WO2024107652A1 - Inhibition de la myostatine - Google Patents

Inhibition de la myostatine Download PDF

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Publication number
WO2024107652A1
WO2024107652A1 PCT/US2023/079519 US2023079519W WO2024107652A1 WO 2024107652 A1 WO2024107652 A1 WO 2024107652A1 US 2023079519 W US2023079519 W US 2023079519W WO 2024107652 A1 WO2024107652 A1 WO 2024107652A1
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seq
insdqualifier
insdseq
insdfeature
oligonucleotide
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PCT/US2023/079519
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John Mansell
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John Mansell
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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
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • 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
    • C12N2310/11—Antisense

Definitions

  • the present disclosure generally relates to compositions and methodologies for the treatment of immobilization- and microgravity-induced muscle atrophy. More specifically this disclosure relates to prophylactic and/or therapeutic utilization of oligonucleotides for the prophylactic treatment of progressive muscle atrophy.
  • Muscle growth is inhibited by myostatin, a 375 amino acid pre-pro-protein that is proteolytically cleaved to a 109 amino acid protein that as a dimer inhibits muscle growth by binding at the activin type 2 receptor and recruiting co-receptors Alk-3 or Alk-4.
  • myostatin inhibits expression of Akt, a kinase associated with muscle hypertrophy, and stimulates ubiquitin-associated protein degradation.
  • Increased expression of Akt and diminished ubiquitin protein degradation through myostatin expression inhibition diminishes the clinically-noted atrophy process of disuse, immobilization or diminished gravity.
  • a formulation comprising an oligonucleotide selected from the group consisting of an oligonucleotide having one of SEQ ID No. 1 through SEQ ID No. 23 or a variant thereof.
  • the oligonucleotide may comprise at least 75% sequence identity to one of SEQ ID No. 1 through SEQ ID No. 23, or at least 85% sequence identity to one of SEQ ID No. 1 through SEQ ID No. 23, or at least 95% sequence identity to one of SEQ ID No. 1 through SEQ ID No. 23, or comprises one of SEQ ID No. 1 through SEQ ID No. 23.
  • the oligonucleotide may be incorporated into a earner system, for example, a liposome, a biodegradable polymer, a hydrogel, or a cyclodextrin, a nucleic acid complex, a virosome, or combinations thereof.
  • a earner system for example, a liposome, a biodegradable polymer, a hydrogel, or a cyclodextrin, a nucleic acid complex, a virosome, or combinations thereof.
  • the method may comprise administering a formulation comprising an oligonucleotide selected from the group consisting of an oligonucleotide having one of SEQ ID No. 1 through SEQ ID No. 3 or a variant thereof to a subject.
  • the oligonucleotide may comprise at least 75% sequence identity to one of SEQ ID No. 1 through SEQ ID No. 23, or at least 85% sequence identity to one of SEQ ID No. 1 through SEQ ID No. 23, or at least 95% sequence identity to one of SEQ ID No. 1 through SEQ ID No. 23, or comprises one of SEQ ID No. 1 through SEQ ID No.:23.
  • Atrophy is the result of disuse, immobilization, or exposure to low gravity environments.
  • treat include alleviating, abating, or ameliorating a disease or condition, or symptoms thereof; managing a disease or condition, or symptoms thereof; preventing additional symptoms; ameliorating or preventing the underlying metabolic causes of symptoms; inhibiting the disease or condition, e.g., arresting the development of the disease or condition; relieving the disease or condition; causing regression of the disease or condition; relieving a symptom caused by the disease or condition; and/or stopping the symptoms of the disease or condition.
  • Treatment as used herein also encompasses any pharmaceutical or medicinal use of the compositions herein.
  • subject refers to an animal which is the object of treatment, observation, or experiment.
  • a subject may be, but is not limited to, a mammal including, but not limited to, a human.
  • tissue to be treated is a commercial growth environment for muscle for food consumption.
  • the subject is administered the compositions disclosed herein in a therapeutically effective amount sufficient for treating, preventing, and/or ameliorating one or the process of muscle atrophy, or accelerating muscle growth.
  • amelioration of the muscle atrophy process by administration of a particular composition of the type disclosed herein refers to any lessening, whether lasting or transient, which can be attributed to or associated with administration of compositions of the type disclosed herein. It is contemplated that the therapeutically effective amount may be optimized by one or more healthcare professionals in consideration of the particular factors affecting a subject.
  • RNA interference refers to the silencing or decreasing of gene expression by iRNA agents (e.g., siRNAs, miRNAs, shRNAs), via the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by an iRNA agent that has a seed region sequence in the iRNA guide strand that is complementary to a sequence of the silenced gene.
  • iRNA agent abbreviation for “interfering RNA agent”
  • interfering RNA agent refers to an RNA agent, or chemically modified RNA, which can down- regulate the expression of a target gene.
  • chemical modification refers to its meaning as is generally accepted in the art.
  • the term refers to any modifications of the chemical structure of the nucleotides that differs from nucleotides of native siRNA or RNA in general.
  • the term “chemical modification” encompasses the addition, substitution, or modification of native siRNA or RNA at the sugar, base, or internucleotide linkage, as described herein or as is otherwise known in the art.
  • the term “chemical modification” can refer to certain forms of RNA that are naturally occurring in certain biological systems, for example 2'-O-methyl modifications or inosine modifications.
  • an iRNA agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA, or pre-transcriptional or pre-translational mechanisms.
  • An iRNA agent can include a single strand (ss) or can include more than one strands, e.g. it can be a double stranded (ds) IRNA agent.
  • ss single strand
  • ds double stranded
  • siRNA refers to a small interfering RNA.
  • siRNAs include short interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30, 15-25 or 19-25 (duplex) nucleotides in length, and is alternatively about 20-24 or about 21-22 or 21-23 (duplex) nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25 or 19-25 nucleotides in length, alternatively about 20-24 or about 21-22, or 21-23 nucleotides in length, alternatively 19-21 nucleotides in length, and the double stranded siRNA is about 15-60, 15-50, 15-40, 15-30, 15-25 or 19-25, alternatively about 20-24, or about 21-22 or 19-21 or 21-23 base pairs in length).
  • siRNA duplexes may comprise 3' overhangs of about 1 to about 4 nucleotides, alternatively about 2 to 3 nucleotides and 5' phosphate termini. In some aspects, the siRNA lacks a terminal phosphate. In some aspects, one or both ends of siRNAs can include single- stranded 3' overhangs that arc two or three nucleotides in length, such as, for example, deoxythymidine (dTdT) or uracil (UU) that are not complementary to the target sequence.
  • dTdT deoxythymidine
  • UU uracil
  • siRNA molecules can include nucleotide analogs (e.g., thiophosphate or G-clamp nucleotide analogs), alternative base linkages (e.g., phosphorothioate, phosphonoacetate, or thiophosphonoacetate) and other modifications useful for enhanced nuclease resistance, enhanced duplex stability, enhanced cellular uptake, or cell targeting.
  • nucleotide analogs e.g., thiophosphate or G-clamp nucleotide analogs
  • alternative base linkages e.g., phosphorothioate, phosphonoacetate, or thiophosphonoacetate
  • the oligonucleotides disclosed herein are used to treat or prevent muscle atrophy processes and thus are designated PMAP.
  • the PMAPs need not be limited to those molecules containing only RNA but may further encompass chemically-modified nucleotides and non-nucleotides.
  • the PMAPs of the present disclosure comprise separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, Van der waals interactions, hydrophobic interactions, and/or stacking interactions.
  • the PMAPs of the present disclosure comprise nucleotide sequence that is complementary to nucleotide sequence of a target gene.
  • the PMAPs of the present disclosure interact with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.
  • “percent modification” refers to the number of nucleotides in the PMAP (e.g., iRNA, or each of the strand of the siRNA or to the collective dsRNA) that have been modified.
  • a 19% modification of the antisense strand of a PAIN refers to the modification of up to 4 nucleotides/bp in a 21-nucleotide sequence (21mer).
  • 100% modification refers to a fully modified dsRNA.
  • the extent of chemical modification will depend upon various factors such as for example, target mRNA, off-target silencing, degree of endonuclease degradation, etc.
  • shRNA or “short hairpin RNAs” refers to individual transcripts that adopt stem-loop structures which are processed into siRNA by RNAi machinery.
  • Typical shRNA molecules comprise two inverted repeats containing the sense and antisense target sequence separated by a loop sequence.
  • the base-paired segment may vary from 17 to 29 nucleotides, wherein one strand of the base-paired stem is complementary to the mRNA of a target gene.
  • the loop of the shRNA stem-loop structure may be any suitable length that allows inactivation of the target gene in vivo. While the loop may be from 3 to 30 nucleotides in length, typically it is 1-10 nucleotides in length.
  • the base paired stem may be perfectly base paired or may have 1 or 2 mismatched base pairs.
  • the duplex portion may, but typically does not. contain one or more bulges consisting of one or more unpaired nucleotides.
  • the shRNA may have non-base- paired 5' and 3' sequences extending from the base-paired stem. Typically, however, there is no 5' extension.
  • the first nucleotide of the shRNA at the 5' end is a G, because this is the first nucleotide transcribed by polymerase III. If G is not present as the first base in the target sequence, a G may be added before the specific target sequence.
  • the 5' G typically forms a portion of the base-paired stem.
  • shRNAs are processed into siRNAs by the conserved cellular RNAi machinery.
  • shRNAs are precursors of siRNAs and are, in general, similarly capable of inhibiting expression of a target mRNA transcript.
  • isolated in the context of an isolated nucleic acid molecule (e.g., PMAP), is one which is altered or removed from the natural state through human intervention.
  • an RNA naturally present in a living animal is not “isolated.”
  • complementary refers to nucleic acid sequences that are capable of base-pairing according to the standard Watson-Crick complementary rules.
  • the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA.
  • G:C cytosine
  • A:T thymine
  • A:U uracil
  • the term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA and/or a polypeptide, or its precursor as well as noncoding sequences (untranslated regions) surrounding the 5' and 3' ends of the coding sequences.
  • the term “gene” encompasses both cDNA and genomic forms of a gene.
  • a functional polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence as long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, antigenic presentation) of the polypeptide are retained.
  • 5' untranslated sequences The sequences which arc located 5' of the coding region and which arc present on the mRNA arc referred to as 5' untranslated sequences (“5'UTR”).
  • 3' untranslated sequences The sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' untranslated sequences, or (“3'UTR”).
  • the term “substantial silencing” means that the mRNA of the targeted gene (e.g., MSTN) is inhibited and/or degraded by the presence of the introduced PMAP, such that expression of the targeted gene is reduced by about 10% to 100% as compared to the level of expression seen when the PMAP is not present.
  • a gene when a gene is substantially silenced, it will have at least 40%, 50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%.
  • the term “substantially normal activity” means the level of expression of a gene when a PMAP has not been introduced.
  • the terms “inhibit,” “down-regulate,” or “reduce” as used herein refers to its meaning as is generally accepted in the art.
  • the term generally refers the reduction in the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, below that observed in the absence of the nucleic acid molecules (e.g., PMAP) of the present disclosure.
  • Down-regulation can also be associated with post-transcriptional silencing, such as, RNAi mediated cleavage or by alteration in DNA methylation patterns or DNA chromatin structure.
  • Inhibition, down-regulation or reduction with a PMAP can be in reference to an inactive molecule, an attenuated molecule, an oligonucleotide with a scrambled sequence, or an oligonucleotide with mismatches or alternatively, it can be in reference to the system in the absence of the oligonucleotide.
  • compositions disclosed herein comprise a PMAP which results in a down-regulation or reduction in the expression of the myostatin prepro-protein encoded by MSTN.
  • the PMAP comprises an oligonucleotide that inhibits expression of the gene coding for the PMAP prepro-protein or alternatively substantially silences the expression of the gene coding for the MSTN protein.
  • the extent of downregulation of MSTN or its respective gene product, myostatin may be determined using any suitable assay.
  • suitable assays include without limitation, e.g., examination of protein or mRNA levels using any suitable technique such as dot blots, northern blots, in situ hybridization, ELISA, microarray hybridization, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.
  • a test sample e.g., a biological sample from organism of interest expressing the target gene(s) or a sample of cells in culture expressing the target gene(s)
  • a PMAP that silences, reduces, or inhibits expression of the target gene(s).
  • Expression of the target gene in the test sample is compared to expression of the target gene in a control sample (e.g., a biological sample from organism of interest expressing the target gene or a sample of cells in culture expressing the target gene) that is not contacted with the PMAP.
  • Control samples i.e., samples expressing the target gene
  • substantial silencing, inhibition, down-regulation or reduction of expression of a target gene is achieved when the value of test the test sample relative to the control sample is about 95%, 90%, 85%, 80%, 75%, 70%, 65%. 60%. 55%, 50%, 45%, 40%. 35%. 30%, 25%, 20%, or 10%.
  • the PMAP is a microRNA (miRNA, miR).
  • miRs refer to single- stranded RNA molecules that are generally 21-23 nucleotides in length which regulate gene expression.
  • MicroRNAs are processed from primary transcripts known as pri-miRNA to short stem-loop structures called precursor (pre)-miRNA and finally to functional, mature microRNA.
  • Mature microRNA molecules are partially complementary to one or more messenger RNA molecules, and their primary function is to down-regulate gene expression through the RNAi pathway.
  • the PMAP is a small interfering RNA (siRNA).
  • RNAi Naturally occurring RNAi, a double-stranded RNA (dsRNA) is cleaved by an RNase Ill/helicase protein, Dicer, into small interfering RNA (siRNA) molecules, a dsRNA of 19-27 nucleotides (nt) with 2-nt overhangs at the 3' ends.
  • siRNAs are incorporated into a multicomponent-ribonuclease called RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • One strand of siRNA remains associated with RISC and guides the complex toward a cognate RNA that has sequence complementary to the guider ss-siRNA in RISC. This siRNA-directed endonuclease digests the RNA, thereby inactivating it.
  • the PMAP is an antisense oligonucleotide.
  • Antisense oligonucleotides are synthetic nucleic acids that bind to a complementary target and suppress function of that target.
  • ASOs are used to reduce or alter expression of RNA targets, particularly messenger RNA (mRNA) or microRNA (miRNA) species.
  • ASOs can suppress gene expression via two different mechanisms of action, including: 1) by steric blocking, wherein the ASO tightly binds the target nucleic acid and inactivates that species, preventing its participation in cellular biology, or 2) by triggering degradation, wherein the ASO binds the target and leads to activation of a cellular nuclease that degrades the targeted nucleic acid species.
  • One class of “target degrading” ASOs are “RNase H active”; formation of heteroduplex nucleic acids by hybridization of the target RNA with a DNA-containing “RNase H active” ASO forms a substrate for the enzyme RNase H.
  • RNase H degrades the RNA portion of the heteroduplex molecule, thereby reducing expression of that species. Degradation of the target RNA releases the ASO, which is not degraded, which is then free to recycle and can bind another RNA target of the same sequence.
  • a PMAP comprises a microRNA, a siRNA, an ASO, an iRNA, an iRNA agent, an shRNA, a functional variant thereof; or combinations thereof.
  • a functional variant of an oligonucleotide disclosed herein comprises at least 70% sequence identity with any sequence disclosed herein, alternatively at least 75%, alternatively at least 80%, alternatively at least 85%, alternatively at least 90% or alternatively at least 95%.
  • identity refers to an exact nucleotide-to-nucleotide correspondence of two oligonucleotides or polynucleotides sequences.
  • Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100.
  • Readily available computer programs can be used to aid in the analysis, such as Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs, which rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.
  • homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
  • Biomolecules of interest that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.
  • SEQ ID No. 1 through SEQ ID No. 23 are representative of the PMAPs described herein.
  • SEQ ID No. 1 through SEQ ID No. 23 are also depicted in Table 1 below.
  • the PMAP comprises an oligonucleotide having any one of SEQ ID No. 1 through SEQ ID No. 23, alternatively a functional variant thereof.
  • INS DS eq_length 42 ⁇ /IN S D
  • S eq_length ⁇ INSDSeq_moltype>RNA ⁇ /INSDSeq_moltype> ⁇ INSDSeq_division>PAT ⁇ /INSDSeq_division>
  • INS DS eq_moltype > RN A ⁇ /IN S DSeq_moltype > ⁇ INSDSeq_division>PAT ⁇ /INSDSeq_division>
  • INS DS eq_length 42 ⁇ /IN S D S eq_length > ⁇ INSDSeq_moltype>RNA ⁇ /INSDSeq_moltype> ⁇ INSDSeq_division>PAT ⁇ /INSDSeq_division>
  • INS DS eq_moltype > RN A ⁇ /IN S DSeq_moltype > ⁇ INSDSeq_division>PAT ⁇ /INSDSeq_division>
  • INS DS eq_moltype > RN A ⁇ /IN S DSeq_moltype > ⁇ INSDSeq_division>PAT ⁇ /INSDSeq_division>
  • the PMAP has from about 20% to about a 90% modification or alternatively from about a 40% to about 60% modification.
  • PMAPs of the present disclosure are chemically synthesized.
  • Oligonucleotides e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides
  • Oligonucleotides are synthesized using protocols known in the art, for example as described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.
  • oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramiditcs at the 3'-cnd.
  • PMAP of the present disclosure that interact with and down-regulate myostatin can be expressed and delivered from a transcript inserted into DNA or RNA vectors.
  • the recombinant vectors can be DNA plasmids or viral vectors.
  • Nonlimiting examples of PMAP expressing viral vectors can be constructed based on adeno-associated virus, retrovirus, adenovirus, or alphavirus.
  • pol III based constructs are used to express PMAPs of the present disclosure. Transcription of the siNA molecule sequences can be driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III), (see for example, Thompson, U.S. Pat. Nos. 5,902,880 and 6,146,886). Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
  • Prokaryotic RNA polymerase promoters may also be used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells
  • exemplary transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors).
  • Vectors used to express the PMAPs of the present disclosure can encode one or both strands of an siNA duplex, or a single self-complementary strand that self hybridizes into an siRNA duplex.
  • the nucleic acid sequences encoding the PMAPs of the present disclosure can be operably linked in a manner that allows expression of the PMAP.
  • the constructs comprising PMAPs may additionally comprise reporter genes (e.g., green fluorescent protein) and selection genes (e.g., for antibiotic resistance).
  • the PMAPs of the present are added directly, or can be complexed with cationic lipids, packaged within liposomes, or as a recombinant plasmid or viral vectors which express the PMAP, or otherwise delivered to target cells or tissues.
  • Nucleic acid molecules can be administered to cells by any suitable methodology, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres, biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors.
  • the present disclosure provides carrier systems containing the PMAPs described herein.
  • the carrier system is a lipid-based carrier system, cationic lipid, or liposome, nucleic acid complexes, a liposome, a micelle, a virosome, a lipid nanoparticle or a mixture thereof.
  • the earner system is a polymer-based carrier system such as a cationic polymer-nucleic acid complex.
  • the carrier system is a cyclodextrin- based carrier system such as a cyclodextrin polymer-nucleic acid complex.
  • the carrier system is a protein-based carrier system such as a cationic peptide-nucleic acid complex.
  • the PMAP is a component of a conjugate or complex provided that can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the present disclosure.
  • the conjugate can comprise polyethylene glycol (PEG) can be covalently attached to a PMAP.
  • PEG polyethylene glycol
  • the attached PEG can be any molecular weight, for example from about 100 to about 50,000 daltons (Da).
  • the PMAP is a component of compositions or formulations comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes, or stealth liposomes) and PMAPs.
  • the siRNA molecules of the present disclosure can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI- PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG- triGAL) derivatives.
  • the PMAPs of this disclosure are prepared into a composition or formulation for administration to a subject.
  • composition or “formulation” as used herein refer to their generally accepted meaning in the art. These terms generally refer to a composition or formulation, such as in a pharmaceutically acceptable carrier or diluent, in a form suitable for administration, e.g., systemic or local administration, into a cell or subject, including, for example, a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, inhalation, or by intravenous, or intramuscular injection.
  • compositions injected into the blood stream should be soluble.
  • Other factors include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.
  • Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant present disclosure include: Lipid Nanoparticles (see for example Semple et al., 2010, Nat Biotechnol., February; 28(2): 172-6); P-glycoprotein inhibitors (such as Pluronic P85); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery (Emerich, D F et al, 1990, Cell Transplant, 8, 47-58); and loaded nanoparticles, such as those made of polybutylcyanoacrylate.
  • Other non-limiting examples of delivers strategies for the nucleic acid molecules of the instant present disclosure include material described in Boado et al., 1998, J. Pharm.
  • a “pharmaceutically acceptable composition” or “pharmaceutically acceptable formulation” can refer to a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant disclosure to the physical location most suitable for their desired activity.
  • the formulation may contain additional ingredients.
  • additional ingredients include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials.
  • the subject is administered a pharmaceutical formulation comprising a PMAP having a sequence as disclosed herein prior to, concomitant with, subsequent to a surgical procedure where the subject was administered a general anesthetic.
  • the general anesthetic may comprise a halogenated gaseous compound such as isoflurane or sevoflurane.
  • these different forms of oligonucleotides would diminish efficient transcription of the MSTN gene and production of myostatin, reduce successful movement of guide strand mRNA to translation and interfere with efficient translation of mRNA which produces myostatin.
  • compositions can be administered via oral, subcutaneous, intrapulmonary, transmucosal, intraperitoneal, intrauterine, sublingual, intrathecal or intramuscular routes.
  • Injectable formulations of the PMAP compositions or formulations of the present disclosure may contain various carriers.
  • Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients.
  • Intramuscular preparations e.g., a sterile formulation of the compounds of the present disclosure can be dissolved and administered in a pharmaceutical excipient was as water-for-injection, 0.9% saline, or 5% glucose solution.
  • formulations disclosed herein would be administered so as to be present in neural and other affected tissues, before, during, and for a period of time after, exposure to anesthetics so as to decrease or prevent anesthetic-triggered apoptotic events.
  • the formulations disclosed herein could be administered at least about 30 minutes prior to exposure to anesthetics, or at least about 1 hour, or at least about 2 hours, or at least about 4 hours, or at least about 8 hours, or at least about 16 hours, or at least about 24 hours, or at least about 32 hours, or at least about 48 hours, or at least about 60 hours, or at least about 72 hours prior to exposure to anesthetics and, additionally or alternatively, substantially contemporaneously with the exposure anesthetics and, additionally or alternatively, about 30 minutes following exposure to anesthetics, or about 1 hour, or about 2 hours, or about 4 hours, or about 8 hours, or about 16 hours, or about 24 hours, or about 32 hours, or at about 48 hours, or about 60 hours, or about 72 hours following exposure to anesthetics.
  • Embodiment No. 1 is a formulation comprising an oligonucleotide selected from the group consisting of an oligonucleotide having one of SEQ ID No. 1 through SEQ ID No. 23 or a variant thereof.
  • Embodiment No. 2 is the formulation of Embodiment No. 1, wherein the oligonucleotide comprises at least 75% sequence identity to one of SEQ ID No. 1 through SEQ ID No. 23.
  • Embodiment No. 3 is the formulation of one of Embodiment Nos. 1-2, wherein the oligonucleotide comprises at least 85% sequence identity to one of SEQ ID No. 1 through SEQ ID No. 23.
  • Embodiment No. 4 is the formulation of one of Embodiment Nos. 1-3, wherein the oligonucleotide comprises at least 95% sequence identity to one of SEQ ID No. 1 through SEQ ID No. 23.
  • Embodiment No. 5 is the formulation of one of Embodiment Nos. 1-4, wherein the oligonucleotide comprises one of SEQ ID No. 1 through SEQ ID No. 23.
  • Embodiment No. 6 is the formulation of one of Embodiment Nos. 1-5, wherein the oligonucleotide is incorporated into a carrier system.
  • Embodiment No. 7 is the formulation of Embodiment No. 6, wherein the earner system comprises a liposome.
  • Embodiment No. 8 is the formulation of one of Embodiment Nos. 6-7, wherein the carrier system comprises a biodegradable polymer, a hydrogel, or a cyclodextrin.
  • Embodiment No. 9 is the formulation of one of Embodiment Nos. 6-8, wherein the carrier system comprises a nucleic acid complex.
  • Embodiment No. 10 is the formulation of one of Embodiment Nos. 6-9, wherein the carrier system comprises a virosome.
  • Embodiment No. 11 is a method of prophylactic treatment of progressive muscle atrophy, the method comprising administering a formulation comprising an oligonucleotide selected from the group consisting of an oligonucleotide having one of SEQ ID No. 1 through SEQ ID No. 23 or a variant thereof to a subject, wherein the formulation is administered prior to, concomitant with, subsequent to, or combinations thereof administration of a general anesthetic comprising a fluorinated compound in anticipation of conditions leading to muscle atrophy or the need for accelerated muscle growth, or the administration of a domain- specific antibody to interfere with the muscle metabolic role of myostatin
  • Embodiment No. 12 is the method of Embodiment No. 11, wherein the oligonucleotide comprises at least 75% sequence identity to one of SEQ ID No. 1 through SEQ ID No. 23.
  • Embodiment No. 13 is the method of one of Embodiment Nos. 11-12, wherein the oligonucleotide comprises at least 85% sequence identity to one of SEQ ID No. 1 through SEQ ID No. 23.
  • Embodiment No. 14 is the method of one of Embodiment Nos. 11-13, wherein the oligonucleotide comprises at least 95% sequence identity to one of SEQ ID No. 1 through SEQ ID No. 23.
  • Embodiment No. 15 is the method of one of Embodiment Nos. 11-14, wherein the oligonucleotide comprises one of SEQ ID No. 1 through SEQ ID No. 23.
  • Embodiment No. 16 is the method of one of Embodiment Nos. 11-15, wherein the oligonucleotide is incorporated into a carrier system.
  • Embodiment No. 17 is the method of Embodiment No. 16, wherein the carrier system comprises a liposome.
  • Embodiment No. 18 is the method of one of Embodiment Nos. 16-17, wherein the carrier system comprises a biodegradable polymer, a hydrogel, or a cyclodextrin.
  • Embodiment No. 19 is the method of one of Embodiment Nos. 16-18, wherein the carrier system comprises a nucleic acid complex.
  • Embodiment No. 20 is the method of one of Embodiment Nos. 16-19, wherein the carrier system comprises a virosome.
  • formulations disclosed herein particularly, formulations including an oligonucleotide having one or more of SEQ ID No. 1 through SEQ ID No. 23 or a variant thereof
  • administration of the formulations disclosed herein substantially contemporaneously with (for example, shortly before, during, or shortly after) the conditions that cause muscle atrophy or the need to accelerate muscle growth.
  • the formulations disclosed herein particularly, formulations including an oligonucleotide having one or more of SEQ ID No. 1 through SEQ ID No. 23 or a variant thereof
  • formulations disclosed herein may be particularly advantageous in the context of Conditions causing muscle atrophy or the need to accelerate muscle growth.
  • formulations disclosed herein may be particularly advantageous in the context of subjects exposed to long term lower gravity environments wherein slow and progressive muscle loss occurs.
  • formulations disclosed herein may be particularly advantageous in the context of growing cultured meat, or in vitro production of meat for consumption as food. These formulations would be a supplement to culture media to facilitate or augment muscle cell growth.
  • any section headings used herein are provided to provide organizational cues. These headings shall not limit or characterize the subject matter set out in any claims that may issue from this disclosure. Specifically, and by way of example, although the headings might refer to a “Field,” the claims should not be limited by the language chosen under this heading to describe the so-called field. Further, a description of a technology in the “Background” is not to be construed as an admission that certain technology is prior art to any subject matter of this disclosure. Neither is the “Summary” to be considered as a limiting characterization of the subject matter set forth in issued claims. In all instances, the scope of the claims shall be considered on their own merits in light of this disclosure but should not be constrained by the headings set forth herein.

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Abstract

L'invention concerne une formulation contenant un oligonucléotide choisi dans le groupe constitué par un oligonucléotide présentant un N° compris entre les N°1 ID SÉQ et N°23 ID SÉQ, ou un variant associé. L'invention concerne également une méthode de traitement prophylactique de l'atrophie musculaire progressive. La méthode peut consister en l'administration de la formulation. La formulation peut être administrée avant, simultanément avec, après l'administration d'un anesthésique général contenant un composé fluoré, ou selon une combinaison de ces modes d'administration. L'oligonucléotide peut être incorporé dans un système vecteur, par exemple un liposome, un polymère biodégradable, un hydrogel ou une cyclodextrine, un complexe d'acides nucléiques, un virosome ou des combinaisons de ces derniers.
PCT/US2023/079519 2022-11-14 2023-11-13 Inhibition de la myostatine WO2024107652A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7534432B2 (en) * 1993-03-19 2009-05-19 The Johns Hopkins University School Of Medicine Methods of treating musculodegenerative disease with an antibody that binds growth differentiation factor-8
US8236751B2 (en) * 2007-03-07 2012-08-07 The Johns Hopkins University Methods of increasing muscle mass using follistatin-like related gene (FLRG)
US8323964B2 (en) * 1993-03-19 2012-12-04 The John Hopkins University School Of Medicine Polynucleotides encoding promyostatin polypeptides
US8426374B1 (en) * 2006-05-04 2013-04-23 Los Angeles Biomedical Research Institute At Harbor-Ucla Medical Center Method for modifying myostatin expression
US20210222174A1 (en) * 2018-06-29 2021-07-22 John Mansell Compositions and methods for the treatment of anesthesia-induced neurotoxicity

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7534432B2 (en) * 1993-03-19 2009-05-19 The Johns Hopkins University School Of Medicine Methods of treating musculodegenerative disease with an antibody that binds growth differentiation factor-8
US8323964B2 (en) * 1993-03-19 2012-12-04 The John Hopkins University School Of Medicine Polynucleotides encoding promyostatin polypeptides
US8426374B1 (en) * 2006-05-04 2013-04-23 Los Angeles Biomedical Research Institute At Harbor-Ucla Medical Center Method for modifying myostatin expression
US8236751B2 (en) * 2007-03-07 2012-08-07 The Johns Hopkins University Methods of increasing muscle mass using follistatin-like related gene (FLRG)
US20210222174A1 (en) * 2018-06-29 2021-07-22 John Mansell Compositions and methods for the treatment of anesthesia-induced neurotoxicity

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