Limits...
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.

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

License
getmorefigures.php?uid=PMC2651870&req=5

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.

Mentions: To test this hypothesis we transduced 293T cells at MOI 1 (based on functional titer) with the four CMV vectors. Cells were passaged and part of each cell culture was harvested at days 3, 6, 14 and 27. Real-time PCR (using primers LV2 and ALB) on DNA isolated from these cells revealed that the proviral load was significantly (P < 0.001) decreased in cells transduced with the d2 × 250 bp vector and the 1.2 kb vector during day 3–6 (on day six the proviral loads of the d2 × 250 bp vector and the 1.2 kb vectors were 28% ± 10% and 37% ± 16%, respectively, of their initial proviral loads measured on day three) (Figure 3). After the initial drop the proviral load stayed a constant low level for at least 27 days (only 3, 6 and 14 days time points are shown in the figure). In vectors containing none or only one insulator-doublet in the reverse transcribed form (CMV.SIN and s2 × 250 bp.CMV) no such drop was observed (Figure 3). These results support the hypothesis that lentiviral vectors carrying two separated copies of the cHS4 insulator in the reverse transcribed form are capable of efficient cell-entry and subsequent reverse transcription (since high levels of viral DNA can be detected 3 days post transduction), but that the transduction process is impaired at some step after reverse transcription but before integration (since the abundance of viral DNA declines during day 3–6 post transduction). Notably, the insulator sequence itself is not deleterious to the vector since we could not detect any decrease in viral load of the s2 × 250 bp.CMV vector from day 3 to 6 (Figure 3). The same was true for our vector containing the SAR element confirming that loss of viral DNA from day 3–6 was not attributable to the size increment of the d2 × 250 bp vector (supplementary figure 3 (Additional file 3: Supplementary figures.pdf)). It is possible that two identical insulator sequences separated by an appropriate spacing as in the d2 × 250 bp.CMV vector and the reversed transcribed form of the 1.2 kb.CMV vector induce the formation of secondary structure incompatible with proper transduction and transgene expression.


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)

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

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

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.
Mentions: To test this hypothesis we transduced 293T cells at MOI 1 (based on functional titer) with the four CMV vectors. Cells were passaged and part of each cell culture was harvested at days 3, 6, 14 and 27. Real-time PCR (using primers LV2 and ALB) on DNA isolated from these cells revealed that the proviral load was significantly (P < 0.001) decreased in cells transduced with the d2 × 250 bp vector and the 1.2 kb vector during day 3–6 (on day six the proviral loads of the d2 × 250 bp vector and the 1.2 kb vectors were 28% ± 10% and 37% ± 16%, respectively, of their initial proviral loads measured on day three) (Figure 3). After the initial drop the proviral load stayed a constant low level for at least 27 days (only 3, 6 and 14 days time points are shown in the figure). In vectors containing none or only one insulator-doublet in the reverse transcribed form (CMV.SIN and s2 × 250 bp.CMV) no such drop was observed (Figure 3). These results support the hypothesis that lentiviral vectors carrying two separated copies of the cHS4 insulator in the reverse transcribed form are capable of efficient cell-entry and subsequent reverse transcription (since high levels of viral DNA can be detected 3 days post transduction), but that the transduction process is impaired at some step after reverse transcription but before integration (since the abundance of viral DNA declines during day 3–6 post transduction). Notably, the insulator sequence itself is not deleterious to the vector since we could not detect any decrease in viral load of the s2 × 250 bp.CMV vector from day 3 to 6 (Figure 3). The same was true for our vector containing the SAR element confirming that loss of viral DNA from day 3–6 was not attributable to the size increment of the d2 × 250 bp vector (supplementary figure 3 (Additional file 3: Supplementary figures.pdf)). It is possible that two identical insulator sequences separated by an appropriate spacing as in the d2 × 250 bp.CMV vector and the reversed transcribed form of the 1.2 kb.CMV vector induce the formation of secondary structure incompatible with proper transduction and transgene expression.

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