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AAV-mediated gene transfer to models of muscular dystrophy: Insights into assembly of multi-subunit membrane proteins.

机译：AAV介导的基因转移到肌肉营养不良模型：深入了解多亚基膜蛋白的组装。

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摘要

The sarcoglycanopathies are a subset of the limb girdle muscular dystrophies (LGMD) caused by mutations in the sarcoglycan genes (α, β, γ and δ). In collaborative studies, δ-sarcoglycan was delivered to deficient hamsters using a recombinant adeno-associated virus (AAV), which rescued muscle biochemically, histologically, and functionally. Murine knockouts for the other sarcoglycans permitted us to pursue AAV-mediated gene delivery. AAV-mediated gene delivery of β-sarcoglycan to deficient mice provided long-term biochemical and histological rescue. AAV-mediated gene delivery of α-sarcoglycan to deficient mice showed initial rescue of biochemical and histological defects, although expression was not persistent. Severe Combined Immune-Deficient (SCID) mouse studies indicated that α-sarcoglycan over-expression leads to cytotoxicity. The apparent cytotoxicity can be interpreted with emerging models of sarcoglycan complex assembly. These studies show that AAV-mediated delivery of even closely related proteins can lead to different outcomes, and aspects of protein biochemistry can alter efficacy of gene delivery.; Inherited muscle disorders typically have defined primary biochemical defects. However, there are likely secondary responses that mitigate gene delivery success. To dissect such variables, we studied the immunostimulatory properties of dystrophic muscle. We hypothesized that immune cell infiltrate accompanying degeneration/regeneration could be immunostimulatory, which could elicit an immune response to delivered transgenes, hampering the success of gene delivery. To study this, we tested antibody response to and persistence of, β-galactosidase in normal and dystrophic muscle. Consistent with our hypothesis, dystrophic muscle showed increased immune surveillance and recognition of β-galactosidase, evidenced by antibody titers and clearance of transduced cells. Furthermore, biochemical rescue of the dystrophy quenched the immune response. This indicated that dystrophic muscle is more prone to immune responses and that aspects of tissue pathology influence the persistence and efficacy of gene delivery. Our results suggest that full biochemical rescue will attenuate immunostimulatory effects.; We also address a hurdle facing AAV-mediated gene therapy; namely, delivery methods. We developed an injection manifold, which was used to safely, accurately, and consistently deliver genes to 20 mm2 regions of muscle.; Taken together, these results more clearly define barriers to gene delivery. Future research will finely tune regulation of transgenes and enable full rescue of biochemical defects.
机译：肌糖蛋白病是由肌糖蛋白基因（α，β，γ和δ）突变引起的四肢腰肌营养不良（LGMD）的一个子集。在合作研究中，使用重组腺相关病毒（AAV）将δ-糖聚糖递送到缺乏的仓鼠中，该病毒从生化，组织学和功能上拯救了肌肉。其他肌糖苷的小鼠基因敲除使我们能够进行AAV介导的基因传递。 AAV介导的β-肌糖蛋白基因向缺陷小鼠的传递提供了长期的生化和组织学挽救。 AAV介导的α-肌糖蛋白基因向缺陷小鼠的传递显示出生化和组织学缺陷的初步挽救，尽管表达并不持久。严重的联合免疫缺陷（SCID）小鼠研究表明，α-肌糖蛋白过度表达会导致细胞毒性。明显的细胞毒性可以用新兴的糖聚糖复合物组装模型来解释。这些研究表明，即使是紧密相关的蛋白质，AAV介导的传递也会导致不同的结果，并且蛋白质生物化学的各个方面可以改变基因传递的功效。遗传性肌肉疾病通常具有明确的主要生化缺陷。但是，可能有一些次要反应会减轻基因传递的成功。为了剖析这些变量，我们研究了营养不良性肌肉的免疫刺激特性。我们假设伴随变性/再生的免疫细胞浸润可能是免疫刺激的，这可能引起对已传递的转基因的免疫反应，从而阻碍了基因传递的成功。为了研究这一点，我们测试了正常肌和营养不良性肌对β-半乳糖苷酶的抗体反应和持久性。与我们的假设一致，营养不良的肌肉表现出增强的免疫监视能力和对β-半乳糖苷酶的识别，这通过抗体滴度和转导细胞清除来证明。此外，对营养不良的生物化学挽救使免疫反应终止。这表明营养不良的肌肉更容易产生免疫反应，并且组织病理学的各个方面影响基因传递的持久性和功效。我们的结果表明，完全的生化挽救将减弱免疫刺激作用。我们还解决了AAV介导的基因治疗面临的障碍；即交付方式。我们开发了一种注射歧管，用于安全，准确且一致地将基因传递至20 mm 2 肌肉区域。综上所述，这些结果更清楚地定义了基因传递的障碍。未来的研究将对转基因的调节进行微调，并能够完全挽救生化缺陷。

著录项

  • 作者

    Dressman, Devin Charles.;

  • 作者单位

    University of Pittsburgh.;

  • 授予单位 University of Pittsburgh.;
  • 学科 Biology Genetics.; Biology Molecular.; Health Sciences Pathology.
  • 学位 Ph.D.
  • 年度 2002
  • 页码 p.4471
  • 总页数 203
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 遗传学;
  • 关键词

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