A novel producer cell line for more efficient manufacturing of viral vector systems

ABSTRACT Significant advances in the specific targeting of delivery vectors and the increased therapeutic efficacy of such vectors for gene delivery have been made, stimulating major interest in the development and commercialization of therapeutic products focused on gene therapy indications. CLAIRIgene LLC focused on the development and optimization of various viral gene-to-cell transfer delivery platforms targeted a broad range of applications, including CRISPR/Cas9 systems. Of the gene therapy products in development, we focused on recombinant Adeno-Associated Virus (AAV)-based vectors and Integrase-Deficient Lentiviral Vectors (IDLVs), as they show the greatest potential for delivery in gene therapy indications. Both systems operate at transient “hit-and-run” mode, associated with minimal integration levels, which substantially reduces the risk of insertional mutagenesis, oncogenesis and other associated toxicities, therefore is highly advantageous for gene therapy applications. Furthermore, we recently evolved these non-integrating vectors onto efficient and safe delivery vehicles for gene-to-cell transfer of CRISPR/Cas9 components into various primary cells and tissues, including post-mitotic neurons in vitro and in vivo. Notwithstanding these advantages, episomal vectors, including IDLV and AAV expressed at relatively low levels, as their genomes are packaged into heterochromatin structures, largely inaccessible by transcription machinery. Consistently, we previously demonstrated that histone deacetylase inhibitors (HDACsi) added during vector production step can dramatically increase its titer and transduction efficiency. Nevertheless, most small-molecule-based HDAC inhibitors are non-selective, and evidently demonstrate high-level of undesirable off-target activities, including cell cycle arrest, changes in cellular differentiation and apoptotic signaling. Therefore, more selective and targeted strategies aimed to downregulate expression of HDAC-of-interest are needed. The current proposal is based on our preliminary data demonstrated that class I of the HDAC family HDAC8 could play an important role in the regulation of life cycle of episomal vectors, including AAV and IDLV. We have shown that vector- producer cells, human embryonic kidney HEK293T knockouted for HDAC8 support the improved packaging and titer of the vectors. In the current proposal, we aim to create (i) a stable, monoclonal producer HDAC8-/- HEK293 cell line for more efficient production of AAV and IDLV vectors. Following characterization and optimization, we aim to certify the product for a range of gene therapy applications using viral vectors as delivery systems. To evaluate whether the developed cells could be used universally for production of other non-integrating systems, we will aim in the future testing them for manufacturing adenovirus (Ad), herpes simplex virus type I (HSV-1), and non- human lentiviral vectors. The growing pipeline of cell and gene therapies has led to a substantial increase in demand to high quality gene delivery vehicles; here, we believe that the improved producer cells will create a significant business opportunity for us to support this demand.