Minimizing transgene clearance

最近的一项研究证实了rAAV介导的ScFv免疫粘附素肌肉内递送的概念 恒河猴可以产生持续高水平的这些抑制物，并保护研究动物 从高剂量的SIV挑战中。然而，在那项研究中，一些接受治疗的动物产生了抗免疫粘附素 抗体反应，导致清除抗病毒抑制剂。免疫清除这一点 Sort已经在rAAV基因治疗文献中得到了很好的记录：来自 在大型动物模型中，rAAVs通常可以诱导强烈的三角基因导向免疫，导致宿主 对表达的转基因进行清除。事实上，免疫清晰度仍然是进一步 RAAV递送疗法的转译发展。因此，有必要更好地定义免疫 促进抗抑制物抗体反应的过程，并开发新的战略来预防 清除已表达的转基因。这些是项目2的主要目标。 我们建议进行以下研究，以达致这些目标： 1)。限制rAAV-免疫粘附素在非预期靶细胞中的转导和抗原提呈 通过评估一组肌肉特异性启动子的启动子强度、组织特异性和 用于包装大小受限的rAAV基因组的可行性。 2)。利用内源性miRNAs靶向抗原提呈细胞转导rAAV 树突状细胞特异性转录后转基因沉默。 3)。亲肝rAAV8介导诱导免疫粘附素表达的持续系统耐受 和肝脏特异性转导。 这些研究将产生AAV载体基因组，以限制表达的转基因的清晰度，并 目的在许多情况下，AAV载体的治疗应用是至关重要的。

A recent study proved the concept that rAAV-mediated intramuscular delivery of scFv immunoadhesins to rhesus macaques can generate sustained high serum levels of these inhibitors, and protect study animals from a high-dose SIV challenge. However, some of the treated animals in that study developed anti-immunoadhesin antibody responses, resulting in clearance of antiviral inhibitors. Immune clearance of this sort has been well documented in the rAAV gene-therapy literature: expression of foreign proteins from rAAVs in large animal models can in general elicit strong trangene-directed immunity, leading to host clearance of the expressed transgene. Indeed immune clearence remains a key challenge to further translational development of rAAV-delivered therapies. It is therefore necessary to better define immune processes contributing to anti-inhibitor antibody responses, and to develop novel strategies to prevent clearance of expressed transgenes. These are the main objectives of Project 2. We proposed the following studies to accomplish these objectives: 1). To limit transduction and antigen presentation of rAAV-immunoadhesin in unintended target cells and tissues by evaluating a panel of muscle specific promoters for promoter strength, tissue specificity and feasibility for use in the packaging size-limited rAAV genome. 2). To detarget rAAV transduction from antigen presenting cells by harnessing endogenous miRNAs for dendritic cell specific post-transcriptional transgene silencing. 3). To induce sustained systemic tolerance to immunoadhesin expression by hepatotropic rAAV8-mediated and liver-specific transduction. These studies will generate an AAV vector genome optimized to limit clearence of expressed transgenes, an objective critical to the therapeutic use of AAV vectors in many contexts.