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。幸运的是，这种肺部疾病可以通过基因添加疗法来预防。然而，这种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。因此，PIZZ相关肝病的基因治疗应着眼于在DNA和/或RNA水平上长期消除/纠正突变蛋白。小干扰RNA(SiRNA)是一种很有前途的抑制PIZ的方法，基于DNA载体的持久siRNA可以将这种效应延长数年(目标1)。腺相关病毒2(AAV2)载体已用于治疗AAT缺乏症的前期和临床试验。在肝脏中，重组(R)AAV2转导可以诱导持续的基因表达；然而，即使在高颗粒与细胞比的情况下，也只有5%-10%的肝细胞被转导。最近的研究，包括我们的研究表明，其他AAV血清型，主要是AAV6，AAV8和AAV9能够比AAV2更有效地转导肝细胞。为了进一步加强这种转导，我们开发了一种新型的AAV载体，它包装了一个双链(DS)基因组。这些载体绕过了第二链合成的限速步骤，导致了转基因表达的增加和更早(比传统的单链(Ss)载体早两周)。特别是在肝脏，我们已经能够用较低的总剂量证明dsAAV2转导高达90%，dsAAV8转导95%以上。尽管AAV转导的肌肉在血液中可以检测到AAT的表达，但肝脏是产生AAT的天然器官，而且AAV转导的肝脏的转基因表达高于肌肉。因此，随着PIZZ基因表达的抑制和成功的基因添加策略，应该分别消除肝脏和肺这两种疾病表现。为了避免siRNA/PIZs引起的wtAAT基因的降解，增加整体AAT的合成，我们将根据遗传密码的简并性创建一个优化的AAT基因(目标2)。应该注意的是，虽然AAV2和AAV8高效地转导肝脏，但总体取向是广泛的，当然不限于肝细胞。细胞特异性转导需要几个步骤，包括病毒结合、内吞、运输和脱壳，我们已经开始使用改组的血清型衣壳文库将结构与功能联系起来。在我们初步数据的支持下，有可能进化出一种实验室AAV毒株，这种毒株可能具有更好的、但受到限制的肝脏转导，以减少其他组织中的siRNA“脱靶”效应。它还表明，可以确定负责有效肝转导的特定衣壳结构域(目标3)。 公共卫生相关性：腺相关病毒(AAV)是一种很有前途的α-抗胰蛋白酶(AAT)缺乏症(Alpha-1)基因治疗载体。AAV能够使治疗性基因长期稳定表达，不会导致任何已知疾病。目前已分离出12种AAV，其中AAV2是最具生物学特性和作为基因输送系统的AAV2。AAV2载体感染后，只有5-10%的肝细胞能表达AAT基因。AAV1、AAV5、AAV8和AAV9感染后，AAT的肝脏表达水平明显升高。这表明不同类型的AAV使用不同的细胞感染途径(即细胞表面结合、病毒在细胞内移动、病毒进入细胞核、病毒核酸从其蛋白质壳中移除)。AAV是一种单链DNA病毒。只有在单链DNA基因组转化为双链(DS)DNA后，这些基因组才会转录。使用dsAAV向量可以克服该速率限制步骤。最近，我们开发了dsAAV载体，与传统的单链AAV载体相比，它能更快、更高地诱导转基因表达。几乎所有的肝细胞都被这些双链AAV载体感染，利用这种双链载体携带治疗性基因对于预防阿尔法-1患者肝病的发展具有非常重要的意义。由于AAT缺乏的肝病是由于肝细胞内存在突变的AAT而引起的。为了破坏这种突变蛋白，我们将使用dsAAV8载体(AIM 1)将针对突变AAT的siRNA转移到肝脏中。在肝细胞中敲除突变型AAT只能预防肝病的发展，而不能预防AAT缺乏引起的肺损伤。为了恢复AAT的表达，我们将使用dsAAV2载体感染肝脏来传递优化的AAT，它不能被突变体AAT/siRNA(AIM 2)降解。目的3描述新的AAV肝脏特异性变异体的开发和特性，该变异体将具有优越的转导能力，特别是对已知血清型的肝细胞。这些新的变异体将通过体外“DNA改组”和肝脏体内选择的方法产生。本研究对设计AAV/AAT载体用于人类临床试验具有重要意义。

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. Fortunately, the lung disease can be prevented by gene addition therapy. However, this strategy could 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. Therefore, gene therapy for the PiZZ-associated liver diseases should be focused on long-term elimination/correction of mutant protein at the DNA and/or RNA levels. Small interference RNA (siRNA) represents a promising approach to suppress PiZZ and a persistent DNA vector-based siRNA could prolong this effect for years (Aim 1). Adeno-associated virus 2 (AAV2) vectors have been utilized for treatment of AAT deficiency in pre- and clinical trials. In the liver, transduction by recombinant (r) AAV2 induces sustained gene expression; however, only 5-10% of liver cells are transduced even at a high particle to cell ratio. Recent studies, including ours, demonstrated that other AAV serotypes, mainly AAV6, AAV8 and AAV9 are able to transduce liver cells more efficiently than AAV2. To further enhance this transduction, we have developed a novel AAV vector that packages a double-stranded (ds) genome. These vectors by-pass the rate limiting step of second-strand synthesis resulting in both increased and earlier transgene expression (up to two weeks earlier than the traditional single-stranded (ss) vector). Specifically in liver, we have been able to demonstrate dsAAV 2 transduction as high as 90%, and dsAAV 8 with over 95% transduction using a lower total dose. Even though AAT expression can be detected in blood from AAV transduced muscles, liver is a natural organ to produce AAT, and higher transgene expression has been observed in AAV transduced liver compared to muscle. Therefore, suppression of PiZZ gene expression along with the successful gene addition strategy should eliminate both disease manifestations, liver and lung respectively. To avoid wtAAT mRNA degradation induced by siRNA/PiZZ and to increase overall AAT synthesis, we will create an optimized AAT gene based on the degeneracy of the genetic code (Aim 2). It should be noted that although AAV2 and AAV8 transduce the liver with high efficiency the overall tropism is broad and certainly not restricted to liver cells. Several steps including viral binding, endocytosis, trafficking and uncoating are required for cell specific transduction and we have begun to relate structure to function using a shuffled serotype capsid library. Supported by our preliminary data, it is possible to evolve a lab strain of AAV that may have superior, yet restricted, liver transduction to reduce siRNA "off-target" effect in other tissues. It also suggests that specific capsid domains responsible for efficient liver transduction can be identified (Aim 3). PUBLIC HEALTH RELEVANCE: Adeno-associated virus (AAV) is a promising delivery vector for alpha-antitrypsin (AAT) deficiency (Alpha-1) gene therapy. AAV is able to confer long-term stable expression of a therapeutic gene and does not cause any known disease. Recently, 12 types of AAV have been isolated and AAV2 is the best characterized for its biology and as a gene delivery system. Only 5-10% of liver cells can express the AAT gene following infection with AAV2 vectors. AAT liver expression is much higher following infection with AAV1, 5, 8 & 9 vectors. This suggests that different types of AAV use different cellular pathways for infection (i.e. cell surface binding, movement of the virus through the cell, entry of the virus into the nucleus, removal of the viral nucleic acid from its protein shell). AAV is a single-stranded DNA virus. Transcription will ensue from these genomes only after the single-stranded DNA genome is converted to double-stranded (ds) DNA. The use of dsAAV vectors can overcomes this rate limiting step. Recently we have developed the dsAAV vectors which can induce transgene expression much faster and higher than conventional single-stranded AAV vectors. Almost all liver cells are infected with these double-stranded AAV vectors, which is very significant for preventing liver disease development in Alpha-1 patients by using this double-stranded vector to carry therapeutic genes. Since liver disease with AAT deficiency is caused by the existence of mutant AAT inside of liver cells. To destroy this mutant protein, we will deliver siRNA specific for mutant AAT into the liver using dsAAV8 vector (Aim 1). Knocking down of mutant AAT in liver cells only prevent liver disease development and cannot prevent lung damage caused by AAT deficiency. To restore AAT expression, we will infect the liver using dsAAV2 vector to deliver the optimized AAT, which cannot be degraded by mutant AAT/siRNA (Aim 2). Aim 3 describes the development and characterization of new AAV liver-specific variants that will possess superior transduction capacity specifically on liver cells to known serotypes. These new variants will be generated using an approach with in vitro "DNA shuffling" and in vivo selection in liver. This study would be very important to design AAV/AAT vector for future human clinical trials.