A cell line assay to measure insertional mutagenesis by integrating gene therapy vectors
Dr Y Takeuchi/ Prof M Collins
Int. Journal of Exp Pathology
Description of Project:
Retroviral vectors have been successfully used for the correction of inherited immunodeficiencies. However, in two recent clinical gene therapy trials for X-linked Severe Combined Immunodeficiency (SCID-X1), several patients developed leukaemia. This was associated with vector integration near cellular proto-oncogenes, leading to their activation. This is known as insertional mutagenesis. The objective of this work is to develop an in vitro assay to measure the frequency with which retroviral and lentiviral vectors cause insertional mutagenesis and to analyse the mechanisms by which this occurs. In the IL-3-dependent cell line Baf3, the frequency of cytokine-independence following vector transduction is measured. The Bcl15 cell line is derived from Baf3 and over-expresses human Bcl2. The lentivector HV, that has wild type HIV-1 Long Terminal Repeats (LTR) and an internal retroviral promoter driving GFP expression, was tested in this system. One IL-3-independent mutant was obtained in the Baf3 target cell line at a frequency of approximately 1 in 10*9 integrants. In the Bcl15 cell line additional IL-3 independent mutants were obtained with the HV lentivector at the higher frequency of approximately 1 in 2x10*7 integrants. Vector integration sites were analysed. Each of the HV mutants had an independent vector integration site into the first intron of the growth hormone receptor (Ghr) gene, in the same orientation as the Ghr transcript. A hybrid HIV-Ghr transcript is detected in all mutants, which originates from the HIV-1 5’-LTR then splices from the HIV-1 major splice donor to the splice acceptor of Ghr exon 2, which is the 1st coding exon of this gene. The mutants express GHR and grow in response to bovine growth hormone (bGH) in the foetal bovine serum of the culture medium. We are currently investigating which components of the HV lentiviral vector are responsible for Ghr gene activation. Two different retroviral vectors, both containing wild type Murine Leukaemia Virus LTRs, were also assayed for insertional mutagenesis in the Bcl15 target cell. IL-3 independent mutants were obtained, again at a frequency of approximately 1 in 2x10*7 integrants. Retroviral vector transformation of the Bcl15 cell line does not occur via Ghr gene activation. Retroviral vectors up-regulate expression of the cytokine interleukin-3 (IL-3), either by insertion into the IL-3 gene or by insertion into other genes, which may act as upstream activators of IL-3 expression. Though we find a similar rate of insertional mutagenesis, different mechanisms seem to be responsible for transforming Baf3/Bcl15 target cells with a lentiviral compared to a retroviral vector in this mutagenesis assay.