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，但更广泛的AAV载体应用于患者的主要问题之一是 中和抗体(NAB)患病率高。在一般人类人口中，超过95%的人有 感染了甲型流感病毒，平均约有50%的人感染了新城疫。已经有几种方法 探索规避AAV的NAb，包括用聚乙二醇或外切体掩蔽表位，不同血清型的AAV 载体、衣壳的合理设计和组合诱变以及生物耗竭 NABS(空衣壳利用、B细胞耗尽、血浆置换和免疫球蛋白裂解酶)。一般来说， 这些方法效率低、副作用大或AAV趋向性改变。因此，迫切需要 制定理想的策略来躲避NAB，但不会因衣壳工程或 药物治疗的负面副作用。最近，我们开发了一种不依赖于载体的 基于蛋白质的策略来普遍阻止NAB，并证明了这种方法对 通过使用一种独特的支原体来源蛋白和它的 类似物，称为蛋白质-M。蛋白-M能够与任何物种的免疫球蛋白相互作用，而不需要 对抗原的依赖。我们发现，蛋白M在体外保护AAV载体的中和能力超过100倍 而在1000倍的小鼠体内过继转移NAB阳性血清，是迄今为止最有效的规避策略 AAV NAB。然而，野生型蛋白M在体温(37摄氏度)下结构不稳定，使其 对临床应用具有挑战性。在体温下展开的蛋白质可能对应于 在体内使用该蛋白时，NAB会被阻断。为了提高PM的稳定性，我们合理地设计了 这是一个由150个单独的蛋白质突变体组成的库，这些突变体是为提高热稳定性而设计的。至少10个突变体 具有较高的热稳定性和较高的生物活性。在这项建议中，我们将进一步 这些突变体在体外和过继转移NAB阳性血清小鼠中的作用 (目标1)。接下来，我们将研究蛋白M在免疫前小鼠再次接种AAV中的作用(目标2)。然后 我们将前进到疾病模型来测试最好的蛋白M变体在体内阻断AAVNAb的能力 AAV载体介导的基因传递用于表型纠正的动物(目标3)。长期目标是 开发一种更有效的策略来规避未来AAV载体在患者基因治疗中的NAB活性 和Nabs在一起。

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.