Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity

Abstract

Retroviral vectors with long terminal repeats (LTRs), which contain strong enhancer/promoter sequences at both ends of their genome, are widely used for stable gene transfer into hematopoietic cells. However, recent clinical data and mouse models point to insertional activation of cellular proto-oncogenes as a dose-limiting side effect of retroviral gene delivery that potentially induces leukemia. Self-inactivating (SIN) retroviral vectors do not contain the terminal repetition of the enhancer/promoter, theoretically attenuating the interaction with neighboring cellular genes. With a new assay based on in vitro expansion of primary murine hematopoietic cells and selection in limiting dilution, we showed that SIN vectors using a strong internal retroviral enhancer/promoter may also transform cells by insertional mutagenesis. Most transformed clones, including those obtained after dose escalation of SIN vectors, showed insertions upstream of the third exon of Evi1 and in reverse orientation to its transcriptional orientation. Normalizing for the vector copy number, we found the transforming capacity of SIN vectors to be significantly reduced when compared with corresponding LTR vectors. Additional modifications of SIN vectors may further increase safety. Improved cell-culture assays will likely play an important role in the evaluation of insertional mutagenesis.

Figure 1.

Experimental setup and vectors. (A) Murine Lin^- cells were isolated, prestimulated, and transduced with cell-free vector supernatants with the use of MOIs, as indicated in Table 1. The cells were expanded as a mass culture for 2 weeks and subsequently selected on a 96-well plate (step 1). Randomly picked clones were further expanded to numbers exceeding 10⁶ for phenotyping and to harvest DNA and RNA (step 2). (B) Retroviral vectors used for transduction shown as proviruses. LTRSF is an LTR-driven retroviral vector that has been previously described (SF91). It contains a splice-competent leader region, including the primer binding site (θ) and the packaging signal (Ψ), and encodes either eGFP or DsRed. The U3 region containing all the enhancer/promoter elements is derived from spleen focus-forming virus (SF). SINSF is a self-inactivating (SIN) retroviral vector. The U3 region is almost completely deleted, leaving only the integrase attachment sites intact. eGFP and DsRed are driven by the SF enhancer/promoter, identical to the cis-elements used in the LTR-driven vector.

Figure 2.

FACS analyses of the transduction, selection, and expansion process. eGFP expression and percentage of positive cells of SIN- and LTR-transduced BM cells 1 day after the second transduction (top panel), after 2 weeks (middle panel), and after selection of a single clone (bottom panel). Representative data obtained in experiment 3 and experiment 4 (FACS analysis of the SIN vector-transduced clone).

Figure 3.

Replating ability of Lin^- cells depends on vector design. Correcting the frequency of replating cells for the average vector copy number detected on day 7 after transduction, LTR vectors showed a significantly increased risk for transforming Lin^- cells to acquire replating potential. The median is indicated as a thick black line. If no clones were obtained, the frequency of 1 in 9550 was taken for calculation; if all 96 wells contained replating cells, the frequency was estimated as (at least) 1 in 22 cells.

Figure 4.

Genetic analyses of insertional mutants obtained after step 2. (A) Representative Southern blot analysis using 10 μg genomic DNA of expanded clones. Only selected clones of each experiment are shown displaying a different band pattern within each blot. The cDNA for eGFP or DsRed or a fragment spanning the wPRE were used for probing. Clone names are indicated above each lane. (B) Location of insertions into the Evi1 allele. The Mds1 locus lies further upstream in the same transcriptional orientation. LTR and SIN vectors recovered in our in vitro studies show the same pattern, consistent with our previous findings made with LTR vectors in dominant or leukemic clones in vivo.,, (C) Quantitative real-time PCR was used to determine the transcript level of Evi1 or HoxA7. Bars represent the relative enhancement compared with expression levels in mock-transduced and expanded cells. Clone 6.4 contains a vector insertion upstream of HoxA7, whereas clone C4 does not. Each PCR was performed in triplicate, and bars represent the mean of 3 CT values. Values for clones 1.5 and a1 represent the average of 2 independent determinates performed in triplicate.

Figure 5.

Phenotype of clones obtained after step 2. The “immortal” clone B has the lowest frequency of differentiating myeloid cells. (A) Cytospin preparations of mock-expanded cells and one “immortal” clone selected after transduction with LTRSFeGFP (May-Grünwald/Giemsa staining). In no case did we detect mature forms of the granulocytic lineage. Images were visualized using an Olympus BX51 upright microscope (Olympus, Hamburg, Germany) equipped with a 40 ×/0.75 numeric aperture objective lens. Images were processed using the Colorview Soft Imaging System and analySIS Five software (Olympus). (B) Clones were subjected to FACS analysis using antibodies as indicated.