AAV Gene Therapy for AAT Deficiency

描述(申请人提供)：α-1抗胰蛋白酶(AAT)缺乏，由于“PIZZ”突变，导致危及生命的儿童和成人的肺和肝脏疾病。目前，基因添加疗法正在测试中，以预防肺部疾病。然而，这一策略并不能阻止肝脏疾病的进展，因为突变的PIZZ蛋白积累在肝细胞的内质网中，而不是正常分泌到血液和体液中。从我们之前两年由ARRA(R01DK084033)资助的R01申请中，我们已经生成了大量的数据集，证明：1)AAV载体可以有效地传递自我互补的shRNA，在mRNA和蛋白质水平上下调PIZAAT的表达，从而改善PIZZ小鼠的肝脏病理。2)替代的组合方法既可以用来抑制PIZZ的表达，也可以用来恢复功能性的循环AAT；这些方法包括肝脏AAV-shRNA递送然后肌肉注射AAV-wtAAT盒，通过单独的shRNA载体和避免基于shRNA的降解的优化AAT盒将双载体注射到肝脏，以及最后递送同时将shRNA和优化的AAT递送到肝脏的单个载体。在这些实验中应用的双重靶向策略同时防止了AAT缺乏时肝脏和肺部疾病的发展。然而，AAV运送shRNA面临三个障碍：衣壳特异性CTL介导的AAV转导的肝细胞的消除，shRNA在非靶向转导组织中潜在的非靶向作用，以及AAV中和抗体(NAB)在人类中的高度流行。针对这些障碍，这项更新建议的主要目标是：(I)研究经scAAV/shRNA载体处理后，AAV衣壳蛋白特异性CTL介导的杀伤作用对PIZZ小鼠AAV转导的肝细胞的影响(Ii)建立专门控制PIZZ小鼠肝脏shRNA表达的新方法(Iii)产生能够对肝脏特异的转导和逃避中和抗体活性的AAV突变体。该提案的长期目标是设计更安全、更有效的AAV载体，用于AAT缺乏症患者的基因治疗。

DESCRIPTION (provided by applicant): Alpha-1 antitrypsin (AAT) deficiency, due to the "piZZ" mutation, results in life-threatening lung and liver diseases in children and adults. Currently, gene addition therapy is being tested to prevent lung disease. However, this strategy does not halt liver disease progression due to the accumulation of mutant PiZZ protein in the endoplasmic reticulum of liver cells rather than normal secretion into the blood and body fluids. From our previous two year R01 application funded from the ARRA (R01DK084033), we have generated a substantial dataset, demonstrating that : 1) AAV vectors can efficiently deliver self-complimentary shRNA to knock down piZZ AAT expression at both mRNA and protein levels, resulting in improved liver pathology of piZZ mice. 2) Alternative combinatorial approaches can be used to both knockdown piZZ expression and restore functional, circulating AAT; These approaches include liver AAV-shRNA delivery followed by muscular injection of an AAV-wtAAT cassette, dual vector administration into the liver with independent vectors for shRNA and an optimized AAT cassette that avoids shRNA-based degradation, and finally delivery of a single vector that simultaneously delivers shRNA and optimized AAT into the liver. The dual-targeting strategy applied in these experiments simultaneously prevents both liver and lung disease development in AAT deficiencies. However, AAV delivery of shRNA faces three obstacles: capsid specific CTL-mediated elimination of AAV-transduced liver cells, potential off-target effects of shRNA in non-targeted, transduced tissue, and the high prevalence of neutralizing antibodies (Nab) to AAV in the human population. In response to these obstacles, the primary objectives of this renewal proposal are to: (i) investigate the effect of AAV capsid specific CTL-mediated killing on AAV-transduced liver cells in piZZ mice after treatment with scAAV/shRNA vector (ii) develop novel approaches to exclusively control shRNA expression in the liver of piZZ mice (iii) generate AAV mutants capable of liver-specific transduction and evasion of neutralizing antibody activity. The long-term goal of the proposal is to design safer and more effective AAV vectors for gene therapy in patients with AAT deficiency.