Optimized Production and Validation of Rationally Designed AAV Vectors for Cockayne Syndrome Gene Therapy

用于科凯恩综合征基因治疗的合理设计的 AAV 载体的优化生产和验证

• 批准号: 10413533
• 负责人: George V Aslanidi
• 金额: $ 60万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10413533
• 关键词: Optimized; Production; Validation; Rationally; Designed

Project Summary- Abstract. Our current proposal is focusing on the production of novel AAV-based gene therapy for ultra-rare Cockayne Syndrome (CS). CS is affected both males and females in equal numbers with the incidence of less than 1 case per 250,000 live births in the U.S. The existing therapies are manly supportive rather than curative, and in severe cases, patients usually do not survive past childhood. The mutations in the ERCC8 (CSA)/ERCC6 (CSB) genes causing disease have been already identified however no disease altering gene therapies are available. On the other hand, Adeno-associated virus (AAV) has been widely acknowledged as a safe and effective clinical-stage vector for gene therapy for a broad spectrum of inherited diseases, and most recently, AAV-based treatments for inherited retinal dystrophy and spinal muscular atrophy (SMA) were approved by the US FDA and became available for patients. Thus our interdisciplinary team of basic, translational and clinical scientists propose to design a continuous AAV production and purification process prototype to address the needs for novel CS gene therapy. To achieve that goal we, first, will collect clinical data and confirmation of mutations. We also will evaluation of the prevalence of serotype-specific anti-AAV neutralizing antibodies (NA) to identify AAV serotype(s) with the potential to be used in the vast majority of CS patients. Second, optimize AAV capsid and expression cassette to ensure robust therapeutic C S A gene expression and test novel vectors on an animal model of CS in vivo. Finally, we will test centrifugation-free continuous AAV production protocol based on c ross-flow filtration followed by dual- chromatography purification. Our robust quality control procedures included state-of-art methods such as cryo- EM discrimination of full and empty AAV capsids with high accuracy and novel noninvasive in vivo tracking of AAV distribution. In summary, our proposal will result in a reliable protocol for AAV purification and quality control which can be easily implemented in GMP applications and ultimately used for AAV-mediated CS gene therapies in the clinic.

项目摘要-摘要。 我们目前的建议是集中在生产新的基于AAV的基因治疗超罕见的科凯恩 综合征（CS）。CS在男性和女性中的发病率相同，发病率低于1例 现有的治疗方法是男性支持性的，而不是治疗性的，在严重的情况下， 在某些情况下，患者通常不能活过童年。ERCC 8（CSA）/ERCC 6（CSB）基因突变 已经确定了致病基因，但是没有可用的改变疾病的基因疗法。上 另一方面，腺相关病毒（Adeno-associated virus，AAV）作为一种安全有效的临床治疗手段， 用于广谱遗传性疾病的基因治疗的载体，以及最近的基于AAV的治疗 用于遗传性视网膜营养不良和脊髓性肌萎缩症（SMA）的药物获得美国FDA批准， 可供患者使用。 因此，我们的跨学科团队的基础，翻译和临床科学家建议设计一个连续的AAV 生产和纯化工艺原型，以满足新型CS基因治疗的需求。实现这一 我们的目标是，首先，收集临床数据和确认突变。我们还将评估 用于鉴定可能使用的AAV血清型的亚型特异性抗AAV中和抗体（NA） 在绝大多数CS患者中。第二，优化AAV衣壳和表达盒，以确保稳定性。 治疗性CS A基因表达和在体内CS动物模型上测试新载体。最后我们 将测试基于交叉流过滤的无离心连续AAV生产方案， 双层析纯化。我们强大的质量控制程序包括最先进的方法，如 高准确性和新型非侵入性活体内完整和空AAV衣壳的冷冻- EM鉴别 跟踪AAV分布。总之，我们的提议将产生用于AAV纯化的可靠方案， - 质量控制，其可以容易地在GMP应用中实施，并最终用于AAV介导的CS 基因治疗在临床上的应用