Novel strategy to block Nabs for AAV gene delivery

腺相关病毒（AAV）载体已应用于不同疾病患者的临床试验。 尽管在临床研究中取得了成功，但对患者更广泛地应用AAV载体的主要关注之一是 中和抗体（Nab）流行率高。在一般人群中，超过95%的个体 被AAV感染，平均约50%的人有Nabs。已经采取了几种方法 探索了逃避AAV Nab的方法，包括用PEG或外泌体掩蔽表位，不同的AAV血清型 载体，衣壳的合理设计和原位组合诱变，以及生物耗竭， Nabs（空衣壳利用、B细胞耗竭、血浆单采术和IgG裂解酶）。一般而言， 这些方法具有低效率或副作用或AAV向性改变。因此，当务之急是 开发出逃避Nabs的理想策略，但不改变来自衣壳工程的组织向性， 药物治疗的副作用。最近，我们开发了一种矢量独立 基于蛋白质的策略来普遍阻断Nabs，并证明这种方法对抗Nabs有效。 广泛的预先存在的Nab浓度，通过使用独特的支原体衍生蛋白， 类似物，称为蛋白质-M。蛋白-M能够与来自任何物种的免疫球蛋白相互作用， 抗原依赖性我们已经发现蛋白M在体外保护AAV载体中和超过100倍 和1000倍的小鼠过继转移的Nab阳性血清，到目前为止，最有效的策略，以逃避 AAV Nabs.然而，野生型蛋白M在体温（37°C）下结构不稳定，使得其 临床应用具有挑战性。蛋白质在体温下解折叠可能对应于减少 当在体内使用蛋白质时的Nab阻断。为了提高永磁同步电机的稳定性，我们对永磁同步电机进行了合理的设计， silico是一个150个单独的蛋白质突变体的文库，这些突变体被工程化以提高热稳定性。至少有10个变种人 被鉴定为具有增强的热稳定性和高生物活性。在这一建议中，我们将进一步 表征这些突变体在体外和具有Nab阳性血清过继转移的小鼠中的功效 (Aim 1）。接下来，我们将研究蛋白M在预先免疫的小鼠中AAV再给药中的作用（目的2）。然后 我们将进一步研究疾病模型，以测试最佳蛋白M变体阻断AAV Nab的能力。 用AAV载体介导的基因递送进行表型校正的动物（Aim 3）。长期目标是 开发一种更有效的策略，以避免在未来的基因治疗中使用AAV载体在患者中的Nab活性 关于Nabs

Adeno-associated virus (AAV) vectors have been applied in clinical trials in patients with different disorders. Although successful in clinical studies, one of major concerns for broader AAV vector application for patients is high prevalence of neutralizing antibody (Nab). In the general human population, over 95% of individuals have been infected by AAV and, on average, approximate 50% of them have Nabs. Several approaches have been explored to evade AAV Nabs, including epitope masking with PEG or exosome, different serotype of AAV vector, rational design and combinatorial mutagenesis of the capsid in situ, as well as biological depletion of Nabs (empty capsid utilization, B cell depletion, plasma-apheresis and IgG cleavage enzymes). Generally, these approaches have low efficiency or side effects or AAV tropism change. Therefore, it is imperative to develop ideal strategies to evade Nabs, but without a change in tissue tropism from capsid engineering or negative side effects from pharmacological treatment. Recently, we have developed a vector independent protein based strategy to universally block Nabs and demonstrated that this approach is effective against a broad range of pre-existing Nab concentrations by use of a unique mycoplasma derived protein and it’s analogues, termed Protein-M. Protein-M is able to interact with immunoglobulin from any species without antigen dependence. We have found that protein M protected AAV vector neutralization over 100 fold in vitro and 1000 fold in mice with adoptive transfer of Nab positive serum, so far, the most effective strategy to evade AAV Nabs. However, the wild type protein M is structurally unstable at body temperature (37°C), making it challenging for clinical application. The protein unfolding at body temperature may correspond with decreased Nab blockade when using the protein in vivo. In order to improve PM stability, we have rationally designed in silico a library of 150 individual protein mutants engineered for improved thermal stability. At least 10 mutants were identified with enhanced thermal stability and high biological activity. In this proposal, we will further characterize the efficacy of these mutants in vitro and in mice with adoptive transfer of Nab positive serum (Aim 1). Next, we will study the role of protein M in AAV re-dosing in mice with pre-immunization (Aim 2). Then we will move forward to a disease model to test the ability of the best protein M variants to block AAV Nabs in animals for phenotypic correction with AAV vector mediated gene delivery (Aim 3). The long term objective is to develop a more effective strategy to evade Nab activity in future gene therapy with AAV vectors in patients with Nabs.