Synthetic Neo-Amplicon Production System for High Capacity Therapeutic Gene Delivery

Neochromosome，Inc.机密 项目摘要 基因治疗旨在通过单一治疗提供持久的临床益处，并有可能治愈广泛的 疾病的范围。基于病毒载体的基因递送占当前基因治疗的大部分 临床试验尽管推动了该领域的发展，但这些载体受到小DNA有效载荷（5- 10 kb）的限制， 载体产生、向性、免疫原性和致癌风险。在这里，我们建议开发下一个- 第二代单纯疱疹病毒（HSV）为基础的载体系统，克服了这些限制。我们的目标是 利用HSV扩增子载体，其可与HSV一起编码高达~ 150 kb的设计DNA序列 包装和复制顺式信号。这个简单的设计，消除了所有的病毒蛋白编码基因， 确保这些载体无毒。此外，与其他HSV载体一样，扩增子在染色体外保留一次， 在细胞内，不存在插入突变的风险。虽然HSV扩增子基因治疗载体已经被广泛使用， 经过多年的开发，它们的使用受到安全问题和制造问题的限制。更 具体地说，所有病毒基因的缺乏需要使用辅助病毒进行扩增子包装;迄今为止， 还没有实现完全排除辅助病毒污染的扩增子包装系统。这里 我们建议建立扩增子包装细胞系，用于可靠地生产100%纯的扩增子载体 没有辅助基因序列的群体。我们将证明扩增子载体的重复传代， 产生高滴度储备液，同时保持100%纯度。我们的设想是，大的有效载荷扩增子介导的 基因递送将使得能够使用包括内含子和转录调控基因在内的全长人类基因座。 序列，允许治疗基因表达的自然空间和时间控制。高容量 可容纳大的和多基因盒的载体，如HSV-1衍生系统，代表了 未来的基因治疗工具，并将是必不可少的治疗复杂的遗传疾病，特别是在 个脑袋

Neochromosome, Inc., Confidential PROJECT SUMMARY Gene therapy aims to provide durable clinical benefit with a single treatment and has potential to cure a broad range of disease conditions. Viral vector-based gene delivery accounts for the majority of current gene therapy clinical trials. Despite advancing the field, these vectors are limited by small DNA payloads (5-10kb), difficult vector production, tropism, immunogenicity and risk of carcinogenesis. Here we propose to develop a next- generation herpes simplex virus (HSV)-based vector system that overcomes these limitations. We aim to leverage HSV amplicon vectors, which can encode up to ~150kb of designer DNA sequence together with HSV packaging and replication cis-signals. This simple design, which eliminates all viral protein-coding genes, ensures these vectors are non-toxic. Further, like other HSV vectors, amplicons remain extrachromosomal once inside cells and pose no risk of insertional mutagenesis. While HSV amplicon gene therapy vectors have been under development for years, their use has been limited by safety concerns and manufacturing issues. More specifically, the absence of all viral genes necessitates the use of helper virus for amplicon packaging; to date an amplicon packaging system that completely excludes helper virus contamination has not been achieved. Here we propose to build an amplicon packaging cell line for reliable production of 100% pure amplicon vector populations devoid of helper gene sequences. We will demonstrate repeated passaging of amplicon vectors to generate high titer stocks while maintaining 100% purity. Our vision is that large payload amplicon-mediated gene delivery will enable the use of full-length human gene loci including introns and transcriptional regulatory sequences, allowing for natural spatial and temporal control of therapeutic gene expression. High capacity vectors that can accommodate large and multigene cassettes, such as HSV-1 derived systems, represent the future of gene therapy tools and will be essential to the treatment of complex genetic diseases particularly in the brain.