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Gene delivery using helper virus-free HSV-1 amplicon vectors
Andrea S Laimbacher1, Cornel Fraefel
1University of Zurich, Zurich, Switzerland.
Current Protocols in Neuroscience
|July 4, 2012
Summary
Herpes simplex virus type 1 (HSV-1) amplicon vectors can be produced free of helper virus. This method uses packaging-defective HSV-1 genomes to package amplicon DNA, ensuring pure vector stocks for research.
Area of Science:
- Molecular Biology
- Virology
- Gene Therapy
Background:
- Herpes simplex virus type 1 (HSV-1)-based amplicon vectors require helper functions for replication and packaging.
- Traditional methods rely on replication-defective or packaging-defective HSV-1 mutants.
Purpose of the Study:
- To describe a method for producing HSV-1 amplicon vectors free of contaminating helper virus.
- To enable efficient packaging of amplicon DNA using modified HSV-1 genomes.
Main Methods:
- Cotransfection of amplicon DNA and cosmid/BAC DNA (representing the HSV-1 genome with deleted packaging signals) into 2-2 cells.
- Cationic liposome-mediated transfection.
- Harvesting of packaged amplicon vector particles.
Main Results:
- Reconstituted viral genomes lacking DNA cleavage/packaging signals provide helper functions but are not packageable.
- This results in stocks of packaged amplicon vectors essentially free of helper virus.
- Protocols for DNA preparation and titer determination are provided.
Conclusions:
- This method allows for the generation of high-titer, helper-virus-free HSV-1 amplicon stocks.
- The described technique is valuable for applications requiring pure amplicon vectors, such as gene therapy and research.
