INTRODUCTION
UCLA researchers in the Department of Microbiology, Immunology, & Molecular Genetics have found that the knockout of four host cell antiviral factors could increase lentiviral titers, allowing the potential to reduce common off target toxicities of gene therapy while lowering the cost of treatment manufacturing.
BACKGROUND
Gene therapy holds the promise of providing lasting therapies or cures for diseases thought to be previously untreatable. In gene therapy, identified defective genes are corrected though viral vector-mediated gene addition or gene editing. A commonly utilized pathway for gene therapy is through the use of lentiviral vectors with autologous hematopoietic stem cells (HSCs). The HSCs are harvested from patients, then grown in culture with a lentiviral vector to transduce and produce gene-modified HSCs that can be reinfused to the patient to produce a therapeutic response. This pathway is often begun with cell culture lines like HEK293T cells as vector packaging cells, to both reduce manufacturing costs and increase transfection efficiency. While mounting clinical success has been found for several diseases through this pathway, the use of complex vectors for inserting larger genes (such as beta-globin) leads to reductions in the amounts of vector made (known as titer). As a consequence, poor titer leads to ineffective gene transfer to HSCs and he high amounts of vector needed per patient dose are often restrictive in manufacturing cost: stagnating the production of a wide catalogue of gene therapies. Therefore, a current unmet need exists to identify a universal methodology that could increase the titer of lentivirus constructs for the purpose of extending gene therapy to more diseases.
INNOVATION
Dr. Kohn and colleagues in the Department of Microbiology, Immunology, & Molecular Genetics at UCLA have identified four host cell antiviral factors, in the virus production cell line, HEK293T cells. These factors regulate the DNA damage response pathway, transcription, and innate immunity in the host cell. As a model system, the researchers used a lentiviral vector for sickle cell disease and knocked out the four cell host factors for transfected HEK293T cells using CRISPR/Cas9. It was observed that the titer for the lentiviral vector increased 2-4 fold. Importantly, the knockout methodology could be applied with any lentiviral construct, allowing the ability to effectively increase titer. The proposed methodology could therefore allow the ability to increase gene therapy efforts to several diseases, thought to be untreatable with gene therapy.
APPLICATIONS
- Gene Therapy
- Genetic Diseases such as Sickle Cell Disease
- Cancer Immunotherapy
ADVANTAGES
- Decrease the cost of lentivirus production
- Increase the successful rate of gene editing
- Ability to package larger transgenes by the lentiviral vector
STATE OF DEVELOPMENT
The study has shown by knocking out antiviral factors in HEK293T cell line, the titer of B-globin lentivirus for the treatment of sickle cell disease increases 2-4-fold.
Related Papers: Improved lentiviral vector titers from a multi-gene knockout packaging line. Han, Jiaying et al. Molecular Therapy - Oncolytics, Volume 23, 582 – 592. https://www.cell.com/molecular-therapy-family/oncolytics/fulltext/S2372-7705(21)00161-3