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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. (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).
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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).

Mentions: To test the transduction potential of these remaining functional vectors, we transduced RN33B cells at an MOI of 1 and 5, this time based on the functional titers. At 7 days after transduction cells were harvested and flow cytometry performed. For the CMV vectors at a MOI of 1, the d2 × 250 bp vector seemed to perform as well as the control vector (Figure 4A and 4B). However, at high MOI, the d2 × 250 bp vector expressed GFP at a much lower level than the control vector (Figure 4A and 4B). When using EF1α vectors the performance of the d2 × 250 bp vector was even worse compared to the control vector (Figure 4C and 4D).


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

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

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).
Mentions: To test the transduction potential of these remaining functional vectors, we transduced RN33B cells at an MOI of 1 and 5, this time based on the functional titers. At 7 days after transduction cells were harvested and flow cytometry performed. For the CMV vectors at a MOI of 1, the d2 × 250 bp vector seemed to perform as well as the control vector (Figure 4A and 4B). However, at high MOI, the d2 × 250 bp vector expressed GFP at a much lower level than the control vector (Figure 4A and 4B). When using EF1α vectors the performance of the d2 × 250 bp vector was even worse compared to the control vector (Figure 4C and 4D).

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