Optimized Gene Replacement for AAT deficiency and Modeling of Clinical Outcomes in small and large animal models

项目概要（项目1） α-1抗胰蛋白酶缺乏症（AATD）是一种常见的单基因疾病，由以下突变引起： SERPINA 1基因通常编码非常丰富的血清抗蛋白酶， 主要功能是保护肺的间质弹性蛋白基质免于降解， 中性粒细胞弹性蛋白酶（NE）。E242 K（PI*Z）突变等位基因在那些 欧洲血统，E342 K纯合子编码一种分泌受损的蛋白质， 导致血清水平不足，导致NE活性不受限制， 弹性蛋白这一过程引发肺部炎症，导致肺部弹性回缩的丧失， 气道阻塞，造成肺气肿的临床表现，这是限制生命的影响， 大多数患者的AATD。目前的AATD治疗包括每周IV输注AAT，但这种方法不适用于AAT。 在前瞻性研究中从未证明治疗有益于患者的肺功能， 这导致了关于先前确定的血清目标水平（11 µM）是否 是否足够，或者恢复这种水平的基因治疗是否有效。本实验室 此前已经开发出rAAV基因治疗载体，并将其用于临床试验。那些 试验未能达到目标，达到略高于0.5 μM的水平，但仍在持续 5年以上患者。我们还使用基于CRISPR的技术生产了第一个 SERPINA 1基因敲除小鼠，并已开始与爱荷华州大学的研究小组合作， 他们创造了SERPINA 1基因敲除和SERPINA 1-E342 K雪貂模型。在 根据目前的建议，将在小鼠和雪貂模型中测试改进和优化的载体。 在雪貂中，它们将与复杂的条件性转基因重组一起进行测试， 确定治疗的最佳目标是11 µM还是25 µM。这些研究将为 为今后的临床产品开发提供了更好的rAAV-AAT载体。 在这个项目中，我们建议研究遗传性肺气肿由于α-1抗胰蛋白酶缺乏症 （AATD）。AATD作为一种遗传性疾病是相当常见的， 慢性阻塞性肺疾病（COPD）的常见原因。在提案中，我们将使用 先进的基因编辑工具，以创建基因定义的AATD动物模型（称为 转基因动物），在小鼠和雪貂中，这是研究肺部疾病的良好模型。 在研究过程中，我们将使用额外的转基因方法和改良的转基因方法。 重组病毒（rAAV）在这些模型中遗传治疗AATD，并检查我们如何 可能会设计评估未来基因治疗遗传性肺气肿的有效性。

Project Summary (Project 1) Alpha-1 antitrypsin deficiency (AATD) is a common single gene disorder caused by mutations in the SERPINA1 gene, which normally encodes a very abundant serum antiprotease, whose primary function is to protect the interstitial elastin matrix of the lung from degradation by neutrophil elastase (NE). The E242K (PI*Z) mutant allele is very common among those of European ancestry, and E342K homozygotes encode a protein with impaired secretion, resulting in deficient serum levels, leading to unrestrained NE activity, degrading pulmonary elastin. This process triggers lung inflammation and leads to loss of lung elastic recoil and airways obstruction, creating the clinical picture of emphysema, which is the life-limiting effect of AATD in most patients. Current AATD therapy consists of weekly IV infusion of AAT, but this therapy has never been proven to benefit lung function in patients in prospective studies, leading to questions about whether the previously identified serum target level (11 µM) is sufficient, or whether gene therapy to restore such a level would be efficacious. Our laboratory has previously developed rAAV gene therapy vectors and used them in clinical trials. Those trials have failed to achieve the target, achieving levels just over 0.5 µM but being sustained over 5 years in patients. We have also use CRISPR-based technology to produce the first SERPINA1-knockout mice and have begun a collaboration with the University of Iowa group, who have created both SERPINA1-knockout and SERPINA1-E342K ferret models. In the current proposal, improved and optimized vectors will be tested in both mice and ferret models. In the ferrets, they will be tested alongside sophisticated conditional transgenic reconstitution to determine whether the optimal target for therapy is 11 µM vs. 25 µM. These studies will pave the way for better rAAV-AAT vectors appropriate for future clinical product development. In this project, we propose to study genetic emphysema due to alpha-1 antitrypsin deficiency (AATD). AATD is both fairly common as a genetic disease and is a model of much more common causes of chronic obstructive pulmonary disease (COPD). In the proposal, we will use advanced gene editing tools to create genetically defined animal models of AATD (known as transgenic animals), both in mice and in ferrets, which are a good model to study lung diseases. In the course of the study, we will use both additional transgenic approaches and modified recombinant viruses (rAAV) to genetically treat AATD in these models and examine how we might design evaluate the effectiveness of future gene therapies for genetic emphysema.