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  • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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
• • 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 N.A.

Definitions

• the present disclosure relates generally to LncMyoD, a LncRNA located in the human about 22 kb upstream of (5 ' to) the MyoD gene, and located in the mouse about 30 kb upstream of the MyoD gene.
• the disclosure particularly pertains to compositions comprising the
• LncMyoD or a LncMyoD-derived polynucleotide, such as a pharmaceutical composition.
• LncMyoD-derived polynucleotides include polynucleotides comprising a portion of the
• LncMyoD sequence such as one or more exons, one or more ORFs, one or more CAUH sequences, one or more fragments or portions, etc.
• the disclosure also pertains to methods of use of LncMyoD or a LncMyoD-derived polynucleotide in increasing muscle growth, treating sarcoma, Rhabdomyosarcoma, or Embryonic Rhabdomyosarcoma, treating other cancers in settings where IMP1 and/or IMP2 are upregulated, and/or in altering expression of other genes, such as c-Myc.
• LncRNA Long non-coding RNA
• LncRNAs play important roles in normal physiology as well as many diseases, including embryonic stem cell maintenance, organ development and cancer progression.
• LncRNAs represent a new class of regulators of stem cell biology. Guttman et al. 2011 Nature 477: 295-300; Klattenhoff et al. 2013 Cell 152: 570-583; and Cesana et al. 2011 Cell 147: 358-369. However, the number of LncRNAs expressed in skeletal muscle stem cells and whether they are biologically important remain largely unknown. Cesana et al. 2011 Cell 147: 358-369.
• LncMyoD An object of this disclosure is to provide mammalian LncMyoD (human sequence, SEQ ID NO: 1; and mouse sequences, SEQ ID NOs: 2 and 3), and polynucleotides comprising these sequences.
• LncMyoD-derived polynucleotides which comprise one or more fragments or portions of the LncMyoD sequence such as, in the human, any one or more of:
• Predicted ORF2 nt 116 to nt 286 of SEQ ID NO: 1 ;
• a LncMyoD-derived polynucleotide can comprise, for example, a portion of
• LncMyoD (SEQ ID NO: 1) which is less than 600 nt long.
• LncMyoD-derived polynucleotides also include polynucleotides which contain a few mismatches from LncMyoD or a fragment or portion thereof.
• a LncMyoD-derived polynucleotide can comprise a portion of SEQ ID NO: 1, e.g., any one or more of: nt 1-20 of SEQ ID NO: l; nt 21-40 of SEQ ID NO: l; nt 41-60 of SEQ ID NO: l; nt 61-80 of SEQ ID NO: l; nt 81-100 of SEQ ID NO: l; nt 101-120 of SEQ ID NO: l; nt 121-140 of SEQ ID NO: l; nt 141-160 of SEQ ID NO: l; nt 161-180 of SEQ ID NO: l; nt 181-200 of SEQ ID NO: l; nt 201- 220 of
• a LncMyoD-derived polynucleotide can comprise a portion of SEQ ID NO: 1, e.g., any one or more of: nt 1-50 of SEQ ID NO: 1; nt 51-100 of SEQ ID NO: 1; nt 101-150 of SEQ ID NO: l; nt 151-200 of SEQ ID NO: 1; nt 201-250 of SEQ ID NO: l; nt 251-300 of SEQ ID NO: l; nt 301-350 of SEQ ID NO: 1; nt 351-400 of SEQ ID NO: 1; nt 401-450 of SEQ ID NO: 1; nt 451-500 of SEQ ID NO: 1; nt 501-550 of SEQ ID NO: 1; and nt 551-600 of SEQ ID NO: 1, wherein no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nt are mismatches from SEQ ID NO: 1 (e.g.,
• the LncMyoD-derived polynucleotide comprises the sequence of LncMyoD (SEQ ID NO: 1) and an exogenous (non-LncMyoD) sequence.
• the exogenous sequence is a promoter, transcriptional enhancer, transcriptional terminator, or marker gene.
• the disclosure pertains to a vector comprising the LncMyoD sequence (SEQ ID NO: 1) or a LncMyoD-derived polynucleotide.
• the disclosure pertains to a cell comprising such a vector.
• the LncMyoD-derived polynucleotide is polyadenylated (has a polyA tail).
• the LncMyoD-derived polynucleotide is not polyadenylated (does not have a polyA tail).
• the LncMyoD-derived polynucleotide is 5' capped.
• the LncMyoD-derived polynucleotide is not 5' capped.
• the LncMyoD (SEQ ID NO: 1) or a LncMyoD-derived polynucleotide is RNA, DNA, a RNA-DNA hybrid, locked nucleic acid (LNA), Morpholino, peptidic nucleic acid (PNA), threose nucleic acid (TNA), or glycol nucleic acid (GNA), unlocked nucleic acid (UNA), wherein the polymeric molecule optionally comprises one or more modification.
• the LncMyoD-derived polynucleotide comprises one or more sequences as described herein and performs at least one function or activity of LncMyoD.
• Functions and activities of LncMyoD include:
• LyncMyoD is required for myoblast differentiation into myotubes
• LncMyoD is required for the up-regulation of differentiation-associated and other genes, including Ckm, Slc2a4, Jphl, Sri, and Col6al, since it is required for differentiation; LncMyoD must inhibit IMPs to allow for differentiation, creating a permissive state for the up-regulation of these genes;
• LncMyoD binds to IMP1 and IMP2;
• LncMyoD regulates IMP2 protein, blocking IMPs binding to proliferation-required genes like Myc and Nras;
• LncMyoD decreases binding of IMP2 to many target m NAs, including Myc, Ccngl, Igflr, Igf2, Nras and Rhla;
• LncMyoD down-regulates NF-kb and FOXOl pathway (not NF-Kb and FoxOl themselves) and up-regulated PGCla/b and other mitochondria pathway.
• a LncMyoD-derived polynucleotide can fulfillment the requirement in any one or more of these roles for LncMyoD; e.g., the LncMyoD-derived polynucleotide can perform or at least partially perform the described function of LncMyoD.
• compositions comprising LncMyoD and/or one or more LncMyoD-derived polynucleotides. These include
• compositions e.g., a pharmaceutical composition comprising LncMyoD and/or a LncMyoD-derived polynucleotide, and a pharmaceutically acceptable carrier.
• the disclosure pertains to a method of increasing myoblast differentiation in a cell or myotube in need there (e.g., a cell or myotube with decreased
• LncMyoD level and/or activity which method comprising the step of increasing the level and/or activity of LncMyoD.
• the disclosure pertains to a method of up-regulation of differentiation-associated and other genes (e.g., Ckm, Slc2a4, Jphl, Sri, and Col6al), which method comprising the step of increasing the level and/or activity of LncMyoD.
• differentiation-associated and other genes e.g., Ckm, Slc2a4, Jphl, Sri, and Col6al
• the disclosure pertains to a method of blocking IMPs (e.g., IMP1 and IMP2) binding to proliferation-required genes (e.g., Myc and Nras), which method comprising the step of increasing the level and/or activity of LncMyoD.
• the disclosure pertains to a method of decreasing the level and/or activity of Myc, which method comprising the step of decreasing the level and/or activity of IMP 1 and/or IMP2 wherein the step of decreasing the level and/or activity of IMP 1 and/or IMP2 comprises the step of introducing exogenous LncMyoD or a LncMyoD-derived polynucleotide into a cell.
• the step of increasing the level and/or activity of LncMyoD comprises the step of introducing exogenous LncMyoD into a cell.
• the disclosure pertains to a method of decreasing tumor cell proliferation in a cell or patient in need there (e.g., a cell or patient with increased IMP1 and/or IMP2 level and/or activity), which method comprising the step of increasing the level and/or activity of LncMyoD to block the action of IMP 1 or IMP2.
• the disclosure pertains to a method of treating sarcoma (e.g., Rhabdomyosarcoma or Embryonic Rhabdomyosarcoma) in a patient in need therefore, which method comprises the step of increasing the level and/or activity of LncMyoD.
• sarcoma e.g., Rhabdomyosarcoma or Embryonic Rhabdomyosarcoma
• the disclosure pertains to a method of treating a cancer in which IMP1 and/or IMP2 are up-regulated in a patient in need therefore, which method comprises the step of increasing the level and/or activity of LncMyoD, and wherein LncMyoD decreases the activity of IMP 1 and/or IMP2.
• LncRNAs are identified from mouse skeletal muscle. Most of these are found to be expressed at similar levels in myoblasts and myotubes (black dots), while subsets are either enriched in undifferentiated myoblasts (pink dots) or differentiated multi-nuclear myotubes (green dots). LncMyoD was one of the top LncRNAs that was found to be highly enriched in myotubes.
• LncMyoD was enriched in myotube nuclei, since about 70% of the spliced LncMyoD located in the nucleus. Both LncMyoD and MyoD mRNA levels are up-regulated during myoblast differentiation. * p ⁇ 0.05. Error bars depict mean ⁇ SEM.
• LncMyoD was predicted to be a non-coding RNA.
• the RNA sequences of LncMyoD, MyoD and HOTAIR are put into Coding Potential Calculator (CPC) program and both LncMyoD and HOTAIR are predicted to be non-coding RNAs while MyoD RNA was identified to code for protein.
• CPC Coding Potential Calculator
• Figure 2 The expression of LncMyoD is highly tissue specific; LncMyoD is a direct downstream target of MyoD.
• Mouse LncMyoD is expressed only detected in myoblasts, early myotubes and the testicle, but not any other tissues examined, including ovary, liver, lung, spleen, embryo, kidney, heart, thymus, brain and (notably) mature skeletal muscle. MyoD is only expressed in myoblasts and myotubes. Error bars depict mean ⁇ SEM.
• FIG. 3 LncMyoD plays important roles in myoblast differentiation.
• LncMyoD shRNAs or NT shRNA treated myotubes are harvested and western blots are performed to detect the proteins of NRAS and MYC.
• a-Tubulin was used as a loading control.
• IMP1 and IMP2 have been shown to be upregulated in a variety of tumors, and required for proliferation of rhabdomyosarcoma.
• LncMyoD can inhibit the function of IMPs, by blocking proliferation-necessary genes such as Myc and Nras, demonstrates that LncMyoD could be used to treat those tumors or cancers in which IMP1 or IMP2 are upregulated or activated, or in which their activity is required for tumor maintenance.
• microRNAs are identified by R A-seq data. Many identified microRNAs, such as Mir I, Mir24, Mir27, Mir 133, Mir205 and Mir296 have well-documented roles in skeletal muscle maintenance and myogenesis.
• LncMyoD is polyA tailed. Total RNA was extracted from myotubes and reverse transcribed using Oligo dT primer. Spliced LncMyoD was steadily detected by RT-PCR, suggesting that LncMyoD has PolyA tail, like mRNAs.
• FIG. 6 Cardiotoxin (CTX)-injury induced muscle regeneration in mouse.
• Skeletal muscles are injected with CTX and samples are harvested at different time- points - from day 0 to day 14.
• the LncMyoD promoter region contains multiple potential MyoD binding sites (E-Box).
• E-Box MyoD binding sites
• Figure 8 LncMyoD regulates the survival and differentiation of myoblasts.
• Figure 9 Top transcriptional factor networks and top biological functions regulated by LncMyoD.
• Top transcriptional factor networks up-regulated by LncMyoD shRNAs include NF-kb, FoxOl while the most down-regulated ones are PGCla/b, MyoCD etc.
• Figure 10 (a) and (b): Mouse LncMyoD contains multiple IMP binding sites.
• Figure 11 Exogenous human LncMyoD expression restored MyHC expression, suggesting a conserved function of LncMyoD between human and mouse even though the sequence similarity of LncMyoD is very low between these two species.
• Figure 12 Function of various mouse LncMyoD fragments; (a) Schematic of mouse LncMyoD full length and truncated variants; (b-c) Mouse LncMyoD shRNA treated myoblast were transfected with empty vector, mouse full length LncMyoD or each truncated variant expression vector. RNA level was analyzed by real-time PCR using different primer sets as indicated in a, protein level was analyzed by western blotting. Superscripted R stands for shRNA resistance. * p ⁇ 0.05. Error bars depict mean ⁇ SEM. mouse LncMyoD Full Length (SEQ ID NO: 9)
• the present disclosure pertains to LncMyoD, and LncMyoD-derived polynucleotides, and methods of their use. [0060] DEFINITIONS
• LncMyoD refers to a novel LncR A located in the human about 22 kb upstream of the MyoD gene, and located in the mouse about 30 kb upstream of the MyoD gene.
• LncMyoD is strongly up-regulated upon differentiation from myoblasts to myotubes. It contains two exons and one intron (Fig. lc) and, like many LncR As, it is polyA tailed. About 70% of the spliced LncMyoD transcript resides in the nucleus.
• the size of the human LncMyoD is 600 nt and the sequence is presented in SEQ ID NO: 1.
• LncMyoD The size of the mouse LncMyoD is 361 bp and the sequence is presented in SEQ ID NO: 2. Consistent with LncMyoD being a non-coding RNA, LncMyoD harbors no open reading frames (ORFs) larger than 150 bp. No evidence of a protein product from LncMyoD using in vitro translation was detected.
• LncMyoD is expressed in the testes. Interestingly, like MyoD, LncMyoD is not expressed in mature skeletal muscles, suggesting that LncMyoD is temporally up-regulated during early differentiation of myoblasts, but eventually turned off as the muscle matures into post-differentiated fibers.
• the mouse LncMyoD has two transcripts. These are transcribed from two different start sites.
• the mouse LncMyoD is represented by a long intron (SEQ ID NO: 2) and a short intron (SEQ ID NO: 3):
• Mouse LncMyoD short intron also known as LncMyoD*
• the LncMyoD-derived polynucleotide comprises one or more sequences as described herein and performs at least one function or activity of LncMyoD.
• LncMyoD Functions and activities of LncMyoD include:
• LyncMyoD is required for myoblast differentiation into myotubes
• LncMyoD is required for the up-regulation of differentiation-associated and other genes, including Ckm, Slc2a4, Jphl, Sri, and Col6al, since it is required for differentiation; LncMyoD must inhibit IMPs to allow for differentiation, creating a permissive state for the up-regulation of these genes;
• LncMyoD binds to IMP1 and IMP2;
• LncMyoD regulates IMP2 protein, blocking IMPs binding to proliferation-required genes like Myc and Nras;
• LncMyoD decreases binding of IMP2 to many target mRNAs, including Myc, Ccngl, Igflr, Igf2, Nras and Rhla;
• LncMyoD down-regulates NF-kb and FOXOl pathway (not NF-Kb and FoxOl themselves) and up-regulated PGCla/b and other mitochondria pathway.
• MyoD or “Myod” is meant the gene or its protein product, a key transcriptional factor regulating myogenesis, also referenced as: MYOD1; Myod, MYF3; MYOD; PUM;
• bHLHcl External IDs: OMIM: 159970 MGI: 97275 HomoloGene: 7857 GeneCards: MYOD1 Gene; Human; Entrez; 4654; Ensembl; ENSG00000129152; UniProt ; P15172; RefSeq (mRNA); NM_002478; RefSeq (protein); NP_002469; Location (UCSC); Chr 11 :17.74 - 17.74 Mb;
• MyoD is a protein with a key role in regulating muscle differentiation. MyoD belongs to a family of proteins known as myogenic regulatory factors (MRFs). These bHLH (basic helix loop helix) transcription factors act sequentially in myogenic differentiation. MRF family members include MyoD, Myf5, myogenin, and MRF4 (Myf6).
• MRFs myogenic regulatory factors
• MyoD is one of the earliest markers of myogenic commitment. MyoD is expressed in activated satellite cells, but not in quiescent satellite cells. Although MyoD marks myoblast commitment, muscle development is not dramatically ablated in mouse mutants lacking the MyoD gene. This is likely to be due to functional redundancy from Myf5. Nevertheless, the combination of MyoD and Myf5 is vital to the success of myogenesis.
• IGF2BP2 the gene or its product also referenced as IGF2BP2; IMP-2; IMP2; VICKZ2; p62; External IDs: OMIM: 608289 MGI: 1890358;
• HomoloGene 4774; GeneCards: IGF2BP2 Gene; Insulin-like growth factor 2 mRNA-binding protein 2.
• This gene encodes a member of the IGF-II mRNA-binding protein (IMP) family.
• the protein encoded by this gene contains several four KH domains and two RRM domains. It functions by binding to the 5' UTR of the insulin- like growth factor 2 (IGF2) mRNA and regulating IGF2 translation.
• IGF2 insulin- like growth factor 2
• the disclosure pertains to a method of decreasing proliferation of a tumor cell in which IMP1 and/or IMP2 are up-regulated, which method comprising the step of increasing the level and/or activity of LncMyoD, and wherein LncMyoD decreases the activity of IMP 1 and/or IMP2.
• the disclosure pertains to a method of decreasing the proliferation of a sarcoma (e.g., Rhabdomyosarcoma or Embryonic Rhabdomyosarcoma) in a patient in need therefore, which method comprises the step of increasing the level and/or activity of LncMyoD.
• a sarcoma e.g., Rhabdomyosarcoma or Embryonic Rhabdomyosarcoma
• the disclosure pertains to a method of treating a cancer in which IMP1 and/or IMP2 are up-regulated, in a patient in need therefore, which method comprises the step of increasing the level and/or activity of LncMyoD, and wherein LncMyoD decreases the activity of IMP 1 and/or IMP2.
• the disclosure pertains to a method of decreasing proliferation of a tumor cell in which IMP1 and/or IMP2 are up-regulated and/or required for tumor cell proliferation and/or survival, which method comprising the step of increasing the level and/or activity of LncMyoD, and wherein LncMyoD decreases the activity of IMP 1 and/or IMP2.
• the disclosure pertains to a method of up- regulation of any one or more of the genes Ckm, Slc2a4, Jphl, Sri, and Col6al, which method comprising the step of decreasing the level and/or activity of IMP 1 or IMP2.
• the disclosure pertains to a method of blocking IMPs binding to genes Myc and Nras, or other mRNAs required for proliferation or tumor cell survival, which method comprising the step of increasing the level and/or activity of LncMyoD.
• the disclosure pertains to a method of decreasing the level and/or activity of Myc, which method comprising the step of decreasing the level and/or activity of IMP 1 and/or IMP2 wherein the step of decreasing the level and/or activity of IMP 1 and/or IMP2 comprises the step of introducing exogenous LncMyoD or a LncMyoD-derived polynucleotide into a cell.
• the disclosure pertains to a method of decreasing the level and/or activity of Myc, which method comprising the step of decreasing the level and/or activity of IMP 1 and/or IMP2, wherein the step of decreasing the level and/or activity of IMP 1 and/or IMP2 comprises the step of introducing exogenous LncMyoD or a LncMyoD-derived polynucleotide into a cell.
• the step of decreasing the level and/or activity of IMP1 and/or IMP2 comprises the step of introducing exogenous LncMyoD or a LncMyoD- derived polynucleotide into a cell.
• any of the LncMyoD or LncMyoD-derived polynucleotides described herein can be used to increase activity and/or level of LncMyoD.
• sarcoma is meant a cancer that arises from transformed cells of mesenchymal origin.
• malignant tumors made of cancerous bone, cartilage, fat, muscle, vascular, or hematopoietic tissues are, by definition, considered sarcomas. This is in contrast to a malignant tumor originating from epithelial cells, which are termed carcinoma.
• Human sarcomas are quite rare. Common malignancies, such as breast, colon, and lung cancer, are almost always carcinoma.
• Sarcomas include, for example, Rhabdomyosarcoma.
• Rhabdomyosarcoma or “RMS” is meant a type of cancer, specifically a sarcoma, in which the cancer cells are thought to arise from skeletal muscle progenitors. It can also be found attached to muscle tissue, wrapped around intestines, or in any anatomic location. Most occur in areas naturally lacking in skeletal muscle, such as the head, neck, and
• Rhabdomyosarcoma Embryonic Rhabdomyosarcoma.
• polynucleotide oligonucleotide or “nucleic acid” and the like is meant a polymeric form of nucleotides or other molecules capable of conveying genetic information, including but not limited to various nucleic acids, including but not limited to RNA, DNA or RNA-DNA hybrids, or forms with an alternative backbone such as locked nucleic acids (LNA), Morpholinos, peptidic nucleic acids (PNA), threose nucleic acid (TNA), or glycol nucleic acid (GNA), arabinose nucleic acid (ANA), 2 ' -fluoroarabinose nucleic acid (FANA), cyclohexene nucleic acid (CeNA), anhydrohexitol nucleic acid (HNA), and/or unlocked nucleic acid (UNA) (a non-nucleotide, acyclic analog wherein the C2'-C3' bond is not present), whether modified or not modified
• LNA locked nucleic
• Modifications include, without limitation, 2'-alkyl, e.g., 2'-OMe, 2'F and 2'-MOE.
• Polynucleotides can thus comprise polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide which is an N- or C- glycoside of a purine or pyrimidine base, and other polymers containing nonnucleotidic backbones such as those described herein.
• a LncMyoD or LncMyoD-derived polynucleotide as disclosed herein can comprise any of these structures, or a combination thereof (e.g., RNA-DNA hybrid; RNA partially substituted with DNA, LNA, TNA, ANA, FANA, CeNA, HNA, PNA, and/or GNA, etc.) in combination with any modification and/or secondary moiety as described herein.
• RNA-DNA hybrid RNA partially substituted with DNA, LNA, TNA, ANA, FANA, CeNA, HNA, PNA, and/or GNA, etc.
• Polynucleotides also include, as non-limiting examples: 3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3' P5' phosphoramidates, 2'-0-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, microRNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or RNA, and also include known types of modifications, for example, labels which are known in the art, methylation, "caps," substitution of one or more of the naturally occurring nucleotides with an analog (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine, C5- propynyluridine, C5-bromouridine, C5-fluorouridine, C5-
• the term also includes locked nucleic acids (e.g., comprising a ribonucleotide that has a methylene bridge between the 2'-oxygen atom and the 4'-carbon atom).
• locked nucleic acids e.g., comprising a ribonucleotide that has a methylene bridge between the 2'-oxygen atom and the 4'-carbon atom.
• Polynucleotides can be single-, double- or triple-stranded or have complex structures involving a variety of double-stranded and single-stranded regions (some which may be loops).
• Polynucleotides can be modified or not modified.
• Modifications include a modified sugar backbone, a phosphorothioate linkage, or a 2'-modified nucleotide.
• 2'-modifications can be selected from the group consisting of: 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl (2'-OMe), 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2 * -0-AP), 2 * -0 -dimethylaminoethyl (2 * -0-DMAOE), 2 * -0-dimethylaminopropyl (2 * -0-DMAP), 2'-0 -dimethylaminoethyloxyethyl (2'-0-DMAEOE), and 2'-0-N-methylacetamido (2'-0 - NMA).
• a polynucleotide can also include at least one modified nucleotide, including but not limited to a 2'-0-methyl modified nucleotide, a nucleoside comprising a 5' phosphorothioate linkage group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2'-deoxy-2'-fluoro modified nucleoside, a 2'-amino-modified nucleoside, 2'-alkyl-modified nucleoside, morpholino nucleoside, an unlocked ribonucleotide (e.g., an acyclic nucleotide monomer, as described in WO 2008/147824), a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof.
• a 2'-0-methyl modified nucleotide a
• Additional examples of modifications include 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl)uracil, 5 -carboxymethylaminomethyl-2-thiouridine, 5 - carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5 -methyl cytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-meth
• a polynucleotide molecule can comprise at least two modifications, or 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 15, at least 20, at least 30, at least 50, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, or more, up to the entire length of the polynucleotide molecule.
• Polynucleotides can be ligated to one or more secondary moiety, including, without limitation, diagnostic compound, reporter group, cross-linking agent, nuclease-resistance conferring moiety, natural or unusual nucleobase, lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin,
• Polynucleotides can be radiolabeled or not.
• LncMyoD and LncMyoD-derived polynucleotides of any sequence disclosed herein (or any portion thereof) can thus have the structure of any polynucleotide known.
• LncMyoD or Lnc-MyoD-derived polynucleotides disclosed herein may be administered with a pharmaceutically-acceptable carrier, e.g., an excipient, carrier, diluent, salt, delivery vehicle or the like, which facilitates entry to the cell.
• a pharmaceutically-acceptable carrier e.g., an excipient, carrier, diluent, salt, delivery vehicle or the like
• pharmaceutically-acceptable carrier e.g., an excipient, carrier, diluent, salt, delivery vehicle or the like
• pharmaceutically-acceptable carrier e.g., an excipient, carrier, diluent, salt, delivery vehicle or the like
• pharmaceutically-acceptable carrier e.g., an excipient, carrier, diluent, salt, delivery vehicle or the like
• pharmaceutically-acceptable carrier e.g., an excipient, carrier, diluent, salt, delivery vehicle or the like
• pharmaceutically-acceptable carrier
• Suitable delivery vehicles include, for example, viral vectors, viral particles, liposome formulations, and lipofectin.
• Pharmaceutically-acceptable carriers are also useful for delivery of inhibitory agents to LncMyoD (e.g., a shR A or siRNA or other polynucleotides targeting LncMyoD), for methods involving the step of decreasing LncMyoD level and/or activity.
• Polynucleotides can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see e.g., Gonzalez et al, Bioconjugate Chem., 10: 1068-1074 (1999); WO 03/47518; and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA
• microspheres see for example U.S. Pat. No. 6,447,796 and U.S. 2002/130430
• biodegradable nanocapsules see for example U.S. Pat. No. 6,447,796 and U.S. 2002/130430
• bioadhesive microspheres or by proteinaceous vectors (WO 00/53722).
• the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump.
• Direct injection of the nucleic acid molecules of the invention can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Corny et al, Clin. Cancer Res., 5: 2330-2337 (1999) and WO 99/31262.
• Polynucleotides may be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues.
• the polynucleotide complexes can be locally administered to relevant tissues ex vivo, or in vivo through direct dermal application, transdermal application, or injection, with or without their incorporation in biopolymers.
• Delivery systems include surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes).
• Polynucleotides may be formulated or complexed with polyethylenimine (e.g., linear or branched PEI) and/or polyethylenimine derivatives, including for example polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI -PEG-GAL) or
• PEI-PEG-triGAL polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine
• grafted PEIs such as galactose PEI, cholesterol PEI, antibody derivatized PEI, and polyethylene glycol PEI (PEG-PEI) derivatives thereof
• Delivery systems may include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as
• solubilizers e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids
• hydrophilic polymers e.g., polycarbophil and polyvinylpyrolidone
• the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer.
• liposomes which can be used in this invention include the following: (1) CellFectin, 1 : 1.5 (M/M) liposome formulation of the cationic lipid ⁇ , ⁇ , ⁇ , ⁇ -tetramethyl- N,NI,NII,NIII-tetrapalmit-y-spermine and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) Cytofectin GSV, 2: 1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research); (3) DOTAP (N-[l-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate) (Boehringer Manheim); and (4) Lipofectamine, 3: 1 (M/M) liposome formulation of the polycationic lipid DOSPA, the neutral lipid DOPE (GIBCO BRL) and Di- Alkylated Amino Acid (DiLA2).
• DOPE
• Polynucleotides may be expressed from transcription units inserted into DNA or RNA vectors.
• Recombinant vectors can be DNA plasmids or viral vectors.
• Nucleic acid molecule expressing viral vectors can be constructed based on, but not limited to, adeno- associated virus, retrovirus, adenovirus, or alphavirus.
• the recombinant vectors are capable of expressing the nucleic acid molecules either permanently or transiently in target cells. Delivery of nucleic acid molecule expressing vectors can be systemic, such as by intravenous, subcutaneous, or intramuscular administration.
• Expression vectors may include a nucleic acid sequence encoding at least one nucleic acid molecule disclosed herein, in a manner which allows expression of the nucleic acid molecule.
• the vector may contain sequence(s) encoding both strands of a nucleic acid molecule that include a duplex.
• the vector can also contain sequence(s) encoding a single nucleic acid molecule that is self-complementary and thus forms a nucleic acid molecule.
• Non- limiting examples of such expression vectors are described in Paul et al, 2002, Nature
• An expression vector may encode one or both strands of a nucleic acid duplex, or a single self-complementary strand that self hybridizes into a nucleic acid duplex.
• the polynucleotides can be operably linked to a transcriptional regulatory element that results expression of the nucleic acid molecule in the target cell.
• Transcriptional regulatory elements may include one or more transcription initiation regions (e.g., eukaryotic pol I, II or III initiation region) and/or transcription termination regions (e.g., eukaryotic pol I, II or III termination region).
• the vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5' side or the 3 '-side of the sequence encoding the nucleic acid molecule; and/or an intron (intervening sequences).
• ORF open reading frame
• the polynucleotides or the vector construct can be introduced into the cell using suitable formulations.
• One preferable formulation is with a lipid formulation such as in
• formulations can also be administered to animals such as by intravenous, intramuscular, or intraperitoneal injection, or orally or by inhalation or other methods as are known in the art.
• the formulation is suitable for administration into animals such as mammals and more specifically humans, the formulation is also pharmaceutically acceptable.
• Pharmaceutically acceptable formulations for administering polynucleotides are known and can be used.
• it may be preferable to formulate dsR A in a buffer or saline solution and directly inject the formulated dsR A into cells, as in studies with oocytes.
• the direct injection of dsRNA duplexes may also be done. Suitable methods of introducing dsRNA are provided, for example, in U.S. 2004/0203145 and U.S. 20070265220.
• Polymeric nanocapsules or microcapsules facilitate transport and release of the encapsulated or bound dsRNA into the cell. They include polymeric and monomeric materials, especially including polybutylcyanoacrylate.
• the polymeric materials which are formed from monomeric and/or oligomeric precursors in the polymerization / nanoparticle generation step, are per se known from the prior art, as are the molecular weights and molecular weight distribution of the polymeric material which a person skilled in the field of manufacturing nanoparticles may suitably select in accordance with the usual skill.
• Polynucleotides may be formulated as a microemulsion.
• a microemulsion is a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution.
• microemulsions are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a 4th component, generally an intermediate chain-length alcohol to form a transparent system.
• Surfactants that may be used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants.
• ionic surfactants non-ionic surfactants
• Brij 96 polyoxyethylene oleyl ethers
• polyglycerol fatty acid esters tetraglycerol monolaurate (
• the cosurfactant usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules.
• a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol
• a pharmaceutical composition comprising a polynucleotide can also optionally comprise a salt or pharmaceutically acceptable salt.
• the term “Pharmaceutically acceptable salt” includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate,
• ethylsuccinate citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para- toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and
• lactobionate salts include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).
• Any polynucleotide described herein can comprise any modification or substitution described herein or known in the art (e.g., DNA, PNA, TNA, etc.), and can be prepared in a pharmaceutical composition comprising any component described herein or known in the art.
• the disclosure thus pertains to a pharmaceutical composition
• a pharmaceutical composition comprising LncMyoD and/or a LncMyoD-derived polynucleotide (or a polynucleotide which inhibits LncMyoD) and a pharmaceutically-acceptable carrier (e.g., any carrier, vehicle, emulsion, salt, etc. described herein or known in the art).
• a pharmaceutically-acceptable carrier e.g., any carrier, vehicle, emulsion, salt, etc. described herein or known in the art.
• a suitable pharmaceutically-acceptable carrier for a polynucleotide is capable of delivery of a therapeutically effective dose or effective amount of a polynucleotide.
• terapéuticaally effective dose refers to an amount of a compound that produces a desired effect.
• a population of cells may be contacted with an effective amount of a compound to study its effect in vitro (e.g., cell culture) or to produce a desired therapeutic effect ex vivo or in vitro.
• An effective amount of a compound may be used to produce a therapeutic effect in a subject, such as preventing or treating a target condition, alleviating symptoms associated with the condition, or producing a desired physiological effect.
• the effective amount of a compound is a "therapeutically effective amount,” “therapeutically effective concentration” or “therapeutically effective dose.”
• the precise effective amount or therapeutically effective amount is an amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject or population of cells. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication) or cells, the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration.
• an effective or therapeutically effective amount may vary depending on whether the compound is administered alone or in combination with another compound, drug, therapy or other therapeutic method or modality.
• One skilled in the clinical and pharmacological arts will be able to determine an effective amount or therapeutically effective amount through routine experimentation, namely by monitoring a cell's or subject's response to administration of a compound and adjusting the dosage accordingly.
• Remington The Science and Practice of Pharmacy, 21st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, Pa., 2005, which is hereby incorporated by reference as if fully set forth herein.
• the polynucleotide can be delivered in a pharmaceutically acceptable carrier in an effective dose alone or in combination with another appropriate agent.
• in combination means in the course of treating the same disease in the same patient using two or more agents, drugs, treatment regimens, treatment modalities or a combination thereof, in any order. This includes simultaneous administration, as well as in a temporally spaced order of up to several days apart. Such combination treatment may also include more than a single administration of any one or more of the agents, drugs, treatment regimens or treatment modalities. Further, the
• administration of the two or more agents, drugs, treatment regimens, treatment modalities or a combination thereof may be by the same or different routes of administration.
• LncRNAs represent a new class of regulators of stem cell biology. Guttman et al. 2011 Nature 477: 295-300; Klattenhoff et al. 2013 Cell 152: 570-583; and Cesana et al. 2011 Cell 147: 358-369. However, the number of LncRNAs expressed in skeletal muscle stem cells and whether they are biologically important remain largely unknown. Cesana et al. 2011 Cell 147: 358-369.
• LncMyoD is strongly up-regulated upon differentiation from myoblasts to myotubes (Fig. la, c). It contains two exons and one intron (Fig. lc) and, like many LncRNAs, it is polyA tailed (Supplementary Fig. lb).
• Cell fractionation followed by quantitative RT-PCR demonstrates that about 70% of the spliced LncMyoD transcript resides in the nucleus (Fig. Id). 5' RACE and 3 'RACE demonstrated that the size of LncMyoD is 361bp, and that it partially overlaps with a previous annotated RNA AK006355 (Fig. le).
• LncMyoD being a non-coding RNA, it harbors no open reading frames (ORFs) larger than 150bp; the CPC (coding potential calculator) computational algorism [Kong et al. 2007 Nucleic Acids Res.
• LncMyoD has a very low coding potential, similar to HOTAIR, a well- known LncRNA (Fig. If ). No evidence of a protein product from LncMyoD is found using in vitro translation.
• LncMyoD is low in uninjured muscle and strongly up-regulated at 3-5 days after muscle injury. It begins to be down-regulated after day 5, when the muscle regeneration enters the late stage (Fig. 2b and Supplementary Fig. 2a). Notably, this expression pattern is almost identical to that of MyoD mRNA (Fig. 2b) [00134]
• the upstream factor (or factors) that is regulating LncMyoD during myogenesis we analyze the 5' and intron sequences of the gene, and find that there are five canonical MyoD binding sites (E-Boxes) in this region, raising the possibility that LncMyoD is a direct target of MyoD. Such a finding would be consistent with the coincident expression profiles of LncMyoD with MyoD. To determine whether MyoD regulates LncMyoD, the
• LncMyoD regulatory element is cloned into a PGL3 luciferase reporter construct.
• the reporter When the reporter is co-transfected with MyoD overexpression construct, it shows dose-dependent activation by MyoD (Fig. 2c), suggesting that MyoD could directly promote LncMyoD transcription.
• the activity of a promoter is usually orientation dependent. Consistent with this, the "reverse reporter" had much weaker baseline activity and it could only be modestly activated by MyoD (Fig. 2c).
• chromatin immunoprecipitation ChoIP
• MyoD is found to strongly bind to a 5' E-Box and 3' E-Box tandem in the LncMyoD promoter (Fig. 2d). Importantly, these bindings of MyoD are significantly weaker in myoblasts than in myotubes, consistent with low expression of LncMyoD in myoblasts (Fig. 2d). Together, these data demonstrate that LncMyoD is a direct MyoD target in vivo.
• LncMyoD is essential for cell cycle withdrawal, a key step in myoblast differentiation. These myoblasts eventually die or go into a senescent state (Supplementary Fig. 4b). Endogenous overexpression of LncMyoD alone in myoblasts does not cause premature differentiation or other obvious phenotypes. Therefore, LncMyoD is necessary but not sufficient in promoting myoblast differentiation.
• LncMyoD shRNAs alters the levels of hundreds of mRNAs (Fig. 3e).
• genes involved in skeletal muscle functions such as muscle contractility and myofibril are among the mostly down-regulated by LncMyoD knockdown.
• targets of myogenic factors such as Myocd and Meox2 are significantly down-regulated.
• targets of mitochondria biogenesis factors such as Pgcla, Pgclfi, EsrrA are also among the most down-regulated genes; muscle differentiation is associated with mitochondria synthesis.
• LncMyoD may regulate cell cycle and myogenesis
• we attempt to identify LncMyoD interacting proteins using a biotinylated-ZncA yoD protein pull-down assay. Both non-biotinylated LncMyoD and biotinylated antisense RNA are used as controls. After screening a set of RNA-binding proteins, IGF2 mRNA binding proteins (IMPs) are found to strongly bind to LncMyoD (Fig. 4a).
• IMPs IGF2 mRNA binding proteins
• the IMP family has three members: IMP1 - 3.
• IMP2 we focus our efforts on IMP2 because we have recently found this protein to be required for myogenesis, as a result of its binding and downregulating the translation of proliferation-relevant target mRNAs, resulting in differentiation. Li et al. 2012 Dev. Cell 23: 1176-1188; and Boudoukha et al. 2010 Mol. Cell. Biol. 30: 5710-5725.
• LncMyoD contains 5 CAUH sequences.
• the direct binding of LncMyoD to IMP2 raises the possibility that LncMyoD may regulate IMP2 functions.
• IMP2 regulates myoblast growth through binding to and enhancing the translation of mRNAs involved in proliferation such as Nras and Myc. Li et al. 2012 Dev. Cell 23: 1176-1188.
• LncMyoD knockdown causes a significant increase in the binding of many target mRNAs to IMP2, including Myc, Ccngl, Igflr, Igfi, Nras and Rhla (Fig. 4e). Consequently, the levels of proteins like NRAS and MYC are maintained at high levels even upon differentiation stimuli (Fig. 4f), which apparently contributes to the failure of terminal differentiation, given the undifferentiated phenotype of the LncMyoD knockdown myoblasts (Fig 3 c).
• Confluent C2C12 myoblast differentation model mRNA-seq raw sequence read data, from Trapnell et al (ref 20436464; data series GSE20846) is downloaded from the sequence read archive. Specifically, data relating to the data series SRX017794 ("-24 hours”; Mouse skeletal muscle C2C12 cells, exponential growth phase in high serum medium, taken as a model of undifferentiated myoblasts) and data series SRX017795 ("+60 hours”; confluent mouse skeletal muscle C2C12 cells, 60 hours post switch to low serum medium, initiating myogenic
• SRA data archives are converted to FASTQ format using the SRA toolkit, and aligned against the mouse genome (build
• Cells are lysed with NP40 buffer (25 mM Hepes, 100 mM NaCl, 5 mM MgC12, 10% glycerol, 0.2% NP-40, phosphatase inhibitor cocktail- 1 and -2 (Sigma) and protease inhibitor cocktail (Roche) for total protein characterization.
• NP40 buffer 25 mM Hepes, 100 mM NaCl, 5 mM MgC12, 10% glycerol, 0.2% NP-40, phosphatase inhibitor cocktail- 1 and -2 (Sigma) and protease inhibitor cocktail (Roche) for total protein characterization.
• NP40 buffer 25 mM Hepes, 100 mM NaCl, 5 mM MgC12, 10% glycerol, 0.2% NP-40, phosphatase inhibitor cocktail- 1 and -2 (Sigma) and protease inhibitor cocktail (Roche) for total protein characterization.
• phosphatase inhibitor cocktail- 1 and -2 Sigma
• Muscle degeneration/regeneration by cardiotoxin (CTX)-mediated injury is performed as previously described 24 .
• CTX cardiotoxin
• Muscle samples are harvested for immunohistochemistry at day 0, day 1 , day 3, day 5, day 7 and day 14 after injection and stained by H&E and specific antibodies. Regeneration is clearly activated in the first 3 days and recovered by 14 days after injury.
• RNAs are isolated by RNeasy Kits (Qiagen) and cDNA is made using an iScript cDNA Synthesis Kit.
• SYBR Green dye based Quantitative Real Time PCR (qRT-PCR) is performed using SYBR Green PCR Master Mix and 7900HT Fast Real-Time PCR System from Applied Biosystems. Individual gene primers are designed and synthesized by Integrated DNA Technologies.
• RNA ligase-mediated rapid amplification of cDNA ends are carried out with total RNA extracted from primary myoblasts culture, and are used to determine the transcription start points and the size of the LncMyoD transcripts. Rapid amplification of 5 Or 3' cDNA ends is carried out using a FirstChoice RLM-RACE kit (Ambion), according to the manufacturer's instructions. Due to the low copy number of LncMyoD in cells, nested PCR is performed for each reaction.
• RNA-binding protein immunoprecipitation is performed using a Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit (Millipore). Briefly, primary mouse myoblasts are harvested by adding RIP lysis buffer. Clear supernatant containing IGF2BP2 protein, IgA beads and IGF2BP2 antibody (or IgG control) are mixed to perform the immunoprecipitation. After washing, RNAs binding to IGF2BP2 are eluted and quantified. Reverse transcription and RT-PCR are performed to examine whether certain mRNAs are co-immunoprecipitated with the IGF2BP2 antibody.
• RIP RNA-binding protein immunoprecipitation
• F10 GIBCO
• FBS Fetal Bovine Serum, GIBCO
• bFGF Invitrogen
• myoblasts are switched to differentiation medium (DMEM supplemented with 5% horse serum).
• LncMyoD forward or reverse promoter regions are cloned into a PGL3 luciferase reporter vector (Promega).
• the mouse Myodl coding region is cloned into the PCDNA3.1(+) vector.
• LncMyoD-Pro-Luciferase, Renilla and MyoD plasmids are transfected into 293 cells seeded in 96 well plates, using the FUGENE® 6 (Roche) transfection reagent, following the manufacturer's protocol. 48 hours later, cells are lysed and luciferase assays are performed using a Dual-Luciferase® Reporter Assay System (Promega) on a luminometer.
• Luciferase readings are taken as singlets. Ratios of Renilla luciferase readings to Firefly luciferase readings are taken for each experiment and triplicates are averaged.
• shRNA sequences targeting LncMyoD are cloned into a Tet-pLKO-puro vector (addgene 21915).
• Lentivirus is produced using a ViraPower Lentiviral Packaging Mix (life technologies) in 293T cells, filtered and used to infect myoblast cells.
• the efficacy of shRNA constructs are screened after Doxycycline treatment for 48 hours followed by RT-PCR.
• a Non-Targeting (NT) shRNA sequence is used as negative control (Adapted from Sigma SHC002). 2 shRNAs achieved more than 80% knockdown efficacy, and are therefore selected for sequential experiments.
• RNA is extracted using the TRIzol reagent (Invitrogen) and purified with Qiagen RNeasy separation columns (Qiagen).
• Qiagen Qiagen RNeasy separation columns
• first-strand cDNA is synthesized and hybridized to GeneChip Mouse Genome 430 2.0 Array (Affymetrix).
• An Active Motifs ChIP-IT® Express Kit is used for the Chromatin Immunoprecipitation experiment according to the manufacturer's instructions. Briefly, Cells are cross-linked with 1% formaldehyde for 10 min at room temperature and lysed in SDS lysis buffer. Samples are then sonicated or enzymatically digested to obtain DNA fragments with an average length of 200-800 bp. Supernatant containing DNA-protein complexes are used for immunoprecipitations using an anti-MyoD antibody (Santa Cruz, sc-760) or a normal rabbit IgG control.
• Immunoprecipitated chromatin is collected using protein G magnet beads and, after washing and elution, reverse crosslinking is carried out with 0.2M NaCl at 65°C overnight. The chromatin is then digested by 20 ⁇ g of Proteinase K (Invitrogen) for lh at 45°C and isolated by phenolchloroform extraction. PCR reactions are performed using SYBR Green PCR Master Mix (Applied Biosystems), and primers against LncMyoD promoter regions. Data are normalized to the input signal and reported to IgG values.
• Mouse C2C12 cells were transfected with either an empty, or a mouse LncMyoDR (siRNA resistant) or a human LncMyoD expression plasmid, in addition to a mouse LncMyoD shRNA inducible expression plasmid. Then cells were induced for differentiation. Half of the cells were induced for mouse LncMyoD shRNA expression. The differentiation marker MyHC was analyzed as a readout for the effect of the siRNA. Mouse LncMyoD downregulation by the shRNA decreased MyHC protein level as expected, and exogenous mouse LncMyoD expression restored the expression of MyHC. Notably, exogenous human LncMyoD expression also restored MyHC expression, suggesting a conserved function of LncMyoD between human and mouse even though the sequence similarity of LncMyoD is very low between these two species.
• Mouse LncMyoD shRNA treated myoblast were transfected with empty vector, mouse full length LncMyoD or each truncated variant expression vector.
• Figure 12 shows:
• RNA level was analyzed by realtime PCR using different primer sets as indicated in a, protein level was analyzed by western blotting. Superscripted R stands for shRNA resistance. * p ⁇ 0.05. Error bars depict mean ⁇ SEM.
• An isolated LncMyoD-derived polynucleotide comprising SEQ ID NO: 1, or one or more portions of SEQ ID NO: 1 selected from:
• polynucleotide is less than 600 nt long.
• polynucleotide comprises a sequence selected from:
• a vector comprising the polynucleotide of embodiment 1.
• a cell comprising the vector of embodiment 7.
• LncMyoD-derived polynucleotide comprises R A, DNA, a R A-DNA hybrid, locked nucleic acid (LNA), Morpholino, peptidic nucleic acid (PNA), threose nucleic acid (TNA), arabinose nucleic acid (ANA), 2 ' -fl uoroarabinose nucleic acid (FANA), cyclohexene nucleic acid (CeNA),
• FTNA anhydrohexitol nucleic acid
• GAA glycol nucleic acid
• polynucleotide is present in a therapeutically effective amount.
• a method of increasing myoblast differentiation in a cell or myotube comprising the step of increasing the level and/or activity of the polynucleotide of embodiment 1.
• a method of up-regulation of any one or more of the genes Ckm, Slc2a4, Jphl, Sri, and Col6al which method comprising the step of increasing the level and/or activity of the polynucleotide of embodiment 1.
• a method of decreasing the level and/or activity of Myc which method comprising the step of increasing the level and/or activity of the polynucleotide of embodiment 1.
• composition comprising a pharmaceutically acceptable carrier and the
• composition of embodiment 25, wherein the polynucleotide is DNA, a RNA-DNA hybrid, locked nucleic acid (LNA), Morpholino, peptidic nucleic acid (PNA), threose nucleic acid (TNA), or glycol nucleic acid (GNA), optionally comprising one or more modification.
• LNA locked nucleic acid
• PNA peptidic nucleic acid
• TAA threose nucleic acid
• GNA glycol nucleic acid
• a method of decreasing proliferation of a tumor cell in which IMP1 and/or IMP2 are up-regulated comprising the step of introducing into the cell or increasing the level and/or activity in the cell of the polynucleotide of embodiment 1, and wherein the polynucleotide decreases the activity of IMP1 and/or IMP2.
• a method of treating a cancer in which IMP1 and/or IMP2 are up-regulated, in a patient in need therefore comprises the step of introducing into the patient or increasing the level and/or activity in the patient of the polynucleotide of embodiment 1, and wherein the polynucleotide decreases the activity of IMP 1 and/or IMP2.
• a method of decreasing proliferation of a tumor cell in which IMP1 and/or IMP2 are up-regulated and/or required for tumor cell proliferation and/or survival comprising the step of introducing into the cell or increasing the level and/or activity in the cell of the polynucleotide of embodiment 1 , and wherein polynucleotide decreases the activity of IMP 1 and/or IMP2.
• a method of up-regulation of any one or more of the genes Ckm, Slc2a4, Jphl , Sri, and Col6al in a cell comprising the step of introducing into the cell or increasing the level and/or activity in the cell of the polynucleotide of embodiment 1, and wherein polynucleotide decreases the level and/or activity of IMP 1 or IMP2.
• a method of blocking IMPs binding to genes Myc and Nras, or other mRNAs required for proliferation or tumor cell survival in a cell comprising the step of introducing into the cell or increasing the level and/or activity in the cell of the polynucleotide of embodiment 1.
• a method of decreasing the level and/or activity of Myc which method comprising the step of introducing into the cell or increasing the level and/or activity in the cell of the polynucleotide of embodiment 1.
• a method of decreasing the level and/or activity of Myc which method comprising the step of decreasing the level and/or activity of IMP 1 and/or IMP2, wherein the step of decreasing the level and/or activity of IMP 1 and/or IMP2 comprises the step of introducing the polynucleotide of embodiment 1 into a cell.

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