vRNAs found to play important role in lentiviral production
Lentiviral vector production is a key bottleneck in the large-scale production of gene-edited therapies, and new research has uncovered new insight into the mechanisms at work at each stage of the product process.
Scientists in California have uncovered new insight into the importance of viral RNA (vRNA) in the production of lentiviral vectors (LVs), which are important for delivering genetic material in the production of gene-edited cells. The team, from UCLA and The Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research (both CA, USA), were studying the mechanisms of titer and defective gene transfer of complex LVs that negatively impact the efficiency of the production process.
In the research, presented today at the American Society of Gene & Cell Therapy Annual Meeting (29 April–May 2, Washington, DC, USA), vRNA transcription efficiency was characterized during the production of a ‘well-behaved’ vector, EFS-ADA (4 kb), and a poorly-performing vector, CCL-βAS3 (8.7 kb). During production of CCL- βAS3, which has a low titer and defective gene transfer, vRNAs were found to be significantly more truncated. In CCL- βAS3, only 9.3% of vRNA was complete, which can be fully reverse transcribed and integrated into the host cell genomes, compared to 75% for EFS-ADA.
However, titers could be improved by increasing the level of complete vRNA LVs with Tat, an HIV accessory protein that enhances the processivity of RNA Pol II. Knocking out protein kinase R (PKR) also rescued translation of viral proteins, improving the titers of vectors with reverse-orientation gene cassettes by 5-7-fold.
Concluding their abstract, the authors commented: “Our findings uncover an important role of complete vRNAs as substrates for each step of lentiviral lifecycle, and reveal that knocking out PKR abolishes the antiviral response against vectors in reverse orientation.
“Current studies are underway to increase the production of complete vRNA by deleting the problematic sequences or manipulating cellular factors.”
Source: Han J, Tam K, Aleshe B, Hollis R, Kohn D. Developing Strategies to Improve the Titers and Gene Transfer of Complex Lentiviral Vectors. Presented at: American Society of Gene & Cell Therapy Annual Meeting. Washington, DC, USA. 2019