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 g-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 ganciclovirmediated 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 g-retroviral or lentiviral vectors.

Chimeric Antigen Receptors: A Cell and Gene Therapy Perspective

“…[60][61][62][63] Another feature of retroviral vectors is their mutation rate incurred during reverse transcription, which is about 5-fold higher for HIV-1 reverse transcriptase compared with that of Moloney murine leukemia virus (Mo-MLV). [64][65][66][67][68][69] DNA Transposons g-retroviral and lentiviral vectors are complex biological reagents that require expensive biosafety testing and storage. Non-viral approaches would be advantageous, provided that they were as effective.…”

Immunotherapeutic Approaches in Prostate Cancer: Combinations and Clinical Integration

“…Our group has shown that the zeta chain-based CARs could induce strong activation capable of sustaining T-cell proliferation and permitting secondary antigenic restimulation in vitro provided that antigen was presented in the context of CD28-mediated costimulation. 55,59,60 In an effort to determine if T cells-particularly human T cells-expanded in this manner could mediate tumor eradication in vivo and if further in vivo costimulation would be needed to sustain their function, three tumors models using severe combined immunodefıciency-beige/ beige mice were developed that showed that PSMA-targeted T cells could effectively eliminate prostate cancer. T cells were transduced with Pz1, a CAR-targeting human PSMA.…”