Tuning VSV-G Expression Improves Baculovirus Integrity, Stability and Mammalian Cell Transduction Efficiency

Area of Science:

• * Molecular biology
• * Virology
• * Gene therapy

Background:

• * Baculoviral vectors (BVs) from *Autographa californica* multiple nucleopolyhedrovirus (AcMNPV) are effective for multigene delivery in mammalian cells, crucial for technologies like CRISPR.
• * Vesicular stomatitis virus G-protein (VSV-G) pseudotyping enhances BV endosomal escape, typically driven by the polH promoter.
• * High VSV-G expression via polH leads to BVs with reduced stability, impaired morphology, and toxicity in mammalian cells.

Purpose of the Study:

• * To investigate alternative viral promoters for optimizing VSV-G expression levels on pseudotyped BVs.
• * To overcome the drawbacks associated with polH-driven VSV-G hyperexpression in BVs.
• * To assess the impact of modulated VSV-G expression on BV integrity and gene delivery efficiency.

Main Methods:

• * Explored five alternative viral promoters to control VSV-G expression in BVs.
• * Quantified VSV-G expression levels relative to the polH promoter.
• * Evaluated BV morphology, stability, and transduction efficiency in human cell lines.
• * Assessed CRISPR-mediated gene editing capabilities of BVs with varying VSV-G levels.

Main Results:

• * Orf-13 and Orf-81 promoters reduced VSV-G expression to <5% of polH levels.
• * This reduction rescued BV morphology and stability, mitigating polH-induced issues.
• * BVs with reduced VSV-G demonstrated efficient gene delivery and CRISPR editing, comparable to polH VSV-G BVs.
• * Modulated VSV-G expression improved BV integrity and structural stability.

Conclusions:

• * VSV-G hyperexpression is unnecessary for efficient BV transduction in mammalian cells.
• * Utilizing alternative promoters like Orf-13 and Orf-81 optimizes VSV-G levels, enhancing BV performance.
• * Reduced VSV-G expression offers a strategy for improved BV stability and gene editing efficacy.