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

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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.