Incorporating double copies of a chromatin insulator into lentiviral vectors results in less viral integrants

Abstract

Background: Lentiviral vectors hold great promise as gene transfer vectors in gene therapeutic settings. However, problems related to the risk of insertional mutagenesis, transgene silencing and positional effects have stalled the use of such vectors in the clinic. Chromatin insulators are boundary elements that can prevent enhancer-promoter interactions, if placed between these elements, and protect transgene cassettes from silencing and positional effects. It has been suggested that insulators can improve the safety and performance of lentiviral vectors. Therefore insulators have been incorporated into lentiviral vectors in order to enhance their safety profile and improve transgene expression. Commonly such insulator vectors are produced at lower titers than control vectors thus limiting their potential use.

Results: In this study we cloned in tandem copies of the chicken beta-globin insulator (cHS4) on both sides of the transgene cassette in order to enhance the insulating effect. Our insulator vectors were produced at significantly lower titers compared to control vectors, and we show that this reduction in titer is due to a block during the transduction process that appears after reverse transcription but before integration of the viral DNA. This non-integrated viral DNA could be detected by PCR and, importantly, prevented efficient transduction of target cells.

Conclusion: These results have importance for the future use of insulator sequences in lentiviral vectors and might limit the use of insulators in vectors for in vivo use. Therefore, a careful analysis of the optimal design must be performed before insulators are included into clinical lentiviral vectors.

Figure 1

Outline of lentiviral vectors. Schematic drawing of vectors used in this study. Vectors A-D utilize the CMV-promoter whereas vectors E-F use the EF1α-promoter. (A, E) Control vectors for the group of CMV and EF1α vectors respectively. Cloning sites for the cis-elements are shown. (B) One tandem repeat (2 × 250) of the core element of chicken β-globin insulator (cHS4) was inserted (sense orientation) prior to the promoter. (C, F) Two copies of the tandem repeat were inserted into the vectors – one just upstream the promoter and one just downstream the expression cassette. (D) The entire cHS4-insulator (1.2 kb) was inserted in the 3' LTR in sense orientation. (A-F) Horizontal lines denote the location of primers (labelled LV2, GFP, WPRE and INS) used for quantitative real-time PCR (qPCR). Legend: LTR – Long Terminal Repeat, ψ – packaging signal, RRE – Rev Responsive Element, cPPT – Central Poly Purine Tract, CMV – Cytomegalo virus promoter, EF1α – Elongation Factor 1α promoter, GFP – Green Fluorescent Protein, WPRE – Woodchuck Posttranscriptional Regulatory Element, 2 × 250 – tandem repeat of the cHS4 core element and 1.2 kb – full length cHS4 insulator sequence.

Figure 2

Expression data of the d2 × 250 bp vectors. Flow cytometric data and corresponding determination of proviral load for d2 × 250 vectors in three different cell types: RN33B (naïve) (A-E), 293T (F-K) and K562 (L-Q). Cells were transduced at MOI 1 and 5 (relative DNA titer) and analysed 7 days after transduction. The figure shows the percentage of GFP positive cells for each vector (A, D, F, I, L and O) along with the corresponding mean fluorescence (MFU) (B, E, G, J, M and P) and proviral load (the latter only for selected vectors) (C, H, K, N and Q). Error bars denote standard deviations.

Figure 3

Persistence of viral DNA in transduced cells during a 14 day time period. 293T cells were transduced at MOI 1 (relative DNA titer) with the four vectors utilizing the CMV-promoter. After 3, 6, 14 and 27 days cells were harvested and the proviral load determined by qPCR using primers LV2 and ALB. The vectors containing two separated copies of the insulator (d2 × 250 bp.CMV and 1.2 kb.CMV) elicit a significant (*, p < 0.05) drop in proviral load within the first 6 days after transduction compared to control vectors, CMV.SIN and s2 × 250 bp.CMV. The experiment was continued for 27 days with no change in proviral load compared to the 14 days time point. Error bars denote standard error of the mean.

Figure 4

Expression data of the d2 × 250 bp vectors. (A-D) Flow cytometric data for the transduction of naïve RN33B cells with d2 × 250 bp vectors and controls at MOI 1 and 5 (functional titer). Cells were analysed 7 days after transduction. The percentage of GFP-positive cells is shown (A and C) along with the mean fluorescence (MFU) (B and D). Error bars denote standard deviations. E-F: Dose-response curves showing CMV.SIN, d2 × 250 bp.CMV and 1.2 kb.CMV vector performance in naïve RN33B cells. The percentage of GFP positive cells (E) and mean fluorescence is shown (F).