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Updated: Jul 4, 2026
Measurement of γHV68 Infection in Mice
Published on: November 22, 2011
Quantitative analysis of clinically relevant mutations occurring in lymphoid cells harboring gamma-retrovirus-encoded
X Wang1, M Olszewska, V Capacio
1Gene Transfer and Somatic Cell Engineering Facility, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Abstract:
The in vivo regulation of T lymphocyte activity by the activation of a suicide mechanism is an essential paradigm for the safety of adoptive cell therapies. In light of reports showing that gamma-retroviral vector-encoded herpes simplex virus thymidine kinase (hsvtk) undergoes recombination, we undertook a thorough investigation of the genomic stability of SFG-based vectors using two variants of the wild-type hsvtk gene. In a large panel of independent clones, we demonstrate that both hsvtk genes undergo recombination with molecular signatures indicative of template switching in GC-rich regions displaying homology at the deletion junctions or RNA splicing. In the absence of ganciclovir selection, the frequency of recombination is 3% per retroviral replication cycle. Our results underscore the importance of the five nucleotide difference between the two hsvtk genes that account for the presence of recombinogenic hot spots in one variant and not the other, indicating that the probability of RNA splicing is influenced by minute nucleotide changes in sequences adjacent to the splice donor and acceptor sites. Furthermore, our mutational analysis in an unbiased panel of human lymphoid cells (that is, without immune or ganciclovir-mediated selective pressure) provides a robust in vitro assay to predict and quantify clinically relevant mutations in hsvtk suicide genes, which can be applied to studying and improving the stability of any transgene expressed in gamma-retroviral or lentiviral vectors.
Insights
Genomic instability in herpes simplex virus thymidine kinase (hsvtk) genes used in adoptive cell therapy can lead to recombination. This study reveals hot spots and influences of nucleotide changes on gene stability, crucial for therapeutic safety.
Area of Science:
- Gene therapy
- Molecular biology
- Immunology
Background:
- Adoptive cell therapies rely on T lymphocyte regulation via suicide mechanisms for safety.
- Previous reports indicated recombination issues with gamma-retroviral vector-encoded herpes simplex virus thymidine kinase (hsvtk).
Purpose of the Study:
- To investigate the genomic stability of SFG-based vectors encoding two variants of the hsvtk gene.
- To identify molecular signatures and frequencies of recombination in hsvtk genes.
- To develop a predictive in vitro assay for clinically relevant mutations.
Main Methods:
- Analysis of a large panel of independent clones expressing hsvtk variants.
- Investigation of recombination signatures, including template switching and RNA splicing.
- Mutational analysis in an unbiased panel of human lymphoid cells without selective pressure.
Main Results:
- Both hsvtk variants showed recombination with signatures of template switching and RNA splicing.
- Recombination frequency was approximately 3% per retroviral replication cycle without ganciclovir selection.
- A five-nucleotide difference between hsvtk variants influenced recombinogenic hot spots and RNA splicing probability.
Conclusions:
- The genomic stability of hsvtk genes is susceptible to recombination, influenced by sequence homology and minor nucleotide changes.
- A robust in vitro assay was developed to predict and quantify mutations in hsvtk suicide genes.
- Findings are critical for improving the safety and stability of transgenes in gamma-retroviral and lentiviral vectors for cell therapies.
