Effective Suicide Gene Therapy for Leukemia in a Model of Insertional Oncogenesis in Mice

Abstract

The safety of gene therapy using hematopoietic stem cells may be increased by including a suicide gene in the therapeutic vector to eliminate adverse events like insertional oncogenesis while retaining the clinical benefits. We have developed a model of experimental insertional oncogenesis by transducing the murine factor-dependent leukemia cell line Ba/F3 with a bicistronic Moloney murine leukemia virus retroviral vector encoding a murine oncogene (cKit(D814V)) in addition to one of three suicide genes: Herpes simplex virus thymidine kinase (HSV-TK); SR39, an HSV-TK mutant with an increased affinity for the drug substrate Ganciclovir (GCV); or sc39, a splice-corrected version of SR39. Following intravenous challenge with transduced Ba/F3 clones and treatment with GCV, leukemia developed in mice given cells expressing HSV-TK, but not SR39 or sc39. In vitro GCV resistance was observed in heterogeneously transduced Ba/F3 pools at 2.5-14%, and single-nucleotide changes or partial loss of the suicide gene were identified as mechanisms of drug escape. However, GCV treatment resulted in 80-100% survival of mice challenged even with pools of partially resistant Ba/F3 cells expressing SR39 or sc39. Thus, in this model of vector-driven insertional oncogenesis, a suicide gene approach was effective for eliminating leukemia using modified HSV-TK variants with improved biological activity.

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

“…This is consistent with half of the clones bearing more than one copy of the vector per cell (Supplementary Figure S2). Our data in conjunction with that of others 4,5,7,10 demonstrate that the genomic stability of g-retroviral vectors is affected by the vector backbones and within the same g-retroviral vector backbone, by minute sequence variations in transgene sequences. The assay described here can be used not only to determine the unbiased frequency of template switching and other rearrangements occurring in suicide genes, but also to guide the design of improved, genetically stable suicide genes, transgenes and vector backbones.…”

Targeting Lentiviral Vectors to Antigen-Specific Immunoglobulins

“…coding herpes simplex virus type 1 thymidine kinase (HSV1-TK), to xenografted tumors in an immunocompromised mouse model. The cells transduced to express HSV1-TK can be deleted by treatment with the prodrug GCV, which is transformed into a toxic metabolite form by HSV1-TK (Blumenthal et al, 2007). Suicide gene therapy based on HSV1-TK has been evaluated clinically by in situ injection of gammaretroviral vectors into solid tumors (Ram et al, 1997;Satoh et al, 2005).…”

Gene therapy for mucopolysaccharidosis

“…Although tumors due to an AAV vector per se have not been reported in other AAV trials, few have involved neonatal transduction with long-term evaluation. Elements that could insulate adjacent sequences from enhancement [94] or could promote death of the cell if needed [95] are being developed, and could improve the safety of any vector.…”