Novel strategy to block Nabs for AAV gene delivery

阻断 Nabs 进行 AAV 基因传递的新策略

基本信息

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
项目类别：
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

项目摘要

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 AAV 载体更广泛应用于患者的主要问题之一是在一般人群中，超过 95% 的人患有中和抗体 (Nab)。被 AAV 感染的人中，平均约有 50% 感染了 Nab。探索逃避 AAV Nabs，包括用 PEG 或外泌体、不同血清型的 AAV 进行表位掩蔽载体、衣壳原位的合理设计和组合诱变，以及生物耗竭 Nab（空衣壳利用、B 细胞去除、血浆分离和 IgG 裂解酶）。这些方法效率较低或有副作用或AAV趋向性改变，因此势在必行。制定逃避 Nabs 的理想策略，但不改变衣壳工程或组织向性最近，我们开发了一种独立于载体的药物治疗的副作用。基于蛋白质的策略普遍阻断 Nab，并证明该方法可有效对抗通过使用独特的支原体衍生蛋白，可实现广泛的预先存在的 Nab 浓度，并且类似物，称为 Protein-M，Protein-M 能够与任何物种的免疫球蛋白相互作用。我们发现蛋白质 M 在体外可保护 AAV 载体中和超过 100 倍。过继转移 Nab 阳性血清的小鼠中的 1000 倍，迄今为止，最有效的逃避策略然而，AAV 蛋白 M 在体温 (37°C) 下结构不稳定，使其体温下解折叠的蛋白质可能与临床应用的减少相对应。体内使用蛋白时的Nab阻断为了提高PM稳定性，我们合理设计了。计算机上包含 150 个单独的蛋白质突变体，经过工程改造，可提高热稳定性至少 10 个突变体。在本提案中，我们将进一步确定其具有增强的热稳定性和高生物活性。表征这些突变体在体外以及在过继转移 Nab 阳性血清的小鼠中的功效（目标 1）接下来，我们将研究蛋白 M 在预免疫小鼠中重新给药中的作用（目标 2）。我们将继续开发疾病模型来测试最佳蛋白 M 变体阻断 AAV Nab 的能力通过 AAV 载体介导的基因传递对动物进行表型校正（目标 3）。制定更有效的策略，在未来使用 AAV 载体对患者进行基因治疗时规避 Nab 活性与纳布斯。