Novel strategy to block Nabs for AAV gene delivery

阻断 Nabs 进行 AAV 基因传递的新策略

• 批准号: 10416627
• 负责人: Chengwen Li
• 金额: $ 57.88万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10416627
• 关键词: Address; Adoptive Transfer; Animal Disease Models; Animal Model; Animals; Antibodies; Antigens; Automobile Driving; B-Lymphocytes; Binding; Biological; Blindness; Blocking Antibodies; Blood Component Removal; Body Temperature; Canis familiaris; Capsid; Cells; Clinical Research; Clinical Trials; Control Groups; Dependence; Dependovirus; Development; Disease; Disease model; Dose; Engineering; Enzymes; Epitopes; Future; Gene Delivery; Hemophilia A; High Prevalence; Human; Immunization; Immunize; Immunoglobulin G; Immunoglobulins; In Situ; In Vitro; Individual; Intravenous Immunoglobulins; Libraries; Light; Masks; Mediating; Modification; Mus; Mutagenesis; Mycoplasma; Nuclear; Patients; Pharmacological Treatment; Phenotype; Plasma; Population; Protein Conformation; Proteins; Role; Safety; Serotyping; Serum; Surface; Testing; Variant; Virion; adeno-associated viral vector; analog; base; clinical application; combinatorial; disease phenotype; exosome; gene therapy; improved; in silico; in vivo; inhibitor; multiple myeloma M Protein; mutant; neutralizing antibody; novel strategies; prevent; rational design; side effect; therapeutic protein; tissue tropism; trafficking; vector; virus tropism

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.

腺相关病毒（AAV）载体已用于不同疾病患者的临床试验中。 尽管在临床研究方面取得了成功，但针对患者的广泛AAV向量应用的主要问题之一是 中和抗体（NAB）的高患病率。在一般人口中，超过95％的人有 被AAV感染，平均有大约50％的NAB。几种方法已经 探索以逃避AAV NAB，包括用钉或外泌体的表位掩盖，AAV的不同血清型 capsid的载体，理性设计和组合诱变原位，以及生物学耗竭 NABS（空的衣壳利用率，B细胞耗竭，等离子体置换和IgG裂解酶）。一般来说， 这些方法的效率低或副作用或AAV的偏向主义改变。因此，必须 制定理想的策略来逃避NAB，但没有改变Capsid工程或 药物治疗的负面影响。最近，我们开发了一个矢量独立 基于蛋白质的策略，以普遍阻止NABS，并证明这种方法有效反对A 通过使用独特的支原体衍生的蛋白质，广泛存在的NAB浓度 类似物，称为蛋白质。蛋白质M能够与任何物种的免疫球蛋白相互作用 抗原依赖性。我们发现蛋白质M保护了100倍的AAV载体神经化的体外 到目前为止，NAB阳性血清的自适应转移的小鼠中有1000倍，到目前为止，逃避的最有效策略 aav nabs。但是，野生型蛋白M在体温（37°C）上在结构上不稳定，使其成为 在临床应用方面具有挑战性。体温下展开的蛋白质可能与改善 使用体内蛋白质时NAB阻滞。为了提高PM稳定性，我们在 硅库由150个单个蛋白突变体的库，用于改善热稳定性。至少10个突变体 鉴定出增强的热稳定性和高生物学活性。在此提案中，我们将进一步 表征这些突变体在体外和小鼠中具有NAB正序的自适应转移的效率 （目标1）。接下来，我们将研究蛋白M在具有前免疫的小鼠中AAV重新剂量中的作用（AIM 2）。然后 我们将继续前进到疾病模型，以测试最佳蛋白质M变种阻断AAV NABS中的能力 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 制定一种更有效的策略，以逃避患者的AAV载体的未来基因疗法中的NAB活性 与nabs。