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

Nielsen TT, Jakobsson J, Rosenqvist N, Lundberg C - BMC Biotechnol. (2009)

Bottom Line: It has been suggested that insulators can improve the safety and performance of lentiviral vectors.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: CNS Gene Therapy Unit, Wallenberg Neuroscience Center, Department of Experimental Medical Sciences, Lund University, Lund, Sweden. troelsn@sund.ku.dk

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.

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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.
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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.

Mentions: When transducing the cell lines with the d2 × 250 bp vectors, we found less GPF-positive cells and lower mean fluorescence (mean fluorescence units – MFU) independent of cell type and promoter choice when comparing with control vectors (Figure 2A–Q), which suggests that the cHS4 core element fails to increase transgene expression. In addition, the insulator was unable to prevent transgene silencing during differentiation of RN33B cells (results not shown). This is in agreement with recent results showing that the 250 bp core element of the cHS4-insulator is insufficient for insulating properties in a retroviral and lentiviral context[15,28]. Surprisingly, however, QPCR analyses of the proviral load in transduced cell cultures revealed significantly lower levels of viral DNA in cultures transduced with the d2 × 250 bp vectors compared to control vectors (Figure 2, C, H, K and 2N), despite the fact that we had normalized the various transductions using the relative DNA titer. This difference in viral DNA accounted for most of the difference in transgene expression. This is surprising since we have previously used the relative DNA titer for titer determination of vectors with another commonly used cis-element, the scaffold attachment region (SAR)[19,29], and subsequently found complete agreement between the proviral loads of cell cultures transduced with the vector containing this cis-element and the control vector (supplementary information (Additional file 1 and 2: Supplementary information.pdf and Supplementary tables.pdf)). In addition, these data confirm that the negative effects on vector functionality observed when inserting the 2 × 250 bp doublet after the WPRE are not caused by the size increment of the vector, since the insertion of the SAR element, which is similar in size compared to the 2 × 250 bp doublet, in this position does not influence the functional titer, the relative DNA titer, transgene expression levels or proviral loads of transduced cultures. Thus the reduced transgene expression from the d2 × 250 bp vectors probably reflects impaired vector functionality with regard to cell entry, integration etc, and this might explain the observed discrepancies between the functional titer and the relative DNA titer.


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

Nielsen TT, Jakobsson J, Rosenqvist N, Lundberg C - BMC Biotechnol. (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651870&req=5

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.
Mentions: When transducing the cell lines with the d2 × 250 bp vectors, we found less GPF-positive cells and lower mean fluorescence (mean fluorescence units – MFU) independent of cell type and promoter choice when comparing with control vectors (Figure 2A–Q), which suggests that the cHS4 core element fails to increase transgene expression. In addition, the insulator was unable to prevent transgene silencing during differentiation of RN33B cells (results not shown). This is in agreement with recent results showing that the 250 bp core element of the cHS4-insulator is insufficient for insulating properties in a retroviral and lentiviral context[15,28]. Surprisingly, however, QPCR analyses of the proviral load in transduced cell cultures revealed significantly lower levels of viral DNA in cultures transduced with the d2 × 250 bp vectors compared to control vectors (Figure 2, C, H, K and 2N), despite the fact that we had normalized the various transductions using the relative DNA titer. This difference in viral DNA accounted for most of the difference in transgene expression. This is surprising since we have previously used the relative DNA titer for titer determination of vectors with another commonly used cis-element, the scaffold attachment region (SAR)[19,29], and subsequently found complete agreement between the proviral loads of cell cultures transduced with the vector containing this cis-element and the control vector (supplementary information (Additional file 1 and 2: Supplementary information.pdf and Supplementary tables.pdf)). In addition, these data confirm that the negative effects on vector functionality observed when inserting the 2 × 250 bp doublet after the WPRE are not caused by the size increment of the vector, since the insertion of the SAR element, which is similar in size compared to the 2 × 250 bp doublet, in this position does not influence the functional titer, the relative DNA titer, transgene expression levels or proviral loads of transduced cultures. Thus the reduced transgene expression from the d2 × 250 bp vectors probably reflects impaired vector functionality with regard to cell entry, integration etc, and this might explain the observed discrepancies between the functional titer and the relative DNA titer.

Bottom Line: It has been suggested that insulators can improve the safety and performance of lentiviral vectors.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: CNS Gene Therapy Unit, Wallenberg Neuroscience Center, Department of Experimental Medical Sciences, Lund University, Lund, Sweden. troelsn@sund.ku.dk

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.

Show MeSH