcium concentration oligonucleotides in IT injected SD rats

[0273] To further investigate the optimal calcium concentration to minimize the acute toxicity of oligonucleotide formulations and to verify the reproducibility of the protocol, specified test articles (i.e. TA10, TA11, TA12, TA13 and TA14, see Table 9) were prepared and administered to adult female SD rats (body weight range: 140-260 g) by IT injection at 6 mg / dose (200 mg / mL, 30 pL). TA10 was prepared by directly dissolving oligonucleotide substance RD-12500 (WuXi Biologies, COA: P220505001-C-MC00655-16-C-Lyo-V01, 5.6% w / w sodium, no calcium) in aCSF and used as a neurotoxicity positive control. Cage-side clinical symptoms and FOB were recorded within 3 hours post-IT injection. The results are shown in Table 10 and Table 11. In the TA10 group, 5 out of 6 rats exhibited acute neurotoxicity, including convulsions, abnormal vocalization and abnormal gait. TA10 was consistent with TA4 in the same formulation except for the different oligonucleotide substance batch (sodium content difference within 1% w / w).

[0274] In TA11 group, all rats showed abnormal clinical signs. In TA12 group, 4 out of 6 rats showed abnormal clinical signs, i.e. convulsion, abnormal vocalization and / or abnormal gait. In TA13 group, 2 out of 6 rats showed abnormal clinical signs. Surprisingly, in TA14 group, no abnormal clinical symptoms were observed when the calcium concentration was increased to 35 mM. TA14 is consistent with TA7, both of which were prepared in the same formulation by Method B. TA14 and TA7 showed reproducibility in reducing acute toxicity in two independent experiments. In summary, the results indicated that oligonucleotide formulations with calcium concentration no less than 10 mM, preferably at least 20 mM, more preferably above 35 mM, can effectively reduce the acute toxicity of oligonucleotide substances and / or their formulations.

[0275] Table 9. Information of oligonucleotide samples and their uses

[0276] Notes: “-” means not applicable.

[0277] n represents the number of rats.

[0278] a: MB-5 means TA was prepared by Method B and the final calcium concentration was 5 mM.

[0279] Table 10. Abnormal clinical symptoms record within 3 hours after single IT administration of TA

[0280] Notes: n represents the number of rats.

[0281] N means not observed, i.e. no symptoms were observed.

[0282] Y means yes, i.e. symptoms were observed.

[0283] “min” means the duration of observed symptoms in minutes.

[0284] Severe pain is manifested as hind limb paralysis, complete lack of pain sensation, and no response to painful stimuli (e.g. pinch of the paw). The animal can only move using its forelimbs.

[0285] Table 11. FOB record within 3 hours after single IT administration of TA

[0286] Notes: n represents the number of rats, 0 means no motor dysfunction signs, 1 means the assessed site has been paralyzed.

[0287] Example 18: Preparation of compound C5x5

[0288] In this example, compound C5x5 is prepared by using the following method.

[0289]

[0290] (1) Preparation of compound 42

[0291] To a solution of methyl methyl 2-(4-fluoro-3-nitrophenyl)acetate (compound 41; 17.3 g, 81 mmol, 1.0 eq) and K2CO3(11.2 g, 81 mmol) in dry DMF (200 mL) was added compound 40 (19.56 g, 81 mmol, 1.0 eq) under nitrogen protection. The reaction mixture was stirred at 55 °C for 6 h, then cold water (100 mL) was added. The mixture was extracted with ethyl acetate for three times, then the organic phase was washed with saturated LiCl solution for three times, brine for once, dried over anhydrous Na2SO4, concentrated under reduced pressure to form compound 42 as a yellow oil, which was used in the next step without further purification.

[0292] (2) Preparation of compound 43

[0293] To a solution of compound 42 (35.18 g, 81 mmol, 1.0 eq) in THF / H2O (9:1, 280 mL) was added HCOONH4(30.67 g, 486 mmol, 6.0 eq) and Zn powder (31.78 g, 486 mmol, 6.0 eq) in an ice bath. After 10 min, the reaction mixture was removed to room temperature and stirred overnight. Then the reaction mixture was filtered and concentrated under reduced pressure. Water (200 mL) was added, and the mixture was extracted with ethyl acetate for three times, and the organic phase was washed with brine for once. After drying over anhydrous Na2SO4and concentrating under reduced pressure, compound 43 was formed and used in the next step without further purification. Compound 43 was characterized by mass spectrometry. MW calculated: 404.34; MW found: 405.3 [M+H] + .

[0294] (3) Preparation of compound 45

[0295] To a solution of compound 43 (19.36 g, 48 mmol, 1.0 eq) in EtOH (200 mL) was added 3-((tert-butyldimethylsilyl)oxy)propanal (compound 44; 9.0 g, 48 mmol, 1.0 eq) and AcOH (11 mL, 192 mmol, 4.0 eq) under nitrogen protection. The reaction mixture was stirred at 80 °C overnight, then concentrated under reduced pressure. Then saturated NaHC03solution (100 mL) was added, the mixture was extracted with ethyl acetate three times, the organic phases were combined, washed with brine, dried over Na2S04, and concentrated. The resulting residue compound 45 was used directly in the next step without further purification.

[0296] (4) Preparation of compound 46

[0297] To a solution of compound 45 (10 g, 17.5 mmol, 1.0 eq) in anhydrous THF (50 mL) was added 1 M TBAF in THF (26.3 mL, 26.3 mmol, 1.5 eq) under nitrogen protection. The reaction mixture was stirred at room temperature for 1 h, then concentrated under reduced pressure. Then water (100 mL) was added, the mixture was extracted with DCM three times, the organic phases were combined, washed with brine, dried over Na2S04, and concentrated. The resulting residue was dissolved in 50 mL of pyridine, and to it was added DMTrCl (7.12 g, 21 mmol, 1.2 eq). The reaction mixture was stirred at room temperature for 6 h, then concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1-3% MeOH / DCM) to give compound 46 (8.1 g, 61% yield) as a yellow solid. The product was characterized by mass spectrometry and 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, + . 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).

[0298] (5) Preparation of compound C5x5

[0299] To a solution of compound 46 (2.7 g, 3.55 mmol, 1.0 eq) in dry THF (20 mL) was added LiAIH4(202 mg, 5.33 mmol, 1.5 eq) under nitrogen protection and ice bath. After 10 min, the mixture was transferred to room temperature and stirred for 1 h. Then the reaction was transferred to ice bath and saturated NaKC solution (20 mL) was slowly added to the mixture. After 30 min, the reaction was extracted with Et20 three times, and then the organic phase was combined, washed with brine, dried over Na2S04, and concentrated. The crude product (300 mg, 0.41 mmol, 1.0 eq) was dissolved in dry DCM (5 mL), and then DIPEA (204 μL, 1.23 mmol, 3.0 eq) and 3-((chloro(diisopropylamino)phosphoryl)oxy)propanenitrile (compound 47; 274 μL, 1.23 mmol, 3.0 eq) were added under nitrogen protection at 25 °C. The mixture was stirred for 1 h, extracted with DCM twice, and then washed with brine, dried over anhydrous Na2S04. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, gradient eluent: 1%-5% MeOH / DCM, 1% Et3N) to give compound C5x5 (299 mg, 78% yield) as a colorless oil. The product was characterized by mass spectrometry and 1 H NMR characterization. 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).

[0300] Example 19: Preparation of conjugated oligonucleotides linked to conjugation groups derived from compound C5x5 of the present disclosure

[0301] According to the general synthetic procedure for oligonucleotides described above, C5x5-conjugated oligonucleotides were produced by using C5x5 as the terminal imidate and using a conjugation group derived from compound C5x5.

[0302] An exemplary structure of a lipid-conjugated oligonucleotide is O1 as shown below:

[0303] O1 (compound C5x5 application)

[0304] As can be seen, in the structure of O1, the conjugation group derived from the delivery-enhancing compound C5x5 is linked to a double-stranded RNA (dsRNA) duplex (including but not limited to a saRNA or siRNA) at the 5' end of the sense strand (S) via a linking moiety, such as -OP(O)2O-, -OP(O)(S)O- or -P(O)-O-, where (S) is the sense strand and (AS) is the antisense strand.

[0305] Example 20. Design and synthesis of oligonucleotides

[0306] To further assess the toxicity of oligonucleotide formulations, oligonucleotides listed in Table 12 with full chemical modification (i.e., 2'-O-methyl, 2'-O-methoxyethyl, 5'-( E )-vinylphosphonate or phosphorothioate (PS) backbone modification) and conjugated to ACO or a lipid (i.e., C5x5), respectively, were designed and synthesized. Oligonucleotide formulations were prepared according to General Procedure B.

[0307] Table 12. Oligonucleotide strand sequences and duplex compositions

[0308] Note: capital letters represent RNA; P(O)(S)O-; f represents 2'-fluoro; m represents 2'-O-methyl (2'-OMe); me represents 2'-O-methoxyethyl (2'MOE); Vp represents 5'-(E)-vinylphosphonate; meC represents 2'-O-methoxyethyl-5-methylcytosine; meU represents 2'-O-methoxyethyl-5-methyluracil; S9 represents a 9-atom long triethyleneglycol.

[0309] Example 21. CNS acute toxicity of oligonucleotides dissolved in 35 mM calcium aCSF in IT injected SD rats

[0310] Lipid-conjugated oligonucleotide or duplex-ACO (e.g., siRNA-ACO) compounds generally showed severe acute toxicity at moderate doses within 3 hours post-IT injection (such as shown in Tables 13 and 14). However, in our observation, proper (preferably optimized) calcium concentration in oligonucleotide formulations can greatly mitigate the acute toxicity derived from oligonucleotide substances such as oligonucleotide lipid conjugates (RD-16234 and RD-16237) or siRNA-ACO (RD-16223, RD-16224, RD-16226, RD-16229, RD-16230 and RD-16236).

[0311] To evaluate the acute toxicity of oligonucleotide formulations, the indicated siRNA-ACO (i.e., RD-16223, RD-16224, RD-16226, RD-16229, RD-16230 and RD-16236, see Table 12) and lipid-conjugated oligonucleotide (i.e., RD-16234 and RD-16237, see Table 12) were dissolved in aCSF and 35 mM calcium aCSF, respectively. The indicated oligonucleotides were administered at 6 mg / dose (200 mg / mL, 30 μL) via IT injection into adult female SD rats, respectively. aCSF was used as solvent and as negative control. The cage-side clinical signs and FOB of oligonucleotides dissolved in commercial aCSF (TOCRIS, batch: 57A) or 35 mM calcium aCSF were recorded within 3 hours post-IT injection, as summarized in Tables 13, 15, 14 and 16. In contrast, oligonucleotide formulations dissolved in aCSF containing 35 mM calcium provided reduced CNS acute toxicity, especially for lipid-conjugated oligonucleotides (i.e., RD-16234 and RD-16237).

[0312] Table 13. Abnormal clinical symptom recording within 3 hours post single IT dosing of oligonucleotides in commercial aCSF

[0313] Note: N means no, i.e., no observation. "min" means the duration of the observed symptom. Indicates the time of death. Severe pain was manifested as hind limb paralysis, complete lack of pain sensation, and no response to painful stimuli (e.g., pinch of the paw). The animal was only able to move using its forelimbs.

[0314] Table 14. Abnormal clinical sign recording within 3 hours post single IT dosing of oligonucleotides

[0315] Formulated in aCSF containing 35 mM calcium

[0316] Note: N means not observed. "min" means the duration of the observed symptom.

[0317] Table 15. FOB recording within 3 hours after single IT administration of oligonucleotide in commercial aCSF

[0318] Note: n means the number of rats, 0 means no motor dysfunction indication, 1 means paralysis of the assessed site.

[0319] Table 16. FOB recording within 3 hours after single IT administration of oligonucleotide in commercial aCSF containing 35 mM calcium

[0320] Note: n means the number of rats, 0 means no motor dysfunction indication, 1 means paralysis of the assessed site.

[0321] Example 22. Preparation of DP-10

[0322] In this example, DP-10 was prepared by using the following methods.

[0323] 1) Solution A1 and Solution B1 were prepared according to the following Table 17.

[0324] Table 17. Summary of Scheme A1 and Scheme B1

[0325] 2) In a 15 mL test tube, the prescribed amount of oligonucleotide substance RD-12500 (500 mg, COA: P220505001-C-MC00655-16-C-Lyo-V01) was dissolved in 2.5 mL of A1 solution, and then 1.0 mL of B1 solution 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. The osmotic pressure was measured, and the result was 278 mOsmol / kg. Then 3.21 mg of sodium chloride was added, so that the final concentration of DP-10 was 100 mg / mL, and the osmotic pressure was 305 mOsmol / kg.

[0326] Example 23. Preparation of DP-15

[0327] In this example, DP-15 was prepared by using the following methods.

[0328] 1) Solution A2 and Solution B2 were prepared according to the following Table 18.

[0329] Table 18. Summary of Scheme A2 and Scheme B2

[0330] 2) In a 15 mL test tube, the prescribed amount of oligonucleotide material RD-12500 (500 mg, WuXi, COA: P220505001-C-MC00655-16-C-Lyo-V01) was dissolved in 2.5 mL of A2 solution, and 1.0 mL of B2 solution 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. The osmolarity was measured, and the result was 263 mOsmol / kg. Then 5.12 mg of sodium chloride was added to make the final concentration of DP-15 100 mg / mL, and the osmolarity was 298 mOsmol / kg.

[0331] Example 24. Preparation of DP-20

[0332] In this example, DP-20 was prepared by using the following method.

[0333] 1) Solution A3 and solution B3 were prepared according to the following Table 19.

[0334] Table 19. Summary of Scheme A3 and Scheme B3

[0335] 2) In a 15 mL test tube, the prescribed amount of oligonucleotide material RD-12500 (500 mg, WuXi, COA: P220505001-C-MC00655-16-C-Lyo-V01) was dissolved in 2.5 mL of A3 solution, and 1.0 mL of B3 solution 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. The osmolarity was measured, and the result was 267 mOsmol / kg. Then 4.12 mg of sodium chloride was added to make the final concentration of DP-20 100 mg / mL, and the osmolarity was 297 mOsmol / kg.

[0336] Example 25. Preparation of DP-25

[0337] In this example, DP-25 was prepared by using the following method.

[0338] 1) Solution A4 and solution B4 were prepared according to the following Table 20.

[0339] Table 20. Summary of Scheme A4 and Scheme B4

[0340] 2) In a 15 mL test tube, the prescribed amount of oligonucleotide substance RD-12500 (500 mg, WuXi, COA: P220505001-C-MC00655-16-C-Lyo-V01) was dissolved in 2.5 mL of A4 solution, and 1.0 mL of B4 solution 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. The osmotic pressure was measured, and the result was 249 mOsmol / kg. Then 6.58 mg of sodium chloride was added to make the final concentration of DP-25 100 mg / mL, and the osmotic pressure was 297 mOsmol / kg.

[0341] Example 26. Preparation of DP-35

[0342] In this example, DP-35 was prepared by using the following method.

[0343] 1) Solution A5 and solution B5 were prepared according to the following Table 21.

[0344] Table 21. Summary of Scheme A5 and Scheme B5

[0345] 2) In a 15 mL test tube, the prescribed amount of oligonucleotide substance RD-12500 (500 mg, WuXi, COA: P220505001-C-MC00655-16-C-Lyo-V01) was dissolved in 2.5 mL of A5 solution, and 1.0 mL of B5 solution 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. The osmotic pressure was measured, and the result was 268 mOsmol / kg. Then 4.37 mg of sodium chloride was added to make the final concentration of DP-35 100 mg / mL, and the osmotic pressure was 305 mOsmol / kg.

[0346] Example 27. CNS acute toxicity of oligonucleotide isotonic formulations in IT injected SD rats

[0347] To evaluate the acute toxicity of oligonucleotide formulations, the specified test samples (i.e., DP-10, DP-15, DP-20, DP-25, and DP-35, see Table 22) were prepared and administered to adult female SD rats (body weight range: 140 g-260 g) at 3 mg / dose (100 mg / mL, 30 μL) by IT injection. The preparation of DP-10, DP-15, DP-20, DP-25, and DP-35 is described in Examples 22, 23, 24, 25, and 26, respectively. The cage side clinical symptoms and FOB were recorded within 3 hours after IT injection, and the results are shown in Table 22 and Table 23. No obvious acute toxicity was observed in each group. The results show that oligonucleotide formulations with an isotonic osmotic pressure range of 280 mOsmol / kg to 320 mOsmol / kg can significantly reduce acute toxicity.

[0348] While the application has been particularly shown and described with reference to preferred embodiments and various alternative embodiments, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the application.

[0349] Table 22. Abnormal clinical sign records within 3 hours after single IT dosing of oligonucleotide

[0350] Note: N indicates not observed. "min" indicates duration of observed symptom.

[0351] Table 23. FOB records within 3 hours after single IT dosing of oligonucleotide

[0352] Note: " / " indicates no data.

Claims

1. An oligonucleotide formulation comprising: (a) a calcium-containing solution having a calcium concentration of at least 15 mM in the formulation; and (b) an oligonucleotide material in the solution.

2. The oligonucleotide formulation of claim 1, wherein the calcium concentration in the formulation is 15 mM to less than 25 mM when the oligonucleotide material is a non-conjugated oligonucleotide; or wherein the calcium concentration in the formulation is 15 mM to 150 mM when the oligonucleotide material is a conjugated oligonucleotide; and / or wherein the osmolarity of the oligonucleotide formulation is in the range of 250 mOsmol / kg to 350 mOsmol / kg.

3. The oligonucleotide formulation of claim 1, wherein the oligonucleotide material comprises a single-stranded oligonucleotide or a double-stranded oligonucleotide or a combination thereof.

4. The oligonucleotide formulation of claim 1, wherein the single-stranded oligonucleotide is one or more antisense oligonucleotides (ASOs) selected from gapmers, mixmers, steric blockers, or splice modulators; and / or the double-stranded oligonucleotide is one or more duplex RNAs selected from siRNAs or saRNAs, or a combination of siRNAs and / or saRNAs, or conjugates of siRNAs and / or saRNAs, or miRNAs.

5. The oligonucleotide formulation of any one of claims 2-4, wherein the conjugated oligonucleotide is conjugated to one or more conjugating moieties; and / or wherein the one or more conjugating moieties are selected from lipids, luminophores, ligands, sugars, peptides, and antibodies; and / or wherein the conjugating moiety is an accessory oligonucleotide (ACO) conjugated to the oligonucleotide; and / or wherein the one or more conjugating moieties are conjugated to the oligonucleotide at one or more terminal ends of the oligonucleotide chain or at an internal position of the oligonucleotide.

6. The oligonucleotide formulation of claim 5, wherein the one or more conjugating moieties are selected from fatty acids, cell penetrating peptides, fluorophores, polyethylene glycols, alkaloids, tryptamines, benzimidazoles, quinolones, amino acids, cholesterols, glucose, and N-acetylgalactosamine; and / or wherein the oligonucleotide material is an oligonucleotide conjugated to an ACO or an oligonucleotide conjugated to a lipid moiety.

9. The oligonucleotide formulation of claim 4, wherein the conjugating moiety is derived from C5x5:

10. The oligonucleotide formulation of claim 9, wherein C5x5 is conjugated to the 5' end of the sense strand or the antisense strand in the oligonucleotide through -OP(O)2O-, -OP(O)(S)O-, and / or -P(O)-O-.

11. The oligonucleotide formulation of claim 5, wherein the accessory oligonucleotide (ACO) is a single-stranded oligonucleotide having a length of at least 6 nucleotides; and / or wherein the accessory oligonucleotide (ACO) is a non-targeting oligonucleotide; and / or ​ ​ ​ ​ ​ 7. The oligonucleotide formulation of claim 5, wherein the lipid moiety is C4-C 30 fatty acid moiety.

8. The oligonucleotide formulation of claim 5, wherein the lipid moiety comprises a saturated or unsaturated C6, C8, C 10 , C 12 , C 14 , C 16 , C 18 , C 20 , C 22 or C 24 carbon chain. ​ (C5x5). ​ ​ ​ wherein the ancillary oligonucleotide (ACO) comprises 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 the backbone, or all of the nucleotides in the backbone are phosphorothioate modified.

12. The oligonucleotide formulation of claim 1, wherein the calcium is in a form selected from the group consisting of molecular calcium, ionic calcium, complexed calcium, or any combination thereof.

13. The oligonucleotide formulation of claim 1, wherein the calcium is provided by calcium chloride, calcium gluconate, calcium lactate, calcium bicarbonate, calcium dihydrogen phosphate, calcium hydrogen phosphate, and any combination thereof.

14. The oligonucleotide formulation of claim 2, wherein when the oligonucleotide agent is a non-conjugated oligonucleotide, the concentration of calcium in the formulation is in the range of 15 mM to 24 mM, 23 mM, 22 mM, 21 mM, 20 mM, 19 mM, 18 mM, 17 mM, or 16 mM, or any sub-range or value within the range; and / or wherein when the oligonucleotide agent is a non-conjugated oligonucleotide, the osmolality of the formulation is in the range of 280 mOsmol / kg to 320 mOsmol / kg.

15. The oligonucleotide formulation of claim 2, wherein, when the oligonucleotide agent is a conjugated oligonucleotide, the concentration of calcium in the formulation 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 concentration of calcium in the formulation is about 20 mM, 35 mM, 47.5 mM, 60 mM, 82.5 mM, 100 mM, 120 mM; and / or wherein the concentration of calcium in the formulation is about 35 mM, 47.4 mM, 60 mM, 100 mM; and / or wherein the concentration of calcium in the formulation is 15 mM to less than 25 mM; and / or wherein the concentration of calcium in the formulation is in the range of 15 mM to 24 mM, 23 mM, 22 mM, 21 mM, 20 mM, 19 mM, 18 mM, 17 mM, or 16 mM, or any sub-range or value within the range; and / or wherein when the oligonucleotide agent is a conjugated oligonucleotide, the osmolality of the formulation is in the range of 280 mOsmol / kg to 320 mOsmol / kg.

16. The oligonucleotide formulation of any one of claims 1-15, wherein at least one nucleotide of the oligonucleotide agent is a chemically modified nucleotide.

17. The oligonucleotide formulation of claim 16, wherein the chemically modified nucleotide is in the antisense strand of the ASO, or in the sense strand, the antisense strand, or both strands of the duplexed RNA, or in the ACO; and / or wherein the chemically modified nucleotides are nucleotides modified at the 5' end, 3' end, both ends, or internally of the strand; and / or wherein at least 50% of the nucleotides in the oligonucleotide agent are chemically modified.

18. The oligonucleotide agent of claim 16, wherein the chemically modified nucleotides are one or more selected from: a 2' sugar modification; a base modification; a phosphorothioate (PS) backbone modification; addition of a 5'-phosphate moiety or a 5-methylcytosine moiety at the 5' end of the nucleotide strand.

19. The oligonucleotide agent of claim 18, wherein the 2' sugar modification is one or more selected from: a 2'-fluoro-2'-deoxynucleoside (2'-F) modification, a 2'-O-methyl (2'-O-Me) modification, and a 2'-O-(2-methoxyethyl) (2'-O-MOE) modification; and / or wherein the addition of a 5'-phosphate moiety is selected from one or more of addition of a (E)-vinylphosphonate moiety at the 5' end of the nucleotide strand.

20. The oligonucleotide agent of any one of claims 1-19, wherein the agent further comprises one or more additional excipients selected from preservatives, wetting agents, emulsifying agents, dispersing agents, isotonic agents, and antioxidants.

21. The oligonucleotide agent of claim 1, wherein the agent comprises one or more selected from a salt, a polyol, a sugar, and / or an alcohol; and / or wherein the carrier in the agent is artificial cerebrospinal fluid (aCSF) or water; and / or wherein the calcium-containing solution is aCSF or an aqueous solution; and / or wherein the agent comprises one or more ingredients selected from NaCl, KCl, MgSO4, KH2PO4, NaHCO3, glucose, and sucrose.

22. The oligonucleotide agent of claim 1, wherein the agent 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.

23. The oligonucleotide agent of claim 1, wherein the agent 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.

24. The oligonucleotide agent of claim 1, wherein the agent comprises ingredients selected from solution sets Al, Bl, A2, B2, A3, B3, A4, B4, A5, and B5 as listed in Tables 17, 18, 19, 20, and 21.

25. The oligonucleotide formulation of claim 1, wherein the oligonucleotide in the oligonucleotide formulation targets SOD1 a gene; and / or wherein the oligonucleotide in the oligonucleotide formulation targets FUS a gene; and / or wherein the oligonucleotide in the oligonucleotide formulation targets a nucleic acid encoding a polypeptide 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 .

26. The oligonucleotide formulation of claim 1, wherein, The oligonucleotides in the oligonucleotide formulation comprise one or more oligonucleotide strands or strand pairs in the oligonucleotide material of Table 1 and / or Table 12; and / or wherein the oligonucleotides in the oligonucleotide formulation comprise one or more oligonucleotide strands or strand pairs having a sequence as set forth in one or more of SEQ ID NOs: 1-15.

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

28. The oligonucleotide formulation of any one of claims 1-27, wherein the formulation is in a form suitable for an administration route selected from the group consisting of subcutaneous injection, intravenous injection, intraocular injection, intradermal injection, intramuscular injection, intraperitoneal injection, intratracheal administration, intraadipose administration, intraarticular administration, intrathecal administration, epidural administration, inhalation, intranasal administration, oral administration, sublingual administration, buccal administration, rectal administration, vaginal administration, intracisternal administration, transdermal administration, and topical administration or administration by local delivery.

29. A method of making the oligonucleotide formulation of any one of claims 1-28, comprising: combining the oligonucleotide material with a calcium-containing material in solution to form the oligonucleotide formulation.

30. The method of claim 29, wherein the calcium is provided by a calcium salt such as calcium chloride (CaCl2), calcium gluconate, calcium lactate, calcium bicarbonate, calcium dihydrogen phosphate, calcium hydrogen phosphate.

31. The method of claim 29, wherein the calcium is included in aCSF used to dissolve the oligonucleotide material; and / or incorporating calcium into the solution by exchanging sodium for calcium in the solution comprising the oligonucleotide material; and / or wherein the concentration of calcium in the formulation is 15 mM to less than 25 mM when the oligonucleotide material is a non-conjugated oligonucleotide; or wherein the concentration of calcium in the oligonucleotide formulation is in the range of 15 mM to 150 mM when the oligonucleotide material is a conjugated oligonucleotide.

32. The method of claim 29, wherein the formulation is made by adding CaCl2 to aCSF solution and dissolving the oligonucleotide material in the aCSF solution containing CaCl2.

33. A product comprising the oligonucleotide formulation of any one of claims 1-28.

34. The product of claim 33, wherein the product is a drug, a vaccine, a diagnostic product, an imaging product, or a kit.

35. Use of the oligonucleotide formulation of any one of claims 1-28 in the manufacture of a product for treating, preventing, or detecting a disease or condition in a subject.

36. A method for treating, preventing, or detecting a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically, preventively, or diagnostically effective amount of the oligonucleotide formulation of any one of claims 1-28.

37. The oligonucleotide formulation of any one of claims 1-28 for use in treating, preventing, or detecting a disease or condition in a subject.

38. The use of claim 35, the method of claim 36, or the oligonucleotide formulation for use according to claim 37, wherein the oligonucleotide formulation has reduced acute toxicity in vivo as compared to a reference oligonucleotide formulation in the absence of calcium in the defined concentration range.

39. The use of claim 35, the method of claim 36, or the oligonucleotide formulation for use according to claim 37, wherein the oligonucleotide formulation has reduced acute toxicity in vivo as compared to a reference oligonucleotide formulation in the absence of calcium in the defined concentration range, and wherein the acute toxicity in vivo is toxicity to the somatic motor nervous system; and / or wherein the oligonucleotide formulation is for use in acting on the somatic motor nervous system; and / or wherein the oligonucleotide formulation is for use in acting on the central nervous system (CNS); and / or wherein the acute toxicity is CNS acute toxicity.

40. The use of claim 35, the method of claim 36, or the oligonucleotide formulation for use according to claim 37, wherein the subject is in need of a drug for treating the disease or condition, a vaccine for preventing the disease or condition, a diagnostic product for diagnosing the disease or condition, and an imaging product for imaging one or more sites of the disease or condition.

41. The use of claim 35, the method of claim 36, or the oligonucleotide formulation for use according to claim 37, wherein the disease or condition is selected from the group consisting of a disease of the brain, a disease of the spinal cord, and a peripheral neuropathy. 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 42. The use of claim 35, the method of claim 36, or the oligonucleotide preparation for use of claim 37, wherein the oligonucleotide targets a sequence selected from the group consisting of: TTR ​ ​ and ​ .

43. The use of claim 35, the method of claim 36, or the oligonucleotide preparation for use of claim 37, wherein the disease or disorder is selected from spinal muscular atrophy (SMA), Duchenne 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 disease, prion disease, Lafora disease, migraine, schizophrenia, depression, pain, and stroke.

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