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。幸运的是，这种肺部疾病可以通过基因补充疗法来预防。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，这种策略不能阻止肝病的进展。因此，pizz相关肝脏疾病的基因治疗应侧重于在DNA和/或RNA水平上长期消除/纠正突变蛋白。小干扰RNA （siRNA）是抑制PiZZ的一种很有前途的方法，基于DNA载体的siRNA可以将这种效果延长数年（Aim 1）。腺相关病毒2 （AAV2）载体已被用于治疗AAT缺乏症的前期和临床试验。在肝脏中，重组(r) AAV2转导诱导持续的基因表达；然而，即使在高颗粒细胞比下，也只有5-10%的肝细胞被转导。包括我们在内的最近研究表明，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。为了进一步增强这种转导，我们开发了一种包装双链基因组的新型AAV载体。这些载体绕过第二链合成的限速步骤，从而增加和提前转基因表达（比传统的单链（ss）载体早两周）。特别是在肝脏中，我们已经能够证明dsAAV 2的转导率高达90%，dsAAV 8的转导率超过95%，使用较低的总剂量。尽管在AAV转导肌肉的血液中可以检测到AAT的表达，但肝脏是产生AAT的天然器官，并且在AAV转导的肝脏中观察到比肌肉更高的转基因表达。因此，抑制PiZZ基因表达以及成功的基因添加策略应该可以分别消除肝脏和肺部的疾病表现。为了避免siRNA/PiZZ诱导的wtAAT mRNA降解，并增加AAT的总体合成，我们将基于遗传密码的简并性创建一个优化的AAT基因（目的2）。值得注意的是，虽然AAV2和AAV8转导肝脏的效率很高，但总体上的向性是广泛的，当然并不局限于肝细胞。细胞特异性转导需要几个步骤，包括病毒结合、内吞、运输和剥离，我们已经开始使用洗刷的血清型衣壳文库将结构与功能联系起来。根据我们的初步数据，有可能进化出一种AAV实验室菌株，它可能具有优越但有限的肝脏转导，以减少siRNA在其他组织中的“脱靶”效应。这也表明可以确定负责有效肝脏转导的特定衣壳结构域（Aim 3）。

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.