Muscular DystrophyNovember 3, 2008Duchenne muscular dystrophyCommon (1/3500 boys)X-linked inheritanceFemale carriers may exhibit somesymptomsProgressive skeletal muscle wastingbeginning at around age 3Characteristic changes in posture;lordosis, pseudohypertrophy of calves“Gower’s maneuver” (climbing up froma sitting position)Usually confined to a wheelchair by age10-12Progressive difficulty breathing due towasting of diaphragm muscleDeath usually before age 30PathologyDefinitive diagnosis by muscle biopsyMuscle fibers variable in sizeNecrotic fibers with significant inflammationHigh serum levels of creatine kinase (alsooften true in asymptomatic female carriers)normal7 yo DMD9 yo DMDPathologyEvidence of regenerating musclefibers; satellite cells proliferate toform new myoblasts, fusion into newmuscle fibers with central nucleinormalnecrotic necrotic regenerating regeneratingDystrophinEnormous gene/protein: >3500 amino acids, ~170 nm long16 hours required for transcription; 79 exons spread over 2.6 MbpMost mutations in large spectrin-like coiled coil domainTissue-specific promoters andalternatively spliced forms:Full-length - muscleDP260 - retinalDP140 - brainDP116 - Schwann cellsDP71 - generalDystrophin distribution in normaland dystrophic muscleNote female carriers often have some affected fibers due to mosaic X inactivation;elevated serum creatine kinase, sometimes muscle weaknessMuscular dystrophies and relateddegenerative diseasesDuchenne muscular dystrophyAll skeletal muscles are affected, usually with cardiac involvementCuased by null or severe hypomorphic mutations in the gene encodingdystrophin, often frame-shift or nonsense mutations or large deletionsAbout 2/3 of cases inherited from carrier mother, 1/3 new mutationsBecker muscular dystrophyLess severe, later onset, about 1/20,000 boysUsually in-frame deletions or point mutations in dystrophinEmery-Dreifuss muscular dystrophyWasting of shoulders and upper arms first, often with cardiomyopathyTwo genetically distinct forms, both nuclear proteins (lamin A/C, emerin)Limb girdle muscular dystrophiesPelvic and shoulder girdles affected firstGenetically heterogeneous group of diseases, several sarcoglycansCongenital muscular dystrophyGenetically heterogeneous group, symptoms present at birth~50% due to deficiency in laminin !2About 40 neuromuscular diseases tracked by mdausa.org includegenetic and autoimmune causesMolecules deficient in variousmuscular dystrophiesIdentified by positional cloning in affected familiesMechanical attachment of skeletal muscle contractile apparatus toextracellular matrixPossible roles in cell signaling via NOSTreatment modalitiesOptions for degenerative diseases are usually poorSupportive therapy includes vitamins, corticosteroids(prednisone), breathing exercisesApproaches in development:Conventional gene therapy with various vectorsMinigenesAlternative gene therapiesAntisense suppressionUtrophin upregulation*Exon skippingTherapies to increase muscle massMyostatin blockadeDeacetylase inhibitionCell-based therapiesMyoblast transplant*Stem cell transplants - bone marrow, ES cells, satellite cells, others?Animal models for DMDmdx mouse (nonsense mutation in exon 23)Pathology does not mimic human diseaseFiber degeneration, compensation by regenerationMyopathy grows less severe later in lifeNormal lifespanCXMD golden retriever dogsSpontaneous mutation, pathologyfirst described in 1988Exon skipping, frame shift due tosplice site mutationSevere muscle degenerationConventional gene therapy for DMDDelivery of full-length gene with adenovirus, herpes simplex virus,or plasmid vectorsHigh transduction levels are required; work OK in regeneratingmouse muscle but persistence is poorInflammatory response is a particular problem since degeneratingmuscle is already inflamedViral vectors not yet used in human patientsMinigenes mimic Becker, can be carried by simpler and moreefficient vectors. Alleviate degeneration in mdx mice.Utrophin as a substitute?Ubiquitously expressed dystrophin paralogFound in mature skeletal muscle fibers at neuromuscular junctions;associates with acetycholine receptorsExpression upregulated in mdx mice and protein relocates to adystrophin-like pattern; double knock-out mice have pathologymore similar to human DMDAlternative genetic approach: exon skippingRelated to mechanism of“reversion” in DMD**Antisense oligonucleotidesblock splice site recognitionTemporary effect in mdx miceand cultured humanmyotubesAartsma-Rus et al., 2003, Hum. Mol. Genet., 12:907A different approach: Target “boostergenes” that affect disease progressionMyostatin as a candidate to increasemuscle mass and regeneration in DMDNote regeneration appears to compensate early in disease progressionCandidate for regulation: MYOSTATINTGF-" family member, cloned by degenerate PCRExpressed exclusively in skeletal muscle at all stages of developmentMcPherron et al., 1997, Nature 387: 83Myostatin knockout supermiceMcPherron et al., 1997, Nature 387: 8330% larger than littermates“Abnormal body shape withpronounced shoulders and hips”Skeletal muscle mass increases 2-3XHyperplasia and hypertrophy (notedifferent dominant negative allelescan separate these two effects)Also mysotatin defects in double-muscled cattleMcPherron and Lee, 1997PNAS 94: 12457And in humans!Myostatin regulates satellite cellproliferation and differentiationSecreted by mature muscle fibersIn satellite cells, represses cyclin-dependent kinase Cdk2 andupregulates expression of CKI p21Muscle fiber regenerationby satellite cell activationMyostatin KO improves musclemass and grip strength in mdx miceBlue - mdxRed - mdx, Mstn-/-Yellow - wild typeGreen - Mstn -/-Wagner et al., 2002, Annal. Neurol. 52:832Blockade with antibody has asimilar beneficial effectDeacetylase inhibitors: indirectregulation of myostatinVarious general deacetylase inhibitors upregulate expressionof the myostatin inhibitor follistatinMinetti et al., 2006, Nature Medicine, 12: 1147TSA = trichostatin ACell-based therapies (transplantation)Myoblasts can be readily cultured from donor biopsysamplesHuman trials; repeated injections into biceps muscle over6 months gave some fibers (1-10%) expressing donor-derived dystrophin but no clinical improvementMendell et al., 1995, NEJM 333: 832Stem cells?Satellite cells are rare and difficult to isolate, possibly heterogeneousBone marrow stem cells????Bone marrow contains multipotent mesenchymal progenitor cells as well ashematopoeitic