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WO2024255846A1 - Formulation d'oligonucléotides - Google Patents

Formulation d'oligonucléotides Download PDF

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Publication number
WO2024255846A1
WO2024255846A1 PCT/CN2024/099219 CN2024099219W WO2024255846A1 WO 2024255846 A1 WO2024255846 A1 WO 2024255846A1 CN 2024099219 W CN2024099219 W CN 2024099219W WO 2024255846 A1 WO2024255846 A1 WO 2024255846A1
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Prior art keywords
oligonucleotide
formulation
calcium
substance
solution
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Inventor
Longcheng Li
Zubao GAN
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Ractigen Therapeutics
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Ractigen Therapeutics
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/53—Methods for regulating/modulating their activity reducing unwanted side-effects

Definitions

  • the present disclosure relates generally to the field of biopharmaceutics and oligonucleotide therapeutics.
  • the present disclosure relates to formulations of oligonucleotide substance with a calcium-containing solution with specific calcium concentrations, as well as specific oligonucleotide formulations with calcium and other components.
  • Oligonucleotide-based therapeutics has emerged as a promising class of therapeutics for neurological diseases.
  • the administration of oligonucleotides to patients, organs, tissues or cells may elicit or impact various biochemical reactions and thus achieve functions such as silencing, inhibiting, activating, and/or modulating gene expression.
  • Single-stranded ASOs in the form of “gapmer” can be used to suppress gene expression by degrading target mRNA via an RNase H mechanism.
  • Another category of ASOs is steric blockers (i.e., splicing modulators) , which are typically composed uniformly of ribonucleotides and bind to pre-mRNA in the nucleus to alter mRNA splicing by blocking the binding of certain splicing factors to the mRNA.
  • Duplex oligonucleotide or double-stranded RNAs sinclude small interfering RNA (siRNA) and small activating RNA (saRNA) , and microRNA (miRNA) , all of which are loaded to argonaute (AGO) proteins in cytoplasm as the initial step before specifically acting on their targets.
  • siRNA binds to target mRNA mainly in the cytoplasm to down-regulate gene expression post-transcriptionally via the RNA interference (RNAi) mechanism.
  • saRNA targets gene regulatory sequences in the nucleus such as gene promoters to upregulate gene expression at the transcriptional level via the RNA activation (RNAa) mechanism.
  • miRNAs are also duplex RNAs that typically bind to the 3’ untranslated region (3’ UTR) of mRNAs with imperfect complementarity following loading into AGO protein and partially inhibit expression via translational repression or/and mRNA degradation.
  • Oligonucleotides e.g., ASO, siRNA or saRNA
  • 2’ -substituted modification such as 2′-fluoro-2′-deoxynucleoside, 2′-O-methyl, or 2′-O- (2-methoxyethyl) modification
  • backbone modification such as phosphorothioate (PS)
  • PS phosphorothioate
  • hydrophilic moiety such as a fatty acid moiety, cholesterol or lipid
  • the chemically modified (such as fluorinated, PS modified) and/or conjugated (such as conjugated to hydrophilic moiety) oligonucleotide/oligonucleotide substance induce dose-limiting acute toxicity resulting in narrow therapeutic windows or even failed clinical trials.
  • the present application provides novel oligonucleotide formulations, methods for the preparation thereof and use thereof.
  • the formulation comprises (a) a calcium-containing solution comprising calcium in a concentration of at least 15 mM; and (b) an oligonucleotide substance in said solution.
  • the calcium concentration in the formulation is from 15 mM to less than 25 mM.
  • the calcium concentration in the formulation is in a range from 15 mM to 24, 23, 22, 21, 20, 19, 18, 17 or 16 mM, or any sub-range or value in the range.
  • the calcium concentration in the formulation is from 15 mM to 150 mM.
  • the concentration of calcium in the formulations is in a range from 15 mM to 150 mM, such as from 20 mM to 150 mM, from 20 mM to 130 mM, from 20 mM to 120 mM, from 30 mM to 110 mM or from 35 mM to 100 mM, for example the calcium concentration in the formulation is about 20, 35, 47.5, 60, 82.5, 100, 120 mM calcium.
  • the calcium concentration in the formulation is from 15 mM to less than 25 mM, such as in a range from 15 mM to 24, 23, 22, 21, 20, 19, 18, 17 or 16 mM, or any sub-range or value in the range.
  • the oligonucleotide formulation has an osmotic pressure in a range from 250 mOsmol/kg to 350 mOsmol/kg.
  • the oligonucleotide is a single-stranded oligonucleotide (such as an antisense RNA (ASO) selected from a gapmer, a steric blocker or a mixmer) or a double-stranded oligonucleotide (such as a duplex RNA selected from a siRNA, a saRNA, or a miRNA, a combination of siRNA and saRNA, or a conjugate of siRNAs and/or saRNAs) or a combination thereof (such as a combination of a single-stranded oligonucleotide and a double-stranded oligonucleotide, e.g., an oligonucleotide agent comprising a targeting duplex and an single-stranded non-targeting accessory oligonucleotide (ACO) conjugated to each other with or without a linker) .
  • ASO antisense RNA
  • ACO single-
  • the oligonucleotide substance is an oligonucleotide conjugate comprising a duplex RNA in conjugation to an ACO.
  • the oligonucleotide is conjugated to one or more conjugation moiety, such as a lipid (e.g., fatty acid) moiety.
  • the oligonucleotide is conjugated to a moiety derived from C5x5.
  • one or more of the nucleotides in the oligonucleotide substance are modified.
  • one or more of the internucleoside linkages in the oligonucleotide or the ACO are selected from the group consisting of phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates.
  • a method for the preparation of the oligonucleotide formulation comprises combining the oligonucleotide substance with calcium-containing substance in a solution to form the oligonucleotide formulation.
  • the calcium is contained in the oligonucleotide substance by exchanging sodium in a solution comprising the oligonucleotide substance with calcium.
  • the formulation is prepared by adding 35 mM of CaCl 2 in artificial cerebrospinal fluid (aCSF) solution, and dissolving the oligonucleotide substance in the CaCl 2 containing aCSF solution.
  • aCSF artificial cerebrospinal fluid
  • a product comprising the oligonucleotide formulation.
  • the product can be selected from a medicament, a vaccine, a diagnostic product, and an imaging product.
  • oligonucleotide formulation in the manufacturing of a product for treatment, prevention, or detection of a disease or disorder in a subject.
  • the product acts in the central nervous system (CNS) .
  • CNS central nervous system
  • a method for treatment, prevention or detection of a disease or disorder in a subject in need thereof comprising administering the oligonucleotide formulation to the subject is provided.
  • an oligonucleotide formulation of the invention for use in treatment, prevention or detection of a disease or disorder in a subject in need thereof is provided.
  • the oligonucleotide targets genes selected from the group consisting of SOD1, FUS, C9orf72, MAPT (Tau) , APP, SMN2, SCN9A, SCN10A, HTT, p21, UTRN, DUX4, SNCA, ATXN1, ATXN2, ATXN3, SCA1, SCA7, SCA8, UCP1, VEGFA, MeCP2, PRNP, DMPK, TARDBP, and TTR.
  • genes selected from the group consisting of SOD1, FUS, C9orf72, MAPT (Tau) , APP, SMN2, SCN9A, SCN10A, HTT, p21, UTRN, DUX4, SNCA, ATXN1, ATXN2, ATXN3, SCA1, SCA7, SCA8, UCP1, VEGFA, MeCP2, PRNP, DMPK, TARDBP, and TTR.
  • the disease or disorder is a disease or disorder in central neuron system (CNS) .
  • the action of the oligonucleotide on CNS is selected from treatment, prevention and/or diagnosis of a disease or disorder in CNS, or imaging site (s) of a disease or disorder in CNS.
  • the disease or disorder in CNS is selected from cerebral disease, myelopathy, and peripheral neuropathy, such as spinal muscular atrophy (SMA) , Duchenne muscular dystrophy (DMD) , amyotrophic lateral sclerosis (ALS) , Alzheimer disease (AD) and Parkinson disease (PD) , Huntington's disease (HD) , multiple sclerosis (MS) , brain tumors, frontotemporal dementia, spinocerebellar, prion, lafora, migraine, schizophrenia, depression, pain, and apoplexy.
  • SMA spinal muscular atrophy
  • DMD Duchenne muscular dystrophy
  • ALS amyotrophic lateral sclerosis
  • AD Alzheimer disease
  • PD Parkinson disease
  • MS multiple sclerosis
  • brain tumors frontotemporal dementia
  • spinocerebellar spinocerebellar
  • prion lafora, migraine, schizophrenia, depression, pain, and apoplexy.
  • the oligonucleotide formulation and product comprising the same can be used to reduce acute toxicity (such as the acute toxicity in CNS) and expand safety window for the oligonucleotide, and thus having great application prospect.
  • FIG. 1 shows purification of calcium exchange oligonucleotide substance (OS) of TA2 (described in Example 1) from free CaCl 2 salt solution which was desalted using size exclusion column by UV and conductivity signal. Fraction 2 was collected and lyophilized to generate calcium OS of TA2.
  • OS calcium exchange oligonucleotide substance
  • FIG. 2 shows purification of calcium exchange OS of TA5 (described in Example 3) from free CaCl 2 salt solution which was desalted using size exclusion column by UV and conductivity signal. Fraction 2 was collected and lyophilized to generate calcium OS of TA5.
  • FIG. 3 shows purification of calcium exchange OS of TA15 (described in Example 4) from free CaCl 2 salt solution which was desalted using size exclusion column by UV and conductivity signal. Fraction 2 was collected and lyophilized to generate calcium OS of TA15.
  • FIG. 4 shows purification of calcium exchange OS of TA6 (described in Example 5) from free CaCl 2 salt solution which was desalted using size exclusion column by UV and conductivity signal. Fraction 2 was collected and lyophilized to generate calcium OS of TA6.
  • FIG. 5 shows purification of calcium exchange OS of TA16 (described in Example 6) from free CaCl 2 salt solution which was desalted using size exclusion column by UV and conductivity signal. Fraction 2 was collected and lyophilized to generate calcium OS of TA16.
  • the term "calcium” refers to calcium existing in the formulation of the oligonucleotide in any form which is capable of reducing in vivo acute toxicity (such as in central nervous system) and expanding in vivo safety window for the oligonucleotide.
  • the calcium is in a form selected from molecular calcium, ionic calcium and complex calcium or any combinations thereof.
  • the calcium (such as in CaCl 2 ) can be incorporated to the oligonucleotide formulation by adding the same into a solution comprising the oligonucleotide (such as in an aCSF solution) or exchanging sodium in a solution comprising the oligonucleotide (such as the synthesized oligonucleotide substance) .
  • calcium-containing solution in the nucleotide formulation refers to a solution comprising calcium in defined concentration and for carrying or carrying the nucleotide of the formulation.
  • Calcium in the formulation may be provided by a calcium source, such as one or more selected from calcium chloride, calcium gluconate, calcium lactate, calcium bicarbonate, calcium dihydrogen phosphate, calcium hydrogen phosphate and any combinations thereof.
  • the concentration of calcium in the formulation is varied.
  • the calcium concentration in the formulation is from 15 mM to less than 25 mM.
  • the calcium concentration in the formulation is in a range from 15 mM to 24, 23, 22, 21, 20, 19, 18, 17 or 16 mM, or any sub-range or value in the range.
  • the calcium concentration in the formulation is from 15 mM to 150 mM.
  • the concentration of calcium in the formulations is in a range from 15 mM to 150 mM, such as from 20 mM to 150 mM, from 20 mM to 130 mM, from 20 mM to 120 mM, from 30 mM to 110 mM or from 35 mM to 100 mM, for example the calcium concentration in the formulation is about 20, 35, 47.5, 60, 82.5, 100, 120 mM calcium.
  • the calcium concentration in the formulation is from 15 mM to less than 25 mM, such as in a range from 15 mM to 24, 23, 22, 21, 20, 19, 18, 17 or 16 mM, or any sub-range or value in the range. Based on the disclosure of the present application, a person skilled in the art could make adjustment to the concentration of calcium in the oligonucleotide formulation according to the need in practice.
  • oligonucleotide substance refers to a substance consists of an oligonucleotide or comprises 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 the gene.
  • 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 forms of isolated oligonucleotide or conjugated oligonucleotide.
  • the single-stranded oligonucleotide can be an antisense RNA (ASO) selected from a gapmer, a steric blocker or a mixmer.
  • ASO antisense RNA
  • 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 siRNAs and/or saRNAs, or a miRNA.
  • Combinations or conjugations of single-stranded oligonucleotides, double-stranded oligonucleotides or single-stranded and double stranded oligonucleotides may also be used in the formulation of the present application.
  • the present disclosure also comprises an oligonucleotide conjugate comprising a targeting duplex and an accessory oligonucleotide (ACO) conjugated to each other with or without a linker.
  • ACO accessory oligonucleotide
  • gapmer refers to a short DNA antisense oligonucleotide (ASO) structure with modified RNA segments on both sides of the central DNA structure.
  • at least one of the modified RNA segments may comprise one or more of modified nucleotides selected from locked nucleic acids (LNA) , and 2'-OMe or 2'-F modified nucleotides to increase affinity to the target, increase nuclease resistance, reduce immunogenicity, and/or decrease toxicity.
  • a gapmer comprises at least one nucleotide modified with a phosphorothioate (PS) group.
  • PS phosphorothioate
  • the gapmer is designed to hybridize to a target fragment of RNA and silence the gene transcript through the induction of RNase H cleavage.
  • LNA refers to a locked nucleic acid in which the 2'-oxygen and 4'-carbon atoms are joined by an extra bridge.
  • BNA refers to a 2'-O and 4'-aminoethylene bridged nucleic acid that can contain a five-membered or six-membered bridged structure with an N-O linkage.
  • PNA refers to a nucleic acid mimic with a pseudopeptide backbone composed of N- (2-aminoethyl) glycine units with the nucleobases attached to the glycine nitrogen via carbonyl methylene linkers.
  • a mixmer refers to an antisense oligonucleotide (ASO) characterized as a mixture of DNA and chemically modified nucleic acid analogs in structure.
  • ASO antisense oligonucleotide
  • a mixmer is composed of fully modified nucleotides or nucleic acid analogs.
  • a mixmer is designed to bind and mask complementary RNA sequence to sterically block proteins, factors, or other RNAs from interacting with targeted RNA.
  • a mixmer is designed to alter pre-mRNA splicing by displacing the spliceosome.
  • a mixmer is designed to bind and sequester microRNAs (miRNAs) , in which it is adopted yet another name called an "antagomir” or an "anti-miR” .
  • splicing modulator refers to an antisense oligonucleotide (ASO) typically composes uniformly of ribonucleotides and binds to pre-mRNA in the nucleus to alter mRNA splicing, such as by blocking the binding of certain splicing factors to the mRNA (asteric blocker) .
  • ASO antisense oligonucleotide
  • dsRNA e.g., siRNA, saRNA
  • duplex refers to the strand having sequence homology or sequence identity with a fragment of the coding strand of the sequence of a target gene.
  • antisense strand of dsRNA e.g., siRNA, saRNA duplex refers to the strand having sequence complementary with the sense strand.
  • overhang refers to non-base-paired nucleotides at the terminus (5'or 3') of an oligonucleotide strand, which is formed by one strand extending out of the other strand in a double-stranded oligonucleotide.
  • a single-stranded region extending out of the 3'terminus and/or 5'terminus of a duplex is referred to as an overhang.
  • the term “natural overhang” as used herein refers to an overhang which consists of one or more nucleotides identical to or complementary to the corresponding position on the target sequence.
  • gene activation As used herein, the terms “gene activation” , “activating gene expression” , “gene upregulation” and “upregulating gene expression” can be used interchangeably, and means an increase or upregulation in transcription, translation, expression or activity of a certain nucleic acid sequence as determined by measuring the transcription level, mRNA level, protein level, enzymatic activity, methylation state, chromatin state or configuration, translation level or the activity or state in a cell or biological system of a gene. These activities or states can be determined directly or indirectly.
  • “gene activation” or “activating gene expression” refers to an increase in activity associated with a nucleic acid sequence, regardless of the mechanism of such activation. For example, gene activation occurs at the transcriptional level to increase transcription into RNA and the RNA is translated into a protein, thereby increasing the expression of the protein.
  • gene silencing As used herein, the terms “gene silencing” , “knockdown of gene expression” , “gene downregulation” and “downregulating gene expression” can be used interchangeably, and means a decrease or downregulation in transcription, translation, expression or activity of a certain nucleic acid sequence as determined by measuring the transcription level, mRNA level, protein level, enzymatic activity, methylation state, chromatin state or configuration, translation level or the activity or state in a cell or biological system of a gene. These activities or states can be determined directly or indirectly.
  • “gene downregulation” or “downregulating gene expression” refers to a decrease in activity associated with a nucleic acid sequence, regardless of the mechanism of such downregulation. For example, gene downregulation occurs at the transcriptional level to decrease or silence transcription into RNA and the RNA is not translated into a protein, thereby decreasing or silencing the expression of the protein.
  • short interfering RNA can be used interchangeably and refer to a ribonucleic acid molecule that can downregulate, knockdown, or silence target gene expression. It can be a double-stranded nucleic acid molecule. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation. siRNA binds to target mRNA mainly in the cytoplasm to down-regulate gene expression post-transcriptionally via the RNA interference (RNAi) mechanism.
  • RNAi RNA interference
  • siRNAs may be designed to target a gene’s mRNA sequence to silence its expression via the RNAi mechanism, such as SOD1, for maximizing treatment outcomes, e.g., for ALS patients.
  • siRNAs are molecules having endogenous RNA bases or chemically modified nucleotides. The modifications do not abolish cellular activity, but rather impart increased stability and/or increased cellular potency. Examples of chemical modifications include phosphorothioate groups, 2'-deoxynucleotide, 2'-OCH 3 -containing ribonucleotides, 2'-F-ribonucleotides, 2'-methoxyethyl ribonucleotides, combinations thereof and the like.
  • the siRNA can have varying lengths (e.g., 10-200 bps) and structures (e.g., hairpins, single/double strands, bulges, nicks/gaps, mismatches) and are processed in cells to provide active gene silencing.
  • a double-stranded siRNA can have the same number of nucleotides on each strand (blunt ends) or asymmetric ends (overhangs) .
  • An overhang of 1-2 nucleotides, for example, can be present on the sense and/or the antisense strand, as well as present on the 5'-and/or the 3'-ends of a given strand.
  • the length of the siRNA molecule is typically about 10 to about 60, about 10 to about 50, about 15 to about 30, about 17 to about 29, about 18 to about 28, about 19 to about 27, about 20 to about 26, about 21 to about 25, and about 22 to about 24 base pairs, and typically about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 23, about 25, about 30, about 40, or about 50 base pairs.
  • small interfering RNA RNA
  • small activating RNA As used herein, the terms “small activating RNA” , “saRNA” and “small activating ribonucleic acid” can be used interchangeably and refer to a ribonucleic acid molecule that can upregulate target gene expression. It can be a double-stranded nucleic acid molecule composed of a first nucleic acid strand containing a ribonucleotide sequence with sequence homology with the non-coding nucleic acid sequence (such as a promoter and an enhancer) of a target gene and a second nucleic acid strand containing a nucleotide sequence complementary with the first strand.
  • a ribonucleic acid molecule that can upregulate target gene expression. It can be a double-stranded nucleic acid molecule composed of a first nucleic acid strand containing a ribonucleotide sequence with sequence homology with the non-coding nucleic acid sequence (such as a promoter and an enhancer
  • the saRNA can also be comprised of a synthesized or vector-expressed single-stranded RNA molecule that prone to form a hairpin structure by two complementary regions within the molecule, wherein the first region contains a ribonucleotide sequence having sequence homology with the target sequence of a promoter of a gene, and a ribonucleotide sequence contained in the second region is complementary with the first region.
  • the length of the duplex region of the saRNA molecule is typically about 10 to about 60, about 10 to about 50, about 10 to about 40, about 12 to about 30, about 14 to about 28, about 16 to about 26, about 18 to about 24, and about 20 to about 22 base pairs, and typically about 10, about 13, about 15, about 17, about 18, about 19, about 20, about 21, about 22, about 25, about 30, about 40, about 50, or about 60 base pairs.
  • the terms "small activating RNA” , "saRNA” and “small activating ribonucleic acid” also contain nucleic acids other than the ribonucleotide, including, but not limited to, modified nucleotides or analogues.
  • the term “complementary” refers to the capability of forming base pairs between two oligonucleotide strands.
  • the base pairs are generally formed through hydrogen bonds between nucleotides in the antiparallel oligonucleotide strands.
  • the bases of the complementary oligonucleotide strands can be paired in the Watson-Crick manner (such as A to T, A to U, and C to G) or in any other manner allowing the formation of a duplex (such as Hoogsteen or reverse Hoogsteen base pairing) .
  • conjugated oligonucleotide or “oligonucleotide conjugate” can be used interchangeably and refers to a chimeric oligonucleotide molecule comprising a targeting oligonucleotide and a non-targeting moiety which, for example, capable of facilitating delivery of the targeting oligonucleotide.
  • the targeting oligonucleotide includes, but is not limited to, double-stranded nucleic acid molecules of DNA, RNA, or DNA/RNA hybrid, oligonucleotide strands containing regularly and irregularly alternating deoxyribosyl portions and/or ribosyl portions, as well as modified and naturally or unnaturally existing frameworks for such oligonucleotides.
  • the targeting oligonucleotide as disclosed herein may be a small inhibiting nucleic acid molecule (siRNA) , a small activating nucleic acid molecule (saRNA) or an antisense oligonucleotide molecule (ASO) .
  • the oligonucleotide conjugate for inhibiting mRNA transcript level of a target gene described herein is a non-targeting moiety conjugated siRNA molecule
  • the oligonucleotide agent for activating transcription of a target gene described herein is a non-targeting moiety conjugated saRNA molecule.
  • non-targeting means that the referenced accessory oligonucleotide (ACO) which conjugates with the targeting oligonucleotide (e.g., siRNA, saRNA, and etc. ) does not specifically complement to the target sequence which the targeting oligonucleotide functions, and/or that the referenced oligonucleotide (i.e., ACO) does not share the same target sequence which the targeting oligonucleotide (e.g., siRNA, saRNA, and etc. ) specifically attends to function to.
  • the targeting oligonucleotide disclosed herein is a nucleic acid sequence that specifically complements to the target sequence or the region thereof.
  • non-targeting oligonucleotide may comprise any referenced oligonucleotide except the “targeting sequence” .
  • the “specifically complementary” may mean that the complementarity between the targeting oligonucleotide and the target sequence or the region thereof is at least about 95%.
  • the non-targeting oligonucleotide i.e., accessory oligonucleotide, or “ACO” used interchangeably
  • ASO oligonucleotide
  • mRNA complementary nucleic acid sequence
  • the non-targeting oligonucleotide i.e., ACO
  • ACO is to facilitate the introduction of the targeting oligonucleotide (e.g., siRNA, saRNA, and etc. ) it conjugates into a certain subject, an organ of the subject, a tissue of the subject, a cell of the subject, or a cell nucleus of the subject, when the oligonucleotide conjugate is administered.
  • the oligonucleotide may further comprise one or more conjugation moieties, such as to facilitate the entry of the oligonucleotide into cell.
  • conjugation moieties modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance.
  • conjugation moieties impart a new property on the attached oligonucleotide, e.g., fluorophores or reporter groups that enable detection of the oligonucleotide.
  • the oligonucleotide is conjugated to one or more conjugation moieties selected from: intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.
  • conjugation moieties selected from: intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phen
  • a conjugation moiety comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S) - (+) -pranoprofen, carprofen, dansylsarcosine, 2, 3, 5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • an active drug substance for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S) - (+) -pranoprofen,
  • the oligonucleotide is conjugated to one or more conjugation moieties selected from: a lipid, a fatty acid, a fluorophore, a ligand, a saccharide, a peptide, and an antibody.
  • a lipid or fatty acid conjugation moiety may comprise a saturated or unsaturated carbon chain having from 4 to 30 carbon atoms, for example, a saturated or unsaturated C 4 , C 6 , C 8 , C 10 , C 12 , C 14 , C 16 , C 18 , C 20 , C 22 , or C 24 carbon chain.
  • conjugation moieties are derived from C5x5 as shown in the present application:
  • the oligonucleotide may further comprise one or more linking moieties or linkers.
  • linking moiety refers to a molecule for covalently joining two molecules, e.g., a non-targeting moiety and a dsRNA (e.g., siRNA or saRNA) , two dsRNAs, etc.
  • dsRNA e.g., siRNA or saRNA
  • the term can include, e.g., a nucleic acid linker, a peptide linker, and the like and also, includes disulfide linkers.
  • the linking moieties or linker can be selected from the group consisting of -O-, -S-, -C (O) -, -NH-, -N ( (C 1 -C 12 ) alkyl) -, -N ( (C 1 -C 12 ) alkyl) -C (O) -O-, -O-C (O) -, -C (O) -O-, -O-C (O) -O-, -C (O) -NH-, -OP (O) 2 O-, -P (O) (O - ) O-, -OP (O) O-, -P (O) -O-, -OP (O) (S) O-, -O-S (O) 2 -O-, -S (O) 2 -O-, -S (O) -O-, - (C 1 -C 22 ) alkylene-, - (C 1 -C 22
  • the conjugation moiety is directly linked with the oligonucleotide when the linking moiety is a direct bond.
  • a conjugation moiety derived from C5x5 may conjugate to 5'-end of a nucleotide strand via -OP (O) 2 O-or -P (O) -O-.
  • nucleotides of the oligonucleotides described herein may be natural, i.e., non-chemically modified nucleotides or at least one nucleotide may be chemically modified.
  • Non-limiting examples of the chemical modification can include one or a combination of the following: modifications to phosphodiester linkages of nucleotides in the nucleotide sequence of a functional oligonucleotide; modification of the 2 '-OH of ribose in the nucleotide sequence of a functional oligonucleotide; and modifications to bases in the nucleotide sequence of a functional oligonucleotide. These modifications may increase the bioavailability of the oligonucleotides, increase affinity for the target sequence, and enhance resistance to nuclease hydrolysis in a cell.
  • modifications of the phosphodiester bond may refer to modifications of oxygen in the phosphodiester bond, including phosphorothioate modifications and boronated phosphate modifications. Both modifications stabilize the oligonucleotide structure, maintaining high specificity and high affinity for base pairing.
  • the ribose modification refers to a modification of the 2'-OH in a nucleotide pentose, i.e., introduction of certain substituents at the hydroxyl position of the ribose, e.g., 2'-fluoro modification, 2'-oxomethyl modification, 2'-oxyethylenemethoxy modification, 2, 4 '-dinitrophenol modification, locked nucleic acid (LNA) , 2'-amino modification, 2'-deoxy modification.
  • LNA locked nucleic acid
  • base modification it is meant a modification of the base of the nucleotide, e.g., 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, 2, 6-diaminopurine modification.
  • synthetic refers to the manner in which oligonucleotides are synthesized, including any means capable of synthesizing or chemically modifying RNA, such as chemical synthesis, in vitro transcription, vector expression, and the like.
  • the terms “subject” and “individual” are used interchangeably herein to mean any living organism that may be treated with agents of the present application.
  • the term “patient” means a human subject or individual, including infants, children and adults.
  • ALS Amyotrophic lateral sclerosis
  • familial ALS fALS
  • sporadic ALS sALS
  • Lou Gehrig's disease diseases associated with mutant genes Chromosome 9 Open Reading Frame 72 gene (C9orf72; 40%) , superoxide dismutase 1 (SOD1; 20%) , transactive response DNA-binding protein 43 (TDP43; 4%) and fused in sarcoma/translocated in liposarcoma (FUS/TLS; 4%) .
  • the target gene is SOD1.
  • target sequence is meant a sequence fragment to which the sense strand or antisense oligonucleotide of the siRNA or saRNA is homologous or complementary.
  • a SOD1 siRNA is homologous or complementary to a target select sequence within human SOD1 transcript.
  • sequence of the sense strand of the SOD1 siRNA is as set forth in SEQ ID NO: 1
  • sequence of the antisense strand of the SOD1 siRNA is as set forth in SEQ ID NO: 2.
  • the target gene is FUS.
  • a FUS siRNA is homologous or complementary to a target select sequence within human FUS transcript.
  • the strands of the FUS siRNA are fully chemically modified.
  • the sense strand of the FUS siRNA comprises a single-stranded accessory oligonucleotide ACO or a lipid conjugation of C5x5.
  • the sequence of the sense strand of the FUS siRNA is as set forth in SEQ ID NO: 3, 5, 6, 8, 10, 11, 13 or 15, and the sequence of the antisense strand of the FUS siRNA is as set forth in SEQ ID NO: 4, 7, 9, 12 or 14.
  • sequences of the sense strand and the antisense strand in a FUS siRNA is selected from (a) SEQ ID NO: 3 and SEQ ID NO: 4; (b) SEQ ID NO: 5 and SEQ ID NO: 4; (c) SEQ ID NO: 6 and SEQ ID NO: 7; (d) SEQ ID NO: 8 and SEQ ID NO: 9; (e) SEQ ID NO: 10 and SEQ ID NO: 9; (f) SEQ ID NO: 11 and SEQ ID NO: 12; (g) SEQ ID NO: 13 and SEQ ID NO: 14; and (h) SEQ ID NO: 15 and SEQ ID NO: 14.
  • an effective amount refers to an amount sufficient to produce the desired effect.
  • a “therapeutically effective amount” or “an effective amount for treatment” of an agent or a composition is an amount sufficient to achieve a desired therapeutic effect, and therefore does not require cure or complete remission.
  • therapeutic efficacy is an improvement in any of the disease indicators, and a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition/symptom in the treated individual.
  • the effective amount may vary depending on such factors as the size and weight of the subject, the type of illness, or the particular agents of the application. For example, the choice of the agent of the application could affect what constitutes an “effective amount. ”
  • One ordinary skill in the art would be able to study the factors contained herein and make the determination regarding the effective amount of the agents of the application without undue experimentation.
  • the regime of administration may affect what constitutes an effective amount.
  • the agent of the application can be administered to the subject either prior to or after the disease diagnosis or condition. Further, several divided dosages, as well as staggered dosages, can be administered daily or sequentially, or the dose can be continuously infused, or can be a bolus injection. Further, the dosages of the agent (s) of the application could be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • treat refers to inhibiting the full development of a disease.
  • the present application is based on the surprising discovery that the existence of calcium in a formulation comprising oligonucleotide can attenuate acute toxicity in vivo induced by the oligonucleotide substance and thus broadening the safety window of the oligonucleotide formulation.
  • oligonucleotide formulation we further screened calcium concentration range in the oligonucleotide formulation and found for non-conjugated oligonucleotide, a calcium concentration of from 15 mM to less than 25 mM, and for conjugated oligonucleotides, a calcium concentration from 10 mM to 210 mM (preferably 35 mM to 100 mM) in the oligonucleotide formulation, is effective in attenuating acute toxicity in vivo, especially in CNS.
  • the oligonucleotide formulation has an osmotic pressure ranging from 250 to 350 mOsmol/kg, more preferably with an isotonic osmotic pressure ranging from 280 to 320 mOsmol/kg.
  • the oligonucleotide formulation is suitable for carrying various oligonucleotide substances, such as for ASO, duplex RNA (e.g., siRNA, saRNA targeting various genes or the combinations thereof) , and relevant conjugate such as lipid conjugate or ACO conjugate.
  • the oligonucleotides target genes (such as SOD1, SMN2, UTRN, FUS) associated with a disease or disorder in CNS, such as spinal muscular atrophy (SMA) , amyotrophic lateral sclerosis (ALS) , Duchenne muscular dystrophy (DMD) .
  • SMA spinal muscular atrophy
  • ALS amyotrophic lateral sclerosis
  • DMD Duchenne muscular dystrophy
  • An oligonucleotide formulation comprising:
  • the calcium concentration in the formulation is from 15 mM to 150 mM;
  • oligonucleotide formulation has an osmotic pressure in a range from 250 mOsmol/kg to 350 mOsmol/kg.
  • ASO antisense oligonucleotide
  • the double-stranded oligonucleotide is one or more duplex RNA selected from a siRNA or a saRNA, or a combination of siRNA and/or saRNA, or a conjugate of siRNA and/or saRNA, or a miRNA.
  • conjugation moieties are selected from: a lipid, a luminophor, a ligand, a saccharide, a peptide, and an antibody; and/or
  • conjugation moiety is an accessory oligonucleotide (ACO) conjugated to the oligonucleotide; and/or
  • conjugation moieties are conjugated to the oligonucleotide at one or more ends of the oligonucleotide strand or at an internal position of the oligonucleotide.
  • oligonucleotide substance is an oligonucleotide conjugated to an ACO or an oligonucleotide conjugated to a lipid moiety.
  • lipid moiety comprises a saturated or unsaturated C 6 , C 8 , C 10 , C 12 , C 14 , C 16 , C 18 , C 20 , C 22 , or C 24 carbon chain.
  • oligonucleotide formulation according to embodiment 5 wherein the accessory oligonucleotide (ACO) is a single stranded oligonucleotide having at least 6 nucleotides in length; and/or
  • accessory oligonucleotide is a non-targeting oligonucleotide
  • accessory oligonucleotide 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 modification on the backbone, or all the nucleotides in the backbone are phosphorothioate modified.
  • oligonucleotide formulation according to embodiment 2 wherein when the oligonucleotide substance is a non-conjugated oligonucleotide, the calcium concentration in the formulation is in a range from 15 mM to 24, 23, 22, 21, 20, 19, 18, 17 or 16 mM, or any sub-range or value in the range; and/or
  • the osmotic pressure of the formulation is in a range from 280 mOsmol/kg to 320 mOsmol/kg.
  • the calcium concentration in the formulation is in a range from 15 mM to 140 mM, 20 mM to 130 mM, from 20 mM to 120 mM, from 30 mM to 110 mM or from 35 mM to 100 mM; and/or, the calcium concentration in the formulation is about 20, 35, 47.5, 60, 82.5, 100 mM, 120 mM; and/or
  • the calcium concentration in the formulation is about 35, 47.4, 60, 100 mM;
  • the calcium concentration in the formulation is from 15 mM to less than 25 mM;
  • the calcium concentration in the formulation is in a range from 15 mM to 24, 23, 22, 21, 20, 19, 18, 17 or 16 mM, or any sub-range or value in the range;
  • the osmotic pressure of the formulation is in a range from 280 mOsmol/kg to 320 mOsmol/kg.
  • oligonucleotide formulation according to any of embodiments 1-15, wherein at least one nucleotide of the oligonucleotide substance is chemically modified nucleotide.
  • oligonucleotide formulation according to embodiment 16 wherein the chemically modified nucleotide locates in an antisense strand of the ASO, or in a sense strand, an antisense strand, or both strands of the duplex RNA, or in an ACO; and/or
  • chemically modified nucleotide is a nucleotide modified at the 5'end, 3'end, both ends, or in internal part of the strand (s) ;
  • oligonucleotide formulation according to embodiment 16 wherein the chemically modified nucleotide is one or more selected from a 2’s ugar modification; base modification; a phosphorothioate (PS) backbone modification; an addition of a 5'-phosophate moiety or a 5-methyl cytosine moiety at the 5’ end of the nucleotide strand.
  • PS phosphorothioate
  • oligonucleotide formulation according to embodiment 18, wherein the 2’s ugar modification is one or more selected from: 2′-fluoro-2′-deoxynucleoside (2′-F) modification, 2′-O-methyl (2′-O-Me) modification, and 2′-O- (2-methoxyethyl) (2′-O-MOE) modification; and/or
  • addition of a 5'-phosophate moiety is one or more selected from an additional of (E)-vinylphosphonate moiety at the 5’ end of the nucleotide strand.
  • oligonucleotide formulation according to any of embodiments 1-19, wherein the formulation further comprises one or more other excipients selected from preservatives, wetting agents, emulsifying agents, dispersing agents, isotonic agents and antioxidant.
  • oligonucleotide formulation according to embodiment 1, wherein the formulation comprises one or more selected from salt, polyol, saccharide and/or alcohol; and/or
  • the carrier in the formulation is artificial cerebrospinal fluid (aCSF) or water; and/or
  • the calcium-containing solution is an aCSF or aqueous solution
  • the formulation comprises one or more ingredients selected from NaCl, KCl, MgSO 4 , KH 2 PO 4 , NaHCO 3 , glucose and sucrose.
  • oligonucleotide formulation according to embodiment 1, wherein the formulation comprises ingredients selected from the solution groups of A1, B1, A2, B2, A3, B3, A4, B4, A5 and B5, as listed in Table 17, Table 18, Table 19, Table 20 and Table 21.
  • oligonucleotide in the oligonucleotide formulation targets FUS gene
  • oligonucleotide in the oligonucleotide formulation targets genes selected from the group consisting of C9orf72, MAPT (Tau) , APP, SMN2, SCN9A, SCN10A, HTT, p21, UTRN, DUX4, SNCA, ATXN1, ATXN2, ATXN3, SCA1, SCA7, SCA8, UCP1, VEGFA, MeCP2, PRNP, DMPK, TARDBP, and TTR.
  • the oligonucleotide in the oligonucleotide formulation comprises one or more oligonucleotide strands or strand pairs having the sequences as shown in one or more of SEQ ID NOs: 1-15.
  • oligonucleotide formulation has an osmotic pressure in a range from 280 mOsmol/kg to 320 mOsmol/kg;
  • oligonucleotide formulation is an isosmotic solution.
  • oligonucleotide formulation according to any of embodiment 1-27, wherein the formulation is in a form suitable for administration route selected from subcutaneous injection, intravenous injection, intraocular injection, intradermal injection, intramuscular injection, intraperitoneal injection, intratracheal administration, intraadiposal 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 via local delivery.
  • a method of preparing the oligonucleotide formulation of any of embodiments 1-28 comprising:
  • oligonucleotide substance with a calcium-containing substance in a solution to form the oligonucleotide formulation.
  • calcium is incorporated to the solution by exchanging sodium in a solution comprising the oligonucleotide substance with calcium;
  • the oligonucleotide substance is a non-conjugated oligonucleotide
  • the calcium concentration in the formulation is from 15 mM to less than 25 mM
  • the concentration of calcium in the oligonucleotide formulation ranges from 15 mM to 150 mM.
  • a product comprising the oligonucleotide formulation of any of embodiments 1-28.
  • oligonucleotide formulation of any of embodiments 1-28 in the manufacturing of a product for treatment, prevention, or detection of a disease or disorder in a subject.
  • a method for treatment, prevention or detection of a disease or disorder in a subject in need thereof comprising administering the oligonucleotide formulation of any of embodiments 1-28 in a treatment, prevention, or detection effective amount to the subject.
  • embodiment 35 the method of 36, or the oligonucleotide formulation for use of embodiment 37, wherein the oligonucleotide formulation has reduced in vivo acute toxicity as compared to a reference oligonucleotide formulation without the presence of calcium in defined concentration range.
  • embodiment 35 the method of 36, or the oligonucleotide formulation for use of embodiment 37, wherein the oligonucleotide formulation has reduced in vivo acute toxicity as compared to a reference oligonucleotide formulation without the presence of calcium in defined concentration range, and wherein the in vivo acute toxicity is the toxicity to the somatic motor nervous system; and/or
  • oligonucleotide formulation is for acting on somatic motor nervous system
  • oligonucleotide formulation is for acting on central nervous system (CNS) ; and/or
  • the acute toxicity is a CNS acute toxicity.
  • embodiment 35 the method of 36, or the oligonucleotide formulation for use of embodiment 37, wherein the subject is in needs of a medicament for the treatment of the disease or disorder, a vaccine for prevention of the disease or disorder, a diagnostic product for diagnosis of the disease or disorder, and an imaging product for imaging site (s) of the disease or disorder.
  • embodiment 35 the method of 36, or the oligonucleotide formulation for use of embodiment 37, wherein the disease or disorder is selected from cerebral disease, myelopathy, and peripheral neuropathy.
  • embodiment 35 the method of 36, or the oligonucleotide formulation for use of embodiment 37, wherein the oligonucleotide targets genes selected from the group consisting of SOD1, FUS, C9orf72, MAPT (Tau) , APP, SMN2, SCN9A, SCN10A, HTT, p21, UTRN, DUX4, SNCA, ATXN1, ATXN2, ATXN3, SCA1, SCA7, SCA8, UCP1, VEGFA, MeCP2, PRNP, DMPK, TARDBP, and TTR.
  • genes selected from the group consisting of SOD1, FUS, C9orf72, MAPT (Tau) , APP, SMN2, SCN9A, SCN10A, HTT, p21, UTRN, DUX4, SNCA, ATXN1, ATXN2, ATXN3, SCA1, SCA7, SCA8, UCP1, VEGFA, MeCP2, PRNP, DMPK, TARD
  • embodiment 35 the method of 36, or the oligonucleotide formulation for use of embodiment 37, wherein the disease or disorder is selected from spinal muscular atrophy (SMA) , Duchenne and Becker muscular dystrophy (DMD &BMD) , amyotrophic lateral sclerosis (ALS) , Alzheimer disease (AD) , Parkinson disease (PD) , Huntington's disease (HD) , multiple sclerosis (MS) , brain tumors, frontotemporal dementia, spinocerebellar, prion, lafora, migraine, schizophrenia, depression, pain, and apoplexy.
  • SMA spinal muscular atrophy
  • DMD &BMD Duchenne and Becker muscular dystrophy
  • ALS amyotrophic lateral sclerosis
  • AD Alzheimer disease
  • PD Parkinson disease
  • HD Huntington's disease
  • MS multiple sclerosis
  • brain tumors frontotemporal dementia
  • spinocerebellar spinocerebellar
  • prion lafora, migraine, schizophrenia, depression
  • reaction products were purchased from commercial sources and used as received unless stated otherwise. Purification of reaction products was performed with column chromatography using silica gel (200-300 mesh) and eluting agents of hexane/ethyl acetate, DCM/MeOH. Thin layer chromatography (TLC) was carried out using pre-coated silica Gel GF plates and visualized using KMnO 4 staining. 1 H-NMR spectra were recorded at 400 or 500 MHz (Varian) using CDCl 3 with TMS.
  • High-resolution mass spectra were recorded on LC/MS (Agilent Technologies 1260 Infinity II/6120 Quadrupole) and a time-of-flight mass spectrometer by ESI or matrix assisted laser desorption/ionization (MALDI) .
  • Single-stranded oligonucleotide was synthesized on a K&ADNA synthesizer (K&ALaborgeraete GbR, chaafheim, Germany) by a solid phase synthesis technique.
  • the starting material was universal solid support or special solid support commercially available or synthesis as disclosure in previous context.
  • phosphoramidite monomers including various linkers and conjugates were added sequentially onto a solid support in the DNA synthesizer to generate the desired full-length oligonucleotides.
  • each cycle of amidite addition consisted of four chemical reactions including detritylation, coupling, oxidation/thiolation and capping.
  • detritylation was performed by using 3%dichloroacetic acid (DCA) in DCM for 45 seconds.
  • DCA 3%dichloroacetic acid
  • phosphoramidite coupling was conducted for 6 minutes for all amidites by 12 eq.
  • oxidation was performed by using 0.02 M iodine in THF: pyridine: water (70: 20: 10, v/v/v) for 1 minute; if phosphorothioate modification was needed then replace oxidation by thiolation which was carried out with 0.1 M solution of xanthane hydride in pyridine: ACN (50: 50, v/v) for 3 minutes.
  • the capping was performed by using a 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 cycles of four chemical reactions were depended by the length of the single stranded oligonucleotide.
  • Deprotection I (Nucleobase Deprotection): after completion of the synthesis, the solid support was transferred to a screw-cap microcentrifuge tube. For a 1 ⁇ mol synthesis scale, 1 ml of a mixture of methylamine and ammonium hydroxide was added. The tube containing the solid support was then heated in an oven at 60°C to 65°C for 15 min and then allowed to cool to room temperature. The cleavage solution was collected and evaporated to dryness in a speed-vac to provide crude single strand of oligonucleotide.
  • Deprotection II Removal of 2’ -TBDMS Group: if the crude RNA oligonucleotide still carried the 2’ -TBDMS groups, then dissolved it in 0.1 ml of DMSO. After adding 1 ml of triethylamine trihydrofluoride, the tube was capped, and the mixture was shaken vigorously to ensure complete dissolution and then heated in an oven at 65°C for 15 minutes. The tube was removed from the oven and cooled down to room temperature. The solution containing the completely desilylated oligonucleotide was cooled on dry ice.
  • the 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, 2M NaCl, pH 7.5) , gradient: 10%B to 60%B in 25 min, flow rate: 1 ml/min.
  • the purified oligonucleotides were collected and desalting by a HiPrep 26/10 Desalting column.
  • duplex After the generation of desalted purified single-stranded solutions, sense strand and antisense strand were mixed in equal volumes at equimolar concentration in the tube. The tube was placed in a heat block at 95°C for 5 min and then cooled to room temperature. Then, the thus obtained duplex was subsequently lyophilized to powder.
  • CaCl 2 (Bidepharm, Lot number: BD119632-500g) was weighed and dissolved in medium [e.g., commercial artificial cerebrospinal fluid (aCSF, TOCRIS, Batch number: 57A) or water] for injection to obtain calcium solutions of 5 mM, 10 mM, 20 mM or 100 mM, respectively.
  • medium e.g., commercial artificial cerebrospinal fluid (aCSF, TOCRIS, Batch number: 57A) or water
  • Oligonucleotide substance RD-12500 (WuXi STA, COA: P220505001-C-MC00759-5-C-Lyo-V04, 6.3%w/w sodium, no calcium) was dissolved in CaCl 2 solution at the indicated concentrations to obtain 20 mg/mL oligonucleotide sample.
  • RD-12500 is a siRNA duplex conjugated to an ACO via a S9 spacer (Table 1) . The thus obtained sample solution was allowed to stand for 30 minutes.
  • oligonucleotide fraction was collected based on the conductivity signals (only collecting the fraction ahead of the valley points of conductivity signal curve which means lowest or free salt) .
  • the collected oligonucleotide with combinational salt was lyophilized, and the sodium (oligo drug substance is sodium salt) and calcium percentage was measured by inductively coupled plasma mass spectrometry (NSF LABORTORY or Medinoah Company, China) .
  • the calcium exchange oligonucleotide was dissolved in aCSF (TOCRIS, Batch number: 57A) to produce test article (TA) at the indicated concentration for intrathecal (IT) injection.
  • TA test article
  • IT intrathecal
  • RNA *, phosphorothioate (PS) backbone modification
  • f 2'-fluoro
  • m 2'-O-methyl (2'-OMe)
  • me 2'-O-methoxyethyl (2’ -MOE)
  • Vp 5’ -
  • E -vinylphosphonate
  • meC 2'-O-methoxyethyl-5-methyl cytosine
  • meU 2'-O-methoxyethyl-5-methyl uracil
  • S9 9 atoms long triethylene glycolyl.
  • CaCl 2 (Bidepharm, Lot number: BD119632-500g) was weighed and dissolved in solvent medium (e.g., aCSF or water) to obtain calcium solutions of 5 mM, 10 mM, 20 mM, 35 mM, 100 mM for intrathecal (IT) injection, respectively.
  • solvent medium e.g., aCSF or water
  • Oligonucleotide with designated concentration was dissolved in the Ca-containing solution to obtain the oligonucleotide formulation.
  • Sprague-Dawley female rats (A102, SPF, China) were purchased from SPF Biotechnology Co., LTD (Suzhou, Jiangsu, China) . All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Ractigen Therapeutics. Fresh formulations for animal treatments were prepared prior to use by dissolving aliquots of lyophilized oligonucleotide into aCSF to create stock solutions for dilution to the intended treatment concentrations. aCSF alone served as a vehicle control. Animals were randomly assigned into study groups (4-6 rats per group) based on body weight (140 g-260 g) .
  • Anesthesia was introduced via administration of 3.0%isoflurane in an induction chamber for continuous 10 minutes. Hair was shaved around the injection site at the base of the tail and the injection site was then cleaned with 75%ethanol. The space between the L5-L6 spinous processes was identified and a 30-gauge needle attached to a microliter syringe containing the appropriate drug formulations was slowly inserted into the intradural space until a tail flick was observed. The needle position was subsequently secured in which 30 ⁇ L total volume of solution was injected over the course of 1 minute.
  • Sprague Dawley rats were administered with test articles at a single IT dose. Within 3 hours post-injection, rats were evaluated for signs of motor deficit using the FOB, which was developed to provide unbiased assessment of the effects of drugs on central and peripheral nervous system based on movement dysfunction.
  • FOB was assigned based on the following 7 different parts of the rat: (1) tail; (2) posterior posture; (3) hind limbs; (4) hind paws; (5) forepaws; (6) anterior posture; (7) head. Based on each of the 7 different parts, each rat was given a sub-score: 0 if no signs of motor dysfunction, 1 if the evaluated part was paralyzed. The sub-scores and mean values were calculated and summarized for each rat after evaluating each part.
  • TA2 was prepared in this Example by using the following procedures as General Method A.
  • Oligonucleotide substance RD-12500 (WuXi STA, COA: P220505001-C-MC00759-5-C-Lyo-V04) was dissolved in 5 mL 5 mM CaCl 2 solution to obtain 20 mg/mL oligonucleotide sample. The obtained TA solution was allowed to stand for 30 minutes. After that, the sample was loaded on desalting column to remove free CaCl 2 , and oligonucleotide fraction was collected based on conductivity signal (FIG. 1, fraction 2) .
  • the calcium exchange oligonucleotide was dissolved in aCSF to obtain TA2 solution at 100 mg/mL concentration.
  • TA3 was prepared in this Example by using the following procedures.
  • Oligonucleotide substance RD-12500 (WuXi STA, COA: P220505001-C-MC00759-5-C-Lyo-V04) was dissolved in Lactated Ringer's solution (Phygene, lot. 20221129) to get TA3 at 100 mg/mL concentration for IT injection.
  • TA5 was prepared in this Example by using the following procedures as General Method A.
  • Oligonucleotide substance RD-12500 (WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01, 5.6%w/w sodium, no calcium) was dissolved in 5 mL 10 mM CaCl 2 solution to obtain 20 mg/mL oligonucleotide sample. The obtained TA solution was allowed to stand for 30 minutes. After that, the sample was loaded on desalting column to remove free CaCl 2 , and oligonucleotide fraction was collected based on conductivity signal (FIG. 2, fraction 2) .
  • the calcium exchange oligonucleotide was dissolved in aCSF to get TA5 at 200 mg/mL concentration.
  • TA15 was prepared in this Example by using the following procedures as General Method A.
  • the oligonucleotide substance RD-12500 was dissolved in 5 mL 20 mM CaCl 2 solution to obtain 50 mg/mL oligonucleotide sample.
  • the obtained TA solution was allowed to stand for 30 minutes. After that, the sample was loaded on desalting column to remove free CaCl 2 , and oligonucleotide fraction was collected based on the conductivity signal (FIG. 3, fraction 2) .
  • the collected oligonucleotide with combinational salt was lyophilized, followed by characterizing sodium and calcium percentage via inductively coupled plasma mass spectrometry (Medinoah Company, China) . The results are summarized in Table 2. Na: 4.5%, Ca: 1.6%.
  • the calcium exchange oligonucleotide was dissolved in aCSF to get TA15 at 200 mg/mL concentration.
  • TA6 was prepared in this Example by using the following procedures as General Method A.
  • the oligonucleotide substance RD-12500 was dissolved in 5 mL 100 mM CaCl 2 solution to obtain 20 mg/mL oligonucleotide sample.
  • the obtained TA solution was allowed to stand for 30 minutes. After that, the sample was loaded on desalting column to remove free CaCl 2 , and oligonucleotide fraction was collected based on conductivity signal (FIG. 4, fraction 2) .
  • the collected oligonucleotide with combinational salt was lyophilized, followed by characterizing sodium and calcium percentage via inductively coupled plasma mass spectrometry (Medinoah Company, China) . The results are summarized in Table 2. Na: 1.8%, Ca: 4.0%.
  • the calcium exchange oligonucleotide was dissolved in aCSF to obtain TA6 at 200 mg/mL concentration.
  • TA16 was prepared in this Example by using the following procedures as General Method A.
  • the oligonucleotide substance RD-12500 was dissolved in water and loaded on size exclusion column.
  • the sample was desalted by loading the sample on desalting column to remove free CaCl 2 , and oligonucleotide fraction was collected based on conductivity signal (FIG. 5, fraction 2) .
  • the collected oligonucleotide with combinational salt was lyophilized, followed by characterizing sodium and calcium percentage via inductively coupled plasma mass spectrometry (Medinoah Company, China) . The result is summarized in Table 2. Na: 0.8%, Ca: 5.3%.
  • the calcium exchange oligonucleotide was dissolved in aCSF to get TA16 at 200 mg/mL concentration.
  • TA7 was prepared in this Example by using the following procedures as General Method B. 3.6 mg CaCl 2 was weighed and dissolved in 1 mL aCSF to obtain 35 mM calcium-containing aCSF solution.
  • the oligonucleotide substance sodium RD-12500 was dissolved in the 35 mM calcium-containing aCSF solution to get TA7 at 200 mg/mL concentration.
  • TA8 was prepared in this Example by using the following procedures as General Method B. 6.4 mg CaCl 2 was weighed and dissolved in 1 mL aCSF to obtain 60 mM calcium-containing aCSF solution.
  • the oligonucleotide substance sodium RD-12500 was dissolved in the 60 mM calcium-containing aCSF solution to get TA8 at 200 mg/mL concentration.
  • TA9 was prepared in this Example by using the following procedures as General Method B. 10.8 mg CaCl 2 was weighed and dissolved in 1 mL aCSF to obtain 100 mM calcium-containing aCSF solution.
  • the oligonucleotide substance sodium RD-12500 was dissolved in the 100 mM calcium-containing aCSF solution to get TA9 at 200 mg/mL concentration.
  • TA11 was prepared in this Example by using the following procedures as General Method B. 0.3 mg CaCl 2 was weighed and dissolved in 1 mL aCSF to obtain 5 mM calcium-containing aCSF solution.
  • the oligonucleotide substance sodium RD-12500 was dissolved in the 5 mM calcium-containing aCSF solution to get TA8 at 200 mg/mL concentration for IT injection.
  • the mean osmotic pressure of TA11 was 703 mOsmol/kg (WuXi AppTec, China) .
  • TA12 was prepared in this Example by using the following procedures as General Method B. 0.8 mg CaCl 2 was weighed and dissolved in 1 mL solvent aCSF to obtain 10 mM calcium-containing aCSF solution.
  • the oligonucleotide substance sodium RD-12500 was dissolved in the 10 mM calcium-containing aCSF solution to get TA12 at 200 mg/mL concentration for IT injection.
  • the mean osmotic pressure of TA12 was 716 mOsmol/kg (WuXi AppTec, China) .
  • TA13 was prepared in this Example by using the following procedures as General Method B.
  • the oligonucleotide substance sodium RD-12500 was dissolved in the 20 mM calcium-containing aCSF solution to get TA13 at 200 mg/mL concentration for IT injection.
  • the mean osmotic pressure of the TA13 was 739 mOsmol/kg (WuXi AppTec, China) .
  • TA14 was prepared in this Example by using the following procedures as General Method B.
  • the oligonucleotide substance sodium RD-12500 was dissolved in the 35 mM calcium-containing aCSF solution to get TA14 at 200 mg/mL concentration for IT injection.
  • the mean osmotic pressure of the TA14 was 794 mOsmol/kg (WuXi AppTec, China) .
  • Example 14 Introducing calcium to oligonucleotide substance by cation exchange (Method A) As summarized in Table 2, the calcium concentrations in the oligonucleotide substance TA5, TA15, TA6 and TA16 were increased from 0.9%to 4%, or calcium exchange percentage increased from 16%to 71%, by utilizing different CaCl 2 concentrations in Method A or a size exclusion column in Method B. In Example 6, TA16 was subjected to the size exclusion column to remove sodium as much as possible, and then used 2 M calcium solution to dilute the oligonucleotide sample on the column, resulting 5.3%OS calcium percentage and 84%calcium exchange percentage. When dissolved TA16 OS in aCSF as oligonucleotide formulation (OF) solution at 200 mg/mL, the calculated calcium concentration reaches 267.5 mM which is extremely high.
  • OF oligonucleotide formulation
  • the calcium exchange percentage with sodium was calculated by Formula I (OS RD-12500 contains 6.3%sodium) :
  • the calculated calcium concentration (mM) was calculated by Formula II (the aCSF comprises 2.5 mM of calcium) :
  • Oligonucleotide Conc. refers to the concentration of oligonucleotide sodium substrate dissolved in CaCl 2 solution in the unit of mg/mL; OS Ca% (i.e., oligonucleotide substrate calcium %) refers to the value detected by inductively coupled plasma mass spectrometry in Table 2.
  • Method A is effective for preparing oligonucleotide solutions with relative low calcium concentration
  • Method B is more suitable for preparing oligonucleotide solutions with mediate and controllable calcium concentration.
  • Example 15 CNS acute toxicity of oligonucleotide by calcium exchange in IT injected SD rats
  • test articles i.e., TA1, TA2 and TA3, see Table 3
  • TA1, TA2 and TA3 were prepared and administrated into adult female SD rats (body weigh arrange: 140 g-260 g) at 3 mg/dose (100 mg/mL, 30 ⁇ L) via IT injection.
  • aCSF served as a vehicle control to establish baseline FOB.
  • the cage-side clinical symptoms and FOB were recorded within 3 hours after IT injection.
  • Results are shown in Table 4 and Table 5.
  • 3 out of 4 rats showed acute neurological toxicity characterized by convulsion, abnormal vocalization, abnormal gait or hind limb weakness. All rats in TA2 group showed no abnormal behavior.
  • the results suggest that the CNS acute toxicity of oligonucleotide was reduced by calcium exchange.
  • n the number of rats.
  • a represents oligonucleotide substance (sodium salt) directly dissolved in aCSF solution.
  • b represents oligonucleotide substance by calcium exchange was dissolved in aCSF solution in Example 1.
  • Lac represents oligonucleotide substance (sodium salt) dissolved in Lactated Ringer's solution in Example 2.
  • d aCSF contains 2.5 mM calcium.
  • n represents the number of rats, 0 represents no signs of motor dysfunction, 1 represents the evaluated part was paralyzed.
  • Example 16 CNS acute toxicity of oligonucleotide by different calcium exchange in IT injected SD rats
  • test articles i.e., TA4, TA5, TA6, TA7, TA8 and TA9, see Table 6) were prepared and administered into adult female SD rats (body weigh arrange: 140 g-260 g) at 6 mg/dose (200 mg/mL, 30 ⁇ L) via IT injection.
  • TA4 was prepared by directly dissolving RD-12500 in aCSF and served as functional observational battery (FOB) positive control. The cage-side clinical symptoms and FOB were recorded within 3 hours after the IT injection.
  • Results are shown in Table 7 and Table 8.
  • 3 out of 6 rats showed acute neurological toxicity characterized by severe conclusion, abnormal vocalization, ear and limbs paleness, hind limb weakness, abnormal gait, or sensitive to touch, and 1 out of 6 rats died around 1 hour after IT dosing.
  • TA5 TA7, TA8 and TA9 groups
  • no abnormal clinical symptoms were observed in the cage-side clinical observations and FOB record.
  • TA6 group when the calcium concentration in the oligonucleotide formulation reached 203 mM, 3 out of 6 rats showed paralysis which was not recovered within 3 hours.
  • Table 8 shows the result of TA5, TA7 and TA9.
  • Method A and Method B are effective in preparing oligonucleotide formulations with desired calcium concentration, meanwhile Method B is more repeatable and an easier process than Method A. More than 10 mM calcium in the oligonucleotide formulation, especially 20 mM or more, e.g., 35 mM calcium is capable of eliminating most of or all acute toxicity from the oligonucleotide substrate administered into CNS by IT injection.
  • n the number of rats.
  • MA-10 represents TA was prepared by method A and the final calcium concentration was 10 mM.
  • MB-35 represents TA was prepared by method B and the final calcium concentration was 35 mM.
  • Example 17 CNS acute toxicity of oligonucleotide with increased calcium concentration in IT injected SD rats
  • test articles i.e., TA10, TA11, TA12, TA13, and TA14, see Table 9
  • TA10 was prepared by directly dissolving into oligonucleotide substance RD-12500 (WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01, 5.6%w/w sodium, no calcium) in aCSF and served as neurotoxicity positive control.
  • TA10 The cage-side clinical symptoms and FOB were recorded within 3 hours after IT injection. Results are shown in Table 10 and Table 11. In TA10 group, 5 out of 6 rats showed acute neurotoxicity including convulsion, abnormal vocalization, and abnormal gait. TA10 was consistent with TA4 in the same formulation except for different oligonucleotide substance batch (within 1%w/w difference of sodium content) .
  • TA11 group all rats showed abnormal clinical signs.
  • TA12 group 4 out of 6 rats showed abnormal clinical signs, i.e., convulsion, abnormal vocalization and/or abnormal gait.
  • TA13 group 2 out of 6 rats showed abnormal clinical signs.
  • TA14 group when the calcium concentration was increased to 35 mM, no abnormal clinical symptoms were observed.
  • TA14 was consistent with TA7, both of which prepared in the same formulation by Method B) .
  • TA14 and TA7 showed reproducibility in reducing acute toxicity in two independent experiments.
  • oligonucleotide formulations with a calcium concentration of no less than 10 mM, preferably at least 20 mM, more preferably 35 mM or higher can effectively reduce the acute toxicity of oligonucleotide substances and/or their formulations.
  • Compound C5x5 was prepared in this Example by using the following procedures.
  • the crude product (300 mg, 0.41 mmol, 1.0 eq) was dissolved in anhydrous DCM (5 mL) then DIPEA (204 ⁇ L, 1.23 mmol, 3.0 eq) , 3- ( (chloro (diisopropylamino) phosphanyl) oxy) propanenitrile compound 47 (274 ⁇ L, 1.23 mmol, 3.0 eq. ) were added under nitrogen atmosphere at 25°C. The reaction mixture was stirred for 1 h. The mixture was extracted two times with DCM, then washed with brine and dried with anhydrous Na 2 SO 4 .
  • Oligonucleotide conjugate with C5x5 was generated by using a conjugation group derived from the compound C5x5 by using C5x5 as terminus amidite according to the above methods of general synthesis method of oligonucleotide.
  • Exemplary structure of the lipid-conjugated oligonucleotide is O1 as illustrated below:
  • the conjugation derived from the delivery enhancing compound C5x5 is linked with double-stranded RNA (dsRNA) duplexes (including but not limited to saRNA or siRNA) at the 5’ -end of the sense strand (S) via a linking moiety, such as –OP (O) 2 O-, –OP (O) (S) O-or –P (O) -O-, wherein (S) is the sense strand and (AS) is the antisense strand.
  • dsRNA double-stranded RNA
  • dsRNA duplexes including but not limited to saRNA or siRNA
  • oligonucleotide formulations were designed and synthesized with fully chemical modification (i.e., 2’ -O-methyl, 2'-O-methoxyethyl, 5’ - (E) -vinylphosphonate, or phosphorothioate (PS) backbone modification) , and conjugated with ACO or lipid (i.e., C5x5) , respectively.
  • Oligonucleotide formulations were prepared following General Method B.
  • RNA *, phosphorothioate (PS) backbone modification
  • f 2'-fluoro
  • m 2'-O-methyl (2'-OMe)
  • me 2'-O-methoxyethyl (2’ MOE)
  • Vp 5’ -
  • E -vinylphosphonate
  • meC 2'-O-methoxyethyl-5-methyl cytosine
  • meU 2'-O-methoxyethyl-5-methyl uracil
  • S9 9 atoms long triethylene glycolyl.
  • Example 21 CNS acute toxicity of oligonucleotide dissolved in 35 mM calcium aCSF in IT injected SD rats
  • Lipid conjugated oligonucleotide or duplex-ACO e.g., siRNA-ACO
  • oligonucleotide lipid conjugate RD-16234 and RD-16237)
  • siRNA-ACOs RD-16223, RD-16224, RD-16226, RD-16229, RD-16230 and RD-16236) .
  • siRNA-ACOs i.e., RD-16223, RD-16224, RD-16226, RD-16229, RD-16230 and RD-16236, see Table 12
  • lipid conjugated oligonucleotides i.e., RD-16234 and RD-16237, see Table 12
  • aCSF was used as a solvent and served as a negative control.
  • oligonucleotides dissolved in commercial aCSF TOCRIS, Batch number: 57A
  • 35 mM calcium aCSF were recorded within 3 hours after IT injection, as summarized in Table 13, Table 15, Table 14 and Table 16.
  • oligonucleotides formulations dissolved in aCSF with 35 mM calcium provided reduced CNS acute toxicity, especially for lipid-conjugated oligonucleotides (i.e., RD-16234 and RD-16237) .
  • DP-10 was prepared in this Example by using the following procedures.
  • oligonucleotide substance RD-12500 500mg, WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01
  • RD-12500 500mg, WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01
  • the mixed solution was transferred to a 5 mL volumetric flask and the volume was adjusted to 5ml using water for injection.
  • the osmotic pressure was measured, yielding a result of 278 mOsmol/kg.
  • 3.21mg of sodium chloride was added to achieve a final concentration of DP-10 at 100mg/mL with an osmotic pressure of 305 mOsmol/kg.
  • DP-15 was prepared in this Example by using the following procedures.
  • oligonucleotide substance RD-12500 500mg, WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01
  • RD-12500 500mg, WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01
  • the mixed solution was transferred to a 5 mL volumetric flask and the volume was adjusted to 5ml using water for injection.
  • the osmotic pressure was measured, yielding a result of 263 mOsm/kg.
  • 5.12 mg of sodium chloride was added to achieve a final concentration of DP-15 at 100mg/mL with an osmotic pressure of 298 mOsmol/kg.
  • DP-20 was prepared in this Example by using the following procedures.
  • oligonucleotide substance RD-12500 500mg, WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01
  • RD-12500 500mg, WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01
  • the mixed solution was transferred to a 5 mL volumetric flask and the volume was adjusted to 5ml using water for injection.
  • the osmotic pressure was measured, yielding a result of 267 mOsmol/kg.
  • 4.12 mg of sodium chloride was added to achieve a final concentration of DP-20 at 100mg/mL with an osmotic pressure of 297 mOsmol/kg.
  • DP-25 was prepared in this Example by using the following procedures.
  • oligonucleotide substance RD-12500 500mg, WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01
  • RD-12500 500mg, WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01
  • the mixed solution was transferred to a 5 mL volumetric flask and the volume was adjusted to 5ml using water for injection.
  • the osmotic pressure was measured, yielding a result of 249 mOsmol/kg.
  • 6.58 mg of sodium chloride was added to achieve a final concentration of DP-25 at 100mg/mL with an osmotic pressure of 297 mOsmol/kg.
  • DP-35 was prepared in this Example by using the following procedures.
  • oligonucleotide substance RD-12500 500mg, WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01
  • RD-12500 500mg, WuXi STA, COA: P220505001-C-MC00655-16-C-Lyo-V01
  • the mixed solution was transferred to a 5 mL volumetric flask and the volume was adjusted to 5ml using water for injection.
  • the osmotic pressure was measured, yielding a result of 268 mOsmol/kg.
  • 4.37 mg of sodium chloride was added to achieve a final concentration of DP-35 at 100mg/mL with an osmotic pressure of 305 mOsmol/kg.
  • Example 27 CNS acute toxicity of oligonucleotide by using an isotonic formulation in IT injected SD rats
  • test articles i.e., DP-10, DP-15, DP-20, DP-25, and DP-35, see Table 22
  • DP-10, DP-15, DP-20, DP-25, and DP-35 were prepared and administered into adult female SD rats (body weigh arrange: 140 g-260 g) at 3 mg/dose (100 mg/mL, 30 ⁇ L) via IT injection.
  • Preparations of DP-10, DP-15, DP-20, DP-25, and DP-35 were described in Example 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. All groups had no obvious acute toxicity.
  • the results demonstrate that oligonucleotide formulations with isotonic osmotic pressure ranging from 280 to 320 mOsmol/kg can mitigate acute toxicity dramatically.

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Abstract

L'invention concerne des formulations d'oligonucléotides. En particulier, l'invention concerne des formulations d'oligonucléotides comprenant des oligonucléotides (tels que l'ASO, le petit ARNi, le petit ARNa) et le calcium, des procédés de préparation de ces formulations, et leur utilisation. Les formulations d'oligonucléotides ont une toxicité aiguë in vivo réduite (en particulier dans le système nerveux central) et une fenêtre de sécurité in vivo étendue pour les oligonucléotides, en particulier le conjugué lipidique, et ont ainsi de grandes perspectives d'application.
PCT/CN2024/099219 2023-06-16 2024-06-14 Formulation d'oligonucléotides Pending WO2024255846A1 (fr)

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CNPCT/CN2023/100743 2023-06-16

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012021985A1 (fr) * 2010-08-20 2012-02-23 Replicor Inc. Complexes chélatés d'oligonucléotide
WO2013170086A2 (fr) * 2012-05-10 2013-11-14 Adynxx, Inc. Formulations pour l'administration de principes actifs
WO2013170386A1 (fr) * 2012-05-18 2013-11-21 Replicor Inc. Compositions de polypeptide-complexe chélaté oligonucléotidique et procédés
WO2023280190A1 (fr) * 2021-07-07 2023-01-12 Ractigen Therapeutics Véhicule d'administration d'agents à base d'oligonucléotides et procédés d'utilisation de ceux-ci

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012021985A1 (fr) * 2010-08-20 2012-02-23 Replicor Inc. Complexes chélatés d'oligonucléotide
WO2013170086A2 (fr) * 2012-05-10 2013-11-14 Adynxx, Inc. Formulations pour l'administration de principes actifs
WO2013170386A1 (fr) * 2012-05-18 2013-11-21 Replicor Inc. Compositions de polypeptide-complexe chélaté oligonucléotidique et procédés
WO2023280190A1 (fr) * 2021-07-07 2023-01-12 Ractigen Therapeutics Véhicule d'administration d'agents à base d'oligonucléotides et procédés d'utilisation de ceux-ci

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