公开(公告)号：US06262035B1

公开(公告)日：2001-07-17

申请号：US09164664

申请日：1998-10-01

申请人： Kevin P. Campbell ,  Kathleen H. Holt ,  Franck Duclos ,  Leland E. Lim ,  Volker Straub ,  Beverly Davidson ,  Roger Williamson

发明人： Kevin P. Campbell ,  Kathleen H. Holt ,  Franck Duclos ,  Leland E. Lim ,  Volker Straub ,  Beverly Davidson ,  Roger Williamson

IPC分类号： A01N4304

CPC分类号： C12N15/8509 ,  A01K67/0276 ,  A01K2207/15 ,  A01K2217/00 ,  A01K2217/075 ,  A01K2227/105 ,  A01K2267/0306 ,  A61K38/1709 ,  A61K48/00

摘要： Disclosed is a method for treating a patient suffering from the disease sarcoglycan-deficient limb-girdle muscular dystrophy by gene replacement therapy. Sarcoglycan gene replacement therapy produces extensive long-term expression of the 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 aspect, 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 aspect, the invention relates to a mouse, and cells derived therefrom, 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 reduced &agr;-dystroglycan in the sarcolemma of skeletal and cardiac muscles, and a reduction of dystrophin in skeletal muscle, when compared to tissue of a mouse lacking a disrupted &agr;-sarcoglycan gene. In another aspect, 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 the determination of therapeutic effects being a reduction or reversal in disease progression, or a restoration of the dystroglycan complex.

摘要翻译： 本发明公开了一种通过基因替代疗法治疗患有疾病肌糖肌苷缺乏症的肢体肌营养不良症的患者的方法。 Sarcoglycan基因替代疗法产生广泛的长期表达的sarcoglycan物种，其恢复整个slegoglycan复合物，导致αphorpha-dystroglycan与肌膜的稳定关联，并消除肢体肌营养不良的形态学标志物。 另一方面，本发明涉及一种用于确定患者组织中的特定缺陷型糖团聚物物种的方法。 该方法包括从患者获得的肌肉细胞的培养，以及将编码每种肌糖原聚糖物质，α，β，γ和δ的表达载体独立引入培养的细胞中，随后测定肌营养不良蛋白 - 糖蛋白复合物的恢复 。 另一方面，本发明涉及一种小鼠及其衍生的细胞，用于破坏的α-丝裂霉素基因是纯合的。 该破坏阻止了小鼠细胞中功能性α-sarcoglycan的合成，并导致突变小鼠在骨骼肌和心脏肌肉的肌膜中不具有可检测的sarcospan，β - ，γ-，δ-色氨酸聚糖和还原的α-二糖聚糖， 和与缺乏α-sarcoglycan基因的小鼠的组织相比，骨骼肌中营养不良蛋白的减少。 另一方面，本发明涉及筛选用于治疗肌糖原缺乏型肢体肌营养不良症的治疗剂的方法。 所述方法包括将候选治疗剂施用于小鼠或由其衍生的细胞，以及测定对小鼠或细胞的治疗效果，其中治疗效果的确定是疾病进展中的减少或逆转，或者肌萎缩糖复合物的恢复 。

公开(公告)号：US06211340B1

公开(公告)日：2001-04-03

申请号：US09119827

申请日：1998-07-21

申请人： Kevin P. Campbell ,  Daniel Jung ,  Franck Duclos ,  Volker Straub ,  John McPherson

发明人： Kevin P. Campbell ,  Daniel Jung ,  Franck Duclos ,  Volker Straub ,  John McPherson

IPC分类号： C07K1618

CPC分类号： C07K14/4707

摘要： Disclosed herein is a substantially pure nucleic acid sequence encoding a mammalian 35 kDa non-dystrophin component (&dgr;-sarcoglycan) of the dystrophin-glycoprotein complex. Also disclosed are the amino acid sequence and an immunogenic peptide of &dgr;-sarcoglycan. The peptide when used to immunize a mammal, stimulates the production of antibodies which bind specifically to the &dgr;-sarcoglycan. Methods to identify mutations in the &dgr;-sarcoglycan gene associated with autosomal recessive limb-girdle muscular dystrophy are also disclosed. The identification of such mutations enables the design of nucleic acid probes which hybridize specifically to a mutant form of &dgr;-sarcoglycan, or the complement thereof, but not to the DNA of the wild-type form of the gene (or the complement thereof), under stringent hybridization conditions. Such probes are useful, for example, in connection with the diagnosis of autosomal recessive limb-girdle muscular dystrophy. In addition, the identification of such mutations enables the diagnosis of autosomal recessive limb-girdle muscular dystrophy through the use of direct DNA sequencing techniques.

摘要翻译： 本文公开了编码肌营养不良蛋白 - 糖蛋白复合物的哺乳动物35kDa非肌营养不良蛋白成分（δ-莽草酸）的基本上纯的核酸序列。 还公开了δ-色氨酸的氨基酸序列和免疫原性肽。 当肽用于免疫哺乳动物时，刺激产生与δ-糖聚糖特异性结合的抗体。 还公开了鉴定与常染色体隐性遗传性肢体肌营养不良症相关的δ-色氨酸聚糖基因突变的方法。 这种突变的鉴定使得能够设计特异性地与δ-色氨酸聚糖或其互补体的突变形式杂交的核酸探针，而不与野生型形式的基因（或其互补物）的DNA杂交， 在严格的杂交条件下。 这样的探针可用于例如与常染色体隐性性腰带肌营养不良症的诊断有关。 此外，通过使用直接DNA测序技术，鉴定这种突变能够诊断常染色体隐性的腰带肌营养不良症。

公开(公告)号：US5837537A

公开(公告)日：1998-11-17

申请号：US719758

申请日：1996-09-25

申请人： Kevin P. Campbell ,  Daniel Jung ,  Franck Duclos ,  Volker Straub ,  John McPherson

发明人： Kevin P. Campbell ,  Daniel Jung ,  Franck Duclos ,  Volker Straub ,  John McPherson

IPC分类号： C07K14/47 ,  C12N15/12 ,  C12N15/63 ,  C12N15/85

CPC分类号： C07K14/4707

摘要： Disclosed herein is a substantially pure nucleic acid sequence encoding a mammalian 35 kDa non-dystrophin component (.delta.-sarcoglycan) of the dystrophin-glycoprotein complex. Also disclosed are the amino acid sequence and an immunogenic peptide of .delta.-sarcoglycan. The peptide when used to immunize a mammal, stimulates the production of antibodies which bind specifically to the .delta.-sarcoglycan. Methods to identify mutations in the .delta.-sarcoglycan gene associated with autosomal recessive limb-girdle muscular dystrophy are also disclosed. The identification of such mutations enables the design of nucleic acid probes which hybridize specifically to a mutant form of .delta.-sarcoglycan, or the complement thereof, but not to the DNA of the wild-type form of the gene (or the complement thereof), under stringent hybridization conditions. Such probes are useful, for example, in connection with the diagnosis of autosomal recessive limb-girdle muscular dystrophy. In addition, the identification of such mutations enables the diagnosis of autosomal recessive limb-girdle muscular dystrophy through the use of direct DNA sequencing techniques.

摘要翻译： 本文公开了编码肌营养不良蛋白 - 糖蛋白复合物的哺乳动物35kDa非肌营养不良蛋白成分（δ-聚糖）的基本上纯的核酸序列。 还公开了δ-聚糖的氨基酸序列和免疫原性肽。 当肽用于免疫哺乳动物时，刺激产生与δ-聚糖结合特异性的抗体。 还公开了鉴定与常染色体隐性遗传性肢体肌营养不良症相关的δ-聚糖基因突变的方法。 这种突变的鉴定使得能够设计与δ-聚糖或其互补体的突变形式特异性杂交的核酸探针，而不与野生型形式的基因（或其补体）的DNA杂交， 在严格的杂交条件下。 这样的探针可用于例如与常染色体隐性性腰带肌营养不良症的诊断有关。 此外，通过使用直接DNA测序技术，鉴定这种突变能够诊断常染色体隐性的腰带肌营养不良症。

公开(公告)号：US6136546A

公开(公告)日：2000-10-24

申请号：US57740

申请日：1998-04-09

申请人： Kevin P. Campbell ,  Valerie Allamand ,  Yoshihide Sunada ,  Volker Straub ,  Mustafa Salih

发明人： Kevin P. Campbell ,  Valerie Allamand ,  Yoshihide Sunada ,  Volker Straub ,  Mustafa Salih

IPC分类号： G01N33/68 ,  G01N33/53 ,  G01N1/30 ,  G01N33/567

CPC分类号： G01N33/6887 ,  G01N2800/2878

摘要： Disclosed are compositions and methods for aiding in the diagnosis of congenital muscular dystrophy associated with in-frame deletion in the laminin-2 .alpha.2 polypeptide chain in an individual. In a preferred diagnostic method embodiment, an experimental muscle tissue sample is provided from the individual and treated if necessary to render components available for antibody binding. The components of the sample are then separated on the basis of molecular weight. The separated protein components are then transferred to a solid support while maintaining the relative positions established in separation step. The transferred components are then stained with an affinity reagent which is known to bind to a C-terminal domain of the laminin-2 .alpha.2 polypeptide chain. Individual afflicted with congenital muscular dystrophy associated with in-frame deletion in the laminin-2 .alpha.2 polypeptide chain on the basis of positive staining in combination with reduced molecular weight of the laminin-2 .alpha.2 polypeptide chain relative to the wild-type laminin-2 .alpha.2 polypeptide chain. A preferred composition is a nucleic acid probe for the detection of merosin deletion-type congenital muscular dystrophy. The preferred nucleic acid probe is characterized by the ability to bind specifically to a mutant merosin nucleic acid sequence, the mutant merosin nucleic acid sequence comprising a T to C substitution at position 3973 +2 of the consensus donor splice site of exon 25.

摘要翻译： 公开了用于帮助诊断个体中层粘连蛋白-2α2多肽链中与框内缺失相关的先天性肌营养不良的组合物和方法。 在优选的诊断方法实施方案中，从个体提供实验肌肉组织样品，并且如果需要进行处理以使组分可用于抗体结合。 然后基于分子量分离样品的组分。 然后将分离的蛋白质组分转移到固体支持物中，同时保持在分离步骤中建立的相对位置。 转移的组分然后用已知结合层粘连蛋白-2α2多肽链的C末端结构域的亲和试剂染色。 基于阳性染色结合层粘连蛋白-2α2多肽链相对于野生型层粘连蛋白的层粘连蛋白-2α2多肽链的分子量降低而与层粘连蛋白-2α2多肽链中的框内缺失相关的先天性肌营养不良患者， 2α2多肽链。 优选的组合物是用于检测梅洛辛缺失型先天性肌营养不良症的核酸探针。 优选的核酸探针的特征在于特异性结合突变型子糖蛋白核酸序列的能力，所述突变蛋清核酸序列包含外显子25的共有供体剪接位点的第3973±2位的T至C取代。