VIRAL DELIVERY INTO NEONATAL AND ADULT MAMMALS

用于将基因转移到骨骼肌的腺病毒载体系统具有 克隆容量较大，生长滴度较高的优势 体外培养，以及转导未分裂细胞群体的能力。 单元1中概述的其他载体修改可能会延长克隆 容纳全长抗肌营养不良蛋白基因的能力。一个主要问题是 在这项技术临床应用之前解决的是需要 对扩散障碍生物学的更好理解 体内的腺病毒递送。微血管内皮细胞代表 在系统性交付中需要克服的主要扩散障碍 腺病毒感染骨骼肌。我们展示了初步数据来支持 假设毛细血管通透性或跨毛细血管的变化 压力梯度会对效率产生巨大影响 腺病毒介导的骨骼肌基因转移。我们提出了一个 研究其他干预措施的试验计划，可提供 进一步改善对横纹肌的基因转移，最大限度地减少不必要的基因转移 运送到有记录的毒性部位。我们将系统地研究 高压灌流和改变对收缩状态的影响 血管内皮细胞跨内皮细胞扩散过程中的标志物 腺病毒。在项目的后半部分，我们将探索如何使用 这些干预结合体外循环支持 (ECMO)正确评估跨内皮细胞的最大程度 腺病毒扩散是可以实现的。所描述的实验有两个 总体目标。在短期内，主要目标是发展 腺病毒递送计划，以帮助整合实验 其他单位的计划，特别是关于生理化验的计划 在动物模型系统中体液输送重组肌营养不良蛋白。 主要的长期目标是开发一种临床适用的方案。 用于全身腺病毒介导的基因转移。这一过程将是 专门针对杜氏肌营养不良症患者。 预计成功的交付计划将有额外的 适用于其他疾病的临床管理状态。

Adenoviral vector systems for gene transfer into skeletal muscle have the advantages of relatively large cloning capacity, growth to high titre in vitro, and the ability to transduce non-dividing cell populations. Additional vector modifications outlined in unit 1 could extend the cloning capacity to accommodate a full-length dystrophin cDNA. A major issue to be addressed prior to clinical application of this technology is the need for an improved understanding of the biology of diffusion barriers to adenoviral delivery in vivo. The microvascular endothelium represents the major diffusion barrier to be overcome in the systemic delivery of adenovirus to skeletal muscle. We show preliminary data to support the hypothesi that changes in either capillary permeability or transcapillary pressure gradient can have a dramatic impact on the efficiency of adenovirus mediated gene transfer into skeletal muscle. We propose an experimental plan for the study of additional interventions that offer to further improve gene transfer to striated muscle and minimize unwanted delivery to sites of documented toxicity. We will systematically study the impact of high pressure perfusion and alteration in the contractile state of endothelial cells on the process of transendothelial diffusion of marker adenovirus. In the second half of the project we will explore the use of these interventions in conjunction with extracorporeal circulatory support (ECMO) to properly evaluate the maximal degree of transendothelial adenoviral diffusion attainable. The experiments described have two overall goals. In the near term, the principal goal is the development of adenoviral delivery schemes to aid ina the integration of the experimental plans of the other units, especially with regard to the physiologic assay of somatically delivered recombinant dystrophin in animal model systems. The primary long term goal is to develop a clinically applicable protocol for systemic adenovirus mediated gene transfer. This process will be specifically targeted towards patients with Duchenne Muscular Dystrophy. It is anticipated that successful delivery schemes will have additional applicability in the clinical management of other diseases states.