Patent Application
20230304007
The disclosure relates to compositions and methods of treatment of muscle disorders by targeting H19X-encoded non-coding RNAs.
Loss of skeletal muscle mass (i.e. muscle atrophy/wasting) is closely associated with poor prognosis in many chronic diseases, including myopathies, muscular dystrophies, cancers, diabetes, sepsis, and heart failure. Muscle loss also contributes to decreased quality of life in aging. Muscle atrophy results from a reduced rate of protein synthesis, accelerated protein degradation, or both. The ubiquitin—proteasome system and autophagy are two major proteolytic mechanisms that are activated in skeletal muscle in various atrophying conditions. The activity of ubiquitin—proteasome system and autophagy is regulated through coordinated activation of several signaling pathways, such as nuclear factor-κB and p38 mitogen-activated protein kinase pathways. Moreover, the canonical transforming growth factor-β family members, such as myostatin and activin that function through activin receptors and activation of SMAD2/3 transcription factors, are important drivers of muscle wasting. In contrast, the IGF1/AKT/mTOR pathway increases the rate of protein synthesis leading to skeletal muscle hypertrophy. Activation of this pathway also inhibits muscle protein degradation through distinct mechanisms. For example, gene expression of two muscle-specific E3 ubiquitin ligases, MuRF1 and MAFbx/Atrogin-1, is regulated by transcription factors FoxO1/3, which are removed from the nucleus upon phosphorylation by AKT. The transforming growth factor-β inhibits AKT activity through stimulating SMAD signaling.
MicroRNAs (miRNAs) are small non-coding RNAs that regulate post-transcriptional gene expression. The abundance of some miRNAs is altered in wasting human muscle biopsies. Eight miRNAs were differentially expressed in rectus abdominis of cachectic and non-cachectic cancer patients. Thirty-two differentially expressed miRNAs were identified in the quadriceps of chronic obstructive pulmonary disease patients with a low fat-free mass index: 26 were down-regulated, while 6 were up-regulated. Remarkably, five of the six up-regulated miRNAs were encoded by the H19X locus located on Xq26.3. H19X-encoded miRNAs are differentially expressed in skeletal muscle in several cachectic/wasting diseases. The H19X-encoded miRNAs-322(424)/miR-503 cluster was highly expressed in bipotent cardiac and skeletal muscle progenitors. During embryonic stem cell differentiation, the cluster plays an essential role in cardiomyocyte specification by targeting and inhibiting neuroectoderm lineage factors. There is a need for therapeutic agents targeting specific portions of the H19X-encoded genes to treat muscle disorders.
Applicant has recognized the shortcomings in the art and the need for therapeutic agents targeting specific portions of the H19X-encoded genes to treat muscle disorders. H19X-encodes a number of non-coding RNAs including miR-424, miR-503, miR503HG, miR-542, miR-450a1, miR-450a2 and miR-450b that are specifically expressed in embryonic heart and muscles. They are not normally express in adult tissues, but they are activated in muscle wasting associated with cancer and chronic diseases, in sarcopenia (age-related muscle wasting), and in heart failure.
Embodiments include methods of preventing or treating a muscle disorder in a subject in need thereof by administering an inhibitor of one or more of miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b. The muscle disorders include muscle wasting, cachexia, sarcopenia, and heart failure. Heart failure can result from cardiomyopathy. The inhibitor can reduce the expression of the miRNA or affect the activity of the miRNA. In certain embodiments, the inhibitor reduces expression of one or more of miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b. The inhibitor can be a nucleic acid selected from a group consisting of a DNA, an RNA, an antagomir, a siRNA, a shRNA, and an oligonucleotide. The equivalent of miR-424 in humans is miR-322 miRNA in mice.
In certain embodiments, the inhibitor affects activity of one or more of miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b. The inhibitor can act by reducing interaction between one or more of miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b and their natural target mRNA. The inhibitor can be a nucleic acid selected from a group consisting of a DNA, an RNA, an antagomir, a siRNA, a shRNA and an oligonucleotide. Specific antisense-based inhibitors of one or more of miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b preserve muscle in muscular disorders, such as muscle wasting, cachexia, sarcopenia, and cardiomyopathy.
The inhibitor can be a nucleic acid binding to all or a portion of a base sequence of one or more of miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b. In an embodiment, the sequence of one or more of miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b is derived from a human, and includes not only a mature sequence but also a precursor sequence. The nucleic acid inhibitor can be an antisense oligonucleotide comprising a sequence partially or completely complementary to the base sequence of one or more of miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b.
Certain embodiments include methods of preventing or treating a muscle disorder in a subject in need thereof by inducing a mutation to reduce expression of one or more of miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b. In some embodiments, the mutation is an insertion mutation to block transcription of one or more of miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b. Certain methods can include inducing a mutation by editing a sequence of one or more of miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b by a nuclease. The nuclease can be one or more of a meganuclease, a zinc-finger nuclease, a transcription activator-like effector nuclease, and a CRISPR/Cas nuclease.
Embodiments of therapeutic agents targeting specific portions of the H19X-encoded genes to treat muscle disorders include antagomirs, RNA-sponges, CRISPR-mediated promoter silencing or gene editing, and other agents targeting H19X non-coding RNAs.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Embodiments will be readily understood by the following detailed description in conjunction with the accompanying drawings. Embodiments are illustrated by way of example and not by way of limitation in the figures of the accompanying drawings.
Muscle wasting and cachexia are complications of many diseases, including but not limited to cancer, sepsis, chronic obstructive pulmonary diseases, neurological disorders, and arthritis. Muscle wasting is also related to aging. Muscle wasting and cachexia increase the rates of fatality in the underlying diseases and they severely diminish life quality. There are no cures for muscle wasting, cachexia, and sarcopenia. Patients are treated by nutrition support and exercises, which are not effective for most patients. Heart failure is among the leading causes of death. Treatment is usually targeted towards reducing the pre- and after-load of the heart. Despite the many drug options, patients still have one of the worst 5-year survival rates among life-threatening diseases.
As disclosed herein, H19X non-coding RNAs are responsible for skeletal muscle wasting and heart failure. Embodiments include compositions and methods of targeting these non-coding RNAs in treating diseases, such as muscle wasting, cachexia, sarcopenia, and heart failure. Certain H19X-encoded non-coding RNAs of interest include one long non-coding RNA (lncRNA) miR503HG and 7 miRNAs—miR-424, miR-503, miR-542, miR450a1, miR-450a2, and miR-450b.
Embodiments of the present disclosure relates to therapeutic agents targeting one or more of these lncRNAs of interest, such as miR503HG, for preventing or treating muscle wasting. Embodiments also include methods for screening such an agent, which include steps of measuring the expression level of one or more of these lncRNAs of interest, such as miR503HG, and identifying an agent that affects the expression level of one or more of these lncRNAs of interest, such as miR503HG. Embodiments also include pharmaceutical compositions containing an inhibitor of one or more of these lncRNAs of interest, such as miR503HG, for preventing or treating a disease characterized by muscle wasting.
Embodiments of the present disclosure relates to therapeutic agents targeting one or more of these miRNAs of interest—miR-424, miR-503, miR-542, miR450a1, miR-450a2, and miR-450b for preventing or treating muscle wasting. Embodiments also include methods for screening such an agent, which include steps of measuring the expression level of one or more of these miRNAs of interest—miR-424, miR-503, miR-542, miR450a1, miR-450a2, and miR-450b, and identifying an agent that affects the expression level of one or more of these miRNAs of interest. Embodiments also include pharmaceutical compositions containing an inhibitor of one or more of these miRNAs of interest—miR-424, miR-503, miR-542, miR450a1, miR-450a2, and miR-450b for preventing or treating a disease characterized by muscle wasting.
In the present disclosure, the ‘miR’ or ‘microRNA (miRNA)’ refers to a naturally occurring single-stranded, non-coding small RNA molecule that controls gene expression by binding to complementary sequences in its target mRNAs, thereby inhibiting translation or inducing mRNA degradation.
The term “complementary” as used herein refers to an antisense oligonucleotide that is sufficiently complementary to the miRNA target under predetermined hybridization conditions or annealing conditions, specifically under physiological conditions, such that it can selectively hybridize to the target, and encompasses both partially or substantially complementary and completely complementary sequences. Substantially complementary means that, although not completely complementary, it has complementarity sufficient to bind to the target sequence and interfere with the activity of the miRNA. The term “nucleic acid” includes an oligonucleotide, a DNA, an RNA, a polynucleotide, and analogs and derivatives thereof. For example, a PNA or a mixture thereof is included.
In the present disclosure, a miRNA inhibitor can inhibit the expression of the specific miRNA or a lncRNA. In certain embodiments, it can inhibit the interaction between miRNA and its lncRNA target. In certain embodiments, it can inhibit the interaction between miRNA and its mRNA target. For example, the mRNA target for miR-424/miR-503 includes the 3′-untranslated region of translation initiation factors, such as eIF4E1, eIF4G1, eIF4B, eIF2B5, eIF3M, IGF1R, BCL2, and CDC25A. The miRNA targets for miR-542, miR-450a1, miR-450a2 and miR-450b include, but are not limited to, SMAD7, SMURF1, RP 523, TIMMDC1, MT-ND2, ACO2, and ATP5B. In the present disclosure, the miRNA inhibitor may inhibit or interfere with the action or function of miRNA or lncRNA in cells. Inhibitors can directly inhibit the binding of the miRNA to its target mRNA. In certain embodiments, these nucleic acid inhibitors can be selected from a group consisting of DNA, RNA, an antagomir (antisense oligonucleotide of miRNA), a RNA sponge, a siRNA, a shRNA and an oligonucleotide. In other embodiments, the miRNA inhibitor includes any agent capable of reducing the expression and/or activity of miRNA. The agent can include, without any limitations, a low-molecular-weight compound, an antagomir, an antisense molecule, a small hairpin RNA (shRNA) molecule, a small interfering RNA (siRNA) molecule, a seed target LNA (locked nucleic acid) oligonucleotide, a decoy oligonucleotide, an aptamer, a ribozyme, or an antibody that recognizes a DNA:RNA hybrid. The miRNA inhibitor can be an antisense oligonucleotide which can inhibit the activity of miRNA by complementarily binding to all or a part of the precursor and/or mature sequence of the miRNA.
An antagomir is a chemically modified single-stranded oligonucleotide and is used to silence one or more of these miRNAs of interest—miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b. The antagomir has a sequence which is at least partially or completely complementary to one or more of these miRNAs of interest—miR-424, miR-503, miR-542, miR450a1, miR-450a2, and miR-450b. The antagomir can include one or more modification (e.g., 2′-O-methyl-sugar modification or 3′-cholesterol modification). The antagomir can contain one or more phosphorothioate linkage and have a phosphorothioate backbone at least in part. In the present disclosure, the appropriate length of the antagomir for inhibiting the expression of miRNA is 7-50 nt (nucleotides), specifically 10-40 nt, more specifically 15-30 nt, more specifically 15-25 nt, more specifically 20-22 nt, although not being limited thereto.
A sponge RNA is a small synthetic RNA that binds to multiple miRNAs, which have the same sequence in their ‘seed region’. When the sponge is expressed at high levels, it specifically inhibits the activity of a family of miRNAs sharing a common seed (miRNA nucleotides 2-7, the major specificity determinant for target recognition).
Mature miRNA sequence with accession number, and the “seed sequence” as underlined text are provided below in Table 1.
Pre-miRNA sequences with accession number are provided below.
Chromosome coordinates of the miRNAs are provided below.
In the present disclosure, the pharmaceutical composition may further contain, in addition to the miRNA inhibitor, one or more active ingredient exhibiting the same, similar or synergistic function for the treatment of related diseases or a compound which maintains/increases the solubility and/or absorbency of the miRNA inhibitor.
The pharmaceutical composition may further contain one or more pharmaceutically acceptable diluent, carrier and/or adjuvant in addition to the above-mentioned therapeutic agents. In addition, it can be formulated into an injectable formulation such as an aqueous solution, a suspension, an emulsion, etc., a pill, a capsule, a granule or a tablet by additionally adding a diluent, a dispersant, a surfactant, a binder and a lubricant, and it can be used by binding a target organ-specific antibody or other ligand with the carrier. The pharmaceutical composition can be formulated into a formulation for intranasal administration, intravenous administration, subcutaneous injection, intrathecal injection, inhalation administration or oral administration. The pharmaceutical composition can be prepared into a variety of unit dosage forms. The pharmaceutical composition may be administered in a pharmaceutically or therapeutically effective amount. The pharmaceutically or therapeutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and an effective dose level will depend on factors including but not limited to the type and severity of the disease, the activity of the therapeutic agent, sensitivity to the therapeutic agent, the time of administration, the route of administration, the rate of excretion, and the duration of the treatment.
In addition, the pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and may be administered either alone or in combination.
Embodiments of therapeutic regimens to treat muscle disorders include inducing specific mutations to decrease expression of one or more of miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b. Some embodiments of the methods include inducing a mutation that is an insertion mutation to block transcription of one or more of miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b. Mutations can be induced by utilizing gene editing tools such as, but not limited to, integrases (recombinases), clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR associated protein (Cas) nucleases, TALAN nucleases, zinc finger nucleases, triplex forming oligonucleotides, or combinations thereof. Certain methods can include inducing a mutation by editing a sequence of one or more of miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b by a nuclease. The nuclease can be one or more of a meganuclease, a zinc-finger nuclease, a transcription activator-like effector nuclease, a CRISPR/Cas9 nuclease, a CRISPR/Cpf1 nuclease, a CRISPR/CasX nuclease, a CRISPR/CasY nuclease, and a Csm1 nuclease.
Embodiments include methods of preventing or treating a muscle wasting disorder in a subject in need thereof by administering an inhibitor of one or more of miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b. The muscle disorders include muscle wasting, cachexia, sarcopenia and heart failure. The term “treatment” as used in the present disclosure means any action to change favorably or improve the symptoms of related diseases by administering the therapeutic agent. The term “prevention” used in the present disclosure means any action to inhibit or delay the onset of related diseases. It will be apparent to those skilled in the art that the related diseases can be prevented if the pharmaceutical composition according to the present disclosure is administered when or before early symptoms appear. Reference to miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, or miR-450b includes a variant of miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, or miR-450b, respectively. A variant includes a nucleic acid with at least 80% sequence similarity to a particular SEQ ID, a nucleic acid with at least 85% sequence similarity to a particular SEQ ID, a nucleic acid with at least 90% sequence similarity to a particular SEQ ID, or a nucleic acid with at least 95% sequence similarity to a particular SEQ ID.
In certain embodiments, the inhibitor reduces expression of one or more of miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b. The inhibitor can be a nucleic acid selected from a group consisting of a DNA, an RNA, an antagomir, a RNA sponge, a siRNA, a shRNA and an oligonucleotide.
In certain embodiments, the inhibitor reduces interaction between one or more of miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b and their natural target. For example, the target nucleic acids for miR-424/-503 code for one or more of IGF1R, BCL2, CDC25A, eIF4E, eIF4G1, eIF4B, eIF2B5 and eIF3M. For example, the target nucleic acids for miR-542 code for one or more of SMAD7, SMURF1, and RPS23. For example, the target nucleic acids for miR-450 code for one or more of TIMMDC1, MT-ND2, ACO2, and ATP5B. In certain embodiments, as the miRNAs interact with the same target, an inhibitor may play a role in reducing interactions of several miRNAs.
The inhibitor can be a nucleic acid selected from a group consisting of a DNA, an RNA, an antagomir, a siRNA, a shRNA and an oligonucleotide. Specific antisense-based inhibitors of miR-424/miR-503 preserves muscle in wasting conditions. Embodiments of therapeutic agents targeting specific portions of the H19X-encoded genes to treat muscle disorders include antagomirs, RNA-sponge, CRISPR-mediated promoter silencing and gene editing, and other agents targeting H19X non-coding RNAs.
The inhibitor can be a nucleic acid binding to all or a part of a base sequence of miR-424. In an embodiment, the sequence of miR-424 is derived from a human, and includes not only a mature sequence but also a precursor sequence, provided by SEQ ID NOS. 1, 2, or 13 or a variant thereof. The nucleic acid can be an antisense oligonucleotide containing a sequence partially or completely complementary to the base sequence of miR-424, provided by SEQ ID NOS. 1, 2, or 13 or a variant thereof.
The inhibitor can be a nucleic acid binding to all or a part of a base sequence of miR-503. In an embodiment, the sequence of this miRNA is derived from a human, and includes not only a mature sequence but also a precursor sequence, provided by SEQ ID NOS. 3, 4, or 14 or a variant thereof. The nucleic acid can be an antisense oligonucleotide containing a sequence partially or completely complementary to the base sequence of miR-503, provided by SEQ ID NOS. 3, 4, or 14 or a variant thereof.
The inhibitor can be a nucleic acid binding to all or a part of a base sequence of miR-542. In an embodiment, the sequence of this miRNA is derived from a human, and includes not only a mature sequence but also a precursor sequence, provided by SEQ ID NOS. 5, 6, or 15 or a variant thereof. The nucleic acid can be an antisense oligonucleotide comprising a sequence partially or completely complementary to the base sequence of miR-542, provided by SEQ ID NOS. 5, 6, or 15 or a variant thereof.
The inhibitor can be a nucleic acid binding to all or a part of a base sequence of miR450a1. In an embodiment, the sequence of this miRNA is derived from a human, and includes not only a mature sequence but also a precursor sequence, provided by SEQ ID NOS. 7, 8, or 16 or a variant thereof. The nucleic acid can be an antisense oligonucleotide comprising a sequence partially or completely complementary to the base sequence of miR450a1, provided by SEQ ID NOS. 7, 8, or 16 or a variant thereof.
The inhibitor can be a nucleic acid binding to all or a part of a base sequence of miR-450a2. In an embodiment, the sequence of this miRNA is derived from a human, and includes not only a mature sequence but also a precursor sequence, provided by SEQ ID NOS. 9, 10, or 17 or a variant thereof. The nucleic acid can be an antisense oligonucleotide comprising a sequence partially or completely complementary to the base sequence of miR-450a2, provided by SEQ ID NOS. 9, 10, or 17 or a variant thereof.
The inhibitor can be a nucleic acid binding to all or a part of a base sequence of miR-450b. In an embodiment, the sequence of this miRNA is derived from a human, and includes not only a mature sequence but also a precursor sequence, provided by SEQ ID NOS. 11, 12, or 18 or a variant thereof. The nucleic acid can be an antisense oligonucleotide comprising a sequence partially or completely complementary to the base sequence of miR-450b, provided by SEQ ID NOS. 11, 12, or 18 or a variant thereof.
The inhibitor can be a nucleic acid binding to all or a part of a base sequence of miR503HG. In an embodiment, the sequence of this miRNA is derived from a human, and includes not only a mature sequence but also a precursor sequence, provided by SEQ ID NO. 19 or a variant thereof. The nucleic acid can be an antisense oligonucleotide comprising a sequence partially or completely complementary to the base sequence of miR503HG, provided by SEQ ID NO. 19. For example, reference to miR503HG includes its variants, which are nucleic acids with at least 80% sequence similarity to a particular SEQ ID NO: 19, nucleic acids with at least 85% sequence similarity to SEQ ID NO: 19, nucleic acids with at least 90% sequence similarity to SEQ ID NO: 19, nucleic acids with at least 95% sequence similarity to SEQ ID NO: 19.
The delivery of the therapeutic agents targeting one or more of these miRNAs of interest—miR-424, miR-503, miR503HG, miR-542, miR450a1, miR-450a2, and miR-450b can be based on viral or non-viral systems. Non-viral delivery systems include one or more cationic lipids and cationic polymers that form cationic liposomes. Viral delivery systems include without limitation adeno-associated viral systems and retroviral systems.
Forced Expression of miR-322/-503 Impedes Muscle Growth
Among the H19X microRNAs, miR-322, miR-503, and miR-542 are most frequently associated with muscle atrophy and cachexia. The miR-322/-503 cluster has been correlated with a broad range of diseases and has higher expression levels than miR-542. Disclosed here are therapeutic agents targeting the role of miR-322/-503 in regulating muscle mass.
Transgenic mouse strain was generated in which the expression of miR-322/-503 is driven by the MCK promoter (
Body masses of transgenic mice became progressively lower than that of wild-type since 4 weeks old.
Histomorphometric analysis of skeletal muscles was performed.
H19X microRNAs were evaluated as negative regulators of skeletal muscle growth by investigating whether their ablation promotes muscle hypertrophy in vivo. The H19X-LacZ mouse strain is defective in transcribing H19X microRNAs, because of an upstream poly-A transcription termination signal (
Furthermore,
Morphometric analysis depicted a shift toward thicker myofiber in skeletal muscle of mutant mice.
Starvation stimulates the expression of miR-322 and miR-542, two microRNAs that are associated with muscle atrophy and cachexia in human chronic diseases. Inactivating the H19X locus can rescue starvation-induced skeletal muscle atrophy. In H19X mutant and wild-type animals, 48-hour starvation resulted in comparable loss of bodyweight (15.4±0.9% in wt and 16.7±0.9% in mt) (
H19X ncRNAs are Elevated in Skeletal Muscle of the Elderly.
H19X ncRNAs were upregulated in atrophying skeletal muscle in animal models. The levels of H19X ncRNAs were evaluated for expression and regulation in skeletal muscle during aging. The expression of miR503 host gene (miR503HG) in skeletal muscles were analyzed from samples collected in the Genotype-Tissue Expression (GTEx) project.
Higher expression of miR503HG was observed in muscles from individuals who suffered a slow death than those who suffered a quick death. The GTEx project uses the 4-point Hardy Scale to categorize speed of death: 1 for violent and fast death; 2 for fast death of natural causes such as myocardial infarction; 3 for intermediate death such as patients who were ill but death was unexpected; 4 for slow death after a long illness and deaths were not unexpected.
Tetracycline-Induced Cardiomyocyte-Specific Expression of miR-322/503 Caused Rapid Development of Dilated Cardiomyopathy
The role of increased miR-322/503 levels in cardiomyopathy were evaluated. An inducible heart-specific transgenic mouse strain was developed, in which ectopic miR-322/503 is triggered by doxycycline (dox) diet. Continuous dox diet induced expression levels comparable to that in embryonic hearts (See
Transgenic animals appeared lethargic as early as 2 weeks after the start of dox diet. No such animals survived beyond 4 weeks (n=8), while transgenic animals (n=10) fed with regular chow were normal and healthy. Assessment of cardiomyopathy molecular markers showed significantly increased expression of ANF and BNP and upregulated β-MHC/α-MHC ratio at 4 weeks.
Echocardiography assessment of cardiac function showed progressive deterioration of ejection fraction (EF) and fraction shortening, starting from 2 weeks on dox diet.
Inhibition of miR-322/-503 can Attenuate Starvation-Induced Atrophy in Cultured Myotubes
Inhibitors of miR-322/-503 augmented myotube growth and resistance to starvation-induced atrophy. C2C12 myoblasts overexpressing antisense inhibitors of miR-322/-503 (miR-322i/-503i) were differentiated into myotubes and then cultured in regular differentiation media or subjected to starvation in low-serum/low-glucose media for 6 hours. In regular differentiation media, miR-322i/-503i-harboring myotubes had greater diameters, with combined miR-322i and miR-503i showing the strongest effect. Low-serum/low-glucose media induced a marked decrease in myotube diameter in control cultures. Remarkably, miR-322i/-503i partially rescued atrophy in cultured myotubes, with both inhibitors being most effective.
Further, amount of eIF4G1, eIF4B, eIF2B5, and eIF3M proteins was markedly higher in the presence of miR-322i/-503i, either in regular differentiation or starvation culture, or both.
Expression of miR-322/miR-503 is Inversely Related to Postnatal Skeletal Muscle Growth.
Expression of miR-322(424)/miR-503 is highly enriched in heart and somites in early embryos. Tissue distribution and expression level of H19X miRNAs change in postnatal life stages.
Among H19X-encoded non-coding RNAs, there are 7 miRNAs of interest—miR-424, miR-503, miR-542, miR450a1, miR-450a2, and miR-450b. Embodiments include antagomirs targeting one or more of these miRNAs. These antagomirs may be delivered as synthetic oligoes or by adeno-associated virus. These miRNAs can also be targeted by RNA-sponge-based technologies. RNA sponges are specially-designed synthetic RNAs that will be targeted by the aforementioned miRNAs. Therefore, these miRNAs will not be able to bind and degrade their natural targets. The RNA-sponges will be delivered as synthetic mRNAs or by adeno-associated virus. H19X-encoded miR503HG is a long non-coding RNA which can be targeted by siRNA-based technologies. Because the entire H19X locus is activated in skeletal muscle wasting, cachexia, sarcopenia and heart failure, the H19X locus can be targeted by gene editing tools such as, but not limited to, integrases (recombinases), clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR associated protein (Cas) nucleases, TALAN nucleases, zinc finger nucleases, triplex forming oligonucleotides, or combinations thereof. In an example, the H19X locus can be targeted by a CRISPR-based genome editing, including CRISPR-mediated promoter silencing and CRISPR-mediated removal of the genes.
While the specific embodiments of the present disclosure have been described in detail above, those skilled of ordinary skill in the art will appreciate that the specific embodiments are merely specific illustrative embodiments and the scope of the present disclosure is not limited by them. It is to be understood that the substantial scope of the disclosure is defined by the appended claims and their equivalents.
This invention was made with government support under grant no. PR162075 awarded by Department of Defense's Congressionally Directed Medical Research Programs. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63269975 | Mar 2022 | US |
