Gene replacement therapy for muscular dystrophy

Details Gene replacement therapy for muscular dystrophy Gene replacement therapy for muscular dystrophy

1998-10-01

2001-07-17

Clark, Deborah J. R. (Department: 1633)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C435S320100, C435S455000

Reexamination Certificate

active

06262035

ABSTRACT:

BACKGROUND OF THE INVENTION
The term muscular dystrophy describes a group of diseases characterized by hereditary progressive muscle weakness and degeneration. Several muscular dystrophies are caused by mutations in genes that encode sarcolemmal proteins, including certain types of limb-girdle muscular dystrophy (LGMD). LGMD is genetically and clinically heterogeneous; it may be inherited in an autosomal dominant or recessive manner, and may have different rates of progression and severity. A unifying theme among the LGMDs is the initial involvement of the shoulder and pelvic girdle muscles, with relative sparing of most other muscle groups (Jackson et al.,
Pediatrics
41, 495-501 (1968); Bushby, K. M.,
Neuromusc Disord
5, 71-74 (1995)).
The pace of discovery in the field of muscular dystrophy research has been rapid since the discovery of the Duchenne and Becker muscular dystrophy (DMD) gene in 1986 (Monaco et al.,
Nature
323, 646-650 (1986)). The DMD gene encodes dystrophin, a large cytoskeletal protein that together with other molecular components makes up the dystrophin-glycoprotein complex (DGC). The dystrophin-glycoprotein complex (DGC) is a large oligomeric complex of sarcolemmal proteins and glycoproteins in skeletal and cardiac muscle (Campbell, K. P.
Cell
80, 675-679 (1995); Ozawa et al.,
Hum. Mol. Genet.
4, 1711-1716 (1995)). This complex consists of dystrophin, a large cytoskeletal protein which binds F-actin; &agr;- and &bgr;-dystroglycan, which bind laminin and the cysteine-rich region of dystrophin, respectively; &agr;-, &bgr;-, &ggr;-, and &dgr;-sarcoglycan (&dgr;-SG), which form a distinct subcomplex; and sarcospan, a 25 kDa protein predicted to span the membrane four times (Crosbie et al.,
J. Biol. Chem.
272, 31221-31224 (1997). The DGC spans the sarcolemma and is believed to play an essential role in maintaining the normal architecture of the muscle sarcolemma by constituting a link between the subsarcolemmal cytoskeleton and the extracellular matrix. This structural linkage is thought to protect muscle fibers from the mechanical stress of contraction.
Mutations in different components of the DGC lead to similar dystrophic features, suggesting that the function of the DGC as a whole is dependent on intact molecular interactions between its individual subunits. The loss of one component destroys the link, and leads to muscle fiber degeneration. Several components of the DGC have been implicated in several human muscular dystrophies (Straub et al.,
Curr. Opin. Neurol.
10, 168-175 (1997)). Mutations in dystrophin cause Duchenne and Becker muscular dystrophy (DMD) (Hoffman et al.,
Cell
51, 919-928 (1987)). Two forms of congenital muscular dystrophy are caused by mutations in the extracellular matrix protein laminin 2 (Helbling-Leclerc et al.,
Nature Genet.
11, 216-218 (1995); Allamand et al.,
Hum. Mol. Genet.
6, 747-752 (1997)). Mutations in each of &agr;-, &bgr;-, &ggr;-, and &dgr;-SG cause autosomal recessive LGMD types 2D, 2E, 2C, and 2F, respectively (Roberds et al.,
Cell
78, 625-633 (1994); Piccolo et al.,
Nature Genet.
10, 243-245 (1995); Lim et al.,
Nature Genet.
11, 257-265 (1995); Bonneman et al.,
Nature Genet.
11, 266-273 (1995); Noguchi et al.,
Science
270, 819-822 (1995); Passos-Bueno et al.,
Hum. Mol. Genet.
5, 815-820 (1996); Nigro et al.,
Hum. Mol. Genet.
5, 1179-1186 (1996); Nigro et al.,
Nature Genet.
14, 195-198 (1996)).
In the sarcoglycan-deficient LGMDs, a mutation effecting the function of a particular sarcoglycan species leads to the loss or dramatic reduction of all four sarcoglycan proteins from the sarcolemma. Four types of LGMD, LGMD2D, LGMD2E, LGMD2C and LGMD2F, are known to be caused by mutations in distinct sarcoglycan genes, &agr;-, &bgr;-, &ggr;- and &dgr;-SG respectively (Roberds et al.,
Cell
78, 625-633 (1994); Piccolo et al.,
Nature Genet.
10, 243-245 (1995); Lim et al.,
Nature Genet.
11, 257-265 (1995); Bönneman et al.,
Nature Genet.
11, 266-273 (1995); Noguchi et al.,
Science
270, 819-822 (1995); Passos-Bueno et al.,
Hum. Mol. Genet.
5, 815-820 (1996); Nigro et al.,
Hum. Mol. Genet.
5, 1179-1186 (1996); Nigro et al.,
Nature Genet.
14, 195-198 (1996)). Unfortunately, functional characterization of the sarcoglycan complex has proven elusive.
The BIO 14.6 hamster is a widely studied animal model of muscular dystrophy and cardiomyopathy (Homburger et al., 1962). In this animal, the sarcoglycan complex is missing and dystrophic features, including central nucleation and necrosis of muscle fibers, are evident. Previous studies have documented a disruption in the integrity of the DGC in skeletal and cardiac muscle of this animal, with a dramatic decrease in the amount of &agr;-dystroglycan (Roberds et al.,
J Biol Chem.
268, 11496-11499 (1993), Iwata et al.,
FEBS
329, 227-231 (1993)). Recently, it was shown that a mutation in the &dgr;-SG gene of BIO 14.6 hamsters leads to sarcoglycan complex disruption and dystrophic changes (Okazaki et al.,
Nature Genet.
13, 87-90 (1996); Nigro et al.,
Hum. Mol. Genet.
6, 601-607 (1997); Sakamoto et al.,
Proc. Natl. Acad. Sci.
94, 13873-13878 (1997)). These findings make the animal a useful model for the study of human sarcoglycan-deficient LGMD.
While the BIO 14.6 hamster model is useful for certain studies, the generation of additional models of sarcoglycan-deficient LGMD would greatly facilitate progress towards understanding of the disease. The use of animal models deficient in the various sarcoglycan species to study the effects of these specific deficiencies, would help identify the exact role of each species in the DGC. Additionally, since there are no known cases of a LGMD from a primary sarcoglycan defect, with concomitant muscular dystrophy and cardiomyopathy, the generation of a phenotypically more accurate model of AR-LGMD would be beneficial to the research effort.
SUMMARY OF THE INVENTION
In one aspect the present invention relates to a method for treating a patient with the disease sarcoglycan-deficient limb-girdle muscular dystrophy by gene replacement therapy. Sarcoglycan gene replacement therapy produces extensive long-term expression of the deficient sarcoglycan species which restores the entire sarcoglycan complex, results in the stable association of alph&agr;-dystroglycan with the sarcolemma, and eliminates the morphological markers of limb-girdle muscular dystrophy.
In another embodiment, the invention relates to a method for determining a specific defective sarcoglycan species in the tissue of a patient. The method involves culture of muscle cells obtained from the patient, and the independent introduction of expression vectors encoding each of the sarcoglycan species, &agr;, &bgr;, &ggr;, and &dgr;, into the cultured cells, with subsequent assaying for restoration of the dystrophin-glycoprotein complex.
In another embodiment, the invention relates to a mouse, and cells derived therefrom, that is homozygous for a disrupted &agr;-sarcoglycan gene. The disruption prevents the synthesis of functional &agr;-sarcoglycan in cells of the mouse and results in the mutant mouse having no detectable sarcospan, &bgr;-, &ggr;-, &dgr;-sarcoglycan, and having reduced &agr;-dystroglycan in the sarcolemma of skeletal and cardiac muscles, as well as a reduction of dystrophin in skeletal muscle, when compared to tissue of a mouse lacking a disrupted &agr;-sarcoglycan gene.
In another embodiment, the invention relates to methods for screening for therapeutic agents useful in the treatment of sarcoglycan-deficient limb-girdle muscular dystrophy. The methods involve administering a candidate therapeutic agent to a mouse, or cells derived therefrom, and assaying for therapeutic effects on the mouse or cells, with therapeutic effects being a reduction or reversal in disease progression, or a restoration of the dystroglycan complex.


REFERENCES:
patent: 95 05738 (1995-03-01), None
patent: 99 43360 (1999-09-01), None
Eck et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, New York, p. 77-1

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