Adenovirus-mediated transfer of shRNA against Elovl6 reduces the progression of hepatocellular carcinoma

Shiau, A L; Su, Y C; Feng, Y H; Huang, Y S; Wu, P; Wu, C L

Abstract

The Factor IX (FIX) gene therapy (BAX335) was produced in small-scale for hemophilia-B patients in clinical phase I/II. In the course of the internal BAX335 process development, the small-scale clinical product was compared to BAX335 developed using a large-scale process. During this comparability study, an additional AAV-subpopulation was identified by analytical ultracentrifugation and found only in the BAX335 produced with the small-scale process. The BAX335 was produced in the HEK293 cell line and was based on triple transient transfection. The small-scale production process employed downstream purification with iodixanol gradient in a benchtop ultracentrifuge. Whereas the large-scale production process employed a proprietary large-scale ultracentrifugation step. The AAV-subpopulation detected in the iodixanol process was separated from the main full AAV fraction and further enriched in order to obtain isolated AAV-subpopulation fraction for analytical characterization (by qPCR, analytical ultracentrifugation, agarose-gel-electrophoresis, in vitro potency [FIX], in vivo potency in FIX ko mice, and SDS-PAGE). The analytical ultracentrifugation of BAX335 from the small-scale process detected the following fractions: full AAV capsids at 80S (sedimentation coefficient), AAV subpopulation at 70S, and empty AAV capsids detected at 50S. The biological activity was evaluated by measuring the in vitro and in vivo potency and was significantly lower in the AAV-subpopulation compared to the full AAV capsids. This AAV-subpopulation with poor activity and can be removed during the downstream process. In contrast to the small-scale iodixanol ultracentrifuge method, this large-scale ultracentrifugation process was shown to be capable of removing the AAV-subpopulations that did not possess the desired biological activity.

Citation

Shiau, A. L., Su, Y. C., Feng, Y. H., Huang, Y. S., Wu, P., & Wu, C. L. (2018, October). Adenovirus-mediated transfer of shRNA against Elovl6 reduces the progression of hepatocellular carcinoma. Paper presented at Changing the Face of Modern Medicine: Stem Cell and Gene Therapy Organized Jointly by the European Society of Gene & Cell Therapy (ESGCT), International Society for Stem Cell Research (ISSCR) and the French Society of Gene and Cell Therapy (SFTCG), Lausanne, Switzerland

• Presentation Conference Type: Conference Paper (unpublished)
• Conference Name: Changing the Face of Modern Medicine: Stem Cell and Gene Therapy Organized Jointly by the European Society of Gene & Cell Therapy (ESGCT), International Society for Stem Cell Research (ISSCR) and the French Society of Gene and Cell Therapy (SFTCG)
• Conference Location: Lausanne, Switzerland
• Start Date: Oct 16, 2018
• End Date: Oct 19, 2018
• Deposit Date: Jun 20, 2023
• Publisher URL: https://www.liebertpub.com/doi/10.1089/hum.2018.29077.abstracts
• Additional Information: Abstract number; P064