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

项目总结(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 SERPINA1基因通常编码一种非常丰富的血清抗蛋白酶，其 主要功能是保护肺间质弹性蛋白基质不被降解 中性粒细胞弹性蛋白酶(NE)。E242K(Pi*Z)突变等位基因在慢性阻塞性肺疾病患者中非常常见 欧洲血统，E342K纯合子编码一种分泌受损的蛋白质， 导致血清水平不足，导致去甲肾上腺素活性不受抑制，降解肺组织 弹性蛋白。这一过程触发了肺部炎症，并导致肺弹性反冲和 呼吸道阻塞，造成肺气肿的临床画面，这是限制生命的影响 AATD在大多数患者中存在。目前的AATD疗法包括每周静脉输注AAT，但这 在前瞻性研究中，治疗从未被证明有利于患者的肺功能， 这导致了之前确定的血清目标水平(11微米)是否为 是否足够，或者基因疗法恢复这样的水平是否有效。我们的实验室 此前开发了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，达到了略高于0.5微米的水平，但仍在持续 患者住院5年以上。我们还使用了基于CRISPR的技术来生产第一个 SERPINA1基因敲除小鼠，并已开始与爱荷华大学的研究小组合作， 他们创造了SERPINA1-淘汰型和SERPINA1-E342K雪貂模型。在 目前的提议、改进和优化的载体将在小鼠和雪貂模型中进行测试。 在雪貂身上，它们将与复杂的有条件的转基因重组一起进行测试，以 确定最佳治疗目标是否为11微米与25微米。这些研究将为 为今后临床产品开发提供更好的rAAV-AAT载体。 在这个项目中，我们建议研究由于α-1抗胰蛋白酶缺乏引起的遗传性肺气肿。 (AATD)。AATD既是一种相当常见的遗传性疾病，也是一种更常见的 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，可创建基因定义的AATD动物模型(称为 转基因动物)，在小鼠和雪貂身上都是如此，这是研究肺部疾病的一个很好的模型。 在研究过程中，我们将使用额外的转基因方法和修改后的方法 重组病毒(RAAV)在这些模型中从基因上治疗AATD，并检查我们是如何 可能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.