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Gene therapy using adeno-associated viral vectors: Enhancing pharmacologic assessment and transgene delivery.

机译：使用腺相关病毒载体进行基因治疗：加强药理学评估和转基因递送。

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摘要

Duchenne muscular dystrophy (DMD) is a progressive and debilitating disease for which there is currently no cure. Gene therapy is one therapeutic approach that is being tested to treat DMD. Most gene therapy strategies aim to introduce a functional copy of the dystrophin mRNA to replace the dysfunctional "dystrophin" gene within the patient's genome. "Vectors" are used to deliver genes to the affected muscle cells for gene therapy. Vectors based on adeno-associated viruses (AAV) have come to the forefront for DMD gene therapy because of their safety, stability, and ability to mediate systemic gene delivery.;While AAV vectors are robust in mice, their use for systemic therapy requires very large amounts of costly vector to be injected. Such doses are not only expensive to produce but also increase the risk of producing an off-target effect and triggering an adaptive immune response against transduced cells. These immune responses reduce the duration and level of therapeutic gene expression in animals and in humans.;This project was directed towards improving the pharmacology of AAV vectors for systemic therapy. We hypothesized that much of an injected dose of AAV is lost to phagocytic cells in the liver known as Kupffer cells. We hypothesized if Kupffer cells destroy large amounts of AAV, that avoiding these cells could allow much lower amounts of vector to be used to mediate the same therapeutic effects. Likewise, avoiding these macrophages could also reduce innate and adaptive immune responses against the vector to increase gene therapy levels and persistence. To evaluate the pharmacology of AAV in mice, we first developed a novel reporter system that utilized Cre recombinase to activate reporter genes in mouse models. With this and other vector and mouse genetic systems, we tested the role of liver Kupffer cells in AAV biology and the tropism of different AAV serotypes after systemic injection. By these approaches, we have demonstrated the unique tropisms of AAV8 and AAV9 vectors after systemic administration. We have also shown that liver Kupffer cells are unlikely to play a large role in absorption of AAV8 serotype vectors. Instead, we hypothesize that this robust systemic vector may be effective by virtue of its ability to avoid Kupffer cells. This work has also uncovered a unique interaction between AAV vectors and adenovirus vectors. Co-administration of the two vectors can lead to 100-fold amplification of AAV vector expression in the liver. These data suggest that future work to understand interactions between AAV, adenovirus, and Kupffer cells may lead to more robust systemic gene therapy with AAV vectors.
机译：杜兴氏肌营养不良症（DMD）是一种进行性和衰弱性疾病，目前尚无法治愈。基因疗法是一种正在测试治疗DMD的治疗方法。大多数基因治疗策略旨在引入肌营养不良蛋白mRNA的功能性副本，以取代患者基因组中功能失调的“肌营养不良蛋白”基因。 “载体”用于将基因递送至受影响的肌肉细胞以进行基因治疗。基于腺相关病毒（AAV）的载体因其安全性，稳定性和介导系统性基因传递的能力而成为DMD基因治疗的最前沿;虽然AAV载体在小鼠中很健壮，但其在全身治疗中的应用却非常需要注入大量昂贵的载体。这样的剂量不仅生产昂贵，而且增加了产生脱靶效应和触发针对转导细胞的适应性免疫应答的风险。这些免疫反应减少了动物和人类中治疗性基因表达的持续时间和水平。该项目旨在改善用于全身治疗的AAV载体的药理学。我们假设大部分注射剂量的AAV会丢失于肝脏中称为Kupffer细胞的吞噬细胞中。我们假设库普弗细胞是否会破坏大量的AAV，避免这些细胞可能会使使用较少量的载体来介导相同的治疗效果。同样，避免这些巨噬细胞也可以减少针对载体的先天性和适应性免疫反应，从而增加基因治疗水平和持久性。为了评估AAV在小鼠中的药理作用，我们首先开发了一种新颖的报告系统，该系统利用Cre重组酶激活小鼠模型中的报告基因。使用该载体以及其他载体和小鼠遗传系统，我们测试了全身注射后肝Kupffer细胞在AAV生物学中的作用以及不同AAV血清型的嗜性。通过这些方法，我们已经证明了全身给药后AAV8和AAV9载体的独特向性。我们还显示，肝库普弗细胞不太可能在AAV8血清型载体的吸收中发挥重要作用。取而代之的是，我们假设这种强大的系统载体可能会避免库普弗细胞，因此是有效的。这项工作还发现了AAV载体和腺病毒载体之间的独特相互作用。两种载体的共同给药可导致肝脏中AAV载体表达的100倍扩增。这些数据表明，了解AAV，腺病毒和Kupffer细胞之间相互作用的未来工作可能会导致使用AAV载体进行更强大的全身基因治疗。

著录项

  • 作者

    Hillestad, Matthew Lee.;

  • 作者单位

    College of Medicine - Mayo Clinic.;

  • 授予单位 College of Medicine - Mayo Clinic.;
  • 学科 Biology Molecular.;Biology General.;Biology Virology.
  • 学位 Ph.D.
  • 年度 2011
  • 页码 105 p.
  • 总页数 105
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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