Louis Kunkel, PhD
Associate Director; Professor of Pediatrics
Harvard Medical School/Children's Hospital
Department of Genetics
MRRC Project(s)
P01 NS40828-01
(Pending)
Gene Expression in Normal and Diseased Muscle during Development - Program
Director and PI, Project 1: Gene Expression & Biochemical Studies
of Filamin/Sarcoglycan-related Dystrophies
The Kunkel laboratory has had a longstanding interest in understanding
the molecular and biochemical basis of common neuromuscular diseases and
on the design of rational therapies for these diseases. The major focus
has been on the muscular dystrophies, which are a heterogeneous group
of genetic disorders. The most common form of muscular dystrophy is caused
by abnormalities in the dystrophin protein, identified more than 10 years
ago in this laboratory. Dystrophin has been shown to be part of a larger
complex of muscle cell proteins, and many of these latter proteins are
themselves altered to cause autosomal recessive and autosomal dominant
forms of muscular dystrophy. The major aim of the laboratory is to understand
the mechanism by which alteration in many different proteins results in
a common mode of pathogenesis and then use this mechanistic understanding
to design rational therapies.
Research Description
Highlights of Major Accomplishments
Major Results
1. Dystrophin-associated proteins and the pathogenesis of muscular dystrophies
2. Cellular/gene therapy for muscular dystrophy
1. Dystrophin-associated proteins and the pathogenesis of muscular dystrophies
Dystrophin is part of a much larger complex of proteins which biochemically co-purify with dystrophin in membrane preparations from skeletal and cardiac muscle. Dystrophin was shown to be a monomer in this complex, contrary to previous predictions based on sequence homologies with other known proteins. The binding sites for two associated proteins, dystrobrevin and syntrophin, were mapped on the C-terminal end of the dystrophin monomer. A sub-complex of proteins called the sarcoglycans was in part cloned and characterized during this period. Each of these sarcoglycans has been shown to be mutated in autosomal recessive limb-girdle muscular dystrophies (LGMD). The organization of the sarcoglycan complex was shown to be composed of a core of b and d sarcoglycan, with g and a-sarcoglycan more loosely associated with this core. These results were corroborated by analysis of the sarcoglycans in LGMD patients. When mutations were detected in b and d-sarcoglycan, it was found that the whole sarcoglycan complex could not be detected at the muscle cell membrane. In contrast, when a and g-sarcoglycan were mutated, the sarcoglycans were found to be more variably localized to the membrane, although the protein bearing the primary mutation was nearly always absent. Interestingly, dystrophin localization at the membrane tends to be preserved in these sarcoglycanopathies, thus preserving the physical linkage between the intracellular actin cytoskeleton and the extracellular matrix. The sarcoglycans have a structure that is reminiscent of a receptor complex. In fact, the extracellular domains of three of the sarcoglycans share homology to the extracellular domains of other proteins with EGF-like repeats. With the yeast two-hybrid system a novel filamin (FLN) was identified as an intracellular sarcoglycan-interacting protein. Based on the function of FLN-1 in non-muscle cells it is predicted that the novel FLN-2 protein is involved in cell signals that re-arrange the actin cytoskeleton in response to stress to the muscle cell membrane.
2. Cellular/gene therapy for muscular dystrophy
Parallel to our work on the pathogenesis of muscular dystrophy have been efforts to design rational approaches to therapy. One approach to therapy has been myoblast transplantation to introduce a normal dystrophin gene to diseased muscle. Although this approach showed early promise in animal studies, initial human trials were somewhat disappointing. Although shown to be safe, few muscle fibers were shown to produce dystrophin, despite the fact that many donor cells were still resident in the recipient muscle as long as 6 months after they were introduced. Moreover, this approach requires local intramuscular injections, since systemic delivery of cells through myoblast transplantation does not appear to occur. Similarly, the delivery of dystrophin to muscle by in vivo gene transfer with viral vectors has resulted in only local restoration of dystrophin. In a disorder with diffuse, severe involvement of muscle, as in muscular dystrophy, focal or multifocal gene delivery is not likely to be clinically useful.
With this background, in collaboration with Dr. Richard Mulligan, a new member of this MRRC and a leader in the field of gene therapy, we have achieved systemic delivery of dystrophin-expressing stem cells with widespread expression in muscle of the mdx mouse, an animal model of Duchenne’s muscular dystrophy. Thus, first we showed that normal hematopoietic stem cells injected intravenously into irradiated mdx animals results in the reconstitution of the hematopoietic compartment of the transplanted recipients, the incorporation of donor-derived nuclei into muscle, and the partial restoration of dystrophin in the affected muscle. Then, using methods analogous for isolating hematopoietic stem cells, a novel method for preparing muscle stem cells was developed and used to prepare muscle stem cells. When injected intravenously these cells were found to have the capability of reconstituting the bone marrow and targeting skeletal muscle from the circulation. These cells also have the ability to systemically deliver dystrophin to muscle and thus have the potential to treat many forms of muscle disease. More recent experiments have shown that these cells will target dystrophic muscle from the circulation in the absence of lethal irradiation.
Publications
Bonnemann CG, Passos-Bueno MR, McNally EM, Vainzof M, de Sa Moreira E, Marie SK, Pavanello RCM, Noguchi S, Ozawa E, Zatz M, Kunkel LM. Genomic screening for b-sarcoglycan gene mutations: Missense mutations may cause severe limb-girdle muscular dystrophy type 2E (LGMD 2E). Hum Molec Genet 1996; 5:1953-1961.
McNally EM, Duggan D, Gorospe JR, Bonnemann CG, Fanin M, Pegoraro E, Lidov HG, Noguchi S, Ozawa E, Finkel RS, Cruse RP, Angelini C, Kunkel LM, Hoffman EP. Mutations that disrupt the carboxyl-terminus of gamma-sarcoglycan cause muscular dystrophy. Hum Molec Genet 1996; 5:1841-7.
Gussoni E, Wang Y, Fraefel C, Miller RG, Blau HM, Geller AI, Kunkel LM. A method to codetect introduced genes and their products in gene therapy protocols. Nature Biotechnol 1996; 14:1012-6.
Vainzof M, Passos-Bueno MR, Canovas M, Moreira ES, Pavanello RC, Marie SK, Anderson LV, Bonnemann CG, McNally EM, Nigro V, Kunkel LM, Zatz M. The sarcoglycan complex in the six autosomal recessive limb-girdle muscular dystrophies. Hum Molec Genet 1996; 5:1963-9.
McNally EM, Passos-Bueno MR, Bonnemann CG, Vainzof M, de Sa Moreira E, Lidov HG, Othmane KB, Denton PH, Vance JM, Zatz M, Kunkel LM. Mild and severe muscular dystrophy caused by a single gamma-sarcoglycan mutation. Am J Hum Genet 1996; 59:1040-7.
Sadoulet-Puccio HM, Kunkel LM. Dystrophin and its isoforms. Brain Pathol 1996; 6:25-35.
Sadoulet-Puccio HM, Khurana TS, Cohen JB, Kunkel LM. Cloning and characterization of the human homologue of a dystrophin related phosphoprotein found at the Torpedo electric organ post-synaptic membrane. Hum Molec Genet 1996; 5:489-96.
McNally EM, Bonnemann CG, Kunkel LM, Bhattacharya SK. Deficiency of adhalin in a patient with muscular dystrophy and cardiomyopathy. N Eng J Med 1996; 334:1610-1.
Ahn AH, Freener CA, Gussoni E, Yoshida M, Ozawa E, Kunkel LM. The three human syntrophin genes are expressed in diverse tissues, have distinct chromosomal locations, and each bind to dystrophin and its relatives. J Biol Chem 1996; 271:2724-30.
Scharf JM, Damron D, Frisella A, Bruno S, Beggs AH, Kunkel LM, Dietrich WF. The mouse region syntenic for human spinal muscular atrophy lies within the Lgn1 critical interval and contains multiple copies of Naip exon 5. Genomics 1996; 38:405-417.
Khurana TS, Specht LA, Beggs AH, Tome FM, Letureq F, Chevallay M, Chafey P, Kunkel LM. The concomitant use of dystrophin and utrophin/dystrophin related protein antibodies to reduce misdiagnosis of Duchenne/Becker muscular dystrophy. Biochem Biophys Res Comm 1997; 241:232-5.
Bonnemann CG, McNally EM, Kunkel LM. Beyond dystrophin: current progress in the muscular dystrophies. Curr Opin Pediatr 1996; 8:569-582.
Peters MF, O'Brien KF, Sadoulet-Puccio HM, Kunkel LM, Adams ME, Froehner SC. Beta-dystrobrevin, a new member of the dystrophin family. Identification, cloning, and protein associations. J Biol Chem 1997; 272:31561-9.
Sadoulet-Puccio HM, Rajala M, Kunkel LM. Dystrobrevin and dystrophin: an interaction through coiled-coil motifs. Proc Natl Acad Sci USA 1997; 94:12413-8.
Lidov HG, Kunkel LM. Dp140: alternatively spliced isoforms in brain and kidney. Genomics 1997; 45:132-9.
Cox GF, Kunkel LM. Dystrophies and heart disease. Curr Opin Cardiol 1997; 12:329-43.
Gussoni E, Blau HM, Kunkel LM. The fate of individual myoblasts after transplantation into muscles of DMD patients. Nature Medicine 1997; 3:970-7.
Chan Y, Kunkel LM. In vitro expressed dystrophin fragments do not associate with each other. FEBS Lett 1997; 410:153-9.
Carter TA, Bonnemann CG, Wang CH, Obici S, Parano E, De Fatima Bonaldo M, Ross BM, Penchaszadeh GK, Mackenzie A, Soares MB, Kunkel LM, Gilliam TC. A multicopy transcription-repair gene, BTF2p44, maps to the SMA region and demonstrates SMA associated deletions. Hum Molec Genet 1997; 6:229-36.
Holm IA, Huang X, Kunkel LM. Mutational analysis of the PEX gene in patients with X-linked hypophosphatemic rickets. Am J Hum Genet 1997; 60:790-7.
Selig S, Lidov HG, Bruno SA, Segal MM, Kunkel LM. Molecular characterization of Br-cadherin, a developmentally regulated, brain-specific cadherin. Proc Natl Acad Sci USA 1997; 94:2398-2403.
Sadoulet-Puccio HM, Feener CA, Schaid DJ, Thibodeau SN, Michels VV, Kunkel LM. The genomic organization of human dystrobrevin. Neurogenetics 1997; 1:37-42.
McNally EM, Ly CT, Kunkel LM. Human e-sarcoglycan is highly related to a-sarcoglycan (adhalin), the limb girdle muscular dystrophy 2D gene. FEBS Lett 1998; 422:27-32.
Chan Y-M, Tong H-Q, Beggs A, Kunkel LM. Human skeletal muscle-specific a-actinin-2 and -3 isoforms form homodimers and heterodimers in vitro and in vivo. Biochem Biophys Res Comm 1998; 248:134-139.
Bonnemann CG, Wong J, Ben Hamida C, Ben Hamida M, Hentati F, Kunkel LM. LGMD 2E in Tunisia is caused by a homozygous missense mutation in b-sarcoglycan exon 3. Neuromusc Dis 1998; 8:193-197.
Chan Y-M, Bonnemann CG, Lidov HGW, Kunkel LM. Molecular organization of sarcoglycan complex in mouse myotubes in culture. J Cell Biol 1998; 143:2033-2044.
Peters MF, Sadoulet-Puccio HM, Grady MR, Kramarcy NR, Kunkel LM, Sanes JR, Sealock R, Froehner SC. Differential membrane localization and intermolecular associations of alpha-dystrobrevin isoforms in skeletal muscle. J Cell Biol 1998; 142:1269-78.
Lidov HG, Kunkel LM. Dystrophin and Dp140 in the adult rodent kidney. Laboratory Invest 1998; 78:1543-51.
Scharf JM, Endrizzi MG, Wetter A, Huang S, Thompson TG, Zerres K, Dietrich WF, Wirth B, Kunkel LM. Identification of a candidate modifying gene for spinal muscular atrophy by comparative genomics. Nature Genetics 1998; 20:83-6.
McNally EM, de Sa Moreira E, Duggan DJ, Bonnemann CG, Lisanti MP, Lidov HGW, Vainzof M, Passos-Bueno MR, Hoffman EP, Zatz M, Kunkel LM. Caveolin-3 in muscular dystrophy. Hum Molec Genet 1998; 7:871-7.
Kunkel LM. Caveolin-3 deficiency as a cause of limb-girdle muscular dystrophy. J Child Neurol 1999; 14:33-34.
Gussoni E, Soneoka Y, Strickland CD, Buzney EA, Khan MK, Flint AF, Kunkel LM, Mulligan RC. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 1999; 401:390-4.
von Deimling F, Scharf
JM, Liehr T, Rothe M, Kelter AR, Albers P, Dietrich WF, Kunkel LM, Wernert
N, Wirth B. Human and mouse RAD17 genes: identification, localization,
genomic structure and histological expression pattern in normal testis
and seminoma. Human Genetics 1999; 105:17-27.
Lacy SE, Bonnemann CG, Buzney EA, Kunkel LM. Identification of FLRT1,
FLRT2, and FLRT3: a novel family of transmembrane leucine-rich repeat
proteins. Genomics 1999: 62:417-426.
Endrizzi M, Huang S, Scharf J, Kelter AR, Wirth B, Kunkel LM, Miller W, Dietrich WF. Comparative sequence analysis of the mouse and human Lgn1/SMA interval. Genomics 1999; 60:137-151.
Thompson TG, Chan YM, Hack AA, Brosius M, Rajala M, Lidov HGW, McNally EM, Watkins S, Kunkel LM. Filamin 2 (FLN2): A muscle-specific sarcoglycan interacting protein. J Cell Biol 2000; 148:115-126.
Bonnemann CG, Cox GF, Shapiro F, Wu J-J, Feener CA, Thompson TG, Anthony DC, Eyre DR, Darras BT, Kunkel LM. A mutation in the a3 chain of type IX collagen causes autosomal dominant multiple epiphyseal dysplasia with mild myopathy. PNAS 2000, 97:1212-1217.
Thompson TG, Kunkel LM. Advances in muscular dystrophy: Exciting new prospects for the millennium. Neuroscience 2000, in press.
See Dr. Kunkel's publications via PubMed
Contact Information
E-mail:
Louis Kunkel, PhD
Associate Director; Professor of Pediatrics
Harvard Medical School/Children's Hospital
Department of Genetics
