Compounds and Methods for the Treatment of Muscular

Disease, and Related Screening Methods

Related Applications

This application claims the benefit of priority to United States Provisional Patent

Application serial number 61/549,940, filed October 21, 2011, the contents of which are hereby incorporated by reference.

Government Support

This invention was made with government support under Grant No. P50 NS040828- 08 awarded by the National Institutes of Health. The government has certain rights in this invention.

Background of the Invention

Muscle diseases include many diseases and ailments that either directly, via intrinsic muscle pathology, or indirectly, via nerve or neuromuscular junction pathology, impair the functioning of the muscles. Muscular dystrophy (MD) is a group of muscle diseases that weaken the musculoskeletal system and hamper locomotion. Muscular dystrophies are characterized by progressive skeletal muscle weakness, defects in muscle proteins, and the death of muscle cells and tissue.

Myotonic Dystrophy (DM) is the most prevalent muscular dystrophy in adults, affecting 1/8000 individuals worldwide, and is incurable. There are multiple clinical manifestations of the disease including disabling musculoskeletal symptoms (myotonia, muscle weakness and wasting), ophthalmologic defects (cataract) and cognitive deficits. The genetic abnormality underlying the predominant form, DM1, is caused by a pathological expansion of CTG repeats in the 3' untranslated region of the DMPK (DM protein Kinase) gene. When transcribed, the expanded CUG entraps trans-acting RNA binding proteins (RBPs) in the cell nucleus, leading to aberrant processing of many downstream RNA targets. The prevailing model mainly focuses on MBNL1 (muscleblind like protein), a splicing factor which is sequestered on the toxic CUG repeats. Mounting evidence from the literature indicates that other factors co-regulate MBNL1 -dependent RNA processing, and thus play a key role in the pathogenesis of DM1.

DM1 is a dominantly inherited neuromuscular disease characterized by progressive myopathy and myotonia, due to large expansions (> 50 and up to 4000) of CTG-repeats in the 3' UTR of the DMPK gene. The disease also results in a combination of heart defects, cataract, GI dysfunction, insulin resistance and cognitive deficits, and is thus complex and generalized. The transcribed CUG-expanded RNAs adopt an abnormal hairpin structure and accumulate in the nucleus as foci, where they entrap, or more indirectly deregulate, R A processing proteins (RBPs). It is well-established that DM1 is in part caused by the dysfunction of two well-characterized RBPs, MBNL1 and CUGBP1 (CUG binding protein 1, aka CELF1), resulting in a global mis-regulation of splicing events (i.e. 'spliceopathy'). Aberrant splicing in DM1 leads to the generation of isoforms that are normally found in a different developmental stage (e.g. fetal isoform produced in adult muscle), or in a different tissue (e.g. non-muscle isoform in muscle), and are not functional in the temporal/local cellular context. In addition to missplicing, abnormal translation, subcellular localization and turn-over of RNA targets have been reported in DM1. Studies of corresponding DM1 mouse models have greatly enhanced understanding of the mechanism of the pathologic cascade that underlies the disease. Despite these major advances, it has become increasingly apparent that MBNL1/CUGPB1 functional imbalance does not fully explain DM1 pathophysiology. For example, in MBNL1 -deficient mice muscle wasting (as observed in DM1 patients) is not recapitulated, nor is the full spectrum of aberrantly spliced exons that is associated with DM1 in humans. A 2-fold elevation of CUGBP1 levels in transgenic mice (comparable to that observed in DM1 muscle) does not induce changes in alternative splicing. Given that RBPs often act in protein complexes, these observations prompted an active search for additional factors that likely contribute to the disease. Among the candidates, hnRNP H, and more recently Staufenl have been shown to modulate the MBNL1/CUGBP1 -dependent aberrant splicing in DM, but other factors have yet to be uncovered.

Isoprenoid antibiotics were originally isolated from the phytopathogenic fungus Ascochyta viciae. (Sasaki, H. et al, Isolation and structure of ascofuranone and ascofranol, antibiotics with hypolipidemic activity. J Antibiot (Tokyo), 1973, 26:676-680). Among them, ascochlorin and ascofuranone have been shown to be non-toxic compounds. Structurally related compounds have been subsequently isolated from other fungi (e.g., Fusarium, Cylindrocladium, Cylindrocladium ilicicola, Nectria coccinea, CoUetotrichum nicotianae, Acremonium luzulae, Cephalosporium diospyri, Verticillium, Cylindrocarpon lucidum, Nigrosabulum globosum, and the insect pathogenic fungus Verticillium hemipterigenum). (Hosono, K. et al, Ll-zl272 alpha epoxide, a precursor of ascochlorin produced by a mutant of ascochyta viciae, J Antibiot (Tokyo), 2009, 62:571-574; Seephonkai, P. et al, A novel ascochlorin glycoside from the insect pathogenic fungus verticillium hemipterigenum bcc 2370, J Antibiot (Tokyo), 2004, 57: 10-16).

Ascochlorin and ascofuranone display antitumorigenic properties, both in vitro and in vivo. (Jeong JH, et al. J Cell Biochem. 2012 Apr;l 13(4): 1302-13; Kang, J. H. et al. J Biol Chem. 2012 May 4;287(19): 15661 -71 ; Jeong, J.H. and Chang, Y.C., Ascochlorin, an isoprenoid antibiotic, induces gl arrest via downregulation of c-myc in a p53 -independent manner, Biochem Biophys Res Commun., 2010, 398:68-73; Magae, J. et al, Antitumor and antimetastatic activity of an antibiotic, ascofuranone, and activation of phagocytes, J Antibiot (Tokyo), 1988, 41 :959-965; Nakajima, H. et al., Aberrant expression of fra-1 in estrogen receptor-negative breast cancers and suppression of their propagation in vivo by ascochlorin, an antibiotic that inhibits cellular activator protein- 1 activity. J Antibiot (Tokyo), 2007, 60:682-689). In a murine model of mammary carcinoma, it was demonstrated that intraperitoneal administration of ascochlorin significantly suppressed cancer growth and prolonged survival. (Nakajima et al, 2007).

In addition to anticancer properties, ascochlorin and its derivatives exhibit a wide range of physiological activities, including antimicrobial/antiviral activity, trypanocidal properties, hypolipidemic activity, suppression of hypertension, improvement of type I and II diabetes, and immunomodulation. (Yabu, Y. et al., The efficacy of ascofuranone in a consecutive treatment on trypanosoma brucei brucei in mice, Parasitol Int. 2003, 52: 155- 164; Hosono et al., 2009).

There exists a need for non-toxic compounds for the treatment of muscle diseases.

Summary of the Invention

One aspect of the invention relates to a method of treating a muscle disease, comprising the step of administering to a mammal in need thereof a therapeutically effective amount of an isoprenoid antibiotic.

Another aspect of the invention relates to a method of improving normal muscle function, comprising the step of administering to a mammal in need thereof a therapeutically effective amount of an isoprenoid antibiotic.

Yet another aspect of the invention relates to a method of upregulating hnRNP L, comprising the step of administering to a mammal in need thereof a therapeutically effective amount of an isoprenoid antibiotic. Another aspect of the invention relates to a method of upregulating a hnRNP L target, comprising the step of administering to a mammal in need thereof a therapeutically effective amount of an isoprenoid antibiotic.

In certain embodiments, the invention relates to a RNA interference (RNAi) agent capable of reducing, attenuating, or abolishing the production of hnRNP L in a cell, wherein the cell is a mammalian myoblast or a mammalian myotube.

In certain embodiments, the invention relates to a method of attenuating hnRNP L gene expression in a cell, comprising the steps of:

contacting a plurality of cells with a recombinant nucleic acid construct or vector, wherein the recombinant nucleic acid construct or vector encodes any one of the aforementioned RNAi agents, and wherein the cells are mammalian myoblasts, thereby producing a subset of cells that express the RNAi in an amount sufficient to attenuate hnRNP L gene expression in the subset of cells.

In certain embodiments, the invention relates to a method comprising the steps of: contacting a plurality of cells with a recombinant nucleic acid construct or vector, wherein the recombinant nucleic acid construct or vector encodes any one of the aforementioned RNAi agents, and wherein the cells are mammalian myoblasts, thereby producing a subset of cells that express the RNAi in an amount sufficient to attenuate hnRNP L gene expression in the subset of cells;

selecting the subset of cells, thereby forming a selected subset of cells;

exposing the selected subset of cells to a compound; and

analyzing the progression of differentiation of the mammalian myoblasts into mammalian myotubes.

Brief Description of the Drawings

Figure 1 shows adult smooth mutant Drosophila flies treated with ascochlorin display prolonged survival time. Smooth mutant Drosophila flies (sm homozygotes, that develop muscle dystrophy) were maintained on paper discs soaked either with ascochlorin (Santa Cruz Biotechnology, 1 mM solution in 9 % sucrose, 10 % ethanol), or with vehicle alone. The treatment was maintained throughout the lifespan of the organism. Unaffected control siblings (sm heterozygotes) were maintained on vehicle. The number of dead flies was recorded daily, and percent survival was plotted as a function of time in days. Figure 2 shows the position of the two amplicons that are amplified by qPCR primer pair 1 and primer pair 2 (red bars) on the corresponding mouse hnRNP L mRNA sequence graphic (from GenBank).

Figure 3 shows melting curves generated for hnRNP L primer pair 1, hnRNP L primer pair 2, and β-actin.

Figure 4 shows a standard curve for hnRNP L primer pair 1 , hnRNP L primer pair 2, and β-actin.

Figure 5 depicts images showing that shRNAi knockdown of hnRNP L in differentiating human myoblasts results in death of the myotubes at Day 4 Differentiation (circle). shLuc: control anti-luciferase; shFiNRP L: anti-hnRNP L.

Figure 6 depicts images showing that shRNAi knockdown of FiNRNP L in differentiating human myoblasts results in disorganized myotubes at Day 14 Differentiation (arrow). ShLuc: anti-luciferase control virus; shFiNRP L: anti-hnRNP L virus.

Figure 7 depicts gels taken at day 7 of differentiation for myotubes that express the shHRNP L virus. The results show a decrease in hnRNP L protein levels, in both the whole cell lysate (Cyto fraction), and nuclear fraction (vs. normal levels in shLuc control virus- expressing myotubes). Gapdh staining demonstrates cytoplasmic lysate loading.

Figure 8 depicts HNRNP interacting with MBNLl . Top panel Co- immunoprecipitation (co-IP) of FLAG-tagged hnRNP L isoforms (589 aa), or (456 aa), with HA-tagged-MBNLl . Immunoprecipitation is performed with HA-beads (MBNLl - HA), and immunoblot with anti-FLAG antibody. Input lysate control immunoblot detecting the anti-HA antibody is shown at bottom. Middle panel. Reverse co-IP using FLAG-beads (HNRNP L). Of note, multiple MBNLl isoforms are pulled. Control immunoblot detecting the anti-FLAG antibody is shown at bottom. Abbreviations, IP: immunoprecipitation; WB: Western blot.

Figure 9 depicts HnRNP L expression in proliferating and differentiating, normal and diseased, human myoblasts. Western Blot analysis (using the 4D11 monoclonal anti- hnRNP L antibody, Sigma) shows a doublet at ~ 60 kDa. The level of the lower hnRNP L band in the doublet appears elevated (as a ratio of the higher band), in differentiated DM1 (mild, or severe, phenotype) muscle cells, but not in DMD (Duchenne muscular dystrophy) cells, (compared to normal muscle cells). Abbreviations, %: percent confluence; D: differentiation day. Figure 10 depicts that DMD exon 71 is aberrantly spliced in hnRNP L-deficient myotubes. Total RNA was prepared from Day 2 differentiation myotubes that express either the shHNRNPL (i.e. hnRNP L KD), or the control shLuc, lentivector. RT-PCR analysis was performed using the same primer pair that was utilized by Nakamori et al. to demonstrate missplicing of DMD E71 in human DM1 muscle. Exclusion of the exon results in the generation of the shorter Δ E71 DMD isoform.

Figure 11 depicts the effect of shRNAi knockdown (KD) of hnRNP L in differentiating human myoblasts. HnRNP L KD results in the death of multi-nucleated myotubes at Day 4 Differentiation (circle). HnRNP L KD results in disorganized myotubes at Day 14 Differentiation (arrow). shLuc: anti-luciferase control virus; shHNRNP L: anti- hnRNP L virus.

Figure 12 tabulates the chemical libraries that will be screened using the hnRNP L- deficient myoblast HTS assay.

Figure 13 depicts a sequence alignment of hnRNPL a and b sequences

Detailed Description of the Invention

Overview

The RBP hnRNP L (heteronuclear ribonucleoprotein L) may play a role in the pathogenesis of DM1, implying that its therapeutic modulation holds promise in the treatment of DM. Downregulation of the fly homolog of hnRNP L, smooth, leads to pronounced muscle degeneration and premature death, in adult flies. In addition, hnRNP L binds to MBNL1 in vitro (in HEK293 cells). hnRNP L (i) is essential for myo fiber survival in vitro, (ii) is differentially expressed in DM1 muscle cell line and (iii) regulates the splicing of exons that are aberrantly processed in DM1.

In certain embodiments, the invention relates to the decrease in hnRNP L activity in DM1 muscle, due to its interaction with sequestered MBNL1. In certain embodiments, the invention relates to a method of increasing hnRNP L levels in a cell comprising contacting the cell with a therapeutically effective amount of a compound.

The splicing factor hnRNP L may be a candidate modifier of myotonic dystrophy/MBNLl function. In certain embodiments, the invention relates to investigations designed to further establish the relevance of this protein in the pathogenic mechanisms underlying DM1. In certain embodiments, the invention relates to the investigation of the spectrum of alternative exons regulated by hnRNP L in human muscle cells, and the definition of the overlap with exons that are aberrantly spliced in DM1 (as identified in previous transcriptome analyses of muscle isolated from DM1 patients). Knowledge of the pathogenic mechanisms that are regulated by hnRNP L in DM can be translated into the identification of drugs that target these processes. Despite major efforts that have focused on myotonic dystrophy, only symptomatic therapies are currently available. In certain embodiments, the invention relates to a cell-based high throughput screen that is designed to enable the identification of compounds that not only lead to symptomatic relief, but can enhance the survival of mature myo fibers that would otherwise degenerate.

In certain embodiments, the invention relates to a cell-based assay that uniquely assesses the survival of multi-nucleated human myofibers, where alternative splicing is de- regulated. Using the cell-based assay, pharmacological compounds that improve myotube survival/maintenance can be identified. Hit compounds may provide novel chemical entities for the development of DM1 therapeutics. Using the cell-based assay, the spectrum of RNA targets that are regulated by hnRNP L in muscle can be defined. A better understanding of hnRNP L-dependent pathways in muscle will provide novel means to fight spliceopathy- induced muscle disease (e.g. DM1).

Link to the splicing factor hnRNP L/downstream RNA targets

Proteome analysis has demonstrated that ascochlorin treatment of human osteosarcoma cells (U20S) results in a > 10 fold increase in the levels of three proteins, including the splicing factor hnRNP L (most up-regulated protein, 12 x), as well as BINl (third most up-regulated protein, 10 x). (Kang, J.H. et al, Proteome analysis of responses to ascochlorin in a human osteosarcoma cell line by 2-d gel electrophoresis and maldi-tof MS, J Proteome Res., 2006, 5:2620-2631). We have determined by bioinformatics analysis that BINl is a candidate target of hnRNP L (Table 1, below). Importantly, mutations in BINl (alternative name, amphiphysin 2) are linked to centronuclear myopathies in humans. (Toussaint, A. et al., Defects in amphiphysin 2 (BINl) and triads in several forms of centronuclear myopathies, Acta Neuropathol., 2011, 121 :253-266).

Recent studies also demonstrated that the methylated derivative of ascochlorin, 4-0- methylascochlorin (MAC), increases the expression of vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT-1). (Jeong, J.H. et al, 4-o-methylascochlorin, methylated derivative of ascochlorin, stabilizes HIF-1 -alpha via AMPK activation, Biochem. Biophys. Res. Commun., 2011, 406:353-358). Both VEGF and GLUT-1 RNAs are well-established targets of hnRNP L. (Hamilton, B.J. et al, HnRNP a2 and hnRNP 1 bind the 3'utr of glucose transporter 1 mRNA and exist as a complex in vivo, Biochem. Biophys. Res. Commun., 1999, 261 :646-651; Ray, P.S. et al, A stress-responsive RNA switch regulates VEGFa expression, Nature, 2009, 457:915-919; Shih, S.C. and Claffey, K.P., Regulation of human vascular endothelial growth factor mRNA stability in hypoxia by heterogeneous nuclear ribonucleoprotein 1, J. Biol. Chem., 1999, 274: 1359-1365).

Previous reports in the literature have demonstrated that hnRNP L is a key regulator of alternative splicing that can promote either the inclusion, or the exclusion, of tissue specific exons. Interestingly, hnRNP L binding motifs were shown to be enriched in alternative splicing events that are associated with myogenic differentiation. We recently carried out a bioinformatic analysis of a compendium of known human alternative splicing events to identify sequences that include the hnRNP L binding motif, and are expressed in skeletal muscle and/or the heart. This analysis revealed 46 conserved putative targets of hnRNP L in muscle and the heart. Importantly, the list includes nine targets, APP, BIN1, DMD, DTNA, MAPT, MEF2A, PPP2R5C, SORBS 1 and TTN, which are known to be aberrantly spliced in DM1 patients. This is a remarkably high number, given that to date, a total of less than 30 splicing defects have been confirmed in DM1 patients.

Table 1: Candidate RNA targets of hnRNP L/SMOOTH in skeletal muscle and/or the heart.

Human Description Associated Muscle / Heart Drosophila

Gene Disease in Humans Homolog

ABM ABL interactor 1/ spectrin SH3 Abi

ACSS2 Acetyl Coenzyme A synthetase AcCoAS

AGAP3 ArfGAP GTPase ankyrin repeat and PH cenGIA domain containing

AGXT2L2 Alanine glyoxylate aminotransferase 2 CG8745

APP Beta amyloid precursor Appl

ATP2B1 Plasma membrane calcium ATPase PMCA

ATP2B4 Plasma membrane calcium ATPase PMCA

BIN 1 Bridging integrator Centronuclear myopathy Amph

BPTF Nucleosome remodeling factor subunit E(bx)

C12orf41 UNKNOWN dgtl

C14orf133 UNKNOWN Vps16B

DMD Dystrophin Duchenne/Becker muscular Dys

dystrophy, Dilated

cardiomyopathy

DTNA Dystrobrevin Dyb

EIF2C2 Argonaute translation factor 2C AG01 EPB41 L2 ! UNKNOWN I cora

FMNL2 i Formin j CG32138

GARNL1 ] Ral GTPase activating protein a subunit I CG5521

ITSN2 ] Intersectin j Dap160

KIAA1217 I Homolog of murine Sickle tail ! CG32809

LRRFIP1 i Leucine rich repeat flightless interacting j CG8578

MAPT ] Microtubule associated Tau ] Tau

MAX ] MYC associated factor X j Max

MEF2A I Myocyte specific enhancer factor ! Mef2

NCAM1 i Neural cell adhesion molecule 1 ! Fas2

PALLD ] Palladin ] zormin

PDLIM7 j PDZ and LIM domain j Zasp52

PPP2R5C I Serine/Threonine Phosphatase 2A ! PP2A-B' j regulatory subunit B56

PTPN3 i Tyrosine phosphatase non receptor i Ptpmeg

RPGR i Retinitis pigmentosa GTPase regulator i ca

RRN3 ] RNA polymerase 1 specific transcription I Tif-IA

] factor

SAD1 I Serine threonine kinase SAD I CG61 14

SAMD4A i Smaug homolog 1 ] Smg

SEMA6D i Semaphorin 6D i Sema-1a

] Sema-1 b

SLC25A3 ] Phosphate carrier protein, mitonchondrial Mitochondrial phosphate carrier I CG9090 deficiency ( muscular

hypotonia/ hypertrophic

cardiomyopathy)

SLC39A9 ] UNKNOWN may act as a zinc I CG2177

\ transporter (by similarity)

SMTN \ Smoothelin I Actn3

SORBS1 i Sorbin and SH3 domain containing 1 ! CAP

STXBP5 ] Tomosyn ] tomosyn

SVIL ] Archvillin; Supervilin, i CG33232

TPM1 I Tropomyosin Hypertrophic cardiomyopathy, ! Tm1

Dilated cardiomyopathy Tm2

TPM3 i Tropomyosin Nemaline myopathy 1 , ! Tm1

Congenital cap myopathy Tm2

TRIM66 ] Tripartite motif containing 66 ] bon TTN Titin Autosomal dominant myopathy bt with fatal cardiomyopathy

Hypertrophic cardiomyopathy,

Dilated cardiomyopathy,

VPS29 Vacuolar protein sorting 29 CG4764

XPNPEP1 j Aminoacyl proline aminopeptidase ApepP

ZMYND8 Protein kinase C binding protein 1 CG1815

Compounds

Formula 1

Table 2:

CHO H (CH ₂ ) ₄ COOH CI H

CHO H CH ₂ COO CH ₃ CI H

CHO H Nicotinoyl CI H

CHO H Benzoyl CI H

CHO H Isonicotinoyl CI H

CHO H OCH ₂ COOC ₂ H ₅ CI H

CHO H OCH ₂ COOCH ₃ CI H

CHO H OCH ₂ COOH CI H

CHO H OCHCH ₃ COOC ₂ H ₅ CI H

CHO H OCHCH ₃ COOC ₄ H ₉ CI H

CHO H OCHCH ₂ CH ₃ COOC ₂ H ₅ CI H

CHO H 0(CH ₂ ) ₃ COOC ₂ H ₅ CI H

CHO H OCHCH ₃ COOH CI H

CHO H 0(CH ₂ ) ₃ COOH CI H

CHO H 0 CI H

CHO H OCOC ₆ H ₄ OCH ₃ CI H

CHO H OCOC ₆ H ₄ COOCH ₃ CI H

CHO H OCON(C ₂ H ₅ ) CI H

CHO H OCOCH ₂ OC ₆ H ₄ Cl CI H

CHO H 0 CI H

CHO H o

CI H

CHO H OCH ₃ CI H

C ₂ H ₂ COCH ₃ H H CI O

CHO H CH ₃ CO H H

CHO H CH ₃ H H 42 C ₂ H ₂ COCH ₃ H CH ₂ COOH CI 0

43 CHO CH ₃ CO CH ₃ CO CI H

44 C(OCH ₃ ) ₂ H CH ₃ CO CI H

45 C(OCH ₂ CH ₃ ) ₂ H CH ₃ CO CI H

46 C(OCH ₂ CH ₃ ) ₂ H CH ₃ CI H

47 C(0(CH ₂ ) ₃ CH H CH ₃ CI H

3)2

48 H CH ₃ CI H

49 CHO H 0(CH ₂ ) ₃ CH ₃ CI H

50 CHO H OCH ₂ CH ₃ CI H

51 CHO H OCH ₂ CHCH ₂ CI H

52 C0 ₂ H H H CI H

Formula 2

Table 3:

Genetically Engineered Cells

In certain embodiments, the invention relates to in vitro cultures of differentiating mammalian myoblasts (e.g., human primary myoblasts or mouse C2C12 myoblasts) in which the splicing factor hnRNP L is downregulated.

RNA silencing agents have received particular interest as research tools and therapeutic agents for their ability to knock down expression of a particular protein with a high degree of sequence specificity. The sequence specificity of RNA silencing agents is particularly useful for allele-specific silencing of dominant, gain-of-function gene mutations.

In certain embodiments, the instant invention provides short hairpin RNA (shRNA) capable of mediating RNA silencing of hnRNP L with enhanced selectivity. shRNAs mimic the natural precursors of micro RNAs (miRNAs) and enter at the top of the gene silencing pathway. For this reason, shRNAs are believed to mediate gene silencing more efficiently by being fed through the entire natural gene silencing pathway.

miRNAs are noncoding RNAs of approximately 22 nucleotides which can regulate gene expression at the post transcriptional or translational level during plant and animal development. One common feature of miRNAs is that they are all excised from an approximately 70 nucleotide precursor RNA stem-loop termed pre -miRNA, probably by Dicer, an RNase Ill-type enzyme, or a homolog thereof. Naturally-occurring miRNA precursors (pre -miRNA) have a single strand that forms a duplex stem including two portions that are generally complementary, and a loop, that connects the two portions of the stem. In typical pre-miRNAs, the stem includes one or more bulges, e.g., extra nucleotides that create a single nucleotide "loop" in one portion of the stem, and/or one or more unpaired nucleotides that create a gap in the hybridization of the two portions of the stem to each other. The shRNAs of the invention are artificial constructs based on these naturally occurring pre -miRNAs, but which are engineered to deliver desired RNA silencing agents. By substituting the stem sequences of the pre-miRNA with sequence complementary to the target mRNA, a shRNA is formed. The shRNA is processed by the entire gene silencing pathway of the cell, thereby efficiently mediating RNAi.

shRNA molecules include a first portion and a second portion, having sufficient complementarity to anneal or hybridize to form a duplex or double-stranded stem portion. The two portions need not be fully or perfectly complementary. The first and second "stem" portions are connected by a portion having a sequence that has insufficient sequence complementarity to anneal or hybridize to other portions of the shRNA. This latter portion is referred to as a "loop" portion in the shRNA molecule. The shRNA molecules are processed to generate siRNAs. shRNAs can also include one or more bulges, i.e., extra nucleotides that create a small nucleotide "loop" in a portion of the stem, for example a one- , two- or three-nucleotide loop. The stem portions can be the same length, or one portion can include an overhang of, for example, 1-5 nucleotides. The overhanging nucleotides can include, for example, uracils (Us), e.g., all Us. Such Us are notably encoded by thymidines (Ts) in the shRNA-encoding DNA which signal the termination of transcription.

In shRNAs, one strand of the stem portion of the shRNA is sufficiently complementary (e.g., antisense) to a target RNA (e.g., mRNA) sequence to mediate degradation or cleavage of said target RNA via RNA interference (RNAi). Thus, engineered RNA precursors include a duplex stem with two portions and a loop connecting the two stem portions. The antisense portion can be on the 5' or 3' end of the stem. The stem portions of a shRNA are preferably about 15 to about 50 nucleotides in length. In certain embodiments, the two stem portions are about 18 or 19 to about 21, 22, 23, 24, 25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length. In certain embodiments, the length of the stem portions should be 21 nucleotides or greater. In certain embodiments, when used in mammalian cells, the length of the stem portions should be less than about 30 nucleotides to avoid provoking non-specific responses. In certain embodiments, in non-mammalian cells, the stem can be longer than 30 nucleotides. In fact, the stem can include much larger sections complementary to the target mRNA (up to, and including the entire mRNA). In fact, a stem portion can include much larger sections complementary to the target mRNA (up to, and including the entire mRNA).

The two portions of the duplex stem must be sufficiently complementary to hybridize to form the duplex stem. Thus, the two portions can be, but need not be, fully or perfectly complementary. In addition, the two stem portions can be the same length, or one portion can include an overhang of 1, 2, 3, or 4 nucleotides. The overhanging nucleotides can include, for example, uracils (Us), e.g., all Us. The loop in the shRNAs may differ from natural pre-miRNA sequences by modifying the loop sequence to increase or decrease the number of paired nucleotides, or replacing all or part of the loop sequence with a tetraloop or other loop sequences. Thus, the loop in the shRNAs can be 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, or more nucleotides in length. The loop in the shR As or engineered RNA precursors may differ from natural pre- miRNA sequences by modifying the loop sequence to increase or decrease the number of paired nucleotides, or replacing all or part of the loop sequence with a tetraloop or other loop sequences. Thus, the loop portion in the shRNA can be about 2 to about 20 nucleotides in length, i.e., about 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, or more nucleotides in length. A preferred loop consists of or comprises a "tetraloop" sequences. Exemplary tetraloop sequences include, but are not limited to, the sequences GNRA, where N is any nucleotide and R is a purine nucleotide, GGGG, and UUUU.

In certain embodiments, shRNAs of the invention include the sequences of a desired siRNA molecule described supra. In other embodiments, the sequence of the antisense portion of a shRNA can be designed essentially as described above or generally by selecting an 18, 19, 20, 21 nucleotide, or longer, sequence from within the target RNA, for example, from a region 100 to 200 or 300 nucleotides upstream or downstream of the start of translation. In general, the sequence can be selected from any portion of the target RNA (e.g., mRNA) including the 5' UTR (untranslated region), coding sequence, or 3' UTR, provided said portion is distant from the site of the gain-of-function mutation. This sequence can optionally follow immediately after a region of the target gene containing two adjacent AA nucleotides. The last two nucleotides of the nucleotide sequence can be selected to be UU. This 21 or so nucleotide sequence is used to create one portion of a duplex stem in the shRNA. This sequence can replace a stem portion of a wild-type pre- miRNA sequence, e.g., enzymatically, or is included in a complete sequence that is synthesized. For example, one can synthesize DNA oligonucleotides that encode the entire stem-loop engineered RNA precursor, or that encode just the portion to be inserted into the duplex stem of the precursor, and using restriction enzymes to build the engineered RNA precursor construct, e.g., from a wild-type pre-miRNA.

Exemplary RNAi Agents, Nucleic Acid Molecules, Recombinant Nucleic Acid Constructs and Vectors, and Methods

In certain embodiments, the invention relates to a RNA interference (RNAi) agent capable of reducing, attenuating, or abolishing the production of hnRNP L in a cell, wherein the cell is a mammalian myoblast or a mammalian myotube.

In certain embodiments, the invention relates to any one of the aforementioned RNAi agents, wherein the RNAi agent is selected from the group consisting of RNAi nucleic acid molecules, RNAi nucleic acid analogue molecules, short interfering nucleic acids, short interfering nucleic acid analogues (siNA), short interfering RNA, short interfering RNA analogues (siRNA), double-stranded RNA, double-stranded RNA analogues (dsRNA), micro-RNA, micro-RNA analogues (miRNA), short hairpin RNA, and short hairpin RNA analogues (shRNA).

In certain embodiments, the invention relates to any one of the aforementioned

RNAi agents, wherein the RNAi agent is shRNA.

In certain embodiments, the invention relates to any one of the aforementioned RNAi agents, wherein the RNAi agent is configured to target the hnRNP L gene of the mammalian myoblast or the mammalian myotube, or a variant of the hnRNP L gene of the mammalian myoblast or the mammalian myotube having at least about 80%, about least about 85%o, at least about 90%>, at least about 95%, at least about 98%>, or at least about 99% sequence identity to the hnRNP L gene of the mammalian myoblast or the mammalian myotube.

Human hnRNP L comes in 2 isoforms,

-Transcript variant 1 (2,129 bp linear mRNA, Accession: NM_001533.2)

-Transcript variant 2 (1,895 bp linear mRNA, Accession: NM 001005335.1)

-Protein variant a (589 aa, Accession: NP_001524.2) (See Figure 13)

-Protein variant b (456 aa, Accession: P_001005335.1) (See Figure 13)

Importantly, both a and b protein isoforms co-precipitate with MBNL1 in vitro.

In certain embodiments, the invention relates to a method of attenuating hnRNP L gene expression in a cell, comprising the steps of:

contacting a plurality of cells with a recombinant nucleic acid construct or vector, wherein the recombinant nucleic acid construct or vector encodes any one of the aforementioned RNAi agents, and wherein the cells are mammalian myoblasts, thereby producing a subset of cells that express the RNAi in an amount sufficient to attenuate hnRNP L gene expression in the subset of cells.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the recombinant nucleic acid construct or vector is a lentiviral vector; and the cells are contacted with the lentiviral vector at a viral titer of about 2 x 10 ⁸ TU/mL.

In certain embodiments, the invention relates to a method comprising the steps of: contacting a plurality of cells with a recombinant nucleic acid construct or vector, wherein the recombinant nucleic acid construct or vector encodes any one of the aforementioned RNAi agents, and wherein the cells are mammalian myoblasts, thereby producing a subset of cells that express the R Ai in an amount sufficient to attenuate hnR P L gene expression in the subset of cells;

selecting the subset of cells, thereby forming a selected subset of cells;

exposing the selected subset of cells to a compound; and

analyzing the progression of differentiation of the mammalian myoblasts into mammalian myotubes.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the recombinant nucleic acid construct or vector is a lentiviral vector; and the cells are contacted with the lentiviral vector at a viral titer of about 2 x 10 ⁸ TU/mL.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the selected subset of cells is exposed to the compound on about day 2 of differentiation of the mammalian myoblasts into mammalian myotubes.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the progression of differentiation of the mammalian myoblasts into mammalian myotubes is analyzed on or after day 4 of differentiation of the mammalian myoblasts into mammalian myotubes.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the progression of differentiation of the mammalian myoblasts into mammalian myotubes is analyzed on day 4 of differentiation of the mammalian myoblasts into mammalian myotubes.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein a plurality of subsets of cells is exposed to a plurality of different compounds in parallel.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the progression of differentiation of the mammalian myoblasts into mammalian myotubes is analyzed by microscopy.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the compound is a small molecule, a peptide, a natural product, or a plant extract.

In certain embodiments, these cultures serve as a sensitive high throughput screen to identify hit compounds or derivatives that rescue/enhance muscle function. In certain embodiments, the hit compounds serve as templates for the development of novel therapeutics targeting muscle disease in mammals. In certain embodiments, the invention relates to a compound, wherein the compound rescues or enhances myotube formation as identified in any one of the aforementioned methods. In certain embodiments, the invention relates to a compound identified by any one of the aforementioned methods.

Experimental

Smooth male mutant flies were collected at 1 day of age, and maintained either on vehicle alone, or on one preselected ascochlorin derivative (2 mM solution in vehicle). After 7 days of treatment, the flies were collected and assessed using a negative geotaxis assay (Allikian, M.J. et al., Reduced life span with heart and muscle dysfunction in drosophila sarcoglycan mutants, Hum Mol Genet., 2007, 16:2933-2943; Naimi, B. et al, A tropomyosin-2 mutation suppresses a troponin i myopathy in drosophila, Mol Biol Cell, 2001, 12: 1529-1539) as follows: flies were gently tapped to the bottom of an empty vial, thus prompting a climbing response. The number of flies crossing a 2 cm-high threshold line in 10 seconds was recorded. Percent Drosophila crossing the threshold was used as an index of locomotor activity. Vehicle exposed flies: n = 12; drug treated flies: n = 14. Each repeat showed the average of three consecutive recordings. Three repeats were performed at 2 minute intervals on the same group of flies (nine values total/pharmaco logic exposure).

We have demonstrated that in vivo down regulation of the Drosophila homolog of hnRNP L, SMOOTH, leads to marked degeneration of the muscles in adult flies. The muscle defects are highly reminiscent of lesions observed in patients affected with muscular dystrophy. As in Duchenne muscular dystrophy (DMD) or myotonic dystrophy (DM) patients, the muscle dystrophy is associated with a marked reduction in motor function, as well as in longevity. Smooth mutation-induced muscle dystrophy is triggered when down regulation of smooth is specifically targeted to the mesoderm, highlighting an important intrinsic role for this splicing factor in muscle physiology. (Draper, I. et al., The evolutionarily conserved RNA binding protein smooth is essential for maintaining normal muscle function, Fly (Austin), 2009, 3:235-246). HnRNP L is abundantly expressed in C2C12 mouse myoblasts and myotubes, as well as in rat primary cardiomyocytes (hnRNP L mRNA levels were assessed by RT-PCR analysis, and protein levels were assessed by Western blot analysis, not shown).

Our data indicates that ascochlorin and structurally related compounds hold promise as potential therapeutics for the treatment of muscle degeneration/wasting linked to muscular dystrophy, myopathy, and aging. This effect might be mediated by an increase in the levels of the RNA binding protein hnRNP L and/or hnRNP L targets (some of them already associated with muscle disease, e.g., BINl), with or without effect on mRNA splicing. These drugs may also be utilized to enhance normal muscle function.

Example 1 - HTS hnRNP L levels in C2C12 myoblast cell line

The aim of the current study is to quantify hnRNP L transcript levels in C2C12 myoblasts in response to drug administration (vs. corresponding levels measured in untreated myoblast), utilizing real time PCR. Compounds that induce differential hnRNP L expression will be considered as candidate template chemical structures for the development of novel therapeutics to treat muscle disorders.

A. Design of qPCR primers corresponding to mouse hnRNP L.

Al. Template Mus musculus heterogeneous nuclear ribonucleoprotein L (HnrnpQ, mRNA, 2,142 bp linear mRNA. Accession: NM_177301.5

A2. Primers. hnRNP L qPCR primers were designed utilizing the NCBI program http://www.ncbi.nlm.nih.gov/tools/primer-blast ⁷ , with the following criteria: amplicon size = 90-150 nt, and exon junction crossed. The two pairs described in the Table 4 below are specific to input template, i.e., no other targets were found in the database. The corresponding amplicons are shown on the graphic of hnRNP L mRNA sequence, in Figure

Table 4

B. C2C12 cell culture.

The C2C12 mouse myoblast cell line is routinely cultured in the laboratory, as previously described in the literature (Dalkilic I. et ah, Loss of FilaminC (FLNc) Results in Severe Defects in Myogenesis and Myotube Structure, MCB, 2006). The cells will be grown in a 96-well plate (high throughput) format. Sub-confluent C2C12 cultures grown in medium supplemented with 10% fetal calf serum maintain the myoblast state. By comparison, switching C2C12 cells that are -80% confluent to medium supplemented with 2% horse serum for six days results in myotube formation. HnRNP L levels will be first assessed in myoblast (in the presence, or absence, of the drug). Follow-up analysis will be done using differentiated myotubes.

C. Optimization of the RT-PCR assay.

CI. Preparation of total RNA/cDNA from C2C12 cultured cells

RNA is prepared at Day 1 after plating. At this time, all cells are in the myoblast stage (typically the first myotubes appear at Day 3, most at Day 7). Total RNA is isolated either with RNeasy Mini kit 50/Qiagen Cat# 74104 (prepl, [RNA] = 0.5μ%/μϊ), or with RNA STAT-60/Tel-Test, Inc. (prep 2, [RNA] > Ιμ μϊ). Reverse transcription (RT) reaction was carried out to generate complementary DNA where [cDNA] = SOng/μΙ (use 2 μΐ cDNA/ one regular PCR reaction).

C2. Generation of standard curves to assess amplification accuracy over a range of [cDNA], as well as to measure hnRNP L qPCR primer efficiency and specificity.

Methodology The primer stocks are prepared at 10 pm/μΐ. The qPCR cocktail is calculated to accommodate (n+1) reactions, e.g., n =7 (6 cDNA serial dilutions, 1 no-template control)

+ 1 extra volume.

Per Rxn per (n+1) Rxs

0.6 μί ( ⁿ +l) ^χ 0.6 μί Forward primer (10 pm/μΐ stock: 300 nM final)

0.6 μΐ, (η+1) χ 0.6 μί Reverse primer (10 pm/μΐ stock: 300 nM final)

6.8 μΐ, (n+1) x 6.8 μΐ, dH ₂ 0

10 uL (n+ 1 ) x 10 μΐ, SYBR 2x Mastermix

18 μΐ (n+1) x 18 μΐ,

18 μί of cocktail is aliquoted per well. 2 μΙ_, of water (no-template control), or of the appropriate cDN A dilution (lx, 4x, 16x, 64x, 256x,1024x ....), is added to each well.

The following amplification profile is run:

95 °C: 10 mn 40 X - 95 °C: 5 s

63 °C: 20 s

Dissociation curve

A melting curve was generated for each primer pair utilized (i.e., hnRNP L primer pair 1, hnRNP L primer pair 2, and β-actin). Results show that each pair generates a single melting curve (at or above 80°C), indicating template specificity. (Figure 3)

In addition, for each primer pair, a standard curve is generated using serial dilutions of the cDNA (i.e., lx, 4x, 16x, 64x, 256x,1024x...). In each case, the amplification efficiency falls in the 100% + 10% range, indicating primers specificity. (Figure 4) Typically, [cDNA] that generate cycle thresholds (Ct) between 20-30 are optimal for qPCR analyses.

D. Adaptation of the approach to a 96-well plate, HTS format.

Dl. Preparation of total RNA from C2C12 cultured cells

High quality, concentrated and purified total RNA (up to 5 μg/well; [RNA] = 0.2 μg/μL, 25 μΙ,ΛνεΙΙ) will be isolated using the ZR-96 Quick-RNA™ technology (Zymo research, Catalog Nos. R1052 & R1053).

D2. RT-PCR reaction

PCR reactions will be performed using the QIAGEN OneStep RT-PCR Kit (Qiagen, cat. nos. 210210, 210212), which allows fast and easy RT-PCR setup. Reverse transcription and PCR are carried out sequentially in the same tube. All reagents required for both reactions are combined in one tube during setup, and the thermal cycler program is started.

OneStep RT-PCR Kit contents Features

HotStarTaq® DNA Polymerase Highly specific products

Omniscript® and Sensiscript® RT Wide range of RNA amounts (1 pg - 2 μg)

High sensitivity

OneStep RT-PCR Buffer Minimal optimization needed

No inhibition of PCR by reverse transcriptases Q-Solution Facilitates amplification of GC-rich templates

(Table as shown in the QIAGEN® OneStep RT-PCR Kit Handbook)

E. Drug screen (utilizing the established high-throughput assay described above).

C2C12 myoblast cell cultures will be treated with ascochlorin, or with derivative compounds (e.g., ascofuranone), as described by Kang and colleagues using the human osteosarcoma U20S cell line (Kang JH et al, Proteome Analysis of Responses to Ascochlorin in a Human Osteosarcoma Cell Line by 2-D Gel Electrophoresis and MALDI- TOF MS, J. Proteome Research, 2006). Alternatively, established libraries of small molecules (e.g., The Prestwick Chemical Library® of 100% FDA approved drugs) will be screened for effects on hnRNP L levels. (Kopin AS et al, Identification of a series of CCK- 2 receptor nonpeptide agonists: sensitivity to stereochemistry and a receptor point mutation, PNAS USA, 2003).

The ability of a drug to induce either a significant increase, or a decrease, in hnRNP L levels will be assessed. As an initial threshold, compounds that induce a > 4-fold difference in hnRNP L transcript levels (as assessed by a > 2-fold cycle threshold, Ct, difference by real time PCR, vs. levels in untreated cells) will be further characterized. Independent biological replicates, and corresponding dose response curves, will be obtained/ analyzed. Complementary follow-up studies will include the quantification of hnRNP L protein levels in treated, vs. untreated, C2C12 myoblasts.

Example 2 - Identification of novel therapeutics for muscle diseases that target hnRNP L pathways using a combination of in vitro screens and in vivo screens

An assay was designed to identify compounds that enhance the survival of myotubes that would otherwise degenerate and die following hnRNP L gene silencing. HTS is done utilizing a human myoblast cell line (from a healthy individual) that downregulates hnRNP L via expression of a lentiviral vector encoding small interfering hnRNP L RNAs. We have shown that hnRNP L deficient myoblasts display marked differentiation defects as follows: although hnRNP L KD myoblasts proliferate normally and differentiate into early myotubes (Day 2 differentiation), newly formed mature myotubes (Day 4), rapidly break down leaving in the culture residual myoblasts (Figures 5 and 6). Using the hnRNP L KD cell line, pharmacological compounds that improve myotube survival and maintenance are identified. Hit compounds are further assessed using a combination of in vitro and in vivo confirmatory assays and secondary screens, including (i) assessment of drug-induced differential hnRNP L expression, (ii) rescue of phenotypes displayed by cultured diseased (e.g., DMD, DM1) myotubes, and (iii) in vivo assessment of drug effects on muscle phenotypes displayed by relevant model organisms (e.g., mutant Drosophila, mouse models). Promising compounds are considered as template chemical structures for the development of novel therapeutics to treat muscle disorders and/or sarcopenia. A. Establishment of a human myoblast cell line that expresses interfering hnRNP L RNA.

Al. Human myoblast cell isolation and culture.

Human skeletal muscle-derived myoblasts were isolated and cultured using well- established protocols (Alexander et al, Skeletal Muscle, 2011; Gharaibeh et al, Nature Protocols, 2008). Sub-confluent cultures grown in medium supplemented with 10% fetal bovine serum maintained the myoblast state. By comparison, switching cells that are -90% confluent to medium supplemented with 2%> FBS for 7-14 days resulted in myotube formation.

A2. Lentivirus-mediated silencing of hnRNP L in normal human myoblasts.

Lentiviral vectors expressing small hairpin RNA (shRNAs) can be used to downregulate gene expression in cultured muscle cells. This approach was applied to hnRNP L gene silencing in human myoblasts/myotubes. The design and cloning of the corresponding lentiviral vector (shHNRNP L), as well as of the control vector (shLuc, the hairpin RNA targets the luciferase gene) were done according to established methodologies (Alexander et al, Skeletal Muscle, 2011; Tiscornia et al, Nature Protocols 2006). Lentiviral infections were performed at a viral titer of approximately 2 x 108 TU/mL. Downregulation of hnRNP L expression in differentiating myoblasts resulted in rapid collapse of the myotubes at Day 4 differentiation (Figure 5, Day 4, shFiNRNP L vs. shLuc controls).

The reserve myoblasts that remained in the shFiNRNP L culture at Day 4 later differentiated into few myotubes that were disorganized (Figure 6, Day 14, arrow). This phenotype is reminiscent of that described for cultured myotubes derived from patients with muscular dystrophy (e.g., DM1, Loro et al, Cell Death and Differentiation, 2010).

ShFiNRNP L knockdown efficiency was determined by Western blot analysis of myotubes at Day 7 Differentiation (utilizing a monoclonal anti-hnRNP L antibody, Figure V).

B. Drug screens

Myoblast cell lines (that stably express the shFiNRNP L lentiviral construct) can readily be grown in a 96-well, or 384-well plate format, amenable to HTS.

Differentiating shHRNP L myoblast cell cultures will be treated with ascochlorin, or derivative compounds (e.g., ascofuranone), as described by Kang and colleagues using the human osteosarcoma U20S cell line (Kang JH et al, Proteome Analysis of Responses to Ascochlorin in a Human Osteosarcoma Cell Line by 2-D Gel Electrophoresis and MALDI- TOF MS, J. Proteome Research, 2006). Compound treatment will be done at Day 2 Differentiation, prior to the observed myotube degeneration/death that occurs at Day 4.

Alternatively, established chemical libraries of small molecules (e.g., The Prestwick Chemical Library® of 100% FDA approved drugs), or larger libraries (public domain or proprietary), will be screened for partial or complete rescue of the differentiation defects displayed by hnRNP L-deficient myoblasts (Figure 12). These libraries include a small molecule library, and a natural product library, both available at the Tufts Medical Center Molecular Pharmacology Research Center (MPRC, Dr. A. Kopin Director). In addition, we will screen the library of FDA-approved 'bioactive' drugs from the Chemical Biology/Novel Therapeutics (CB/NT) Outreach Lab at the Broad Institute.

The shFTNRNPL myoblasts will be seeded in the 384-well plates and grown until 90% confluence (two days). The cells will be subsequently differentiated by changing the media from 20% to 2% FBS. Pharmacological treatment will be done at Day 2 differentiation, when the nascent myotubes form. Each compound will be added at 10 ^"5 M (30 nL/100 media in each well, the final DMSO concentration is 0.33%). Treatment with DMSO solvent only (no drug) will provide controls. At Day 4 the cells will be washed to remove the debris, and the effect of the drug on myotube survival will be assessed. An automated imaging program will be used to detect drug-induced rescue of multi-nucleated myotubes myotubes. Negative controls (treated with DMSO, no drug) will show only residual myoblasts, reflecting the degenerative phenotype due to hnRNP L KD. Positive control (shLuc myoblast, hnRNP L levels are normal) show multi-nucleated myotubes.

Automated imaging System. A high throughput phenotype imaging and analysis program, developed at Tufts University Study Center on the Immunogenetics of Infectious Disease (SCIID) to assess myoblast to myotube differentiation in 96-well plates, will be utilized. The strategy uses the high content based ImageXpress Micro Imaging System (Molecular Devices Corporation), with modifications to the MetaXpress program. This workflow can assess each myotube, identified by Troponin T immuno staining, for the following cellular parameters: area, perimeter length, breadth, shape factor, and integrated intensity. Nuclear parameters (e.g. total number of nuclei and nuclear area) also measured. The program will be adapted to a 384-well plate format.

The objectives of the pilot screens are as follows: (1) establishment of assay quality (determination of z' -factors, inter- well and day-to-day variability), (2) potential identification of hits that will be followed as discussed below, and (3) validation of the assay to a point where it is suitable to HTS of a much larger library.

C. In vitro and in vivo confirmatory assays and secondary screens.

Pending positive results, the activity /toxicity of hits/derivative of hits will be further assessed in in vitro and in vivo confirmatory assays, and secondary screens. For each "hit", independent biological replicates, and corresponding dose response curves will be obtained/analyzed. The follow-up studies will include:

1. Western blot analysis of hnRNP L protein levels and/or real-time PCR quantification of hnRNP L mRNA levels, in drug treated, vs. untreated, shFiNRNP L myoblasts.

2. Assessment of drug effects on primary myoblast cell lines isolated from DM1 muscle tissue (see Example 3). The ability of the drug to rescue/improve the phenotype displayed by DM1 myotubes (e.g. disorganization, low abundance), will be assessed. 3. The in vitro primary screen will be followed by in vivo assessment of candidate hits utilizing a whole animal model (i.e. Drosophila). We have previously shown that Drosophila that downregulate smooth, the fly homo log of mammalian hnRNP L, display shortened lifespan and marked muscle degeneration. The in vivo studies using smooth- deficient flies expedite assessment of delivery, activity and toxicity of any candidate compound identified in the primary screen. In parallel, compounds will also be tested for their ability to rescue the phenotypes of established fly models of DM1.

Additional considerations (i) Library compounds that are cytotoxic will act against the selection in our screen (i.e. compounds are selected for their ability to restore/improve survival of muscle fibers). Toxic drugs will thus 'self- eliminate', (ii) A hit compound may improve/rescue myofiber viability via a mechanism which is either hnRNP L-dependent, or -independent (i.e. the drug may not necessarily affect hnRNP L levels). In either case, compounds will provide promising templates for the development of future therapeutics that lessen DM1 associated-muscle degeneration, (iii) Backup screen: In the event that the original phenotypic readout (i.e. survival of hnRNP L-deficient Day 4 myotubes) is too stringent to enable identification of hits, the libraries will be re-screened using as readout the milder phenotype displayed by hnRNP L-deficient Day 14 myotubes. Day 14 fibers are observed in the culture, albeit less numerous and disorganized compared to controls. This less stringent assay may enable the identification of effective drugs that would otherwise be missed in the initial screen, (iv) We are aware that elevating the levels of a global splicing factor (i.e. hnRNP L) might have deleterious physiological consequences. Preliminary studies from our lab, as well as reports in the literature, suggest however that over- expression of smooth (the fly homolog of hnRNP L) in Drosophila, or of hnRNP L in Xenopus, is well-tolerated by the organism. Smoot/z-overexpressing flies display normal gross morphology, locomotor activity, fertility and lifespan. Notably, overexpression of MBNL1 in mice had also no apparent adverse effect.

Collectively, our studies open the possibility of identifying novel candidate pre- therapeutic leads that could be further developed for the treatment of muscle diseases, including myotonic dystrophy. In future studies, the biodistribution, activity/efficacy and toxicity of the most promising candidate drugs will be examined in rodent models of DM1. Example 3 - Characterization of hnRNP L function in normal human and DM1 muscle cell lines

HnRNP L is a multifunctional RNA-binding protein involved in mRNA alternative splicing, export and stability. This conserved splicing factor is expressed in a wide range of tissues, including muscle. Decreased level of smooth (the fly homolog of hnRNP L) in Drosophila, or of hnRNP L in zebrafish results in marked muscle defects. In addition, we and others have recently shown that hnRNP L physically interacts with MBLN1 (Fig. 8), one of the known RBPs that underlies DM pathogenesis. This led us to hypothesize that hnRNP L plays an important role in both normal and myotonic human muscle cells. To begin to define the molecular function of hnRNP L in human skeletal muscle, we propose a combination of in vitro cell culture analyses, using banked normal and DM1 muscle cells in a series of overexpression and knockdown of hnRNP L experiments. Of note, three DM1 myoblast cell lines (# 9886, 10008 and 10009) were obtained from the Telethon Cell Bank, and established in the Kunkel lab.

A. The expression profile of hnRNP L in normal vs. DM1 human myoblasts and myotubes will be determined (Differentiation Day 0 through Day 14). The ratio of known hnRNP L isoforms and subcellular localization will be compared.

The ratio of known hnRNP L isoforms and subcellular localization will be determined during myogenic differentiation (days 0 through 14). The levels of hnRNP L isoforms (variant 1 : 589 aa, 64kDa vs. variant 2: 456 aa, 50kDa) will be determined by quantitative RT-PCR (using isoform-specific primers), and Western blot analysis (using the 4D11 mouse monoclonal, Sigma), in normal and DM1 myoblasts and myotubes. The two hnRNP L RNA isoforms differ in their 5' UTR/start codon region. Variant 1 is the predominant isoform in all tissues (including muscle); whereas we have only seen potential evidence of the shorter hnR P L during fetal myogenesis (data not shown). We will also determine the subcellular localization of hnRNP L (cytoplasmic vs. nuclear) via immunohistochemical analysis as described before. Of note, preliminary analysis of hnRNP L expression in differentiating normal and diseased human myoblasts shows a moderate increase in DM1, vs. normal, muscle cells (Fig. 9). This is reminiscent of findings recently published for another RBP, Staufenl, which up-regulation is postulated to be protective in DM1.

B. The effects of lentivirus-mediated hnRNP L knockdown, or overexpression, on myogenic proliferation/ differentiation will be assessed, in normal vs. DM1 human cell lines. The effects on cell morphology/organization, as well as on the expression of myogenic/myofibrillar markers, will be assessed. In addition, we will analyze the expression of exons that are misspliced in DM1 patients (e.g. DMD exon 71, IR exon 11) to define the role of hnRNP L in this process.

Lentiviral vectors expressing small hairpin RNA (shRNAs) are routinely used to downregulate (KD) gene expression, or full-length cDNA to overexpress (OE) gene expression, in cultured muscle cells. Following lentiviral-mediated KD, or OE, of hnRNP L levels, the analyses (i)-(iv), will be performed.

(i) Myoblast proliferation and differentiation will be measured by MTS assay, as previously described. Proliferation rate of the myoblasts over a 5 -day period will be expressed as a ratio of hnRNP L levels (as determined by Western blot).

(ii) Myogenic differentiation rates will be determined via assessment of myogenic fusion indices based on the expression levels myosin heavy chain (MyHC. a well- characterized myogenic differentiation marker).

(Hi) Myogenic fusion indices will be determined using the ratio of MyHC to nuclei per field, and per multi-nucleated myotube. Additional differentiation markers (e.g. myogenin, Mef2c, and dystrophin) will be analyzed in Western blot and immunostaining, to determine the rate of myogenic differentiation following hnRNP L OK or KD.

(iv) The alternative splicing of exons that are known to be aberrantly processed in DM1 patients will be assessed. To date, these missplicing events correspond to -28 RNA target. Our preliminary data show that DMD (dystrophin) exon 71 is aberrantly processed in hnRNP L KD (vs. normal) muscle cells (Fig. 10). DMD exon 71 is known to be aberrantly spliced in DM1. The proposed experiments will begin to define the role hnRNP L in regulating normal muscle cell growth, differentiation, proliferation and viability. In addition the analysis of hnRNP L-dependent processing of exons that are known to be misspliced in DM1 (e.g. DMD exon 71, Fig. 10), will potentially implicate this factor in DM1 pathogenic processes. Finally, these studies will provide a baseline of information on which to better understand the effects of candidate pre-therapeutic leads, as identified in our screen.

C. The spectrum of RNA targets processed by hnRNP L in normal human muscle cells will be defined. We will carry out high throughput RNA Sequencing (RNA-seq) of total RNA extracted from hnRNP L-silenced (vs. control) Day 2 myotubes. Comparison will be made between missplicing events resulting from hnRNP L knockdown and those known to be associated with DM1 (potentially implicating hnRNP L in the pathogenesis of these events).

Previous reports in the literature have demonstrated that hnRNP L is a key regulator of alternative splicing that can promote either the inclusion, or the exclusion, of tissue- specific exons. Interestingly, hnRNP L binding motifs were shown to be enriched in alternative splicing events that are associated with myogenic differentiation. We recently carried out a bioinformatic analysis of a compendium of known human alternative splicing events to identify sequences that include the hnRNP L binding motif, and are expressed in skeletal muscle and/or the heart. This analysis revealed 46 conserved putative targets of hnRNP L in muscle and the heart. Importantly, the list includes nine targets, APP, BIN1, DMD, DTNA, MAPT, MEF2A, PPP2R5C, SORBS 1 and TTN, which are known to be aberrantly spliced in DM1 patients. This is a remarkably high number, given that to date, a total of less than 30 splicing defects have been confirmed in DM1 patients. These findings, together with data from our lab and others showing that hnRNP L and MBNL 1 form a complex (Fig. 8), provide a strong rationale to define the full complement of RNA targets that are processed by hnRNP L in human muscle. To achieve this objective, we propose to utilize genome-wide RNA based deep-sequencing (RNA-Seq), of total RNA prepared from normal human muscle cells in which hnRNP L is downregulated (KD, vs. controls). Although hnRNP L KD myoblasts differentiate into nascent myotubes, hnRNP L KD multi- nucleated myotubes degenerate rapidly (Fig. 5 and Fig. 11). Our in vitro assay provides a unique system to define the global changes in hnRNP L-dependent alternative splicing that affect the formation and/or maintenance of differentiated muscle cells, with potential relevance to DM1 pathology. Total RNA will be isolated from early (i.e. differentiation Day 2) myotubes that express the hsHNRNPL interference construct (i.e. hnRNP L KD), as well as from Day 2 myotubes that express the hsLuc construct (i.e. controls). For each genotype, three independent RNA samples will be prepared. RNA-Seq will be carried out at the Tufts University Genomics Core (http://genomics.med.tufts.edu) under the supervision of Core manager, Dr. A. Tai, who is a consultant on the proposed project (please see letter of support). The Core offers investigators state of the art technologies for RNA-Seq analysis including (i) quality control of the input RNA (Agilent bioanalyzer) (ii) library preparation (TruSeq Stranded Total RNA Sample Prep Kit), (iii) sequencing (Illumina HiSeq 2000 using the TruSeq SBS Kit v3-HS chemistry), and (iv) data analysis (i.e. mapping, expression and splice variants analysis of the transcriptome) using the Galaxy server (galaxy.med.tufts.edu).

The proposed investigations will potentially unravel (i) early markers of the degenerative muscle process, and (ii) novel therapeutic targets for DM. Comparison will be made with candidate gene lists generated in previous transcriptome analyses of muscle isolated from DM1 patient, or from rodent models of DM1, to determine the common as well as distinct sets of genes/exons that are aberrantly processed following de-regulation of the splicing factors. The subset of targets that are both consistently misspliced across the analyses, and evolutionarily conserved, will be prioritized for further study. RT-PCR analysis will be performed (utilizing exon-specific primers) to confirm the differential splicing of the exon in hnRNP L-deficient, vs. control, myotubes. Although likely be beyond the timeline of this proposal, subsequent studies in Drosophila will offer an expedited means to begin to investigate the functional relevance of the new candidate targets in muscle physiology/disease. Future studies in human cell lines will focus on the most promising among these targets.

Incorporation by Reference

All of the cited U.S. Patents, U.S. patent application publications, and PCT patent application publications designating the U.S., are hereby incorporated by reference in their entirety.

Equivalents

While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto; the invention may be practiced otherwise than as specifically described and claimed.