AAV Gene Therapy for AAT Deficiency

描述（由申请人提供）：由于“piZZ”突变，α -1抗胰蛋白酶（AAT）缺乏导致儿童和成人危及生命的肺部和肝脏疾病。目前，基因附加疗法正在进行测试，以预防肺部疾病。然而，由于突变PiZZ蛋白在肝细胞内质网的积累，而不是正常分泌到血液和体液中，这种策略并不能阻止肝病的进展。从我们前两年ARRA资助的R01申请（R01DK084033）中，我们已经产生了大量的数据集，证明：1)AAV载体可以有效地传递自我补充的shRNA，在mRNA和蛋白质水平上降低piZZ AAT的表达，从而改善piZZ小鼠的肝脏病理。2)替代组合方法可用于敲除piZZ表达和恢复功能性循环AAT；这些方法包括肝脏递送AAV-shRNA，然后肌肉注射AAV-wtAAT盒，用独立的shRNA载体和优化的AAT盒双重载体给药到肝脏，以避免基于shRNA的降解，最后将单个载体同时递送shRNA和优化的AAT到肝脏。在这些实验中应用的双靶向策略可以同时预防AAT缺乏症中肝脏和肺部疾病的发展。然而，AAV递送shRNA面临三个障碍：衣壳特异性ctl介导的AAV转导肝细胞的消除，shRNA在非靶向转导组织中的潜在脱靶效应，以及人群中AAV中和抗体（Nab）的高流行率。针对这些障碍，本更新提案的主要目标是：(i)研究用scAAV/shRNA载体治疗后，AAV衣壳特异性ctl介导的杀伤对piZZ小鼠AAV转导肝细胞的影响；（ii）开发新的方法来专门控制piZZ小鼠肝脏中shRNA的表达；（iii）产生能够肝脏特异性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.