Efficient Strategy for Synthesizing Vector-Free and Oncolytic Herpes Simplex Type 1 Viruses

Area of Science:

• Virology
• Molecular Biology
• Gene Synthesis

Background:

• Viral genome synthesis is crucial for virology research, vaccine development, and antiviral drug discovery.
• Herpes Simplex Virus type 1 (HSV-1) is a large DNA virus utilized in oncolytic virotherapy.
• Previous HSV-1 genome synthesis methods were inefficient and incorporated vector sequences impacting replication.

Purpose of the Study:

• To develop an efficient, vector-free strategy for the synthesis and rescue of synthetic HSV-1.
• To generate a synthetic HSV-1 virus with similar replication and morphogenesis to the parental strain.
• To engineer an oncolytic HSV-1 variant for cancer therapy.

Main Methods:

• Utilized transformation-associated recombination (TAR) in yeast to synthesize overlapping HSV-1 fragments.
• Linearized and co-transfected yeast-synthesized fragments into mammalian cells for virus rescue.
• Employed in vitro CRISPR/Cas9 gene editing to delete specific viral genes (ICP6, ICP34.5, ICP47).

Main Results:

• Successfully generated a synthetic HSV-1 strain (F-Syn) using the vector-free strategy.
• F-Syn exhibited comparable replication dynamics and morphogenesis to the wild-type parental virus.
• Created an engineered oncolytic HSV-1 variant (F-Syn-O) demonstrating dose-dependent cytotoxic effects on human tumor cell lines in vitro.

Conclusions:

• The developed vector-free synthesis and rescue strategy facilitates convenient and systemic manipulation of HSV-1 genomes.
• This approach is applicable to the design and construction of novel oncolytic herpesviruses.
• The engineered oncolytic HSV-1 (F-Syn-O) shows promise for cancer virotherapy applications.