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Updated: Sep 5, 2025
Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus
Published on: May 13, 2021
Evaluation of parameters for efficient purification and long-term storage of herpes simplex virus-based vectors
Seiji Kuroda1, Yoshitaka Miyagawa1, Makoto Sukegawa1,2
1Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan.
Abstract:
Replication competent oncolytic herpes simplex virus (HSV) vectors have been used extensively to treat solid tumors with promising results. However, highly defective HSV vectors will be needed for applications that require sustained therapeutic gene expression in the absence of vector-related toxicity or inflammation. These vectors require complementing cell lines for their manufacture, creating significant challenges to achieve high yields of infectious virus particles. We recently described an improved upstream process for the production of a non-cytotoxic HSV vector for gene therapy applications. Here, we sought to optimize the downstream conditions for purification and long-term storage of the same vector, JΔNI5. We compared different methods to remove cellular impurities and concentrate the vector by monitoring both physical and biological titers, resulting in the establishment of optimal conditions for vector production. To optimize the long-term storage parameters for non-cytotoxic HSV vectors, we evaluated vector stability at low temperature and sensitivity to freeze-thaw cycles. We report that suboptimal purification and storage methods resulted in loss of vector viability. Our results describe effective and reproducible protocols for purification and storage of HSV vectors for pre-clinical studies.
Insights
Optimizing purification and storage for defective herpes simplex virus (HSV) vectors is crucial for gene therapy. This study establishes effective protocols to ensure vector viability and yield for pre-clinical applications.
Area of Science:
- Gene Therapy
- Virology
- Bioprocessing
Background:
- Replication-competent oncolytic herpes simplex virus (HSV) vectors show promise for solid tumor treatment.
- Highly defective HSV vectors are required for sustained gene expression without toxicity, necessitating complementary cell lines for production.
- Manufacturing these defective vectors presents challenges in achieving high yields of infectious particles.
Purpose of the Study:
- To optimize downstream purification and long-term storage conditions for a non-cytotoxic HSV vector (JΔNI5).
- To establish reproducible protocols for producing viable HSV vectors for pre-clinical studies.
Main Methods:
- Compared various methods for cellular impurity removal and vector concentration, assessing physical and biological titers.
- Evaluated vector stability at low temperatures and sensitivity to freeze-thaw cycles to determine optimal storage parameters.
- Focused on optimizing downstream processing for a previously improved upstream process for JΔNI5 vector production.
Main Results:
- Established optimal conditions for purification and concentration, balancing impurity removal with vector yield.
- Identified key parameters for long-term storage, including temperature and freeze-thaw cycle resistance.
- Demonstrated that suboptimal purification and storage significantly reduce vector viability.
Conclusions:
- Effective and reproducible protocols for the purification and storage of non-cytotoxic HSV vectors have been developed.
- These optimized protocols are essential for maintaining vector viability and ensuring successful pre-clinical studies.
- The findings address critical manufacturing challenges for defective HSV vectors used in gene therapy applications.
