Quantitative analysis of clinically relevant mutations occurring in lymphoid cells harboring γ-retrovirus-encoded hsvtk suicide genes Journal Article

Authors: Wang, X.; Olszewska, M.; Capacio, V.; Stefanski, J.; Przybylowski, M.; Samakoglu, S.; Chang, A. H.; Sadelain, M.; Riviere, I.
Article Title: Quantitative analysis of clinically relevant mutations occurring in lymphoid cells harboring γ-retrovirus-encoded hsvtk suicide genes
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 γ-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 γ-retroviral or lentiviral vectors.
Keywords: controlled study; unclassified drug; gene mutation; human cell; mutation; t-lymphocytes; gene expression; cell line; in vitro study; mutational analysis; wild type; genetic vectors; transduction, genetic; gene rearrangement; genetic recombination; genetic engineering; reverse transcriptase polymerase chain reaction; recombination, genetic; genomic instability; quantitative analysis; nucleotide sequence; lentivirus vector; retrovirus vector; transgene; simplexvirus; thymidine kinase; virus replication; clone cells; ganciclovir; rna splicing; rna, viral; retrovirus; antiviral agents; suicide gene; lymphoid cell; herpes simplex virus thymidine kinase; gammaretrovirus; genes, transgenic, suicide
Journal Title: Gene Therapy
Volume: 15
Issue: 21
ISSN: 0969-7128
Publisher: Nature Publishing Group  
Date Published: 2008-11-01
Start Page: 1454
End Page: 1459
Language: English
DOI: 10.1038/gt.2008.103
PUBMED: 18563185
PROVIDER: scopus
PMCID: PMC4528371
Notes: --- - "Cited By (since 1996): 1" - "Export Date: 17 November 2011" - "CODEN: GETHE" - "Molecular Sequence Numbers: GENBANK: AY437640, J02224, V00467;" - "Source: Scopus"
Citation Impact
MSK Authors
  1. Alex Hongsheng Chang
    9 Chang
  2. Michel W J Sadelain
    562 Sadelain
  3. Isabelle C Riviere
    235 Riviere
  4. Xiuyan Wang
    111 Wang