The Effect of a Chromatin Insulator (5′cHS4) on Enhanced Expression of the LMO2 Proto-Oncogene by an Oncoretroviral Long Terminal Repeat.

Activation of the LMO2 proto-oncogene by the insertional mutagenic effect of an oncoretroviral vector contributed to leukemia in two patients otherwise successfully treated for Severe Combined Immunodeficiency (SCID) in a gene therapy trial (Science 302: 415–419, 2003). Various facets of vector redesign have been proposed to improve the safety profile of retroviral gene therapy. We recreated the insertional mutation that occurred in one of the SCID patients by introducing an expression cassette into the first intron of the LMO2 gene in a human T-cell leukemia line (Jurkat) using recombinant adeno-associated viral (rAAV) vector mediated gene targeting. The rAAV targeting vector was constructed to have 1.3 kb of LMO2 intron sequences from both upstream and downstream of the desired insertion site flanking an expression cassette consisting of GFP coding sequences under the control of a single LTR. The expression cassette was in a reverse orientation to the LMO2 gene and flanked by LoxP sites (floxed). Jurkat cells were transduced with the rAAV targeting vector, GFP expressing cells were selected by FACS and single cell clones were recovered. Four clones showed the predicted bands on PCR and Southern blot analysis in addition to wildtype bands indicating successful targeting into one of the LMO2 alleles. As measured by real-time quantitative RT-PCR (qRT-PCR), expression of LMO2 mRNA was at a very low level in untransduced Jurkat cells but was upregulated in all 4 clones ranging from an 1100 to 3000-fold increase over baseline. LMO2 protein was highly expressed in all 4 clones, as shown by Western blot analysis, but was not detected in control Jurkat cells. Cells containing the floxed LTR-GFP cassette in the LMO2 first intron were transduced with a rAAV Cre expression cassette. Twenty-four hours later, the cells were transfected with a plasmid containing a floxed LTR-DsRed expression cassette and GFP−, DsRed+ cells were identified. Four clones were documented to have undergone Cre-mediated cassette exchange by Southern blot and PCR analysis. The LMO2 gene remained highly expressed in these clones. To explore the effect of an insulator element on proto-oncogene activation, a 1.2 kb fragment containing the 5′ DNase Hypersensitive Site 4 (5′cHS4) from the chicken β-globin locus was placed on each side of the retroviral LTR-GFP expression cassette. LoxP sites were located outside of the 5′ and 3′ insulator fragments. DsRed expressing Jurkat cells were transduced with the rAAV-Cre vector and then transfected with the plasmid containing the LoxP-5′cHS4-GFP-LTR-5′cHS4-LoxP cassette. GFP positive clones were isolated and nine were shown to have undergone cassette exchange by PCR and Southern blot analysis. Six clones were studied by qRT-PCR and/or Western blot analysis and all expressed LMO2 gene at much higher levels than the control Jurkat cells although expression was decreased 2- to 10-fold compared to clones containing the LTR-GFP or LTR-DsRed expression cassette. Our results indicated that the 5′cHS4 insulator could reduce LMO2 expression, but was not sufficient to prevent gene activation by the retroviral LTR suggesting that other safety measures should be considered when designing vectors. rAAV-mediated gene targeting and Cre-mediated cassette exchange provide a useful strategy for evaluating the potential safety profile of various retroviral expression cassettes in planning future gene therapy trials.