# Patent

## Title
Compositions and methods for characterizing and treating muscular dystrophy

## Abstract

Compositions and methods for identifying new treatments for Facioscapulohumeral muscular dystrophy (FSHD), and uses thereof.

## Background
CLAIM OF PRIORITY
The present application claims the benefit of U.S. Provisional Patent Application No. 61/622,942, filed on Apr. 11, 2012. The entire contents of the foregoing are incorporated by reference herein.


FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with Government support under Grant No. U54 HD060848 awarded by the National Institutes of Health. The Government has certain rights in the invention.


BACKGROUND OF THE INVENTION
Facioscapulohumeral muscular dystrophy (FSHD) is a progressive neuromuscular disorder caused by contractions of repetitive elements within the macrosatellite D4Z4 on chromosome 4q35. There is currently no effective treatment available for FSHD and clinical trials with novel therapeutics have been discouraged by the lack of a recognized mouse model. Clinical trials have also been discouraged by the fact that FSHD is a highly variable and slowly progressing disease whereas the efficacy of therapeutic interventions is ideally established over short periods of time. Therefore, molecular biomarkers of FSHD that could be used to assay responsiveness to therapy would greatly facilitate FSHD therapeutic development and clinical research. High-density oligonucleotide arrays reliably quantify the expression levels of thousands of genes simultaneously and enable identification of such biomarkers.
SUMMARY OF THE INVENTION
As described below, the present invention features panels of biomarkers useful in diagnosing muscular dystrophy (e.g., FSHD) in a subject, as well as cellular compositions and chimeric animals useful in drug screening.
Thus, in a first aspect, the invention provides methods for identifying a candidate compound for treatment of Facioscapulohumeral muscular dystrophy (FSHD). The methods include contacting a sample comprising a cell derived from an FSHD affected subject with a test compound; determining a level of expression of a gene selected from the group consisting of PRAMEF1, SLC34A2, TRIM49, TRIM43, CD177, NAAA, HSPA6, TC2N, CD34, ATP2A1, PAX7, MYF5, MRAP2, DAG1, CLYBL, CALCRL, ZNF445, and SPATA17, or at least two genes selected from the group consisting of SLC34A2, TRIM49, TRIM43, PRAMEF1, CD177, NAAA, HSPA6, TC2N, CD34, ATP2A1, PAX7, MYF5, MRAP2, DAG1, CLYBL, CALCRL, ZNF445, and SPATA17 in the sample; and selecting as a candidate compound a test compound that reduces the level of expression of one or more of SLC34A2, TRIM49, TRIM43, PRAMEF1, CD177, NAAA, HSPA6, TC2N, or CD34, or a test compound that increases the level of expression of one or more of ATP2A1, PAX7, MYF5, MRAP2, DAG1, CLYBL, CALCRL, ZNF445, or SPATA17. In some embodiments, where expression of only a single gene is determined, that gene is not PRAMEF1 or TRIM43. In some embodiments where expression of only two genes is determined, the genes are not PRAMEF1 and TRIM43. Thus, in some embodiments, where PRAMEF1 or TRIM43 are determined, at least one other gene that is not PRAMEF1 or TRIM43 is also determined.
In some embodiments, the methods include determining a level of expression of at least one gene shown in Table 4 that is upregulated in FSHD, optionally wherein the gene is selected from the group consisting of PRAMEF1; TRIM43; SLC34A2; TRIM49 and CD34, in a sample comprising a cell from the subject; and determining a level of expression of at least one gene shown in Table 4 that is downregulated in FSHD, optionally wherein the gene is selected from the group consisting of PAX7; MYF5; ATP2A1; DAG1; and MRAP2; in the sample; and selecting as a candidate compound a test compound that reduces the level of expression of a gene shown in Table 4 that is upregulated in FSHD and increases the level of expression of a gene shown in Table 4 that is downregulated in FSHD.
In some embodiments, the methods include administering the selected candidate compound to an animal model of FSHD, wherein the animal model comprises at least one chimeric muscle tissue comprising cells from a subject affected with FSHD; performing an assay to determine a level of expression of at least one gene shown in Table 4; comparing the level of expression of the at least one gene to a reference level of expression that represents a level of expression in the absence of the candidate compound; and selecting as a candidate therapeutic compound a candidate compound that reduces the level of expression of a gene shown in Table 4 that is upregulated in FSHD and increases the level of expression of a gene shown in Table 4 that is downregulated in FSHD.
In some embodiments, the level of expression of a gene shown in Table 4 that is upregulated in FSHD is reduced to a level that is nearly or substantially the same as, i.e., not statistically significantly different from, a level in a control cell that is not derived from an FSHD affected subject, or an animal model that comprises at least one chimeric muscle tissue comprising cells from a control subject who is not affected with FSHD.
In some embodiments, the level of expression of a gene shown in Table 4 that is downregulated in FSHD is increased to a level that is nearly or substantially the same as, i.e., not statistically significantly different from, a level in a control cell that is not derived from an FSHD affected subject, or an animal model that comprises at least one chimeric muscle tissue comprising cells from a control subject who is not affected with FSHD.
In some embodiments, levels of expression are determined using quantitative PCR (qPCR).
In some embodiments, the control cell is derived from a first degree relative of the FSHD affected subject.
In another aspect, the invention provides methods (e.g., computer-implemented methods) for identifying a candidate compound for treatment of Facioscapulohumeral muscular dystrophy (FSHD). The methods include contacting a sample comprising a cell derived from an FSHD affected subject with a test compound; determining a level of expression of at least one gene shown in Table 4 that is upregulated in FSHD, optionally wherein the gene is selected from the group consisting of PRAMEF1; TRIM43; SLC34A2; TRIM49 and CD34, in the sample, to determine a value [GeneUP]; determining a level of expression of at least one gene shown in Table 4 that is downregulated in FSHD, optionally wherein the gene is selected from the group consisting of PAX7; MYF5; ATP2A1; DAG1; and MRAP2; in the sample, to determine a value [GeneDOWN]; using the value [GeneDOWN] and the value for [GeneUP] to calculate a classifier for the test compound; comparing the classifier to a reference classifier that represents a classifier in a cell that is from a control subject who is not affected with FSHD; and selecting as a candidate compound a test compound that has a classifier that is not statistically different from the reference classifier.
In some embodiments, [GeneUP] is a level of PRAMEF1 in the sample.
In some embodiments, [GeneDOWN] is a level of PAX7 in the sample.
In some embodiments, the classifier is calculated as:

[GeneUP]−[GeneDOWN]=classifier

In some embodiments, the test compound is an inhibitory nucleic acid.
In some embodiments, the methods include administering the selected candidate compound to an animal model of FSHD, wherein the animal model comprises at least one chimeric muscle tissue comprising cells from a subject affected with FSHD; performing an assay to determine a level of expression of at least one gene selected from the group consisting of SLC34A2, TRIM49, TRIM43, PRAMEF1, CD177, NAAA, HSPA6, TC2N, CD34, ATP2A1, PAX7, MYF5, MRAP2, DAG1, CLYBL, CALCRL, ZNF445, SPATA17; comparing the level of expression of the at least one gene to a reference level of expression that represents a level of expression in the absence of the candidate compound; selecting as a candidate therapeutic compound a candidate compound that reduces the level of expression of one or more of SLC34A2, TRIM49, TRIM43, PRAMEF1, CD177, NAAA, HSPA6, TC2N, or CD34, and increases the level of expression one or more of ATP2A1, PAX7, MYF5, MRAP2, DAG1, CLYBL, CALCRL, ZNF445, or SPATA17.
In some embodiments, the methods include administering the selected candidate compound to an animal model of FSHD, wherein the animal model comprises at least one chimeric muscle tissue comprising cells from a subject affected with FSHD; evaluating an effect of the candidate compound on a biological function associated with FSHD in the animal model; and selecting as a candidate therapeutic compound a candidate compound that improves the biological function (i.e., effects a return to normal or near normal function) in the animal model.
In some embodiments, biological function is assayed using live cell imaging, muscle fiber turnover, the number of muscle stem cells, or biomarker expression.
In another aspect, the invention provides methods for treating FSHD in a subject, the method comprising administering to the subject one or more inhibitory nucleic acids targeting one or more of SLC34A2, TRIM49, TRIM43, CD177, NAAA, HSPA6, TC2N, or CD34. In an additional aspect, the invention provides methods for treating FSHD in a subject, the method comprising administering to the subject two or more inhibitory nucleic acids targeting two or more of SLC34A2, TRIM49, TRIM43, PRAMEF1, CD177, NAAA, HSPA6, TC2N, or CD34. In some embodiments, the inhibitory nucleic acid is a double-stranded RNA, siRNA, shRNA, or antisense oligonucleotide, e.g., a morpholino oligonucleotide.
Also provided herein are inhibitory nucleic acids targeting SLC34A2, TRIM49, TRIM43, CD177, NAAA, HSPA6, TC2N, or CD34 for treating FSHD, and the use of such inhibitory nucleic acids for treating FSHD, as well as for the manufacture of a medicament for the treatment of FSHD.
In another aspect, the invention provides cell lines, e.g., shown in FIG. 1, optionally selected from the group consisting of cell lines designated 07A, 07U, 09A, 09U, 12A, 12U, 15A, 15B, 15V, 21B, or 21U, where A and B designate cells from genetically affected persons with FSHD, and U and V designate genetically unaffected family members of the persons with FSHD.
In another aspect, the invention provides kits including a plurality of cell lines, e.g., a pair or trio of cell lines, from a family cohort as shown in FIG. 1, wherein the kit includes at least one cell line from a genetically affected person with FSHD, and at least one cell line from a genetically unaffected family member, e.g., a first degree relative, of the person with FSHD. In some embodiments, the kit comprises pairs or trios of cell lines selected from the group consisting of: 07A, 07U; 09A, 09U; 12A, 12U; 15A, 15 B, 15V; and 21B, 21U; where A and B designate cells from genetically affected persons with FSHD, and U and V designate genetically unaffected family members of the persons with FSHD.
In one aspect, the invention features a panel of isolated biomarkers including a DUX4 nucleic acid molecule and one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) additional biomarkers including any one or more of the biomarkers listed in Table 2 or 4.
In another aspect, the invention features a microarray containing at least a DUX4 nucleic acid molecule and one or more polynucleotides listed in Table 2 or 4 or their encoded polypeptides, or fragments thereof, fixed to a solid support. In one embodiment, the solid support is a membrane, bead, biochip, multiwell microtiter plate, or a resin.
In another aspect, the invention features a method of characterizing Facioscapulohumeral muscular dystrophy (FSHD) in a cell derived from an FSHD affected subject, the method involving determining the level of expression of one or more nucleic acid molecules listed in Table 2 or 4 or their encoded polypeptides in the cell relative to the level of expression of the nucleic acid molecules or polypeptides in a cell obtained from a first degree relative of the subject who does not have FSHD, thereby characterizing FSHD in the cell. In one embodiment, the method identifies the molecular biomarker profile of the cell. In another embodiment, the cells are in vitro or in vivo. In another embodiment, the FSHD subject is identified as having a contracted 4q D4Z4 region in combination with a 4qA telomeric allele.
In another aspect, the invention features a set of cell cultures, containing one culture containing cells derived from a subject identified as having FSHD and at least one control culture containing cells derived from a first degree relative of the subject that does not have FSHD. In one embodiment, the set comprises two, three or four control cultures obtained from first degree relatives of the subject. In another embodiment, the cell cultures are enriched for myogenic cells. In another embodiment, the cells are isolated by selecting cells positive for human CD56. In another embodiment, the cells are obtained from skeletal muscle biopsies. In another embodiment, the biopsy is of a bicep or deltoid muscle. In another embodiment, the FSHD subject is identified as having a contracted 4q D4Z4 region in combination with a 4qA telomeric allele, and the first degree relative does not have the contracted 4q D4Z4 region.
In another aspect, the invention features a collection containing two or more sets of the cell cultures of any previous aspect or any other aspect of the invention delineated herein, where each set comprises a culture containing cells obtained from a distinct FSHD affected subject and at least one control culture containing cells obtained from that FSHD affected subject's first degree relatives.
In another aspect, the invention features a method for identifying an FSHD biomarker, the method involving comparing the expression of one or more polynucleotides in cells derived from a subject having FSHD relative to the expression of the polynucleotide in control cells derived from a first degree relative of the subject, where an increase or decrease in the polynucleotides relative to the control identifies the polynucleotide as an FSHD biomarker.
In another aspect, the invention features a chimeric mouse containing at least one human cell derived from an FSHD affected subject or a first degree relative thereof.
In another aspect, the invention features a set of chimeric mice including one mouse containing a human cell of an FSHD affected subject, and at least one mouse containing a human cell derived from a first degree relative of the FSHD affected subject.
In another aspect, the invention features a method of identifying an agent that ameliorates FSHD in a subject in need thereof, the method involving contacting a cell derived from an FSHD affected subject with a candidate agent, and comparing the cell's biological function or the level of expression of a nucleic acid molecule of Table 2 or 4 with the biological activity or the level of expression of the nucleic acid molecule in a control cell, where an agent that normalizes the expression of the nucleic acid molecule or enhances biological function ameliorates FSHD. In one embodiment, the control cell is derived from a first degree relative of the affected.
In another aspect, the invention features a method of identifying an agent that ameliorates FSHD in a subject in need thereof, the method involving administering the agent to the chimeric mouse of any previous aspect, and comparing the biological function of a human cell of the mouse before and after treatment, where an agent that enhances the biological function of the cell is identified as ameliorating FSHD.
In another aspect, the invention features a method of identifying an agent that ameliorates FSHD in a subject in need thereof, the method involving administering the agent to the chimeric mouse of any previous aspect, and comparing the level of expression of a nucleic acid molecule of Table 2 or 4 in a human cell of the mouse relative to the level in an untreated control cell, where an agent that normalizes expression in the cell is identified as ameliorating FSHD.
In another aspect, the invention features a method of identifying an inhibitory nucleic acid that ameliorates FSHD in a subject in need thereof, the method involving contacting a cell derived from an FSHD affected subject with an inhibitory nucleic acid molecule that targets a polynucleotide over expressed in FSHD, and comparing the level of expression of the polynucleotide relative to the level in a reference, where an inhibitory nucleic acid molecule that reduces expression of the polynucleotide ameliorates FSHD.
In another aspect, the invention features a method of identifying an inhibitory nucleic acid that ameliorates FSHD in a subject in need thereof, the method involving contacting a cell derived from an FSHD affected subject with an inhibitory nucleic acid molecule that targets a polynucleotide over expressed in FSHD, and comparing the biological function of a human cell of the mouse before and after treatment, where an agent that enhances the biological function of the cell is identified as ameliorating FSHD.
In another aspect, the invention features a method of diagnosing a subject as having, or having a propensity to develop, Facioscapulohumeral muscular dystrophy (FSHD), the method involving determining the level of expression of one or more nucleic acid molecules listed in Table 2 or 4 or their encoded polypeptides in a biological sample of the subject relative to the level of expression of the nucleic acid molecules or polypeptides in a reference, where an alteration in the level of expression is indicative of FSHD.
In various embodiments of the previous aspects or any other aspect of the invention delineated herein, the panel includes polynucleotide or polypeptide biomarkers that are any one or more of DUX4, tripartite motif containing 43 (TRIM43), TRIM49, tandem C2 domains, nuclear (TC2N), PRAME family member 13 (PRAMEF13), PRAMEF2, PRAMEF1, solute carrier family 34 (SLC34A2), heat shock 70 kDa protein 6 (HSP70B), FLJ44674 protein, CD177, and chromosome 9 open reading frame 4 (C9orf4). In one embodiment, the panel includes or consists of DUX4 and one or more additional upregulated biomarkers selected from the group consisting of TRIM43, PRAMEF13, PRAMEF2, PRAMEF1, SLC34A2, TRIM49, CCNA1, and TNXA. In another embodiment, the panel comprises DUX4 and a downregulated biomarker selected from the group consisting of microRNA 30b (MIR30B), dystroglycan 1 (DAG1), melanocortin 2 receptor accessory protein (MRAP2), chromosome 9 open reading frame 153 (C9orf153), ATPase, Ca++ transporting, cardiac (ATP2A1), citrate lyase beta like (CLYBL), calcitonin receptor-like (CALCRL), cytochrome P450, family 39, subfamily (CYP39A1), mastermind-like 3 (MAML3), adrenergic, beta, receptor kinase 2 (ADRBK2), Rho guanine nucleotide exchange factor (ARHGEF7), microRNA 95 (miR95), spermatogenesis associated 17 (SPATA17), islet cell autoantigen 1.69 kDa-like (ICA1L), GABRR1, gamma-aminobutyric acid (GABA) KIAA1217, zinc finger protein 445 (ZNF445), and chromosome 14 open reading frame 39 (C14orf39. In another embodiment, the panel comprises or consists of DUX4 and a downregulated biomarker selected from the group consisting of CALCRL, ATP2A1, MYLK4, E2F8, RGS13, MYOZ2, LRRC39, C6orf142, and MYOZ1. In other embodiments, the human cell is a skeletal muscle cell, muscle stem cell, or differentiated muscle fiber. In other embodiments, the human cells replace 1-100% (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%) of the cells present in a muscle of the mouse. In one embodiment, the human cells replace cells present in the tibialis anterior. In still other embodiments, biological function is assayed using live cell imaging, muscle fiber turnover, the number of muscle stem cells, or biomarker expression.
The invention provides compositions and methods for characterizing FSHD in a subject, as well as compositions and methods useful in drug screening. Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.
DEFINITIONS
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
By “agent” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
By “alteration” is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.”
By “analog” is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.
By “biomarker” is meant a polypeptide, polynucleotide, or clinical criteria associated with a disease or condition. For example, an alteration in the presence, level of expression, or sequence of a biomarker may be associated with or diagnostic of a disease or condition.
In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
By “cell culture” is meant a cell or cells in vitro. A cell culture includes a cell growing or capable of growing in vitro. Thus, a cell culture includes frozen cells capable of growth in vitro.
“Detect” refers to identifying the presence, absence or amount of the analyte to be detected.
By “detectable label” is meant a composition that when linked to a molecule of interest renders the latter detectable. Exemplary methods used to detect a detectable label, include spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
By “effective amount” is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount.
The invention provides a number of targets that are useful for the development of highly specific drugs to treat a condition or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.
By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
“Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
By “inhibitory nucleic acid” is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.
The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
By “Marker profile” is meant a characterization of the expression or expression level of two or more polypeptides or polynucleotides.
As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.
“Primer set” means a set of oligonucleotides that may be used, for example, for PCR. A primer set would consist of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600, or more primers. In particular embodiments, primers of the invention are useful in amplifying a gene listed in Table 2 or 4.
By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.
By “reference” is meant a standard or control condition. For example, the level of a polynucleotide or polypeptide of the invention (e.g., a polynucleotide listed in Table 2 or 4 or the encoded polypeptide) in a subject that is not affected with FSHD, such as a first degree relative of the subject.
A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.
By “a set” is meant a group having more than one member. The group may be composed of 2, 4, 5, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, or 300 polypeptide, nucleic acid molecule, cell culture, or chimeric mouse members.
By “siRNA” is meant a double stranded RNA. Optimally, an siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3′ end. These dsRNAs can be introduced to an individual cell or to a whole animal; for example, they may be introduced systemically via the bloodstream. Such siRNAs are used to downregulate mRNA levels or promoter activity. In one embodiment, the invention provides siRNA that target a polynucleotide of the invention (e.g., a polynucleotide upregulated in FSHD).
By “specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention. In one embodiment, the invention provides antibodies against polypeptides, or fragments thereof, encoded by a gene listed in Table 2 or 4.
Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., more preferably of at least about 42° C., and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e−3 and e−100 indicating a closely related sequence.
By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a table of cell lines as described herein. Each cohort was composed of at least one affected individual with genetically and clinically verified FSHD (designated A or B), and at least one unaffected first degree relative with unshortened D4Z4 alleles and normal strength (designated U or V).



DETAILED DESCRIPTION OF THE INVENTION
The invention provides compositions and methods that are useful treating Facioscapulohumeral muscular dystrophy (FSHD), and methods of identifying new treatments for FSHD.
The invention is based, at least in part, on the discovery of genes whose expression is aberrantly regulated in cells derived from subjects having FSHD (e.g., genes listed in Tables 2 and 4). In certain embodiments, a subset of genes is identified whose expression is most robustly altered in FSHD affected subjects (e.g. 20 genes in Table 2 with smallest p-values among those genes upregulated in FSHD and 20 genes in Table 2 with smallest p-values among those genes downregulated in FSHD). Genes whose expression is altered in FSHD are useful as biomarkers in methods for diagnosing or characterizing FSHD. Thus, the invention provides panels comprising FSHD biomarkers, as well as polynucleotide and polypeptide microarrays comprising such biomarkers.
The discovery of FSHD biomarkers was made possible using a unique collection of cultured cells derived from the skeletal muscles of subjects affected by FSHD, as well as of their first degree relatives. These “FSHD paired cultures” provide a unique advantage not only in identifying genes that are aberrantly regulated in FSHD, but also in identifying and/or assessing the efficacy of therapeutic agents useful in ameliorating FSHD or symptoms thereof. These FSHD paired cultures provide a unique advantage over other cells derived from FSHD affected subjects because they control for familial relationships by comparing expression differences in related FSHD affected subjects and controls, thereby diminishing the effects of interindividual variation on gene expression. Therefore, the expression differences observed between FSHD and control muscles in these FSHD paired cultures likely reflect true pathogenic gene expression profiles suitable for developing into disease biomarkers. The invention further provides screening methods using collections of FSHD paired cultures to identify agents that modify the expression of genes and/or proteins that are aberrantly regulated in FSHD.
In other embodiments, the invention provides pairs of chimeric mice, wherein one member of the pair comprises cells derived from a subject affected by FSHD, and the other member of the pair comprises a cell derived from a first degree relative of the subject. In other embodiments, the invention provides two, three, four or more mice, where one mouse comprises cells from an FSHD affected subject, and the other mice comprises cells derived from one or more of the first degree relatives of that subject. Preferably, certain skeletal muscle cells of the mouse are derived from an FSHD subject or first degree relative of such a subject. Thus, the invention provides a mouse model that is uniquely suited for the identification and characterization of agents useful in treating and/or ameliorating FSHD, and or symptoms thereof.
In still other embodiments, the invention provides panels of biomarkers comprising at least 2, 3, 5, 10, 15, 20, or more of the genes listed in Table 2 or 4. In one embodiment, the panel comprises those genes identified as upregulated in FSHD. In another embodiment, the panel comprises those genes identified as downregulated in FSHD.
Facioscapulohumeral Muscular Dystrophy
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular dystrophy affecting approximately 1 in 7,000-20,000 individuals. It is characterized by progressive weakness and wasting of facial, shoulder girdle and upper arm muscles from which the disease takes its name, and also trunk, hip and leg muscles in some patients. One of the hallmarks of FSHD is asymmetrical and selective degeneration of skeletal muscles. For example, biceps muscle is involved early and severely, whereas the proximal deltoid muscle is relatively spared. The underlying mechanism of this distinctive sparing of certain muscle types is unknown. In addition to muscle degeneration, abnormalities in retinal vasculature and hearing loss are observed in up to 49% and 64% respectively in some populations.
FSHD is caused by partial deletion of a critical number of repeat elements within the highly polymorphic macrosatellite D4Z4 on the subtelomeric region of chromosome 4q. In unaffected individuals, the D4Z4 array consists of 11 to 100 repeats (corresponding to EcoRI fragments of 41 to 350 kb), whereas FSHD patients carry 1 to 10 repeats (corresponding to EcoRI fragments of 10 to 35 kb). Longer residual repeat sizes are often associated with later onset or milder disease severity. In addition to reduction of the tandemly arranged D4Z4 3.3 kb repeat units, the disease causing deletions must occur on chromosomal allele 4qA, whereas deletions on the equally common 4qB allele do not result in FSHD. Although the genetic lesion responsible for 95% of FSHD cases was identified two decades ago, the molecular mechanisms leading to disease progression have long been controversial. The predominantly held position-effect variegation hypothesis proposed that contraction of the D4Z4 repeats induces depression of one or more genes adjacent to D4Z4 with myopathic potential. Several genes (FRG1, FRG2, SLC25A4) residing in the vicinity of D4Z4 have been evaluated using various quantitative approaches by numerous studies but no consistent deregulation of these genes have been demonstrated in human muscle (Winokur et al., (2003) Hum Genet, 12, 2895-2907; Osborne (2007) Neurology, 68, 569-577; Masny et al., (2010) Eur J Hum Genet, 18, 448-456).
DUX4
Several studies have demonstrated the myopathic potential of DUX4, a gene located within each repeat element, in skeletal muscle cells. Overexpression of DUX4, as a result of chromatin relaxation within D4Z4, was initially proposed to induce toxicity to muscle cells, potentially leading to muscle degeneration in vivo. Subsequent studies demonstrated further evidence to support this finding. Recently, genetic analysis of rare families carrying translocations between 4q and 10q chromosomes identified single nucleotide polymorphisms (SNPs) in the pLAM region adjacent to the distal D4Z4 repeat that segregate with FSHD. These SNPs create a canonical polyadenylation signal on the permissive chromosomal allele, whereas the non-permissive alleles lack these SNPs. DUX4 transcripts expressed from the distal-most repeat extends into the pLAM sequence and are polyadenylated when the poly(A) signal SNPs are incorporated into the transcripts, thus increasing their intracellular stability. DUX4, a double homeodomain containing protein, shares similarities with transcription factors PAX3 and PAX7 and is proposed to interfere with transcriptional networks regulated by PAX3/7. It has yet to be determined whether DUX4 overexpression results in global gene misexpression, and in particular it is of considerable interest to determine whether the expression of PAX3/7 target genes are compromised in FSHD muscles, as these transcription factors play an important role in muscle development and maintenance. In view of these findings, agents that reduce DUX4 expression are of interest in treating FSHD and/or ameliorating symptoms associated with FSHD. The analysis of such agents has been hampered by the lack of suitable in vitro and in vivo models systems useful for assaying the efficacy of such agents on FSHD. Thus, the invention provides cell and animal models useful for analysing the agents that treat FSHD. In particular, FSHD paired cultures are useful for analysing the effect of such agents on the expression of genes that are aberrantly regulated in FSHD. In other embodiments, chimeric FSHD mice of the invention are useful for assaying the efficacy of such agents on muscle cells affected with FSHD. In particular, the invention provides methods for assaying the effects of agents that reduce DUX4 expression on genes that are aberrantly regulated in FSHD (e.g., genes listed in Table 2 or 4).
FSHD Cell Cultures and Collections
While the results reported herein provide specific examples of the isolation of muscle cells from subjects identified as having FSHD (or their first degree relatives) during the course of a muscle biopsy, the invention is not so limited. The unpurified source of cells for use in the methods of the invention may be any tissue known in the art obtained from an FSHD subject, although preferably, muscle cells derived from FSHD affected subjects are used. In various embodiments, cells of the invention are isolated from muscle tissue whose biological function is reduced in FSHD (e.g., adult biceps or deltoid skeletal muscles). In one embodiment, the FSHD subject is identified as having a contracted 4q D4Z4 region in combination with a 4qA telomeric allele and the first degree relative is identified as lacking such genetic abnormalities.
The invention provides for the generation of primary muscle cell cultures. Such cultures are obtained by enzymatic dissociation of the tissue using, for example, collagenase IV, dispase and other enzymes known in the art. The cells can be selected against dead cells, by employing dyes associated with dead cells such as propidium iodide (PI). Preferably, the cells are collected in a medium comprising fetal calf serum (FCS) or bovine serum albumin (BSA) or any other suitable, preferably sterile, isotonic medium. Dissociated cells are cultured under standard conditions using cell culture media (e.g., Ham's F10 medium supplemented with fetal bovine serum and/or chicken embryo extract) suitable for maintaining cultures of primary muscle cells. Examples of suitable media for incubating cells of the invention include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), RPMI media or other medias known in the art. The media may be supplemented with fetal calf serum (FCS) or fetal bovine serum (FBS), as well as antibiotics, growth factors, amino acids, inhibitors or the like, which is well within the general knowledge of the skilled artisan.
Cultures are expanded to increase cell number (e.g., to about 50%, 60%, 70%, 80% confluence). Cells are harvested and selected for myogenic cells using standard methods. Such methods include a positive selection for cells expressing one or more myogenic markers. Monoclonal antibodies are particularly useful for identifying markers associated with the desired cells. If desired, negative selection methods can be used in conjunction with the methods of the invention to reduce the number of irrelevant cells present in a population of cells selected for a myogenic phenotype.
In one approach, fluorescence-activated cell sorting (FACS) is carried out to identify cells that are positive for human CD56 (BD Biosciences), MYOD, PAX7, or MYF5. In another approach, magnetic-activated cell sorting (MACS) is used to select for the desired cell type. Other procedures which may be used for selection of cells of interest include, but are not limited to, fluorescence based cell sorting, density gradient centrifugation, flow cytometry, magnetic separation with antibody-coated magnetic beads, cytotoxic agents joined to or used in conjunction with a mAb, including, but not limited to, complement and cytotoxins; and panning with antibody attached to a solid matrix or any other convenient technique.
CD56-positive myogenic cells obtained by FACS are incubated in culture. Cells derived from the skeletal muscles of subjects affected by FSHD, as well as of their first degree relatives are termed “FSHD paired cultures.” In one embodiment, such paired cultures are useful in identifying markers that are aberrantly regulated in FSHD. In another embodiment, such cells are useful in identifying and/or assessing the efficacy of therapeutic agents useful in ameliorating FSHD or symptoms thereof. These FSHD paired cultures provide for the analysis of expression differences in related FSHD affected subjects and controls, thereby diminishing the effects of interindividual variation on gene expression.
Selected cells of the invention may be employed in methods of the invention following isolation and/or growth in vitro.
In one approach, the invention provides paired cell cultures, where one culture is derived from a subject having FSHD and the other culture is obtained from a first degree relative of the subject. Such paired cell cultures comprise skeletal muscle cells isolated from the subject or his relative during muscle biopsy. Such cells are then cultured in vitro to obtain sufficient cells for drug screening or marker expression analysis. The invention further provides a collection of such paired cell cultures. Desirably, the collection includes cell samples from two, three, four, five, six, seven, eight, nine, ten or more FSHD affected subjects and paired control cultures obtained from one or more of the subjects first degree relatives. In certain embodiments, the invention provides a frozen collection of cells suitable for paired culture. Frozen cell compositions typically comprise cryoprotective agents that provide for cell viability when the cells are frozen for a period of months or years and then subsequently thawed.
FSHD Chimeric Animals
The invention further provides chimeric animals that comprise human cells obtained from an FSHD affected. Preferably, the invention provides pairs of chimeric mice, wherein one member of the pair comprises human cells obtained from an FSHD affected and the other member of the pair comprises human cells obtained from a first degree relative of the FSHD affected.
In one embodiment, skeletal muscle cells of a mouse are injured or destroyed, for example, using cardiotoxin. The skeletal muscle cells of the injured mouse are replaced with at least about 10%, 20%, 30%, 50%, 75% or even 100% human cells derived from an FSHD subject. In one embodiment, the mouse's endogenous tibialis anterior is replaced, at least to some degree, with human muscle cells derived from an FSHD affected or a first degree relative thereof. If desired, such cells are genetically modified to express a detectable reporter (e.g., GFP, YFP, RFP, luciferase).
In one embodiment, the method provides chimeric animals, wherein one animal comprises cells of an FSHD affected and one or more other animals comprises cells of a first degree relative of the affected individual. Such chimeric animals are useful in identifying markers that are aberrantly regulated in FSHD. The invention provides a collection of such paired chimeric mice. Desirably, the collection includes cell samples from two, three, four, five, six, seven, eight, nine, ten or more FSHD affected subjects and paired control chimeric mice comprising cells obtained from one or more of the subjects' first degree relatives.
Diagnostics
The present invention features diagnostic assays for the detection of FSHD or the propensity to develop such conditions. In one embodiment, levels of any one or more of the markers listed in Table 2 or 4 are measured in a subject sample and used to characterize FSHD or the propensity to develop FSHD. In other embodiments, levels of markers listed in Table 2 or 4, are characterized in a subject sample. Standard methods may be used to measure levels of a marker in any biological sample. Biological samples include tissue samples (e.g., cell samples, biopsy samples) or biological fluid samples that include markers of the invention (e.g., blood, serum, plasma, urine). Methods for measuring levels of polypeptide biomarkers of the invention (e.g., markers listed in Table 2 or 4) include immunoassay, ELISA, western blotting and radioimmunoassay. The increase in marker levels may be altered (e.g., increased, decreased) by at least about 10%, 25%, 50%, 75% or more relative to levels of markers found in a corresponding control sample (e.g., samples obtained from a normal subject unaffected by FSHD). In one embodiment, any increase or decrease in a marker of the invention, i.e., a marker listed in Table 2 or 4, is indicative of FSHD.
Any suitable method can be used to detect one or more of the markers described herein. Successful practice of the invention can be achieved with one or a combination of methods that can detect and, preferably, quantify the markers. These methods include, without limitation, hybridization-based methods, including those employed in biochip arrays, mass spectrometry (e.g., laser desorption/ionization mass spectrometry), fluorescence (e.g. sandwich immunoassay), surface plasmon resonance, ellipsometry and atomic force microscopy. Expression levels of markers (e.g., polynucleotides or polypeptides) are compared by procedures well known in the art, such as RT-PCR, Northern blotting, Western blotting, flow cytometry, immunocytochemistry, binding to magnetic and/or antibody-coated beads, in situ hybridization, fluorescence in situ hybridization (FISH), flow chamber adhesion assay, ELISA, microarray analysis, or colorimetric assays. Methods may further include, one or more of electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS, ESI-MS/(MS)n, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS)n, atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS/MS, and APPI-(MS)n, quadrupole mass spectrometry, fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry, where n is an integer greater than zero.
Detection methods may include use of a biochip array. Biochip arrays useful in the invention include protein and polynucleotide arrays. One or more markers are captured on the biochip array and subjected to analysis to detect the level of the markers in a sample.
Markers may be captured with capture reagents immobilized to a solid support, such as a biochip, a multiwell microtiter plate, a resin, or a nitrocellulose membrane that is subsequently probed for the presence or level of a marker. Capture can be on a chromatographic surface or a biospecific surface. For example, a sample containing the markers, such as serum, may be used to contact the active surface of a biochip for a sufficient time to allow binding. Unbound molecules are washed from the surface using a suitable eluant, such as phosphate buffered saline. In general, the more stringent the eluant, the more tightly the proteins must be bound to be retained after the wash.
Upon capture on a biochip, analytes can be detected by a variety of detection methods selected from, for example, a gas phase ion spectrometry method, an optical method, an electrochemical method, atomic force microscopy and a radio frequency method. In one embodiment, mass spectrometry, and in particular, SELDI, is used. Optical methods include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry). Optical methods include microscopy (both confocal and non-confocal), imaging methods and non-imaging methods. Immunoassays in various formats (e.g., ELISA) are popular methods for detection of analytes captured on a solid phase. Electrochemical methods include voltametry and amperometry methods. Radio frequency methods include multipolar resonance spectroscopy.
Mass spectrometry (MS) is a well-known tool for analyzing chemical compounds. Thus, in one embodiment, the methods of the present invention comprise performing quantitative MS to measure the serum peptide marker. The method may be performed in an automated (Villanueva, et al., Nature Protocols (2006) 1(2):880-891) or semi-automated format. This can be accomplished, for example with MS operably linked to a liquid chromatography device (LC-MS/MS or LC-MS) or gas chromatography device (GC-MS or GC-MS/MS). Methods for performing MS are known in the field and have been disclosed, for example, in US Patent Application Publication Nos: 20050023454; 20050035286; U.S. Pat. No. 5,800,979 and references disclosed therein.
The protein fragments, whether they are peptides derived from the main chain of the protein or are residues of a side-chain, are collected on the collection layer. They may then be analyzed by a spectroscopic method based on matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI). The preferred procedure is MALDI with time of flight (TOF) analysis, known as MALDI-TOF MS. This involves forming a matrix on the membrane, e.g. as described in the literature, with an agent which absorbs the incident light strongly at the particular wavelength employed. The sample is excited by UV, or IR laser light into the vapour phase in the MALDI mass spectrometer. Ions are generated by the vaporization and form an ion plume. The ions are accelerated in an electric field and separated according to their time of travel along a given distance, giving a mass/charge (m/z) reading which is very accurate and sensitive. MALDI spectrometers are commercially available from PerSeptive Biosystems, Inc. (Frazingham, Mass., USA) and are described in the literature, e.g. M. Kussmann and P. Roepstorff, cited above.
Magnetic-based serum processing can be combined with traditional MALDI-TOF. Through this approach, improved peptide capture is achieved prior to matrix mixture and deposition of the sample on MALDI target plates. Accordingly, methods of peptide capture are enhanced through the use of derivatized magnetic bead based sample processing.
MALDI-TOF MS allows scanning of the fragments of many proteins at once. Thus, many proteins can be run simultaneously on a polyacrylamide gel, subjected to a method of the invention to produce an array of spots on the collecting membrane, and the array may be analyzed. Subsequently, automated output of the results is provided by using the ExPASy server, as at present used for MIDI-TOF MS and to generate the data in a form suitable for computers.
Other techniques for improving the mass accuracy and sensitivity of the MALDI-TOF MS can be used to analyze the fragments of protein obtained on the collection membrane. These include the use of delayed ion extraction, energy reflectors and ion-trap modules. In addition, post source decay and MS-MS analysis are useful to provide further structural analysis. With ESI, the sample is in the liquid phase and the analysis can be by ion-trap, TOF, single quadrupole or multi-quadrupole mass spectrometers. The use of such devices (other than a single quadrupole) allows MS-MS or MSn analysis to be performed. Tandem mass spectrometry allows multiple reactions to be monitored at the same time.
Capillary infusion may be employed to introduce the marker to a desired MS implementation, for instance, because it can efficiently introduce small quantities of a sample into a mass spectrometer without destroying the vacuum. Capillary columns are routinely used to interface the ionization source of a MS with other separation techniques including gas chromatography (GC) and liquid chromatography (LC). GC and LC can serve to separate a solution into its different components prior to mass analysis. Such techniques are readily combined with MS, for instance. One variation of the technique is that high performance liquid chromatography (HPLC) can now be directly coupled to mass spectrometer for integrated sample separation/and mass spectrometer analysis.
Quadrupole mass analyzers may also be employed as needed to practice the invention. Fourier-transform ion cyclotron resonance (FTMS) can also be used for some invention embodiments. It offers high resolution and the ability of tandem MS experiments. FTMS is based on the principle of a charged particle orbiting in the presence of a magnetic field. Coupled to ESI and MALDI, FTMS offers high accuracy with errors as low as 0.001%.
In one embodiment, the marker qualification methods of the invention may further comprise identifying significant peaks from combined spectra. The methods may also further comprise searching for outlier spectra. In another embodiment, the method of the invention further comprises determining distant dependent K-nearest neighbors.
In another embodiment of the method of the invention, an ion mobility spectrometer can be used to detect and characterize FSHD markers. The principle of ion mobility spectrometry is based on different mobility of ions. Specifically, ions of a sample produced by ionization move at different rates, due to their difference in, e.g., mass, charge, or shape, through a tube under the influence of an electric field. The ions (typically in the form of a current) are registered at the detector which can then be used to identify a marker or other substances in a sample. One advantage of ion mobility spectrometry is that it can operate at atmospheric pressure.
In an additional embodiment of the methods of the present invention, multiple markers are measured. The use of multiple markers increases the predictive value of the test and provides greater utility in diagnosis, toxicology, patient stratification and patient monitoring. The process called “Pattern recognition” detects the patterns formed by multiple markers greatly improves the sensitivity and specificity of clinical proteomics for predictive medicine. Subtle variations in data from clinical samples indicate that certain patterns of protein expression can predict phenotypes such as the presence or absence of FSHD.
Expression levels of particular nucleic acids or polypeptides are correlated with FSHD, and thus are useful in diagnosis. Antibodies that bind a polypeptide described herein, oligonucleotides or longer fragments derived from a nucleic acid sequence described herein (e.g., one or more Markers listed in Table 2 or 4), or any other method known in the art may be used to monitor expression of a polynucleotide or polypeptide of interest. Detection of an alteration relative to a normal, reference sample can be used as a diagnostic indicator of FSHD. In particular embodiments, the expression of one or more Markers listed in Table 2 or 4 is indicative of FSHD or the propensity to develop FSHD. In other embodiments, a 2, 3, 4, 5, or 6-fold change in the level of a marker of the invention is indicative of FSHD. In yet another embodiment, an expression profile that characterizes alterations in the expression of two, three, four, five, ten, fifteen, twenty, thirty, or forty markers is correlated with a particular disease state (e.g., FSHD). Such correlations are indicative of FSHD or the propensity to develop FSHD. In one embodiment, FSHD can be monitored using the methods and compositions of the invention.
In one embodiment, the level of one or more markers is measured on at least two different occasions and an alteration in the levels as compared to normal reference levels over time is used as an indicator of FSHD or the propensity to develop FSHD. The level of marker in a subject having FSHD or the propensity to develop such a condition may be altered by as little as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, or 90% or more relative to the level of such marker in a normal control. In general, levels of Markers listed in Table 2 or 4 are present at low or undetectable levels in a healthy subject (i.e., those who do not have and/or who will not develop FSHD). In one embodiment, a subject sample of a skeletal muscle (e.g., bicep) is collected prior to the onset of symptoms of FSHD or early on in the progression of FSHD.
The diagnostic methods described herein can be used individually or in combination with any other diagnostic method described herein for a more accurate diagnosis of the presence or severity of FSHD.
The diagnostic methods described herein can also be used to monitor and manage FSHD, or to reliably distinguish FSHD from other degenerative diseases or diseases having symptoms that are similar to or overlap with FSHD.
As indicated above, the invention provides methods for aiding a muscular dystrophy (e.g., FSHD) diagnosis using one or more markers, as specified herein. These markers can be used alone, in combination with other markers in any set, or with entirely different markers in aiding human muscular dystrophy (e.g., FSHD) diagnosis. The markers are differentially present in samples of a human FSHD patient and a normal subject (e.g., first degree relative of an FSHD subject) in whom FSHD is undetectable. Therefore, detection of one or more of these markers in a person would provide useful information regarding the probability that the person may have FSHD or have a propensity to develop FSHD.
The detection of one or more peptide marker is then correlated with a probable diagnosis of FSHD. In some embodiments, the detection of the mere presence of a marker (e.g., a marker listed in Table 2 or 4), without quantifying the amount thereof, is useful and can be correlated with a probable diagnosis of FSHD. The measurement of markers may also involve quantifying the markers to correlate the detection of markers with a probable diagnosis of FSHD. Thus, if the amount of the markers detected in a subject being tested is different compared to a control amount (i.e., higher or lower than the control), then the subject being tested has a higher probability of having FSHD.
The correlation may take into account the amount of the marker or markers in the sample compared to a control amount of the marker or markers (e.g., in normal subjects). A control can be, e.g., the average or median amount of marker present in comparable samples of normal subjects. The control amount is measured under the same or substantially similar experimental conditions as in measuring the test amount. As a result, the control can be employed as a reference standard, where each result can be compared to that standard, rather than re-running a control.
Accordingly, a marker profile may be obtained from a subject sample and compared to a reference marker profile, so that it is possible to classify the subject as having or not having FSHD. The correlation may take into account the presence or absence of the markers in a test sample and the frequency of detection of the same markers in a control. The correlation may take into account both of such factors to facilitate determination of FSHD status.
In certain embodiments of the invention, the methods further comprise managing subject treatment based on the status.
The markers of the present invention have a number of other uses. For example, they can be used to identify agents useful in methods of treating or ameliorating FSHD. In yet another example, the markers can be used in heredity studies. For instance, certain markers associated with FSHD may be genetically associated with the disease. This can be determined by, e.g., analyzing samples from a population of human subjects whose families have a history of FSHD. The results can then be compared with data obtained from, e.g., subjects whose families do not have a history of FSHD. The markers that are genetically linked may be used as a tool to determine if a subject whose family has a history of FSHD is pre-disposed to having FSHD.
While individual markers are useful diagnostic markers, in some instances, a combination of markers provides greater predictive value than a single marker alone. The detection of a plurality of markers (or absence thereof, as the case may be) in a sample can increase the percentage of true positive and true negative diagnoses and decrease the percentage of false positive or false negative diagnoses. Thus, preferred methods of the present invention comprise the measurement of more than one marker.
Microarrays
As reported herein, a number of markers (e.g., a marker listed in Table 2 or 4) have been identified that are associated with FSHD. Methods for assaying the expression of these polypeptides are useful for characterizing FSHD. In particular, the invention provides diagnostic methods and compositions useful for identifying a polypeptide expression profile that identifies a subject as having or having a propensity to develop FSHD. Such assays can be used to measure an alteration in the level of a polypeptide.
The polypeptides and nucleic acid molecules of the invention are useful as hybridizable array elements in a microarray. The array elements are organized in an ordered fashion such that each element is present at a specified location on the substrate. Useful substrate materials include membranes, composed of paper, nylon or other materials, filters, chips, glass slides, and other solid supports. The ordered arrangement of the array elements allows hybridization patterns and intensities to be interpreted as expression levels of particular genes or proteins. Methods for making nucleic acid microarrays are known to the skilled artisan and are described, for example, in U.S. Pat. No. 5,837,832, Lockhart, et al. (Nat. Biotech. 14:1675-1680, 1996), and Schena, et al. (Proc. Natl. Acad. Sci. 93:10614-10619, 1996), herein incorporated by reference. Methods for making polypeptide microarrays are described, for example, by Ge (Nucleic Acids Res. 28: e3. i-e3. vii, 2000), MacBeath et al., (Science 289:1760-1763, 2000), Zhu et al. (Nature Genet. 26:283-289), and in U.S. Pat. No. 6,436,665, hereby incorporated by reference.
Protein Microarrays
Proteins (e.g., proteins encoded by genes listed in Table 2 or 4) may be analyzed using protein microarrays. Such arrays are useful in high-throughput low-cost screens to identify alterations in the expression or post-translation modification of a polypeptide of the invention, or a fragment thereof. In particular, such microarrays are useful to identify a protein whose expression is altered in FSHD. In one embodiment, a protein microarray of the invention binds a marker present in a subject sample and detects an alteration in the level of the marker. Typically, a protein microarray features a protein, or fragment thereof, bound to a solid support. Suitable solid supports include membranes (e.g., membranes composed of nitrocellulose, paper, or other material), polymer-based films (e.g., polystyrene), beads, or glass slides. For some applications, proteins (e.g., antibodies that bind a marker of the invention) are spotted on a substrate using any convenient method known to the skilled artisan (e.g., by hand or by inkjet printer).
The protein microarray is hybridized with a detectable probe. Such probes can be polypeptide, nucleic acid molecules, antibodies, or small molecules. For some applications, polypeptide and nucleic acid molecule probes are derived from a biological sample taken from a patient, such as a homogenized tissue sample (e.g. a tissue sample obtained by muscle biopsy); or a cell isolated from a patient sample. Probes can also include antibodies, candidate peptides, nucleic acids, or small molecule compounds derived from a peptide, nucleic acid, or chemical library. Hybridization conditions (e.g., temperature, pH, protein concentration, and ionic strength) are optimized to promote specific interactions. Such conditions are known to the skilled artisan and are described, for example, in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual. 1998, New York: Cold Spring Harbor Laboratories. After removal of non-specific probes, specifically bound probes are detected, for example, by fluorescence, enzyme activity (e.g., an enzyme-linked calorimetric assay), direct immunoassay, radiometric assay, or any other suitable detectable method known to the skilled artisan.
Nucleic Acid Microarrays
To produce a nucleic acid microarray, oligonucleotides may be synthesized or bound to the surface of a substrate using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.), incorporated herein by reference. Alternatively, a gridded array may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedure.
A nucleic acid molecule (e.g. RNA or DNA) derived from a biological sample may be used to produce a hybridization probe as described herein. The biological samples are generally derived from a patient, as a tissue sample (e.g. a tissue sample obtained by muscle biopsy). For some applications, cultured cells or other tissue preparations may be used. The mRNA is isolated according to standard methods, and cDNA is produced and used as a template to make complementary RNA suitable for hybridization. Such methods are known in the art. The RNA is amplified in the presence of fluorescent nucleotides, and the labeled probes are then incubated with the microarray to allow the probe sequence to hybridize to complementary oligonucleotides bound to the microarray.
Incubation conditions are adjusted such that hybridization occurs with precise complementary matches or with various degrees of less complementarity depending on the degree of stringency employed. For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30 C., more preferably of at least about 37 C., and most preferably of at least about 42 C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30 C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37 C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42 C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
The removal of nonhybridized probes may be accomplished, for example, by washing. The washing steps that follow hybridization can also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25 C., more preferably of at least about 42° C., and most preferably of at least about 68 C. In a preferred embodiment, wash steps will occur at 25 C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, wash steps will occur at 68 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.
A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct nucleic acid sequences simultaneously (e.g., Heller et al., Proc. Natl. Acad. Sci. 94:2150-2155, 1997). Preferably, a scanner is used to determine the levels and patterns of fluorescence.
Diagnostic Kits
The invention provides kits for diagnosing or monitoring FSHD. In one embodiment, the kit includes a composition containing at least one agent that binds a polypeptide or polynucleotide whose expression is increased in FSHD. In another embodiment, the invention provides a kit that contains an agent that binds a nucleic acid molecule whose expression is altered in FSHD. In some embodiments, the kit comprises a sterile container which contains the binding agent; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
If desired the kit is provided together with instructions for using the kit to diagnose FSHD. The instructions will generally include information about the use of the composition for diagnosing a subject as having FSHD or having a propensity to develop FSHD. In other embodiments, the instructions include at least one of the following: description of the binding agent; warnings; indications; counter-indications; animal study data; clinical study data; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
Screening Assays
As discussed herein, the expression of genes listed in Tables 2 and 4 is altered in FSHD. Based on this discovery, compositions of the invention are useful for the high-throughput low-cost screening of candidate agents to identify those that modulate the expression of genes that are aberrantly expressed in FSHD. In one embodiment, the effects of candidate agents on genes expressed in Tables 2 and 4 are assayed using microarrays, cell compositions, and/or chimeric animals of the invention.
Those genes identified in Tables 2 or 4 whose expression is inappropriately increased in FSHD are targets for therapeutic intervention. The genes TRIM43 and PRAMEF1 are of particular interest. The inappropriate activation of one or more genes upregulated in FSHD likely contributes to the pathology observed in FSHD. Therefore, agents that reduce the expression of genes that are over-expressed in FSHD are useful in the methods of the invention. Such agents include, for example, inhibitory nucleic acids that reduce or eliminate the expression of such genes, as well as proteins (e.g., antibodies and fragments thereof) and small compounds that interfere with the expression or biological activity of the genes or the proteins that they encode. The present methods can be used to identify such agents.
Those genes identified in Table 2 or 4 whose expression is inappropriately decreased in FSHD are also targets for therapeutic intervention. Such agents include, for example, small compounds that increase the biological activity or expression of a gene listed in Table 2 or 4 or of the protein that gene encodes. In other embodiments, agents (e.g., expression vectors encoding proteins downregulated in FSHD) are useful to increase the expression of such genes, particularly in skeletal muscle. Such expression would be expected to ameliorate FSHD or symptoms associated with FSHD. The present methods can be used to identify such agents.
A number of methods are available for carrying out screening assays to identify candidate agents that reduce the expression of genes that are overexpressed in FSHD, or that increase the expression of a gene that is downregulated in FSHD. In one example, candidate agents are added at varying concentrations to the culture medium of cultured cells (e.g., FSHD paired cultures) expressing one of the nucleic acid sequences of the invention. Gene expression is then measured, for example, by microarray analysis, Northern blot analysis (Ausubel et al., supra), reverse transcriptase PCR, quantitative real-time PCR, or any other method known in the art using any appropriate fragment prepared from the nucleic acid molecule as a hybridization probe. The level of gene expression in the FSHD derived cells in the presence of the candidate agent is compared to the level measured in a control culture. In one embodiment, the control culture is a culture of FSHD derived cells that lack the agent. In another embodiment, the control culture is the paired culture of cells obtained from a first degree relative of the FSHD affected. An agent that normalizes or promotes the normalization of expression of aberrantly regulated genes is considered useful in the invention. Such an agent may be used, for example, as a therapeutic to treat FSHD in a human patient. An agent that “normalizes” the expression of an aberrantly regulated gene restores the expression of that gene to a level that is substantially normal. An agent that “promotes normalization” reduces the extent of the disregulation.
In one example, the effect of candidate agents is measured at the level of polypeptide production using the same general approach and standard immunological techniques, such as Western blotting or immunoprecipitation with an antibody specific for a polypeptide encoded by a gene listed in Table 2 or 4. For example, immunoassays may be used to detect or monitor the expression of at least one of the polypeptides of the invention in an organism. Polyclonal or monoclonal antibodies, (produced as described above) that are capable of binding to such a polypeptide may be used in any standard immunoassay format (e.g., ELISA, Western blot, or RIA assay) to measure the level of the polypeptide. In some embodiments, a agent that normalizes or promotes normalization of the expression or biological activity of an aberrantly regulated polypeptide is considered useful. Again, such an agent may be used, for example, as a therapeutic to delay, ameliorate, or treat FSHD disorder, or the symptoms of FSHD, in a human patient.
In yet another working example, candidate agents may be screened for those that specifically bind to a polypeptide encoded by a gene listed in Table 2 or 4. The efficacy of such a candidate agent is dependent upon its ability to interact with such a polypeptide or a functional equivalent thereof. Such an interaction can be readily assayed using any number of standard binding techniques and functional assays (e.g., those described in Ausubel et al., supra). In one embodiment, a candidate agent may be tested in vitro for its ability to specifically bind a polypeptide of the invention. In another embodiment, a candidate agent is tested for its ability to normalize or promote the normalization of the biological activity of a polypeptide described herein. The biological activity of a polypeptide may be assayed using any standard method.
In another example, a gene described herein (e.g., listed in Table 2 or 4) is expressed as a transcriptional or translational fusion with a detectable reporter, and expressed in an isolated cell (e.g., mammalian or insect cell) under the control of a heterologous promoter, such as an inducible promoter. The cell expressing the fusion protein is then contacted with a candidate agent, and the expression of the detectable reporter in that cell is compared to the expression of the detectable reporter in an untreated control cell. A candidate agent that alters (e.g., normalizes or promotes normalization) the expression of the detectable reporter is an agent that is useful for the treatment of FSHD. In preferred embodiments, the candidate agent increases the expression of a reporter gene fused to a gene that is downregulated in FSHD.
In one particular working example, a candidate agent that binds to a polypeptide encoded by a gene listed in Table 2 or 4 may be identified using a chromatography-based technique. For example, a recombinant polypeptide of the invention may be purified by standard techniques from cells engineered to express the polypeptide (e.g., those described above) and may be immobilized on a column. A solution of candidate agents is then passed through the column, and an agent specific for the polypeptide encoded by a nucleic acid molecule listed in Table 2 or 4 is identified on the basis of its ability to bind to the polypeptide and be immobilized on the column. To isolate the agent, the column is washed to remove non-specifically bound molecules, and the agent of interest is then released from the column and collected. Similar methods may be used to isolate an agent bound to a polypeptide microarray. Agents isolated by this method (or any other appropriate method) may, if desired, be further purified (e.g., by high performance liquid chromatography). In addition, these candidate agents may be tested for their ability to increase the activity of gene whose expression is downregulated in FSHD. Agents isolated by this approach may also be used, for example, as therapeutics to treat FSHD in a human patient. Agents that are identified as binding to a polypeptide of the invention with an affinity constant less than or equal to 10 mM are considered particularly useful in the invention. Alternatively, any in vivo protein interaction detection system, for example, any two-hybrid assay may be utilized.
Potential agonists and antagonists include organic molecules, peptides, peptide mimetics, polypeptides, nucleic acid molecules, and antibodies that bind to a nucleic acid sequence or polypeptide of the invention. (e.g., those listed in Table 2 or 4). For those nucleic acid molecules or polypeptides whose expression is decreased in a patient having FSHD, agonists would be particularly useful in the methods of the invention. For those nucleic acid molecules or polypeptides whose expression is increased in a patient having FSHD, antagonists would be particularly useful in the methods of the invention.
Each of the DNA sequences identified herein may be used in the discovery and development of a therapeutic agent for the treatment of FSHD. The encoded protein, upon expression, can be used as a target for the screening of drugs. Additionally, the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct sequences that promote the expression of the coding sequence of interest. Such sequences may be isolated by standard techniques (Ausubel et al., supra). Optionally, agents identified in any of the above-described assays may be confirmed as useful in cell culture or in a chimeric animal of the invention. Small molecules of the invention preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons. It is preferred that these small molecules are organic molecules.
Test Agents and Extracts
In general, agents capable of normalizing or promoting the normalization of expression of a gene listed in Table 2 or 4 are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries or from polypeptide or nucleic acid libraries (e.g., Table 2 or 4), according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or agents is not critical to the screening procedure(s) of the invention. Agents used in screens may include known agents (for example, known therapeutics used for other diseases or disorders). Alternatively, virtually any number of unknown chemical extracts or agents can be screened using the methods described herein. Examples of such extracts or agents include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic agents, as well as modification of existing agents. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical agents, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based agents. Synthetic agent libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of natural agents in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or agent is readily modified using standard chemical, physical, or biochemical methods.
In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their activity should be employed whenever possible.
When a crude extract is found to normalize or promote normalization of the activity of a polypeptide that is aberrantly regulated in FSHD, further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract that increases the expression or activity of the polypeptide. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, agents shown to be useful as therapeutics for the treatment of human FSHD are chemically modified according to methods known in the art.
Inhibitory Nucleic Acids
Inhibitory nucleic acid molecules are those oligonucleotides that inhibit the expression or activity of a polypeptide that is overexpressed in FSHD (e.g., a polypeptide encoded by a gene listed in Table 2 or 4). Such oligonucleotides include single and double stranded nucleic acid molecules (e.g., DNA, RNA, and analogs thereof) that bind a nucleic acid molecule that encodes a polypeptide that is overexpressed in FSHD (e.g., antisense molecules, siRNA, shRNA) as well as nucleic acid molecules that bind directly to the polypeptide to modulate its biological activity (e.g., aptamers).
MOE Gapmers
In one embodiment, the invention provides methods for characterizing the effects of RNaseH1-activating antisense oligonucleotides (ASO's) (“MOE gapmers”) on markers of the invention. The RNAseH1 ASO chemistry provides for a 20 nucleotide phosphorothioate backbone (5-10-5 gapmer). In particular, the oligonucleotide comprises five nucleotides at each end with the 2′-O-(2-methoxyethyl) (MOE) modification and ten central deoxyribonucleotides for activation of RNase H1. In one embodiment, cells derived from an FSHD affected and paired control cells are contacted with ASO's targeting DUX4. The effect of the downregulation of DUX4 on markers of the invention (e.g., markers listed in Table 2 or 4) is assayed. In another embodiment, a marker of the invention (e.g., a marker upregulated in FSHD) is targeted, and the effect of such targeting is assessed on the levels of other markers of the invention.
siRNA
Short twenty-one to twenty-five nucleotide double-stranded RNAs are effective at down-regulating gene expression (Zamore et al., Cell 101: 25-33; Elbashir et al., Nature 411: 494-498, 2001, hereby incorporated by reference). The therapeutic effectiveness of an sirNA approach in mammals was demonstrated in vivo by McCaffrey et al. (Nature 418: 38-39.2002).
Given the sequence of a target gene, siRNAs may be designed to inactivate that gene. Such siRNAs, for example, could be administered directly to an affected tissue, or administered systemically. The nucleic acid sequence of a gene can be used to design small interfering RNAs (siRNAs). The 21 to 25 nucleotide siRNAs may be used, for example, as therapeutics to treat FSHD.
The inhibitory nucleic acid molecules of the present invention may be employed as double-stranded RNAs for RNA interference (RNAi)-mediated knock-down of expression. In one embodiment, expression of a gene listed in Table 2 or 4 is reduced in a skeletal muscle cell. RNAi is a method for decreasing the cellular expression of specific proteins of interest (reviewed in Tuschl, Chembiochem 2:239-245, 2001; Sharp, Genes & Devel. 15:485-490, 2000; Hutvagner and Zamore, Curr. Opin. Genet. Devel. 12:225-232, 2002; and Hannon, Nature 418:244-251, 2002). The introduction of siRNAs into cells either by transfection of dsRNAs or through expression of siRNAs using a plasmid-based expression system is increasingly being used to create loss-of-function phenotypes in mammalian cells.
In one embodiment of the invention, a double-stranded RNA (dsRNA) molecule is made that includes between eight and nineteen consecutive nucleobases of a nucleobase oligomer of the invention. The dsRNA can be two distinct strands of RNA that have duplexed, or a single RNA strand that has self-duplexed (small hairpin (sh)RNA). Typically, dsRNAs are about 21 or 22 base pairs, but may be shorter or longer (up to about 29 nucleobases) if desired. dsRNA can be made using standard techniques (e.g., chemical synthesis or in vitro transcription). Kits are available, for example, from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al. Science 296:550-553, 2002; Paddison et al. Genes & Devel. 16:948-958, 2002. Paul et al. Nature Biotechnol. 20:505-508, 2002; Sui et al. Proc. Natl. Acad. Sci. USA 99:5515-5520, 2002; Yu et al. Proc. Natl. Acad. Sci. USA 99:6047-6052, 2002; Miyagishi et al. Nature Biotechnol. 20:497-500, 2002; and Lee et al. Nature Biotechnol. 20:500-505 2002, each of which is hereby incorporated by reference.
Small hairpin RNAs (shRNAs) comprise an RNA sequence having a stem-loop structure. A “stem-loop structure” refers to a nucleic acid having a secondary structure that includes a region of nucleotides which are known or predicted to form a double strand or duplex (stem portion) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion). The term “hairpin” is also used herein to refer to stem-loop structures. Such structures are well known in the art and the term is used consistently with its known meaning in the art. As is known in the art, the secondary structure does not require exact base-pairing. Thus, the stem can include one or more base mismatches or bulges. Alternatively, the base-pairing can be exact, i.e. not include any mismatches. The multiple stem-loop structures can be linked to one another through a linker, such as, for example, a nucleic acid linker, other molecule, or some combination thereof.
As used herein, the term “small hairpin RNA” includes a conventional stem-loop shRNA, which forms a precursor miRNA (pre-miRNA). While there may be some variation in range, a conventional stem-loop shRNA can comprise a stem ranging from 19 to 29 bp, and a loop ranging from 4 to 30 bp. “shRNA” also includes micro-RNA embedded shRNAs (miRNA-based shRNAs), wherein the guide strand and the passenger strand of the miRNA duplex are incorporated into an existing (or natural) miRNA or into a modified or synthetic (designed) miRNA. In some instances the precursor miRNA molecule can include more than one stem-loop structure. MicroRNAs are endogenously encoded RNA molecules that are about 22-nucleotides long and generally expressed in a highly tissue- or developmental-stage-specific fashion and that post-transcriptionally regulate target genes. More than 200 distinct miRNAs have been identified in plants and animals. These small regulatory RNAs are believed to serve important biological functions by two prevailing modes of action: (1) by repressing the translation of target mRNAs, and (2) through RNA interference (RNAi), that is, cleavage and degradation of mRNAs. In the latter case, miRNAs function analogously to small interfering RNAs (siRNAs). Thus, one can design and express artificial miRNAs based on the features of existing miRNA genes.
shRNAs can be expressed from DNA vectors to provide sustained silencing and high yield delivery into almost any cell type. In some embodiments, the vector is a viral vector. Exemplary viral vectors include retroviral, including lentiviral, adenoviral, baculoviral and avian viral vectors, and including such vectors allowing for stable, single-copy genomic integrations. Retroviruses from which the retroviral plasmid vectors can be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. A retroviral plasmid vector can be employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which can be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14x, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990), which is incorporated herein by reference in its entirety. The vector can transduce the packaging cells through any means known in the art. A producer cell line generates infectious retroviral vector particles which include polynucleotide encoding a DNA replication protein. Such retroviral vector particles then can be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express a DNA replication protein.
Catalytic RNA molecules or ribozymes that include an antisense sequence of the present invention can be used to inhibit expression of a nucleic acid molecule in vivo (e.g., a nucleic acid molecule listed in Table 2 or 4). The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature 334:585-591. 1988, and U.S. Patent Application Publication No. 2003/0003469 A1, each of which is incorporated by reference.
Accordingly, the invention also features a catalytic RNA molecule that includes, in the binding arm, an antisense RNA having between eight and nineteen consecutive nucleobases. In preferred embodiments of this invention, the catalytic nucleic acid molecule is formed in a hammerhead or hairpin motif. Examples of such hammerhead motifs are described by Rossi et al., Aids Research and Human Retroviruses, 8:183, 1992. Example of hairpin motifs are described by Hampel et al., “RNA Catalyst for Cleaving Specific RNA Sequences,” filed Sep. 20, 1989, which is a continuation-in-part of U.S. Ser. No. 07/247,100 filed Sep. 20, 1988, Hampel and Tritz, Biochemistry, 28:4929, 1989, and Hampel et al., Nucleic Acids Research, 18: 299, 1990. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.
Essentially any method for introducing a nucleic acid construct into cells can be employed. Physical methods of introducing nucleic acids include injection of a solution containing the construct, bombardment by particles covered by the construct, soaking a cell, tissue sample or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the construct. A viral construct packaged into a viral particle can be used to accomplish both efficient introduction of an expression construct into the cell and transcription of the encoded shRNA. Other methods known in the art for introducing nucleic acids to cells can be used, such as lipid-mediated carrier transport, chemical mediated transport, such as calcium phosphate, and the like. Thus the shRNA-encoding nucleic acid construct can be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, promote annealing of the duplex strands, stabilize the annealed strands, or otherwise increase inhibition of the target gene.
For expression within cells, DNA vectors, for example plasmid vectors comprising either an RNA polymerase II or RNA polymerase III promoter can be employed. Expression of endogenous miRNAs is controlled by RNA polymerase II (Pol II) promoters and in some cases, shRNAs are most efficiently driven by Pol II promoters, as compared to RNA polymerase III promoters (Dickins et al., 2005, Nat. Genet. 39: 914-921). In some embodiments, expression of the shRNA can be controlled by an inducible promoter or a conditional expression system, including, without limitation, RNA polymerase type II promoters. Examples of useful promoters in the context of the invention are tetracycline-inducible promoters (including TRE-tight), IPTG-inducible promoters, tetracycline transactivator systems, and reverse tetracycline transactivator (rtTA) systems. Constitutive promoters can also be used, as can cell- or tissue-specific promoters. Many promoters will be ubiquitous, such that they are expressed in all cell and tissue types. A certain embodiment uses tetracycline-responsive promoters, one of the most effective conditional gene expression systems in in vitro and in vivo studies. See International Patent Application PCT/US2003/030901 (Publication No. WO 2004-029219 A2) and Fewell et al., 2006, Drug Discovery Today 11: 975-982, for a description of inducible shRNA.
Modified Nucleic Acids
At least two types of oligonucleotides induce the cleavage of RNA by RNase H: polydeoxynucleotides with phosphodiester (PO) or phosphorothioate (PS) linkages. Although 2′-OMe-RNA sequences exhibit a high affinity for RNA targets, these sequences are not substrates for RNase H. A desirable oligonucleotide is one based on 2′-modified oligonucleotides containing oligodeoxynucleotide gaps with some or all internucleotide linkages modified to phosphorothioates for nuclease resistance. The presence of methylphosphonate modifications increases the affinity of the oligonucleotide for its target RNA and thus reduces the IC50. This modification also increases the nuclease resistance of the modified oligonucleotide. It is understood that the methods and reagents of the present invention may be used in conjunction with any technologies that may be developed, including covalently-closed multiple antisense (CMAS) oligonucleotides (Moon et al., Biochem J. 346:295-303, 2000; PCT Publication No. WO 00/61595), ribbon-type antisense (RiAS) oligonucleotides (Moon et al., J. Biol. Chem. 275:4647-4653, 2000; PCT Publication No. WO 00/61595), and large circular antisense oligonucleotides (U.S. Patent Application Publication No. US 2002/0168631 A1).
As is known in the art, a nucleoside is a nucleobase-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric structure can be further joined to form a circular structure; open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.
Specific examples of preferred nucleobase oligomers useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, nucleobase oligomers having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are also considered to be nucleobase oligomers.
Nucleobase oligomers that have modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriest-ers, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity, wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included. Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference.
Nucleobase oligomers having modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH.sub.2 component parts. Representative United States patents that teach the preparation of the above oligonucleotides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.
In other nucleobase oligomers, both the sugar and the internucleoside linkage, i.e., the backbone, are replaced with novel groups. The nucleobase units are maintained for hybridization with a gene listed in Table 2 or 4. One such nucleobase oligomer, is referred to as a Peptide Nucleic Acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Methods for making and using these nucleobase oligomers are described, for example, in “Peptide Nucleic Acids: Protocols and Applications” Ed. P. E. Nielsen, Horizon Press, Norfolk, United Kingdom, 1999. Representative United States patents that teach the preparation of PNAs include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
In particular embodiments of the invention, the nucleobase oligomers have phosphorothioate backbones and nucleosides with heteroatom backbones, and in particular —CH2—NH—O—CH2—, —CH2—N(CH3)—O—CH2— (known as a methylene (methylimino) or MMI backbone), —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2—, and —O—N(CH3)—CH2—CH2—. In some embodiments, the oligonucleotides have morpholino backbone structures described in U.S. Pat. No. 5,034,506.
Nucleobase oligomers may also contain one or more substituted sugar moieties. Nucleobase oligomers comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]nCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other preferred nucleobase oligomers include one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of a nucleobase oligomer, or a group for improving the pharmacodynamic properties of an nucleobase oligomer, and other substituents having similar properties. Preferred modifications are 2′-O-methyl and 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE). Another desirable modification is 2′-dimethylaminooxyethoxy (i.e., O(CH2)2ON(CH3)2), also known as 2′-DMAOE. Other modifications include, 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on an oligonucleotide or other nucleobase oligomer, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Nucleobase oligomers may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is herein incorporated by reference in its entirety.
Nucleobase oligomers may also include nucleobase modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyl uracil and cytosine; 6-azo uracil, cytosine and thymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines; 5-halo (e.g., 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deazaguanine and 7-deazaadenine; and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of an antisense oligonucleotide of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are desirable base substitutions, even more particularly when combined with 2′-O-methoxyethyl or 2′-O-methyl sugar modifications. Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; and 5,750,692, each of which is herein incorporated by reference.
Another modification of a nucleobase oligomer of the invention involves chemically linking to the nucleobase oligomer one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 86:6553-6556, 1989), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let, 4:1053-1060, 1994), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 660:306-309, 1992; Manoharan et al., Bioorg. Med. Chem. Let., 3:2765-2770, 1993), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 20:533-538: 1992), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 10:1111-1118, 1991; Kabanov et al., FEBS Lett., 259:327-330, 1990; Svinarchuk et al., Biochimie, 75:49-54, 1993), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 36:3651-3654, 1995; Shea et al., Nucl. Acids Res., 18:3777-3783, 1990), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 14:969-973, 1995), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 36:3651-3654, 1995), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1264:229-237, 1995), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 277:923-937, 1996. Representative United States patents that teach the preparation of such nucleobase oligomer conjugates include U.S. Pat. Nos. 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,828,979; 4,835,263; 4,876,335; 4,904,582; 4,948,882; 4,958,013; 5,082,830; 5,109,124; 5,112,963; 5,118,802; 5,138,045; 5,214,136; 5,218,105; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,414,077; 5,416,203, 5,451,463; 5,486,603; 5,510,475; 5,512,439; 5,512,667; 5,514,785; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,565,552; 5,567,810; 5,574,142; 5,578,717; 5,578,718; 5,580,731; 5,585,481; 5,587,371; 5,591,584; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,608,046; and 5,688,941, each of which is herein incorporated by reference.
The present invention also includes nucleobase oligomers that are chimeric compounds. “Chimeric” nucleobase oligomers are nucleobase oligomers, particularly oligonucleotides, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide. These nucleobase oligomers typically contain at least one region where the nucleobase oligomer is modified to confer, upon the nucleobase oligomer, increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the nucleobase oligomer may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of nucleobase oligomer inhibition of gene expression. Consequently, comparable results can often be obtained with shorter nucleobase oligomers when chimeric nucleobase oligomers are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region.
Chimeric nucleobase oligomers of the invention may be formed as composite structures of two or more nucleobase oligomers as described above. Such nucleobase oligomers, when oligonucleotides, have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference in its entirety.
The nucleobase oligomers used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.
The nucleobase oligomers of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.
Delivery of Polynucleotides
Naked polynucleotides, or analogs thereof, are capable of entering mammalian cells and inhibiting expression of a gene of interest. Nonetheless, it may be desirable to utilize a formulation that aids in the delivery of oligonucleotides or other nucleobase oligomers to cells (see, e.g., U.S. Pat. Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is hereby incorporated by reference).
Polynucleotide Therapy for FSHD
Polynucleotide therapy is one therapeutic approach for preventing or ameliorating FSHD associated with the reduced expression of a nucleic acid molecule listed in Table 2 or 4. Such nucleic acid molecules can be delivered to cells that lack sufficient, normal protein expression or biological activity. The nucleic acid molecules must be delivered to those cells in a form in which they can be taken up by the cells and so that sufficient levels of protein can be produced to increase protein expression or function in a patient having FSHD.
Transducing viral (e.g., retroviral, adenoviral, and adeno-associated viral) vectors can be used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). For example, a full length gene (e.g., a nucleic acid molecule listed in Table 2 or 4), or a portion thereof, can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from a retroviral long terminal repeat, or from a promoter specific for a target cell type of interest (e.g., a skeletal muscle cell). Promoters useful in the methods of the invention include, for example, myoD.
Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77 S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346). Most preferably, a viral vector is used to administer the gene of interest (e.g., nucleic acid molecules listed in Table 2 or 4) systemically or to a skeletal muscle.
Non-viral approaches can also be employed for the introduction of therapeutic agent to a cell of an FSHD affected. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by micro-injection under surgical conditions (Wolff et al., Science 247:1465, 1990). Preferably the nucleic acids are administered in combination with a liposome and protamine.
Gene transfer can also be achieved using non-viral means involving transfection in vitro. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a patient can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue.
cDNA expression for use in gene therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element. For example, if desired, enhancers known to preferentially direct gene expression in specific cell types, such as cells of the central nervous system or their associated glial cells, can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.
Another therapeutic approach included in the invention involves administration of a recombinant therapeutic, such as a recombinant polypeptide encoded by a gene downregulated in FSHD. In one embodiment, the protein is either administered directly to a disease-affected tissue (for example, by injection into the muscle) or systemically (for example, by any conventional recombinant protein administration technique). The dosage of the administered protein depends on a number of factors, including the size and health of the individual patient. For any particular subject, the specific dosage regimes should be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions. Generally, between 0.1 mg and 100 mg, is administered per day to an adult in any pharmaceutically acceptable formulation.
Pharmaceutical Therapeutics
The invention provides a simple means for identifying agents (including nucleic acid molecules, inhibitory nucleic acid molecules, peptides, small molecules, and mimetics) capable of acting as therapeutics for the treatment of FSHD. Accordingly, a chemical entity discovered to have medicinal value using the methods described herein is useful as a drug or as information for structural modification of existing agents, e.g., by rational drug design.
For therapeutic uses, the agents identified using the methods disclosed herein may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Preferable routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections that provide continuous, sustained levels of the drug in the patient. Treatment of human patients or other animals will be carried out using a therapeutically effective amount of FSHD therapeutic in a physiologically-acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin. The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of FSHD. An agent is administered at a dosage that controls the clinical or physiological symptoms of FSHD as determined by clinical evaluation or by a diagnostic method of the invention that assays the expression of a nucleic acid molecule listed in Table 2 or 4, or the biological activity of a polypeptide encoded by such a nucleic acid molecule.
Formulation of Pharmaceutical Compositions
The administration of an agent for the treatment of FSHD may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing FSHD. The agent may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).
Pharmaceutical compositions according to the invention may be formulated to release the active agent substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in the central nervous system or cerebrospinal fluid; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target FSHD by using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type (e.g., skeletal muscle cell) whose function is perturbed in FSHD. For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.
Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the agent in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the FSHD therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the FSHD therapeutic in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.
Parenteral Compositions
The pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.
Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active therapeutic (s), the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic (s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.
As indicated above, the pharmaceutical compositions according to the invention may be in a form suitable for sterile injection. To prepare such a composition, the suitable active therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the agents is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.
Solid Dosage Forms for Oral Use
Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. Such formulations are known to the skilled artisan. Excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.
The tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby providing a sustained action over a longer period. The coating may be adapted to release the active drug in a predetermined pattern (e.g., in order to achieve a controlled release formulation) or it may be adapted not to release the active drug until after passage of the stomach (enteric coating). The coating may be a sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose, methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and/or ethylcellulose). Furthermore, a time delay material such as, e.g., glyceryl monostearate or glyceryl distearate may be employed.
The solid tablet compositions may include a coating adapted to protect the composition from unwanted chemical changes, (e.g., chemical degradation prior to the release of the active therapeutic substance). The coating may be applied on the solid dosage form in a similar manner as that described in Encyclopedia of Pharmaceutical Technology, supra. Formulations for oral use may also be presented as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders and granulates may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.
The present invention provides methods of treating FSHD or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the formulae herein to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to FSHD or a symptom thereof. The method includes the step of administering to the mammal a therapeutic amount of a compound herein sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.
The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).
As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the compounds herein, such as a compound of the formulae herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like). The compounds herein may be also used in the treatment of any other disorders in which muscular dystrophy may be implicated.
In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., any target delineated herein modulated by a compound herein, a protein or indicator thereof, etc.) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to FSHD, in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
Example 1
Microarray Analysis Identifies Genes that are Differentially Regulated in FSHD
Open muscle biopsy was performed on both the biceps and deltoid muscles of 6 FSHD affected and 5 unaffected subjects that are first degree relatives of the FSHD affected subjects. Characteristics of the donors are provided in Table 1.







TABLE 1







Clinical characteristics of FSHD subjects and unaffected donors.
















Age at






Familial

Enrollment
EcoRI/Blnl
Deltoid
Biceps


Donor*
Relations
Gender
(yrs)
Allele
Strength**
Strength





07A
proband
F
18
29 kb
4+/5 at 90°
full


07U
mother of
F
49
34 kb (4qB),
full
full



proband


53 kb (4qA) 


09A
proband
F
31
25 kb
 5/5 at 45°
4+/5


09U
mother of 09A
F
57
47 kb
full
full


12A
proband
M
49
18 kb
4+/5 at 90°
4+/5


12U
sister of 12A
F
45
>112 kb 
full
full


15A
proband
M
67
28 kb
 5/5 at 90°
 5/5


15B
brother of 15A
M
69
28 kb
full
full


15V
sister of 15A
F
60
107 kb 
full
full


21B
proband
F
59
34 kb
5/5
4+/5


21U
sister of 21B
F
48
150 kb 
full
full





*Donors are designated by cohort (family) number (07, 09, etc.) followed by A, B for the FSHD donors or U, V for the unaffected 1st degree relative(s) of the FSHD subject(s) in the cohort. Each cohort was composed of at least one affected individual with genetically and clinically verified FSHD, and at least one unaffected first degree relative with unshortened D4Z4 alleles and normal strength.


**Muscle strength is presented using a modified MRC scale where 5/5 is full strength for right/left sides.






Molecular diagnosis of FSHD was confirmed by the University of Iowa Diagnostic Laboratories and indicated that each donor with a clinical diagnosis of FSHD also had a contracted 4q D4Z4 region in combination with a 4qA telomeric allele (Table 1).
Primary Cell Culture.
Primary muscle cell strains were established from open muscle biopsies following collagenase IV and dispase dissociation as previously described (Stadler et al., 2011). Cells were cultured at 37° C. in 5% CO2 on 0.1% gelatin-coated dishes and propagated by daily feeding with HMP growth medium consisting of Ham's F10 medium (Cellgro) supplemented with 20% characterized fetal bovine serum (Hyclone), 0.5% chicken embryo extract, 1.2 mM CaCl2, and 1% antibiotics/antimycotics (Cellgro). Cultures were incubated until cells reached 50-70% confluence, at which time cells were harvested after dissociation with TrypLE Express (Gibco), counted, and expanded for fluorescence-activated cell sorting (FACS) or frozen storage.
The initial primary cultures were enriched for myogenic cells by using a FacsAria instrument (BD Biosciences) to select cells based on positive staining with APC-conjugated anti-human CD56 (BD Biosciences). For FACS, cells were trypsinized, counted, and collected by centrifugation, after which  ̃1×106 cells were resuspended in 0.1 ml 10% fetal bovine serum (Hyclone) in PBS and incubated with the CD56 antibody according to manufacturer's instructions. As a control, cells were incubated with APC-conjugated mouse IgG1 K isotype antibody (BD Biosciences). Cells were incubated for 30-60 min on ice, collected by centrifugation, washed twice with 10% fetal bovine serum in PBS, and resuspended in 0.5-1.0 ml 10% fetal bovine serum in PBS and subjected to FACS to select CD56-positive cells.
The CD56-positive populations of myogenic cells that were obtained by FACS were seeded on dishes coated with 0.1% gelatin (Sigma) and incubated at 37° C. and 5% CO2, with each cell strain grown independently. Cells were propagated by daily feeding with HMP growth medium consisting of Ham's F10 medium (Cellgro) supplemented with 20% characterized FBS (Hyclone), 1% chicken embryo extract, 120 mM CaCl2, and 1% antibiotics/antimycotics (Cellgro). Cultures were incubated until cells reached 50-70% confluence, at which time cells were harvested after dissociation with TrypLE Express (Gibco), counted, and used for expansion or for frozen storage. For all experiments described here, cultures were examined at 20-35 population doublings after the initial isolation, which was at least 10-15 population doublings prior to loss of proliferative capacity.
FIG. 1 provides a table showing cell lines produced using these methods.
Primary Cell Cultures for RNA Isolation.
To initiate cultures, CD56-positive cells were seeded at  ̃4000 cells/cm2 and cultured with daily feeding with LHCN growth medium consisting of 4:1 DMEM:Medium 199 supplemented with 15% characterized FBS (Hyclone), 0.02M HEPES (Sigma-Aldrich), 0.03 μg/ml ZnSO4 (Sigma-Aldrich), 1.4 ug/ml Vitamin B12 (Sigma-Aldrich), 0.055 ug/ml dexamethasone (Sigma-Aldrich), 1% antibiotics/antimycotics (Cellgro), 2.5 ng/ml hepatocyte growth factor (Chemicon International) and 10 ng/ml basic fibroblast growth factor (Millipore). To induce differentiation, cells were propagated by daily feeding with LHCN growth medium until  ̃95% confluent, at which time cultures were switched to a low serum differentiation medium (DM) consisting of 4:1 DMEM:Medium 199 supplemented with 2% horse serum (Hyclone), 2 mM L-glutamine (Gibco), 1% antibiotics/antimycotics (Cellgro), 10 mM HEPES (Gibco), and 1 mM sodium pyruvate (Gibco). For RNA isolation for microarray analysis, cultures were harvested at two different stages of culture: (1) after two days of proliferation in growth medium, at which point cells were sub-confluent (GM); (2) after four days in differentiation medium (DM). Cells were harvested by rinsing culture dishes 2× with PBS and removing the cells with cell lifters (Costar), after which the cells were collected by centrifugation, snap frozen in liquid nitrogen, and stored at −80° C.
RNA Isolation and Microarray Analysis.
Total RNA was isolated from frozen cell pellets using 1 ml TRIzol reagent (Invitrogen). RNA concentration was quantified with UV absorption at 260 nm using NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific) and the RNA integrity was assessed using the RNA 6000 Nano chip on the Agilent 2100 Bioanalyzer (Agilent Technologies). Gene expression profiling was carried out using the Affymetrix GeneChip Human Gene 1.0 ST arrays. The current format of these arrays interrogates 28,869 annotated genes in the human genome with approximately twenty six 25-mer oligonucleotide probes spread across the full length of the transcript. Microarray data was collected at Expression Analysis, Inc. (Durham, N.C.). Biotin-labeled target for the microarray experiment was prepared using 100 ng of total RNA and cDNA was synthesized using the GeneChip WT (Whole Transcript) Sense Target Labeling and Control Reagents kit as described by the manufacturer (Affymetrix). The sense cDNA was then fragmented by UDG (uracil DNA glycosylase) and APE 1 (apurinic/apyrimidic endonuclease 1) and biotin-labeled with TdT (terminal deoxynucleotidyl transferase) using the GeneChip WT Terminal labeling kit (Affymetrix). Hybridization was performed using 5 micrograms of biotinylated target, which was incubated with the GeneChip Human Gene 1.0 ST array (Affymetrix) at 45° C. for 16-20 hours. Following hybridization, non-specifically bound material was removed by washing and detection of specifically bound target was performed using the GeneChip Hybridization, Wash and Stain kit, and the GeneChip Fluidics Station 450 (Affymetrix). The arrays were scanned using the GeneChip Scanner 3000 7G (Affymetrix) and raw data was extracted from the scanned images and analyzed with the Affymetrix GeneChip Command Console Software (Affymetrix).
Microarray Data Analysis.
The raw array data was preprocessed and normalized using the Robust Multichip Average (RMA) method. This procedure includes background correction and quantile normalization of the arrays at the probe level, followed by robust summarization of expression at the transcript level. Differential expression between classes was calculated using linear models with the limma package from the Bioconductor project (Smyth, G. K. (2004). Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology 3, No. 1, Article 3). The linear model used was “ ̃0+Class:Muscle:Medium+Cohort”, where Class, Muscle, and Medium are each two-level factors with levels FSHD & Control; Biceps & Deltoid; and GM & DM, respectively; and the factor Cohort has one level for each cohort. The interaction terms (denoted “:”) for the three two-level factors model changes between FSHD and Control expression levels that may vary for each of the four combinations of muscle type and medium, and the additive Cohort factor models different baseline expression levels for samples from different cohorts. To control for multiple hypothesis testing false discovery rates (FDRs) were computed based on the p-values from empirical Bayes moderated t-statistics for differential expression. The reported results are based on only those probesets annotated with Entrez gene IDs, and in cases of multiple probesets with the same Entrez ID on only the probeset with the largest interquartile range; after this filtering probesets corresponding to 19,983 genes were left. FSHD typically affects biceps more severely than deltoid, and differences between FSHD and control cell-cultures were stronger in DM than in GM.
Table 2 lists 142 genes for which the expression difference between FSHD and control biceps in DM had p-value at most 0.001, which corresponded to an FDR of 0.15. The columns labeled AvsU.DM.B.pval and AvsU.DM.B. gives the p-value and FDR, respectively, and the column labeled AvsU.DM.B.lfc gives the log 2(fold-change) between FSHD and control expression levels, with positive scores indicating higher expression in FSHD samples relative to controls, and negative scores indicating lower expression in FSHD samples relative to controls (Table 2).















TABLE 2











AvsU.
AvsU.DM.B.
AvsU.DM.


Probeset
SYMBOL
UNIGENE
ENTREZID
REFSEQ
GENENAME
DM.B.lfc
pval
B.fdr























7933733
FAM13C
Hs.499704
220965
NM_001001971.2
family with sequence
−0.85
5.30E−07
0.011







similarity 13, member C


8153065
MIR30B
NA
407030
NR_029666.1
microRNA 30b
−0.9
1.70E−06
0.017


8079753
DAG1
Hs.76111
1605
NM_001165928.3
dystroglycan 1
−0.59
1.20E−05
0.071







(dystrophin-associated







glycoprotein 1)


7910923
FMN2
Hs.24889
56776
NM_020066.4
formin 2
0.77
1.40E−05
0.071


7927876
TET1
Hs.567594
80312
NM_030625.2
tet oncogene 1
−0.65
1.80E−05
0.072


8075673
RBFOX2
Hs.282998
23543
NM_001031695.2
RNA binding protein,
−0.43
4.10E−05
0.1







fox-1 homolog (C. elegans) 2


7980891
TC2N
Hs.510262
123036
NM_001128595.1
tandem C2 domains,
0.72
5.20E−05
0.1







nuclear


8126770
CYP39A1
Hs.387367
51302
NM_016593.3
cytochrome P450,
−0.51
6.60E−05
0.1







family 39, subfamily







A, polypeptide 1


8054041
TRIM43
Hs.232026
129868
NM_138800.1
tripartite motif
2.59
7.30E−05
0.1







containing 43


8034099
MIR199A1
NA
406976
NR_029586.1
microRNA 199a-1
0.88
7.50E−05
0.1


8057578
CALCRL
Hs.470882
10203
NM_005795.5
calcitonin receptor-like
−1.17
8.40E−05
0.1


7898537
PAX7
Hs.113253
5081
NM_001135254.1
paired box 7
−0.75
8.40E−05
0.1


8084100
USP13
Hs.175322
8975
NM_003940.2
ubiquitin specific
−0.75
8.40E−05
0.1







peptidase 13







(isopeptidase T-3)


7994463
ATP2A1
Hs.657344
487
NM_004320.4
ATPase, Ca++
−1.82
8.70E−05
0.1







transporting, cardiac







muscle, fast twitch 1


7958174
TXNRD1
Hs.728817
7296
NM_001093771.2
thioredoxin reductase 1
0.49
8.80E−05
0.1


7982000
SNORD116-
NA
100033438
NR_003340.2
small nucleolar RNA,
−0.78
9.10E−05
0.1



26



C/D box 116-26


7973580
FITM1
Hs.128060
161247
NM_203402.2
fat storage-inducing
−0.72
9.90E−05
0.1







transmembrane protein 1


7928661
MBL1P
Hs.102310
8512
NR_002724.2
mannose-binding
−0.67
1.00E−04
0.1







lectin (protein A) 1,







pseudogene


8053984
ANKRD23
Hs.643430
200539
NM_144994.7
ankyrin repeat domain
−0.45
0.00011
0.1







23


7941761
RHOD
Hs.15114
29984
NM_014578.3
ras homolog gene
0.45
0.00011
0.1







family, member D


8072015
ADRBK2
Hs.657494
157
NM_005160.3
adrenergic, beta,
−0.78
0.00012
0.1







receptor kinase 2


8027674
ZNF302
Hs.436350
55900
NM_001012320.1
zinc finger protein 302
−0.36
0.00012
0.1


8120961
MRAP2
Hs.370055
112609
NM_138409.2
melanocortin 2
−0.93
0.00013
0.1







receptor accessory







protein 2


7960865
SLC2A3
Hs.419240
6515
NM_006931.2
solute carrier family 2
0.64
0.00013
0.1







(facilitated glucose







transporter), member 3


7947052
IGSF22
Hs.434152
283284
NM_173588.3
immunoglobulin
−0.58
0.00014
0.1







superfamily, member







22


8093665
GRK4
Hs.32959
2868
NM_001004056.1
G protein-coupled
−0.46
0.00014
0.1







receptor kinase 4


8162132
C9orf153
Hs.632073
389766
NM_001010907.1
chromosome 9 open
−0.52
0.00014
0.1







reading frame 153


8008664
AKAP1
Hs.463506
8165
NM_003488.3
A kinase (PRKA)
−0.64
0.00015
0.11







anchor protein 1


8101086
NAAA
Hs.437365
27163
NM_001042402.1
N-acylethanolamine
0.52
0.00016
0.11







acid amidase


7915261
TRIT1
Hs.356554
54802
NM_017646.4
tRNA
−0.48
0.00016
0.11







isopentenyltransferase 1


8058570
C2orf67
Hs.282260
151050
NM_152519.2
chromosome 2 open
−0.56
0.00018
0.11







reading frame 67


7912595
PRAMEF13
Hs.531192
400736
NM_001024661.1
PRAME family
1.52
0.00019
0.11







member 13


7978932
SOS2
Hs.291533
6655
NM_006939.2
son of sevenless
−0.27
0.00019
0.11







homolog 2







(Drosophila)


8023121
ST8SIA5
Hs.465025
29906
NM_013305.4
ST8 alpha-N-acetyl-
−0.61
0.00021
0.12







neuraminide alpha-2,8-







sialyltransferase 5


7934945
PANK1
Hs.376351
53354
NM_138316.3
pantothenate kinase 1
−0.66
0.00021
0.12


7979483
C14orf39
Hs.335754
317761
NM_174978.2
chromosome 14 open
−0.79
0.00022
0.12







reading frame 39


7923978
CD34
Hs.374990
947
NM_001025109.1
CD34 molecule
0.79
0.00023
0.12


7920552
KCNN3
Hs.490765
3782
NM_001204087.1
potassium
−0.88
0.00024
0.12







intermediate/small







conductance calcium-







activated channel,







subfamily N, member 3


8082003
EAF2
Hs.477325
55840
NM_018456.4
ELL associated factor 2
−0.69
0.00024
0.12


8024518
ZNF555
Hs.47712
148254
NM_001172775.1
zinc finger protein 555
−0.82
0.00026
0.12


8151074
PDE7A
Hs.527119
5150
NM_002603.3
phosphodiesterase 7A
−0.61
0.00026
0.12


8130071
C15orf29
Hs.633566
79768
NM_024713.2
chromosome 15 open
−0.76
0.00027
0.12







reading frame 29


8123584
MYLK4
Hs.127830
340156
NM_001012418.3
myosin light chain
−1.18
0.00028
0.12







kinase family, member 4


7906764
HSPA6
Hs.654614
3310
NM_002155.3
heat shock 70 kDa
0.49
0.00029
0.12







protein 6 (HSP70B′)


7897987
PRAMEF2
Hs.104991
65122
NM_023014.1
PRAME family
1.59
0.00029
0.12







member 2


7926679
KIAA1217
Hs.445885
56243
NM_001098500.1
KIAA1217
−0.58
0.00031
0.12


8163733
CDK5RAP2
Hs.269560
55755
NM_001011649.2
CDK5 regulatory
−0.4
0.00032
0.12







subunit associated







protein 2


8050443
SMC6
Hs.526728
79677
NM_001142286.1
structural maintenance
−0.5
0.00033
0.12







of chromosomes 6


7947110
E2F8
Hs.523526
79733
NM_024680.3
E2F transcription
−1.38
0.00033
0.12







factor 8


8073943
ZBED4
Hs.475208
9889
NM_014838.2
zinc finger, BED-type
−0.36
0.00034
0.12







containing 4


7958884
OAS1
Hs.524760
4938
NM_001032409.1
2′,5′-oligoadenylate
0.63
0.00035
0.12







synthetase 1,







40/46 kDa


8133477
GTF2IRD1
Hs.647056
9569
NM_001199207.1
GTF2I repeat domain
−0.51
0.00035
0.12







containing 1


7944955
PKNOX2
Hs.278564
63876
NM_022062.2
PBX/knotted 1
−0.62
0.00036
0.12







homeobox 2


8020068
ANKRD12
Hs.464585
23253
NM_001083625.2
ankyrin repeat domain
−0.43
0.00037
0.12







12


7983704
GLDN
Hs.526441
342035
NM_181789.2
gliomedin
−0.45
0.00037
0.12


8131803
IL6
Hs.654458
3569
NM_000600.3
interleukin 6
1.12
0.00037
0.12







(interferon, beta 2)


7909730
KCNK2
Hs.497745
3776
NM_001017424.2
potassium channel,
1.18
0.00037
0.12







subfamily K, member 2


7908397
RGS13
Hs.497220
6003
NM_002927.4
regulator of G-protein
−1.02
0.00037
0.12







signaling 13


8072170
KREMEN1
Hs.229335
83999
NM_001039570.2
kringle containing
−0.53
0.00037
0.12







transmembrane protein 1


8002020
TPPP3
Hs.534458
51673
NM_015964.2
tubulin
−0.61
0.00039
0.12







polymerization-







promoting protein







family member 3


7897978
PRAMEF1
Hs.454859
65121
NM_023013.2
PRAME family
1.33
0.00039
0.12







member 1


7909545
TRAF5
Hs.523930
7188
NM_001033910.2
TNF receptor-
−0.64
0.00039
0.12







associated factor 5


8094441
SLC34A2
Hs.479372
10568
NM_001177998.1
solute carrier family
2.23
4.00E−04
0.12







34 (sodium







phosphate), member 2


8137670
PDGFA
Hs.535898
5154
NM_002607.5
platelet-derived
−0.58
4.00E−04
0.12







growth factor alpha







polypeptide


8086482
ZNF445
Hs.250481
353274
NM_181489.5
zinc finger protein 445
−0.31
0.00041
0.12


7964646
PPM1H
Hs.435479
57460
NM_020700.1
protein phosphatase,
−0.42
0.00041
0.12







Mg2+/Mn2+







dependent, 1H


8027312
ZNF429
Hs.572567
353088
NM_001001415.2
zinc finger protein 429
−0.58
0.00042
0.12


7969815
CLYBL
Hs.655642
171425
NM_206808.2
citrate lyase beta like
−0.57
0.00043
0.12


8099302
MIR95
NA
407052
NR_029511.1
microRNA 95
−1
0.00045
0.12


7971653
DLEU2
Hs.547964
8847
NR_002612.1
deleted in lymphocytic
−0.53
0.00045
0.12







leukemia 2 (non-







protein coding)


8069991
TCP10L
Hs.728804
140290
NM_144659.5
t-complex 10 (mouse)-
−0.4
0.00047
0.12







like


7970111
ARHGEF7
Hs.508738
8874
NM_001113511.1
Rho guanine
−0.4
0.00047
0.12







nucleotide exchange







factor (GEF) 7


7995440
FLJ44674
Hs.514338
400535
XR_041153.1
FLJ44674 protein
0.35
5.00E−04
0.12


7898211
DDI2
Hs.718857
84301
NM_032341.4
DNA-damage
−0.48
5.00E−04
0.12







inducible 1 homolog 2







(S. cerevisiae)


8163109
C9orf4
Hs.347537
23732
NM_014334.2
chromosome 9 open
0.4
0.00052
0.12







reading frame 4


7918552
C1orf183
Hs.193406
55924
NM_019099.4
chromosome 1 open
−0.43
0.00052
0.12







reading frame 183


7960850
SLC2A14
Hs.655169
144195
NM_153449.2
solute carrier family 2
0.49
0.00053
0.12







(facilitated glucose







transporter), member







14


8050658
ATAD2B
Hs.467862
54454
NM_017552.2
ATPase family, AAA
−0.33
0.00053
0.12







domain containing 2B


8124502
ZNF184
Hs.158174
7738
NM_007149.2
zinc finger protein 184
−0.35
0.00053
0.12


8060813
MCM8
Hs.597484
84515
NM_032485.4
minichromosome
−0.39
0.00053
0.12







maintenance complex







component 8


8097086
MYOZ2
Hs.381047
51778
NM_016599.4
myozenin 2
−1.2
0.00054
0.12


8044008
IL1RL2
Hs.659863
8808
NM_003854.2
interleukin 1 receptor-
0.38
0.00054
0.12







like 2


8054664
ZC3H8
Hs.418416
84524
NM_032494.2
zinc finger CCCH-type
−0.4
0.00055
0.12







containing 8


8097256
FGF2
Hs.284244
2247
NM_002006.4
fibroblast growth
0.88
0.00056
0.12







factor 2 (basic)


8100312
LRRC66
Hs.661450
339977
NM_001024611.1
leucine rich repeat
−0.77
0.00056
0.12







containing 66


8102352
PITX2
Hs.643588
5308
NM_000325.5
paired-like
−0.53
0.00056
0.12







homeodomain 2


8015590
STAT5B
Hs.595276
6777
NM_012448.3
signal transducer and
−0.54
0.00056
0.12







activator of







transcription 5B


8069348
PCNT
Hs.474069
5116
NM_006031.5
pericentrin
−0.34
0.00057
0.12


8136235
CPA1
Hs.2879
1357
NM_001868.2
carboxypeptidase A1
−0.4
0.00058
0.12







(pancreatic)


7968883
C13orf31
Hs.210586
144811
NM_001128303.1
chromosome 13 open
0.94
0.00058
0.12







reading frame 31


7950955
TRIM49
Hs.534218
57093
NM_020358.2
tripartite motif
1.68
0.00058
0.12







containing 49


7957126
KCNMB4
Hs.525529
27345
NM_014505.5
potassium large
−0.82
0.00059
0.12







conductance calcium-







activated channel,







subfamily M, beta







member 4


8102862
MAML3
Hs.586165
55534
NM_018717.4
mastermind-like 3
−0.54
0.00059
0.12







(Drosophila)


7951781
C11orf71
Hs.715083
54494
NM_019021.3
chromosome 11 open
−0.37
6.00E−04
0.12







reading frame 71


7909768
SPATA17
Hs.171130
128153
NM_138796.2
spermatogenesis
−0.49
0.00061
0.12







associated 17


8094778
UCHL1
Hs.518731
7345
NM_004181.4
ubiquitin carboxyl-
0.77
0.00061
0.12







terminal esterase L1







(ubiquitin







thiolesterase)


8164580
PTGES
Hs.146688
9536
NM_004878.4
prostaglandin E
0.94
0.00061
0.12







synthase


8104163
LRRC14B
Hs.683662
389257
NM_001080478.1
leucine rich repeat
−0.57
0.00062
0.12







containing 14B


8036406
ZNF571
Hs.590944
51276
NM_016536.3
zinc finger protein 571
−0.41
0.00062
0.12


8128087
GABRR1
Hs.99927
2569
NM_002042.4
gamma-aminobutyric
−0.63
0.00063
0.12







acid (GABA) receptor,







rho 1


8028219
ZNF420
Hs.444992
147923
NM_144689.3
zinc finger protein 420
−0.39
0.00064
0.13


8057771
STAT4
Hs.80642
6775
NM_003151.3
signal transducer and
0.67
0.00067
0.13







activator of







transcription 4


8058350
ICA1L
Hs.516629
130026
NM_138468.4
islet cell autoantigen
−0.46
0.00068
0.13







1.69 kDa-like


8018922
CYTH1
Hs.191215
9267
NM_004762.2
cytohesin 1
−0.47
0.00068
0.13


7968637
CCNA1
Hs.417050
8900
NM_001111045.1
cyclin A1
2.12
0.00073
0.14


7974771
C14orf135
Hs.509499
64430
NM_022495.5
chromosome 14 open
−0.37
0.00076
0.14







reading frame 135


7907790
CEP350
Hs.413045
9857
NM_014810.4
centrosomal protein
−0.44
0.00076
0.14







350 kDa


8030823
IGLON5
Hs.546636
402665
NM_001101372.1
IgLON family member 5
0.45
0.00077
0.14


7917996
LRRC39
Hs.44277
127495
NM_144620.3
leucine rich repeat
−1.23
0.00078
0.14







containing 39


7905986
FDPS
Hs.335918
2224
NM_001135821.1
farnesyl diphosphate
0.59
0.00079
0.14







synthase


8097867
KIAA0922
Hs.205572
23240
NM_001131007.1
KIAA0922
−0.4
0.00079
0.14


8120300
C6orf142
Hs.449276
90523
NM_138569.2
chromosome 6 open
−1.71
0.00079
0.14







reading frame 142


8068220
C21orf49
Hs.54725
54067
NR_024622.1
chromosome 21 open
−0.43
8.00E−04
0.14







reading frame 49


8037298
CD177
Hs.232165
57126
NM_020406.2
CD177 molecule
0.83
8.00E−04
0.14


8148501
PTP4A3
Hs.43666
11156
NM_007079.2
protein tyrosine
−0.93
0.00081
0.14







phosphatase type IVA,







member 3


7954012
LOH12CR1
Hs.720779
118426
NM_058169.3
loss of heterozygosity,
−0.43
0.00084
0.14







12, chromosomal







region 1


8108708
PCDHB7
Hs.203830
56129
NM_018940.2
protocadherin beta 7
−0.37
0.00085
0.14


8116595
WRNIP1
Hs.236828
56897
NM_020135.2
Werner helicase
−0.31
0.00085
0.14







interacting protein 1


7934434
MYOZ1
Hs.238756
58529
NM_021245.3
myozenin 1
−1.55
0.00085
0.14


8024909
KDM4B
Hs.654816
23030
NM_015015.2
lysine (K)-specific
−0.26
0.00086
0.14







demethylase 4B


8144812
PCM1
Hs.491148
5108
NM_006197.3
pericentriolar material 1
−0.33
0.00086
0.14


7933092
ZNF248
Hs.528423
57209
NM_021045.2
zinc finger protein 248
−0.5
0.00086
0.14


7928705
TSPAN14
Hs.310453
81619
NM_001128309.1
tetraspanin 14
−0.62
0.00086
0.14


8151457
HEY1
Hs.234434
23462
NM_001040708.1
hairy/enhancer-of-split
−0.58
0.00087
0.14







related with YRPW







motif 1


7934442
SYNPO2L
Hs.645273
79933
NM_001114133.1
synaptopodin 2-like
−0.78
0.00088
0.14


8033241
CD70
Hs.501497
970
NM_001252.3
CD70 molecule
0.32
0.00088
0.14


7921955
RXRG
Hs.26550
6258
NM_006917.4
retinoid X receptor,
−0.58
0.00089
0.14







gamma


8167603
CLCN5
Hs.166486
1184
NM_000084.3
chloride channel 5
−0.57
9.00E−04
0.14


8089647
KIAA2018
Hs.632570
205717
NM_001009899.2
KIAA2018
−0.4
0.00091
0.14


8139160
FAM183B
Hs.144075
340286
NR_028347.1
acyloxyacyl hydrolase
0.35
0.00091
0.14







(neutrophil)


7957379
MYF5
Hs.178023
4617
NM_005593.2
myogenic factor 5
−0.91
0.00094
0.14


8144082
C7orf13
Hs.647014
129790
NR_026865.1
chromosome 7 open
−0.39
0.00095
0.14







reading frame 13


7986004
ZNF774
Hs.55307
342132
NM_001004309.2
zinc finger protein 774
−0.28
0.00095
0.14


8045198
CFC1B
Hs.503733
653275
NM_001079530.1
cripto, FRL-1, cryptic
0.26
0.00096
0.14







family 1B


8125289
TNXA
Hs.708061
7146
NR_001284.2
tenascin XA
1.87
0.00097
0.14







pseudogene


7915277
MYCL1
Hs.437922
4610
NM_001033081.2
v-myc
−0.77
0.00098
0.14







myelocytomatosis







viral oncogene







homolog 1, lung







carcinoma derived







(avian)


8002303
NQO1
Hs.406515
1728
NM_000903.2
NAD(P)H
0.67
0.001
0.15







dehydrogenase,







quinone 1


8033362
INSR
Hs.465744
3643
NM_000208.2
insulin receptor
−0.59
0.001
0.15


8025672
SLC44A2
Hs.534560
57153
NM_001145056.1
solute carrier family
−0.35
0.001
0.15







44, member 2


7965510
TMCC3
Hs.370410
57458
NM_020698.2
transmembrane and
−0.42
0.001
0.15







coiled-coil domain







family 3


8118644
RPS18
Hs.627414
6222
NM_022551.2
ribosomal protein S18
0.34
0.001
0.15


7940824
NAA40
Hs.523753
79829
NM_024771.2
N(alpha)-
−0.33
0.001
0.15







acetyltransferase 40,







NatD catalytic subunit,







homolog (S. cerevisiae)









Example 2
A Humanized Mouse Model of FSHD
Both FSHD- and control-derived myoblasts from multiple cohorts (described in Homma et al., European Journal of Human Genetics (2012) 20, 404-410) engrafted and formed human muscle fibers after 30 days in vivo. All mouse experiments were performed using BBRI IACUC-approved protocols. Nonobese diabetic Rag1 and IL2rγ null (NOD-Rag1 null IL2r null or RAG, Jax stock number 007799) mice were used as recipients for human cell transplantations. Adult muscle, composed of multinucleated terminally differentiated myofibers, has a very low rate of cellular turnover under normal conditions. However, it has a remarkable capacity to regenerate in response to injury due to the presence of quiescent satellite cells. A regenerating muscle, which is in the process of incorporating newly differentiating cells, provides a favourable environment to receive a cell graft. Recipient tibialis anterior (TA) muscles were injected with 10 μM cardiotoxin to induce a muscle degeneration/regeneration cycle. 1×106 FSHD myoblasts (from five different family cohorts), maintained in culture between 15 and 20 population doublings, were injected into surgically-exposed TA muscles 6 hours after cardiotoxin injection; following surgery, mice were monitored for recovery from anaesthesia and provided analgesics as required. Mice were sacrificed 4 weeks after transplantation and injected TA muscles, as well as non-injected gastrocnemius muscles were dissected and frozen in nitrogen-cooled isopentane. Entire muscle samples were cut into 10 μm transverse cryostat sections and analyzed by immunofluorescence.
Visualization of engrafted fibers was performed via immunofluorescence using antibodies against the human specific sarcolemmal protein spectrin and the human specific nuclear protein lamin A/C. As shown in FIG. 1, immunofluorescence using human specific antibodies demonstrated high engraftment efficiency. To date, 36 xenografted mice have been generated and investigated. Histological analyses have confirmed that injected human FSHD myoblasts participate in the regeneration of murine muscle to form “humanized” fibers within the host TA. Quantifications have revealed that engraftment rates of greater than 100,000 human nuclei can be achieved in host muscle. These engraftments are of a sufficient magnitude to conduct morphological and molecular phenotype analyses of xenografted muscles. It is hypothesized that prior irradiation of host mice enriches engraftment of human myoblasts.
Example 3
DUX4-fl Expressing FSHD Cells Engraft
Five cell strains (described in Homma et al., European Journal of Human Genetics (2012) 20, 404-410) were used for engraftment studies. Recent breakthroughs in the field suggest that DUX4, a gene identified inside D4Z4 repeats, is inappropriately expressed in the muscles of patients with FSHD. The disease could arise though a toxic gain of function. The precise molecular and cellular pathological mechanism involving DUX4 remains to be uncovered. Recent studies described the detection of two DUX4 transcripts, a long form (or full-length, fl) and a short form, and while the role of the short form is still unclear, the long form was specifically detected in FSHD samples, suggesting a central role in the pathogenic mechanism.
Based on engraftability and expression of DUX4-fl, cell strain selection for engraftment was refined to consist of three strains derived from the biceps of patients affected by FSHD, and three cell strains from corresponding unaffected first degree relatives. DUX4-fl transcript and protein were detected in cultured, differentiated myotubes for each of the three FSHD cell strains, and was absent in each control. Two control cell strains possessed at least one permissive allele for the disease (4qA), but repressed DUX4 transcription. The third control strain did not contain the permissive allele (i.e. was genotyped as 4qB/4qB), and was therefore an ideal negative control for these studies.
Current theory predicts that DUX4 is actively transcribed in an average of 1 out of a 1,000 FSHD-derived nuclei at a given time. Recent engraftment trials have established that over 100,000 human myonuclei can be integrated with murine muscle. Adapting current theory to the invention's xenograft model, DUX4 might be expressed in greater than 100 nuclei in sizeable xenografts. This represents an amount of DUX4 mRNA detectable using 55 cycles of nested PCR; therefore, DUX4 expression at these levels should be detectable in xenografts from FSHD-derived myoblasts. Currently the expression of DUX4 at the mRNA and/or protein level is being assessed in FSHD- and control-transplanted TAs.
Example 4
Xenograft Integration with the Murine Skeletal Muscle Environment
Innervation of Human Fibers
Injecting cultured human myoblasts into murine skeletal muscle imposes a drastic environmental change. The ability of human myoblasts to assimilate successfully with host muscle is one important feature of a disease model. Immunohistological assays have confirmed that injected myoblasts successfully adapted to the murine microenvironment and integrated with the host muscle. Innervation of engrafted fibers by the nervous system of the host is important to prevent atrophy. Immunohistology studies using antibodies against neurofilament and Synaptic Vesicle protein 2 (SV2) were used to visualize afferent murine neurons in transverse sections. SV2 immunofluorescence at the pre-synaptic cleft was coupled with bungarotoxin-rhodamine staining at corresponding post-synaptic acetylcholine receptors to demonstrate an active neuromuscular junction (FIG. 2). Neuromuscular junction dispersion was observed throughout the muscle in specific patterns, directly innervating fibers in their vicinity without appearing to discriminate between mouse and human. Neuromuscular junctions on human and mouse fibers had no noticeable morphological differences. It is likely that resulting human fibers are successfully integrating with the murine musculature and nervous system.
Example 5
Xenograft Integration with Murine Skeletal Muscle Environment: Satellite Cell Pool Replenishment
The ability of injected cells to contribute to long-term muscle regeneration is dependent upon their inclusion into the satellite cell pool of host muscle. Satellite cells are muscle progenitor cells located beneath the basal lamina of myofibers. They are activated in response to damage, causing them to proliferate and fuse to form new myofibers during the repair process. Satellite cells can be identified by the expression of the transcription factor PAX7 and their anatomical location beneath the basal lamina. Using antibodies against these distinct features coupled with human LaminA/C, human nuclei that express PAX7 were identified. This indicates that these cells have assumed a satellite cell identity (FIG. 3).
Example 6
Development of a Tracking Strategy to Follow the Transplant Over Time
In vivo imaging provides a powerful tool to track the growth and survival of implanted muscle cells over time. Lentiviral particles are highly efficient at infection and stable integration of a gene of interest into a cell system. Lentiviral particles expressing a firefly luciferase (Luc) reporter gene provide a simple, long-term cell tracking system. Live small animal in vivo imaging techniques can then be performed to follow the destiny of transplanted Luc+ cells over time. These techniques have been used successfully to track the evolution of muscle cell transplantations. Accordingly, a commercial lentiviral vector carrying a luciferase reporter gene under the control of a CMV promoter (SABiosciences, FIG. 4) was used to develop stable Luc+ FSHD and control myoblast cell lines.
To develop cell lines that could be tracked in vivo following engraftment, FSHD and their matching control cells were seeded on day 0 and lentivirus infection was performed on day 2 according to Manufacturer's directions. Cells were transduced using a 4-hour infection with a Multiplicity of Infection (MOI) of 50. Cells were further amplified and maintained in culture under proliferative conditions where they showed normal signs of proliferation and differentiation. In vitro luciferase assays demonstrated luciferase activity, confirming development of cell models that can be used to track the destiny of the engrafted cells in vivo using bioluminescence imaging techniques.
In short, these results demonstrate the successful engraftment of FSHD cells into murine muscle with high efficiency as well as the development of a method to track the implanted cells in vivo. Live whole animal imaging experiments will be carried out to investigate how engrafted FSHD cells survive and regenerate compared to controls, and to identify biomarkers specific to FSHD.
Luciferase-expressing FSHD cells are engrafted into injured TA muscles, and their growth and differentiation assayed over time in vivo using Bioluminescence Imaging (BLI). Cell number is assessed as the bioluminescence signal derived from constitutive luciferase activity, and the linearity, sensitivity, and reproducibility of the bioluminescence assay for quantifying cell numbers will be first validated both in vitro and in vivo.
For BLI studies, cell-transplanted animals are anesthetized prior to receiving an intraperitoneal injection of luciferin (15 mg/ml at a dose of 130 mg/kg body weight recommended) and assayed in an imaging chamber with a Xenogen device. Images are acquired continuously for 30 minutes, and the same mice are imaged repeatedly over time once a month for up to 6 months. It has been shown that the dynamics of muscle cell behavior during muscle repair can be followed using this imaging technique. In vivo BLI of same mice imaged repeatedly over time has established the ability of transplanted satellite cells to respond to serial injury with successive waves of progenitor expansion and regeneration of muscle fibers. The magnitude of the regeneration response to sequential cardiotoxin injection, as monitored by imaging luciferase activity, reflects the persistence and renewal of stem cells over time. The relative regenerative responses of FSHD versus control muscles over time will test whether satellite cell regenerative capacity is impaired as an FSHD disease mechanism.
Live in vivo imaging technologies provide a unique technology to evaluate the role of satellite cell regenerative potential and muscle fiber survival in FSHD disease progression. In addition to engraftment studies of affected FSHD subjects, gene expression and regeneration and survival are evaluated in xenografts of myogenic cells from non-manifesting FSHD subjects (i.e. individuals with shortened D4Z4 arrays but no detectable signs of muscle weakness). While cell culture studies have suggested that these non-manifesting cells behave similarly to cells from subjects with clinically diagnosed FSHD (e.g. expression of DUX4-fl), it is possible that their in vivo characteristics will show reduced pathology, providing opportunities to investigate modifiers of disease progression.
Example 7
RNaseH1-Active Antisense Oligonucleotides (ASOs)
As indicated in Tables 2 and 4, certain markers are increased in subjects with FSHD relative to the levels of those markers in first degree unaffected subjects. Therapeutic effects are achieved by reducing the levels or biological activity of markers whose expression is upregulated in FSHD. In particular the invention provides targeted for degradation using RNaseH1-activating antisense oligonucleotides (ASO's) (“MOE gapmers”). The RNAseH1 ASO chemistry provides for a 20 nucleotide phosphorothioate backbone (5-10-5 gapmer). In particular, the oligonucleotide comprises five nucleotides at each end with the 2′-O-(2-methoxyethyl) (MOE) modification and ten central deoxyribonucleotides for activation of RNase H1.
For screening purposes, cell cultures of the invention are contacted with ASOs and the cells assayed for an amelioration of FSHD phenotype. In particular, the cells are assayed for an increase in the biological function of the cell or for an increase in the levels of one or more markers down-regulated in FSHD. In another embodiment, ASOs are administered to a chimeric mouse comprising a human FSHD cell. The chimeric mouse is then assayed for an increase in the biological activity of a human FSHD cell or an increase in the level of expression of a marker down-regulated in FSHD. In one embodiment, 25 mg/kg of the ASOs are administered by sub-cutaneous injection at least about 2× per week for 4 weeks or more.
In particular embodiments, the effects of ASOs on cells or chimeric mice of the invention are assayed using live cell imaging, muscle fiber turnover, or biomarker expression. In one embodiment, nude mice are treated to eliminate or reduce the number of muscle stem cells and/or differentiated muscle cell fibers and muscle stem cell replacement of muscle fiber turnover is assayed.
Example 8
Validation with qPCR
Of the 142 genes identified as candidate biomarkers in the microarray study described above, 18 genes (9 of which were up-regulated in FSHD vs. control myotubes and 9 which were down-regulated in FSHD vs. control myotubes) have now been evaluated on a larger collection of samples using quantitative real-time PCR (qPCR). The samples are derived from four of the five families from the microarray study and four additional families. Clinical information for the samples is given in Table 3. The qPCR experiments were performed using the BioMark 96.96 Dynamic Array (Fluidigm) platform with TaqMan Gene Expression Assays (Applied Biosystems).







TABLE 3







Samples used in qPCR study.


















Deltoid
Biceps



Familial


EcoRI/
strength
strength


Subject
relations
Gender
Age
BlnI (kb)
(R, L)
(R, L)





01A
proband
M
42
>40, 18 
4+, 5 
4+, 3−


01U
brother of
M
46
>40, >40
5, 5
5, 5



01A


03A
proband
F
40
>40, 20 
5, 5
4+, 4+


03U
sister of
F
42
157, 80 
5, 5
5, 5



03A


05A
proband
F
55
67, 25
5, 5
5, 5


05C
brother of
M
49
67, 25
5, 5
5, 5



05A


05V
son of 05A
M
18
67
5, 5
5, 5


09A
proband
F
31
>112, 25 
5, 5
4+, 4+


09U
mother of
F
57
>112, 47 
5, 5
5, 5



09A


12A
daughter of
F
22
63, 18
4+, 4+
4+, 4+



12B


12U
daughter of
F
24
>112, >112
5, 5
5, 5



12B


15A
proband
M
66
>112, 28 
5, 5
4+, 4+


15V
sister of
F
60
>145, 107 
5, 5
5, 5



15A


16A
proband
F
56
97, 20
5−, 5−
4−, 4+


16U
sister of
F
60
97, 93, 56
5, 5
5, 5



16A


21B
daughter of
F
59
26, 40
5, 5
4+, 4+



21A


21U
daughter of
F
48
142, 63 
5, 5
5, 5



21A









The 18 genes assessed with qPCR are listed in Table 4 below, along with their log(base 2) fold-change (LFC) between FSHD and control myotubes and the associated statistical significance (P-value) of this difference using qPCR. Table 2 also includes columns for the LFC and P-value from the original microarray study for comparison.








TABLE 4





Genes tested with qPCR.
















Log (base 2) fold-change (LFC) for FSHD vs. control myotubes and the associated p-values are shown for qPCR and also for the original microarray study. Negative values indicate that the gene is downregulated in FSHD.


Asterisks (*) in qPCR columns indicate that the transcript was not detected in at least one sample. In these cases the LFC may be inaccurate and a non-parametric sign test rather than a t-test was used for computing the p-value.






Cycle threshold (Ct) values for each gene in each sample were computed as the median Ct value of three technical qPCR replicates, and were then normalized by additive scaling of all Cts for each sample so that the average Ct of three reference genes M6PR, HPRT1, and PPIA was identical across samples (and equal to the un-normalized mean of these three genes across all samples). Transcripts of four genes (PRAMEF1, TRIM43, SLC34A2, TRIM49, highlighted in Table 4) were not detected with qPCR in one or more of the samples. In these cases the normalized Ct value was set to 40, which represents 2^0.67=1.6-fold lower transcript abundance than the highest observed Ct of 39.33. The LFC estimates may be inaccurate for these genes, and these estimates are flagged with asterisks in the LFC column. Also, because this treatment of non-detected transcripts may violate the assumption of normality in t-tests, non-parametric sign tests were used on the paired (by family) differences between FSHD and control myotubes for these cases, indicated by asterisks in the p-value column. Multiple FSHD samples in a single family were replaced by their median value. In this test non-detected transcripts are considered to have lower expression than detected transcripts, but results do not otherwise depend on the precise Ct value assigned the non-detected transcripts. For genes that were detected in all the samples, p-values are bases on t-tests of the contrast FSHD vs. control from linear models with additive fixed effects for FSHD status and for family. This generalized a usual paired t-test by accommodating families with more than one FSHD subject.

All 9 genes that were up-regulated in FSHD in the microarray study were also upregulated in the qPCR study (positive LFC in both cases), and all 9 genes that were downregulated in FSHD in the microarray study were also down-regulated in the qPCR study (positive LFC in both cases). This overall concordance is directionally of change is significantly better than random (p=3.8e-6 by binomial test), and 6 of the genes individually showed significant differences between FSHD and control myotubes in the qPCR study at the p<0.01 level. Note that in the microarray analysis, to moderate the effect of outliers when ranking the more-than 20,000 genes, a statistical model with a pooled estimate of variance across the myoblasts and myotubes derived from biceps and deltoid biopsies was used, which further shrunk estimates of variance across different genes towards a common mean (by use of empirical Bayes moderated t-statistics). In the present qPCR analysis self-contained statistical tests were performed on myotubes derived from biceps, with no reference to myotube or deltoid samples, and sharing of information across genes. These factors may explain why more of the genes did not attain p<0.01 in the qPCR study.
Note that for each of the six genes with p<0.01 in the qPCR study (PRAMEF1, TRIM43, SLC34A2, TRIM49, TC2N, and DAG1) the FSHD vs. control paired differences showed the same direction for all of the cohorts: For the first five of these genes, each FSHD sample had a lower Ct value (higher expression) than its paired control sample, and for DAG1 each FSHD sample had a higher Ct value (lower expression) than its paired control sample. A stronger result held for PRAME1, TRIM43 and SLC34A2: for these three genes each FSHD sample had a lower Ct value (higher expression) than all of the control samples, not just the sample from the paired first-degree relative. This property is appealing for a biomarker since scores can then be assigned to individuals without the requirement of first-degree relatives as controls. However, the margin between the highest Ct values of FSHD samples and lowest Ct value of control samples was fairly small for these genes (0.56 Ct for TRIM43, 1.06 Cts for SLC34A2, 1.68 Cts for PRAMEF1).
It was then tested whether the difference of Ct values between two genes would provide discrimination between the FSHD and control samples with a larger margin, and thus more likely to generalize to other samples. The use of a simple difference rather than a more complex combination involving more genes makes the test simpler, and also removes the reliance on the choice of “housekeeping” gene(s), as these terms would cancel out so the difference is self-normalizing. The precise cutoffs for biomarkers would still depend on qPCR primers and efficiency of qPCR reactions, however, so should be recalibrated if these change.
Because the genes in the qPCR were selected on the basis of differential expression in the microarray study, assessing discriminants using the samples present in the microarrays will be biased. Moreover, searching over all pairs of genes introduces multiple hypotheses and the potential for overfitting. To address these issues, the pair of genes to use, and the cutoff on their difference to use as a discriminant, were selected based only on the qPCR data for the eight samples present in the microarray, so that the qPCR data for the nine samples not present in the microarray study could serve as an independent validation set. By examining all pairs of the 18 genes with qPCR data, the difference (Ct for PRAMEF1)−(Ct for PAX7) provided the maximum margin between FSHD and control samples, of 4.49 Cts. (Non-detected transcripts were assigned Ct of 40 during this maximization, and in application of the discriminant rule.)
The midpoint of the gap between FSHD and control samples for this difference was 7.05, yielding the discriminant rule of: classify as FSHD if (Ct for PRAMEF1)−(Ct for PAX7)<7.05, and classify as control otherwise. This rule correctly classified all nine samples (five FSHD and four control) that were not represented in the microarray experiment (and hence played no role in selecting the genes PRAMEF1 or PAX7, or the cutoff of 7.05). This is significantly better than random guessing (p=0.002 by binomial test). The margin between FSHD and control samples was slightly reduced when these additional nine samples were included, but was still 3.32 Ct, roughly twice the best margin (1.68) for Cts of any single gene when normalized by the reference genes M6PR, HPRT1, and PPIA.
Note the there are other pairwise differences that give larger margins that 1.68, and in the above we have focused just on the single maximal example chosen using a subset of the samples to avoid multiple-hypothesis testing on the validation samples. Other pairs with large margin are typically differences between one gene up-regulated in FSHD vs. controls and one gene down regulated in FSHD vs. control.
Example 9
Using FSHD Biomarkers to Identify and Evaluate the Efficacy of Antisense Oligonucleotide-Morpholino Drugs Using FSHD Myogenic Cells and Xenograft Muscles
Antisense oligonucleotides conjugated to morpholinos are developed as inhibitors of the expression of FSHD disease genes, using cultured FSHD myogenic cells (prepared as described above in Example 1 and in Homma et al.) and FSHD xenograft muscle derived by engraftment and differentiation of FSHD myogenic cells into regenerating mouse muscles as described above. Antisense oligonucleotide mopholinos are designed that have nucleotide sequences designed to disrupt translation initiation, polyadenylation, and/or RNA splicing to knockdown expression of targeted FSHD disease mRNAs and block production of their encoded disease proteins. Specific antisense oligonucleotide drugs will first be tested by introduction into FSHD myogenic cells by electroporation or transfection with EndoPorter (Gene Tools). Drug-treated FSHD and control cells are monitored for evidence of cytotoxicity and changes in cell morphology, myofiber differentiation, and the expression of muscle protein biomarkers (desmin, MyoD, myogenin, MyHC). The efficacy of selected antisense oligonucleotides to block expression of targeted FSHD disease gene RNAs and proteins is evaluated by qPCR and immunoblotting assays. The efficacy of the antisense oligonucleotides as candidate FSHD drugs is evaluated by quantitative assays of the expression of FSHD disease biomarkers using qPCR, as established above. Promising candidate antisense FSHD drugs are identified by their activities to restore expression of FSHD biomarkers to levels produced by control cells derived from unaffected individuals.
Promising candidates are then tested in FSHD xenograft muscles by localized muscle injection and electroporation or systemic injection of antisense oligonucleotides, followed by qPCR assays of the expression of FSHD biomarkers and evaluation of hepatotoxic and immunostimulatory side effects over the time course of treatment. Antisense drugs with promising therapeutic value are identified by their activities to restore expression of FSHD biomarkers in both FSHD cells and xenograft muscles to levels observed in control myogenic cells and xenograft muscles derived from unaffected individuals.
Other Embodiments
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

## Claims

1. A chimeric mouse comprising human cells derived from an FSHD affected subject or a first degree relative thereof, wherein the human cells replace 25-50% or more of the cells present in a skeletal muscle of the mouse.

2. The mouse of claim 1, wherein the human cells replace cells present in the tibialis anterior.

3. A set of chimeric mice comprising one mouse comprising human cells derived from an FSHD affected subject, wherein the human cells replace 25-50% or more of the cells present in a skeletal muscle of the mouse, and at least one mouse comprising human cells derived from a first degree relative of the FSHD affected subject, wherein the human cells replace 25-50% or more of the cells present in a skeletal muscle of the mouse.

4. A method of identifying an agent that ameliorates FSHD in a subject in need thereof, the method comprising administering the agent to the chimeric mouse of claim 1, and comparing the level of expression of a nucleic acid molecule of Table 2 or 4 in a human cell of the mouse relative to the level in an untreated control cell, wherein an agent that normalizes expression in said cell is identified as ameliorating FSHD.

5. The mouse of claim 1, wherein the human cells are enriched for myogenic cells.

6. The mouse of claim 1, wherein the human cells are isolated by selecting for cells positive for human CD 56.

7. The mouse of claim 1, wherein the human cells are skeletal muscle cells, muscle stem cells, or differentiated muscle fiber.

8. The mouse of claim 1, wherein the human cells are obtained from skeletal muscle biopsies.

9. The mouse of claim 8, wherein the biopsy is of a bicep or deltoid muscle.

10. The method of claim 4, wherein the nucleic acid molecule is selected from the group consisting of PRAMEF1, TRIM43, SLC34A2, TRIM49, TC2N, CD34, NAAA, HSPA6, and CD177.

11. A chimeric mouse created by a method comprising injecting a skeletal muscle of a mouse with at least or about 1×106 human FSHD myoblasts derived from an FSHD affected subject or a first degree relative thereof.

12. The chimeric mouse of claim 11, wherein the myoblasts are derived from skeletal muscle.

13. The chimeric mouse of claim 11, wherein the myoblasts are derived from a bicep or deltoid muscle.

14. A chimeric mouse comprising an engraftment in a skeletal muscle of at least or about 100,000 human nuclei derived from an FSHD affected subject or a first degree relative thereof.

15. The chimeric mouse of claim 14, wherein the nuclei are derived from skeletal muscle.

16. The chimeric mouse of claim 14, wherein the nuclei are derived from a bicep or deltoid muscle.