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Lentiviral vectors containing an enhancer-less ubiquitously acting chromatin opening element (UCOE) provide highly reproducible and stable transgene expression in hematopoietic cells.

Zhang F, Thornhill SI, Howe SJ, Ulaganathan M, Schambach A, Sinclair J, Kinnon C, Gaspar HB, Antoniou M, Thrasher AJ - Blood (2007)

Bottom Line: Furthermore, an A2UCOE-IL2RG vector fully restored the IL-2 signaling pathway within IL2RG-deficient human cells in vitro and successfully rescued the X-linked severe combined immunodeficiency (SCID-X1) phenotype in a mouse model of this disease.These data indicate that the A2UCOE displays highly reliable transcriptional activity within a lentiviral vector, largely overcoming insertion-site position effects and giving rise to therapeutically relevant levels of gene expression.These properties are achieved in the absence of classic enhancer activity and therefore may confer a high safety profile.

View Article: PubMed Central - PubMed

Affiliation: Centre for Immunodeficiency, Molecular Immunology Unit, Institute of Child Health, University College London, London, United Kingdom.

ABSTRACT
Ubiquitously acting chromatin opening elements (UCOEs) consist of methylation-free CpG islands encompassing dual divergently transcribed promoters of housekeeping genes that have been shown to confer resistance to transcriptional silencing and to produce consistent and stable transgene expression in tissue culture systems. To develop improved strategies for hematopoietic cell gene therapy, we have assessed the potential of the novel human HNRPA2B1-CBX3 UCOE (A2UCOE) within the context of a self-inactivating (SIN) lentiviral vector. Unlike viral promoters, the enhancer-less A2UCOE gave rise to populations of cells that expressed a reporter transgene at a highly reproducible level. The efficiency of expression per vector genome was also markedly increased in vivo compared with vectors incorporating either spleen focus-forming virus (SFFV) or cytomegalovirus (CMV) promoters, suggesting a relative resistance to silencing. Furthermore, an A2UCOE-IL2RG vector fully restored the IL-2 signaling pathway within IL2RG-deficient human cells in vitro and successfully rescued the X-linked severe combined immunodeficiency (SCID-X1) phenotype in a mouse model of this disease. These data indicate that the A2UCOE displays highly reliable transcriptional activity within a lentiviral vector, largely overcoming insertion-site position effects and giving rise to therapeutically relevant levels of gene expression. These properties are achieved in the absence of classic enhancer activity and therefore may confer a high safety profile.

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A2UCOE-EGFP gives stable, high efficiency expression per vector copy. (A) Genomic DNA derived from bone marrow of mice that received transplants ex vivo of HSCs transduced with the CMV-EGFP, SFFV-EGFP, and A2UCOE-EGFP vectors (Figure 4) was subjected to standard PCR for the presence of transgene (EGFP) and endogenous murine titin (Ttn) sequences and products were resolved by agarose gel electrophoresis. M indicates DNA size markers; C, mock control mouse bone marrow sample. (B) Summary of real-time quantitative PCR analysis of the same samples shown in panel A to determine lentiviral vector copy number. Error bars denote 1 standard deviation about the mean. (C) Determination of vector copy number in subpopulations of EGFP-expressing total bone marrow cells transduced with the A2UCOE-EGFP lentiviral vector. (Left) A representative sample of A2UCOE-EGFP vector–transduced bone marrow cells was sorted by FACS to isolate either low (gate 1) or high (gate 2) EGFP fluorescence intensity cells. DNA was then isolated from the sorted pools of cells and analyzed by QPCR as in panel B. (Right) Profile showing the sorting gates and corresponding mean fluorescence intensity (MFI). Average lentiviral vector copy number per cell is indicated. Note that the A2UCOE gives a higher number of EGFP-positive cells at a lower vector copy number than either the SFFV or CMV promoters (summarized in Table 1), with a clear trend toward copy number–dependent expression.
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Figure 5: A2UCOE-EGFP gives stable, high efficiency expression per vector copy. (A) Genomic DNA derived from bone marrow of mice that received transplants ex vivo of HSCs transduced with the CMV-EGFP, SFFV-EGFP, and A2UCOE-EGFP vectors (Figure 4) was subjected to standard PCR for the presence of transgene (EGFP) and endogenous murine titin (Ttn) sequences and products were resolved by agarose gel electrophoresis. M indicates DNA size markers; C, mock control mouse bone marrow sample. (B) Summary of real-time quantitative PCR analysis of the same samples shown in panel A to determine lentiviral vector copy number. Error bars denote 1 standard deviation about the mean. (C) Determination of vector copy number in subpopulations of EGFP-expressing total bone marrow cells transduced with the A2UCOE-EGFP lentiviral vector. (Left) A representative sample of A2UCOE-EGFP vector–transduced bone marrow cells was sorted by FACS to isolate either low (gate 1) or high (gate 2) EGFP fluorescence intensity cells. DNA was then isolated from the sorted pools of cells and analyzed by QPCR as in panel B. (Right) Profile showing the sorting gates and corresponding mean fluorescence intensity (MFI). Average lentiviral vector copy number per cell is indicated. Note that the A2UCOE gives a higher number of EGFP-positive cells at a lower vector copy number than either the SFFV or CMV promoters (summarized in Table 1), with a clear trend toward copy number–dependent expression.

Mentions: We quantified the efficiency of A2UCOE-EGFP expression in comparison to the SFFV and CMV constructs by determining vector copy number and correlated this to the mean percentage of cells expressing EGFP within bone marrow of mice. Genomic DNA from bone marrow of all transplant recipients (Figure 4) was assayed by QPCR employing primers for the EGFP gene. Simultaneous amplification of murine Ttn sequences acted as an endogenous 2-copy gene control. Initial PCR analysis showed that the transgene is present in all recipients (Figure 5A). Following QPCR, comparison of the EGFP and Ttn amplification products showed that, in general, the average vector copy number in recipients of HSCs transduced with the A2UCOE-EGFP construct were significantly lower than those harboring the SFFV- and CMV-EGFP vectors (Figure 5B; Table 1). The average copy number across all samples for each vector was 0.25 per cell (range, 0.13 to 0.44 per cell) for the A2UCOE group, 0.96 per cell (range, 0.02 to 1.3 per cell) for SFFV, and 3.7 per cell (range, 1.2 to 6.5 per cell) for CMV vector recipients. A comparison of average vector copy number with average EFGP expression in bone marrow (Table 1) reveals a 1:1 correlation for the EGFP transgene regulated by the A2UCOE. In marked contrast, the ratio of expression to vector copy number is 1:9 for the SFFV and 1:90 for the CMV vectors (Table 1), which is indicative of either very low levels of expression per transgene or, more likely, extensive transgene silencing.17,18


Lentiviral vectors containing an enhancer-less ubiquitously acting chromatin opening element (UCOE) provide highly reproducible and stable transgene expression in hematopoietic cells.

Zhang F, Thornhill SI, Howe SJ, Ulaganathan M, Schambach A, Sinclair J, Kinnon C, Gaspar HB, Antoniou M, Thrasher AJ - Blood (2007)

A2UCOE-EGFP gives stable, high efficiency expression per vector copy. (A) Genomic DNA derived from bone marrow of mice that received transplants ex vivo of HSCs transduced with the CMV-EGFP, SFFV-EGFP, and A2UCOE-EGFP vectors (Figure 4) was subjected to standard PCR for the presence of transgene (EGFP) and endogenous murine titin (Ttn) sequences and products were resolved by agarose gel electrophoresis. M indicates DNA size markers; C, mock control mouse bone marrow sample. (B) Summary of real-time quantitative PCR analysis of the same samples shown in panel A to determine lentiviral vector copy number. Error bars denote 1 standard deviation about the mean. (C) Determination of vector copy number in subpopulations of EGFP-expressing total bone marrow cells transduced with the A2UCOE-EGFP lentiviral vector. (Left) A representative sample of A2UCOE-EGFP vector–transduced bone marrow cells was sorted by FACS to isolate either low (gate 1) or high (gate 2) EGFP fluorescence intensity cells. DNA was then isolated from the sorted pools of cells and analyzed by QPCR as in panel B. (Right) Profile showing the sorting gates and corresponding mean fluorescence intensity (MFI). Average lentiviral vector copy number per cell is indicated. Note that the A2UCOE gives a higher number of EGFP-positive cells at a lower vector copy number than either the SFFV or CMV promoters (summarized in Table 1), with a clear trend toward copy number–dependent expression.
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Related In: Results  -  Collection

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Figure 5: A2UCOE-EGFP gives stable, high efficiency expression per vector copy. (A) Genomic DNA derived from bone marrow of mice that received transplants ex vivo of HSCs transduced with the CMV-EGFP, SFFV-EGFP, and A2UCOE-EGFP vectors (Figure 4) was subjected to standard PCR for the presence of transgene (EGFP) and endogenous murine titin (Ttn) sequences and products were resolved by agarose gel electrophoresis. M indicates DNA size markers; C, mock control mouse bone marrow sample. (B) Summary of real-time quantitative PCR analysis of the same samples shown in panel A to determine lentiviral vector copy number. Error bars denote 1 standard deviation about the mean. (C) Determination of vector copy number in subpopulations of EGFP-expressing total bone marrow cells transduced with the A2UCOE-EGFP lentiviral vector. (Left) A representative sample of A2UCOE-EGFP vector–transduced bone marrow cells was sorted by FACS to isolate either low (gate 1) or high (gate 2) EGFP fluorescence intensity cells. DNA was then isolated from the sorted pools of cells and analyzed by QPCR as in panel B. (Right) Profile showing the sorting gates and corresponding mean fluorescence intensity (MFI). Average lentiviral vector copy number per cell is indicated. Note that the A2UCOE gives a higher number of EGFP-positive cells at a lower vector copy number than either the SFFV or CMV promoters (summarized in Table 1), with a clear trend toward copy number–dependent expression.
Mentions: We quantified the efficiency of A2UCOE-EGFP expression in comparison to the SFFV and CMV constructs by determining vector copy number and correlated this to the mean percentage of cells expressing EGFP within bone marrow of mice. Genomic DNA from bone marrow of all transplant recipients (Figure 4) was assayed by QPCR employing primers for the EGFP gene. Simultaneous amplification of murine Ttn sequences acted as an endogenous 2-copy gene control. Initial PCR analysis showed that the transgene is present in all recipients (Figure 5A). Following QPCR, comparison of the EGFP and Ttn amplification products showed that, in general, the average vector copy number in recipients of HSCs transduced with the A2UCOE-EGFP construct were significantly lower than those harboring the SFFV- and CMV-EGFP vectors (Figure 5B; Table 1). The average copy number across all samples for each vector was 0.25 per cell (range, 0.13 to 0.44 per cell) for the A2UCOE group, 0.96 per cell (range, 0.02 to 1.3 per cell) for SFFV, and 3.7 per cell (range, 1.2 to 6.5 per cell) for CMV vector recipients. A comparison of average vector copy number with average EFGP expression in bone marrow (Table 1) reveals a 1:1 correlation for the EGFP transgene regulated by the A2UCOE. In marked contrast, the ratio of expression to vector copy number is 1:9 for the SFFV and 1:90 for the CMV vectors (Table 1), which is indicative of either very low levels of expression per transgene or, more likely, extensive transgene silencing.17,18

Bottom Line: Furthermore, an A2UCOE-IL2RG vector fully restored the IL-2 signaling pathway within IL2RG-deficient human cells in vitro and successfully rescued the X-linked severe combined immunodeficiency (SCID-X1) phenotype in a mouse model of this disease.These data indicate that the A2UCOE displays highly reliable transcriptional activity within a lentiviral vector, largely overcoming insertion-site position effects and giving rise to therapeutically relevant levels of gene expression.These properties are achieved in the absence of classic enhancer activity and therefore may confer a high safety profile.

View Article: PubMed Central - PubMed

Affiliation: Centre for Immunodeficiency, Molecular Immunology Unit, Institute of Child Health, University College London, London, United Kingdom.

ABSTRACT
Ubiquitously acting chromatin opening elements (UCOEs) consist of methylation-free CpG islands encompassing dual divergently transcribed promoters of housekeeping genes that have been shown to confer resistance to transcriptional silencing and to produce consistent and stable transgene expression in tissue culture systems. To develop improved strategies for hematopoietic cell gene therapy, we have assessed the potential of the novel human HNRPA2B1-CBX3 UCOE (A2UCOE) within the context of a self-inactivating (SIN) lentiviral vector. Unlike viral promoters, the enhancer-less A2UCOE gave rise to populations of cells that expressed a reporter transgene at a highly reproducible level. The efficiency of expression per vector genome was also markedly increased in vivo compared with vectors incorporating either spleen focus-forming virus (SFFV) or cytomegalovirus (CMV) promoters, suggesting a relative resistance to silencing. Furthermore, an A2UCOE-IL2RG vector fully restored the IL-2 signaling pathway within IL2RG-deficient human cells in vitro and successfully rescued the X-linked severe combined immunodeficiency (SCID-X1) phenotype in a mouse model of this disease. These data indicate that the A2UCOE displays highly reliable transcriptional activity within a lentiviral vector, largely overcoming insertion-site position effects and giving rise to therapeutically relevant levels of gene expression. These properties are achieved in the absence of classic enhancer activity and therefore may confer a high safety profile.

Show MeSH
Related in: MedlinePlus