Nuclease free gene editing approaches to treat alpha-1 antitrypsin disease

α-1抗胰蛋白酶缺乏症（AATD）是一种常见的遗传性疾病，可导致肝和肺 这种疾病目前影响着全球约340万患者。α-1抗胰蛋白酶（AAT）编码 由SERPINA 1产生，主要由肝细胞分泌，使其成为最丰富的血清抗蛋白酶。一 AATD中最常见的疾病变体是导致谷氨酸变为赖氨酸（Glu 342 Lys）的突变 PiZ等位基因或Z-AAT。与正常PiM等位基因（M-AAT）相比，Z-AAT蛋白 易于聚合，因此直接用于蛋白水解或在内质网中聚集 肝细胞网。高达85%的AAT蛋白以聚合物的形式保留或在细胞中降解， 肝脏，它为功能丧失（肺）和毒性功能获得（肝脏）疾病奠定了基础， AATD患者。正常情况下，AAT分泌并扩散到整个身体器官，在那里它保护组织 来自蛋白酶的“未检查的”或“脱靶的”活性。在肺中，功能丧失表型是由于 蛋白酶/抗蛋白酶稳态的不平衡。具体来说，蛋白酶称为嗜中性粒细胞弹性蛋白酶， 它是由中性粒细胞分泌的一种先天免疫形式，不受控制，多年来导致 肺结构的退化。这最终表现为慢性阻塞性肺疾病 （COPD）和肺气肿。相比之下，Z-AAT聚集和聚合通过一种毒性的免疫抑制剂引起肝脏疾病。 由于肝细胞中错误折叠蛋白质的积累而导致的功能获得机制， PiZ纯合子患者患有从暴发性肝衰竭和肝硬化到肝硬化的临床肝病。 肝细胞癌我们的团队已经开发出同时进行基因扩增的策略， 本发明涉及用双功能载体减少肺和肝疾病的突变基因，但仍存在未满足的需求。 我们需要解决年轻的肝脏疾病，积极分裂的肝脏，就像AATD肝病的情况一样。 儿科人群。此外，基因编辑方法可以提供比附加型AAV更长期的解决方案。 基因疗法用于治疗因疾病而缓慢转变的成人肝脏。 有两个显着的进步，将支持这第二代的发展 用于肝病的基于rAAV的疗法。这些进展中的第一个是，我们现在认识到， 与AAV的重组（HR）可以在不使用核酸酶的情况下以足够高的效率实现，以具有与AAV的重组（HR）。 对肝脏疾病有意义的临床影响。第二个重要的发展是在AAV介导的基因 编辑字段是某些AAV血清型更好地实现无核酸酶同源重组的实现。 重组最近，从造血干细胞中分离出了称为AAV-HSC的进化枝f的新AAV成员。 干细胞，并且这些载体已经显示出高HR活性。因此，该基金旨在开发新的核酸酶- 免费的基因编辑策略，将通过使用AAV介导的 同源重组到白蛋白或Serpina 1基因座中。我们假设无核酸酶的AAV- 介导的基因编辑是治疗α-1抗胰蛋白酶的可行、有效和安全的治疗方法 缺陷 我们将通过以下三个目标来检验这一假设。在目标1中，我们将使用具有人类肝脏的小鼠 异种移植物以鉴定具有人肝细胞向性和增加的同源性的rAAV-HSC衣壳 重组活性在目标2中，我们将比较无核酸酶的AAV介导的基因编辑与AAV 3b， 在非人灵长类动物模型中的白蛋白或SerpinA 1基因座。最后，在目标3中，我们将进行头对- Aim 1中鉴定的最佳AAV-HSC衣壳与AAV 3b在更有效基因座的头部比较 在目标2中确定。 总的来说，该基金旨在确定实现AAV的理想基因座和最佳衣壳。 介导的无核酸酶基因编辑，用于α-1抗胰蛋白酶缺乏症。应当指出， 这项研究也将直接影响CRISPR/Cas9和ZFN领域，因为目前最重要的是， 使用核酸酶依赖性基因编辑方法为HDR提供DNA模板的有效方法仍然是 依赖于rAAV。因此，我们认为我们的数据将作为HDR基准，从其中核酸酶- 通过将DNA断裂和切口结合到方法中，可以改进依赖方法。

Alpha-1 antitrypsin deficiency (AATD) is a common genetic disorder that can lead to both liver and lung disease and currently affects an estimated 3.4 million patients worldwide. Alpha-1 antitrypsin (AAT) is encoded by SERPINA1 and is primarily secreted by hepatocytes making it the most abundant serum antiprotease. One of the most common disease variants in AATD is a mutation resulting in a glutamate to lysine (Glu342Lys) substitution known as the PiZ allele or Z-AAT. In contrast to the normal PiM allele (M-AAT), the Z-AAT protein is prone to polymerization and consequently is either directed for proteolysis or aggregates in the endoplasmic reticulum of hepatocytes. With up to 85% of the AAT protein being retained as polymers or degraded in the liver, it sets the stage for both the loss-of-function (lung) and toxic gain-of-function (liver) diseases observed in AATD patients. Normally AAT is secreted and diffuses throughout the bodily organs where it protects tissue from the `unchecked' or `off-target' activity of proteases. In the lungs, the loss-of-function phenotype is due to the imbalance of protease/antiprotease homeostasis. Specifically, the protease known as neutrophil elastase, which is secreted by neutrophils as a form of innate immunity, goes unchecked and over years leads to the degradation of the lung architecture. This eventually manifests as chronic obstructive pulmonary disease (COPD) and emphysema. In contrast, Z-AAT aggregation and polymerization causes liver disease by a toxic gain-of-function mechanism due to accumulation of misfolded protein in the hepatocytes whereby 10-20% of PiZ homozygote patients suffer from clinical liver disease ranging from fulminant liver failure and cirrhosis to hepatocellular carcinoma. Our group has developed strategies for simultaneous gene augmentation with mutant gene reduction for both lung and liver disease with dual function vectors, but an unmet need remains. We need to address liver disease in a young, actively dividing liver as is the case of AATD liver disease in the pediatric population. Furthermore, gene editing approaches may offer longer-term solutions over episomal AAV gene therapy for adult livers that are slowly turning over due to disease. There are two notable advancements that will support the development of this second generation of rAAV-based therapies for liver disease. The first of these advances is that we now appreciate that homologous recombination (HR) with AAV can be achieved at high enough efficiency without the use of nuclease to have a meaningful clinical impact for liver disorders. The second important development in the AAV-mediated gene editing field is the realization that certain AAV serotypes are better at achieving nuclease-free homologous recombination. Recently new AAV members of clade f known as AAV-HSCs were isolated form hematopoietic stem cells, and these vectors have shown high HR activity. Thus, this grant aims to develop novel nuclease- free gene-editing strategies that will address the permanent correction of AATD by using AAV-mediated homologous recombination into either the Albumin or Serpina1 locus. We hypothesize that nuclease-free AAV- mediated gene editing is feasible, efficient and a safe therapeutic approach for treating alpha-1 antitrypsin deficiency. We will test this hypothesis with the following three aims. In Aim 1 we will use mice with human liver xenografts to identity rAAV-HSC capsids that have a human hepatocyte tropism and increased homologous recombination activity. In Aim 2 we will compare nuclease-free AAV-mediated, gene editing with AAV3b at either the Albumin or SerpinA1 locus in a non-human primate model. Finally in Aim 3 we will do a head-to- head comparison of the optimal AAV-HSC capsid identified in Aim1 against AAV3b at the more efficient locus identified in Aim 2. Overall, the grant aims to determine the ideal locus and best capsid with which to achieve AAV– mediated, nuclease-free gene editing for alpha-1 antitrypsin deficiency. It should be noted that the results of this research would also directly impact the CRISPR/Cas9 and ZFN fields broadly, as currently the most efficient way of delivering DNA templates for HDR with the nuclease-dependent gene editing approaches still relies on rAAV. Thus, we feel that our data will serve as a HDR benchmark from which the nuclease- dependent approaches can improve upon by incorporating DNA breaks and nicks into the approach.