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Development and validation of non-integrative, self-limited, and replicating minicircles for safe reporter gene imaging of cell-based therapies.

Ronald JA, Cusso L, Chuang HY, Yan X, Dragulescu-Andrasi A, Gambhir SS - PLoS ONE (2013)

Bottom Line: To address this issue, we have developed non-integrative, replicating minicircles (MCs) as an alternative platform for safer monitoring of cells in living subjects.To monitor cell proliferation in vivo, 1.5 × 10(6) cells carrying the S/MAR minicircle were implanted subcutaneously into mice (n = 5) and as tumors developed significantly more bioluminescence signal was noted at day 35 and 43 compared to day 7 post-implant (p<0.05).This will lead to safe tools to assess treatment response at earlier time points and improve the precision of cell-based therapies.

View Article: PubMed Central - PubMed

Affiliation: Molecular Imaging Program at Stanford, Stanford University, Stanford, California, United States of America ; Department of Radiology, Stanford University, Stanford, California, United States of America.

ABSTRACT
Reporter gene (RG) imaging of cell-based therapies provides a direct readout of therapeutic efficacy by assessing the fate of implanted cells. To permit long-term cellular imaging, RGs are traditionally required to be integrated into the cellular genome. This poses a potential safety risk and regulatory bottleneck for clinical translation as integration can lead to cellular transformation. To address this issue, we have developed non-integrative, replicating minicircles (MCs) as an alternative platform for safer monitoring of cells in living subjects. We developed both plasmids and minicircles containing the scaffold/matrix attachment regions (S/MAR) of the human interferon-beta gene, driven by the CMV promoter, and expressing the bioluminescence RG firefly luciferase. Constructs were transfected into breast cancer cells, and expanded S/MAR minicircle clones showed luciferase signal for greater than 3 months in culture and minicircles remained as episomes. Importantly, luciferase activity in clonal populations was slowly lost over time and this corresponded to a loss of episome, providing a way to reversibly label cells. To monitor cell proliferation in vivo, 1.5 × 10(6) cells carrying the S/MAR minicircle were implanted subcutaneously into mice (n = 5) and as tumors developed significantly more bioluminescence signal was noted at day 35 and 43 compared to day 7 post-implant (p<0.05). To our knowledge, this is the first work examining the use of episomal, self-limited, replicating minicircles to track the proliferation of cells using non-invasive imaging in living subjects. Continued development of S/MAR minicircles will provide a broadly applicable vector platform amenable with any of the numerous RG technologies available to allow therapeutic cell fate to be assessed in individual patients, and to achieve this without the need to manipulate the cell's genome so that safety concerns are minimized. This will lead to safe tools to assess treatment response at earlier time points and improve the precision of cell-based therapies.

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Related in: MedlinePlus

S/MAR MCs can label cells in culture for extended periods of time and remain episomal.A) pCMV-Luc2-S/MAR PP and MC constructs were transfected into MDA-MB-231 cells, grown in the absence of antibiotic selection, and BLI was performed over the course of 9 days. On day 6, both MC and PP showed strong BLI signal within the cells. However, by day 9, the MC-labeled cells continued to display strong signal and the signal from PP-labeled cells began to disappear. On this day MC-labeled clones displaying strong luminescent signal were isolated and expanded. B) Two clones (3–5 and 3–7) were cultured over the course of 91 days post-transfection and continued to be imaged. Both clones continued to show luminescent signal over the entire 3-month period. C) Southern blot analysis was performed on total DNA isolated from control cells, from control cells spiked with 200 pg of S/MAR MC, and from an S/MAR MC clonal population (clone 3-7) 47 days after transfection. Total DNA (40 µg) was digested with a single cutting enzyme and probed with a Luc2 probe. A single band at the correct size (4.5 kb) was detectable only in the lanes with control DNA spiked with the original construct (lane 1) and the S/MAR MC clone (lane 3), confirming the episomal nature of the construct.
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pone-0073138-g002: S/MAR MCs can label cells in culture for extended periods of time and remain episomal.A) pCMV-Luc2-S/MAR PP and MC constructs were transfected into MDA-MB-231 cells, grown in the absence of antibiotic selection, and BLI was performed over the course of 9 days. On day 6, both MC and PP showed strong BLI signal within the cells. However, by day 9, the MC-labeled cells continued to display strong signal and the signal from PP-labeled cells began to disappear. On this day MC-labeled clones displaying strong luminescent signal were isolated and expanded. B) Two clones (3–5 and 3–7) were cultured over the course of 91 days post-transfection and continued to be imaged. Both clones continued to show luminescent signal over the entire 3-month period. C) Southern blot analysis was performed on total DNA isolated from control cells, from control cells spiked with 200 pg of S/MAR MC, and from an S/MAR MC clonal population (clone 3-7) 47 days after transfection. Total DNA (40 µg) was digested with a single cutting enzyme and probed with a Luc2 probe. A single band at the correct size (4.5 kb) was detectable only in the lanes with control DNA spiked with the original construct (lane 1) and the S/MAR MC clone (lane 3), confirming the episomal nature of the construct.

Mentions: Our first experimental objective was to establish the ability of our S/MAR MCs to label cultured cells with RGs for extended periods of time. MDA-MB-231 breast cancer cells were transfected with either PP or MC, grown without antibiotic selection, and imaged at both day 6 and 9 after transfection (Figure 2A). Unlike S/MAR PPs, S/MAR MCs do not require antibiotic selection to become established as replicating episomes. Therefore we expect that luciferase activity will be lost over time using S/MAR PPs but better maintained with S/MAR MCs. Cells were transfected with equal mass of PP and MC and therefore due to the inherent differences in transfection efficiencies we focused our comparisons of relative Fluc levels over time to changes within rather than between PP and MC cell populations. On day 6, both PP and MC showed strong bioluminescent signal. In contrast, at day 9, after several days of continued cell growth, the MC signal began to show foci of strong luminescent signal, whereas the PP signal began to disappear (Figure 2A). At this point individual S/MAR MC cell colonies that displayed high levels of bioluminescent signal were isolated and expanded to generate clonal cell populations. Several of these clones (clone 2-1, 3-5, and 3-7) were maintained in culture for extended periods of time (∼4 months) and serial BLI was performed (Figure 2B). As seen in Figure 2B, each clone continued to display bioluminescent signal for at least 3 months following transfection, indicating the ability to express RG in cells with S/MAR MCs for extended periods of time.


Development and validation of non-integrative, self-limited, and replicating minicircles for safe reporter gene imaging of cell-based therapies.

Ronald JA, Cusso L, Chuang HY, Yan X, Dragulescu-Andrasi A, Gambhir SS - PLoS ONE (2013)

S/MAR MCs can label cells in culture for extended periods of time and remain episomal.A) pCMV-Luc2-S/MAR PP and MC constructs were transfected into MDA-MB-231 cells, grown in the absence of antibiotic selection, and BLI was performed over the course of 9 days. On day 6, both MC and PP showed strong BLI signal within the cells. However, by day 9, the MC-labeled cells continued to display strong signal and the signal from PP-labeled cells began to disappear. On this day MC-labeled clones displaying strong luminescent signal were isolated and expanded. B) Two clones (3–5 and 3–7) were cultured over the course of 91 days post-transfection and continued to be imaged. Both clones continued to show luminescent signal over the entire 3-month period. C) Southern blot analysis was performed on total DNA isolated from control cells, from control cells spiked with 200 pg of S/MAR MC, and from an S/MAR MC clonal population (clone 3-7) 47 days after transfection. Total DNA (40 µg) was digested with a single cutting enzyme and probed with a Luc2 probe. A single band at the correct size (4.5 kb) was detectable only in the lanes with control DNA spiked with the original construct (lane 1) and the S/MAR MC clone (lane 3), confirming the episomal nature of the construct.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756008&req=5

pone-0073138-g002: S/MAR MCs can label cells in culture for extended periods of time and remain episomal.A) pCMV-Luc2-S/MAR PP and MC constructs were transfected into MDA-MB-231 cells, grown in the absence of antibiotic selection, and BLI was performed over the course of 9 days. On day 6, both MC and PP showed strong BLI signal within the cells. However, by day 9, the MC-labeled cells continued to display strong signal and the signal from PP-labeled cells began to disappear. On this day MC-labeled clones displaying strong luminescent signal were isolated and expanded. B) Two clones (3–5 and 3–7) were cultured over the course of 91 days post-transfection and continued to be imaged. Both clones continued to show luminescent signal over the entire 3-month period. C) Southern blot analysis was performed on total DNA isolated from control cells, from control cells spiked with 200 pg of S/MAR MC, and from an S/MAR MC clonal population (clone 3-7) 47 days after transfection. Total DNA (40 µg) was digested with a single cutting enzyme and probed with a Luc2 probe. A single band at the correct size (4.5 kb) was detectable only in the lanes with control DNA spiked with the original construct (lane 1) and the S/MAR MC clone (lane 3), confirming the episomal nature of the construct.
Mentions: Our first experimental objective was to establish the ability of our S/MAR MCs to label cultured cells with RGs for extended periods of time. MDA-MB-231 breast cancer cells were transfected with either PP or MC, grown without antibiotic selection, and imaged at both day 6 and 9 after transfection (Figure 2A). Unlike S/MAR PPs, S/MAR MCs do not require antibiotic selection to become established as replicating episomes. Therefore we expect that luciferase activity will be lost over time using S/MAR PPs but better maintained with S/MAR MCs. Cells were transfected with equal mass of PP and MC and therefore due to the inherent differences in transfection efficiencies we focused our comparisons of relative Fluc levels over time to changes within rather than between PP and MC cell populations. On day 6, both PP and MC showed strong bioluminescent signal. In contrast, at day 9, after several days of continued cell growth, the MC signal began to show foci of strong luminescent signal, whereas the PP signal began to disappear (Figure 2A). At this point individual S/MAR MC cell colonies that displayed high levels of bioluminescent signal were isolated and expanded to generate clonal cell populations. Several of these clones (clone 2-1, 3-5, and 3-7) were maintained in culture for extended periods of time (∼4 months) and serial BLI was performed (Figure 2B). As seen in Figure 2B, each clone continued to display bioluminescent signal for at least 3 months following transfection, indicating the ability to express RG in cells with S/MAR MCs for extended periods of time.

Bottom Line: To address this issue, we have developed non-integrative, replicating minicircles (MCs) as an alternative platform for safer monitoring of cells in living subjects.To monitor cell proliferation in vivo, 1.5 × 10(6) cells carrying the S/MAR minicircle were implanted subcutaneously into mice (n = 5) and as tumors developed significantly more bioluminescence signal was noted at day 35 and 43 compared to day 7 post-implant (p<0.05).This will lead to safe tools to assess treatment response at earlier time points and improve the precision of cell-based therapies.

View Article: PubMed Central - PubMed

Affiliation: Molecular Imaging Program at Stanford, Stanford University, Stanford, California, United States of America ; Department of Radiology, Stanford University, Stanford, California, United States of America.

ABSTRACT
Reporter gene (RG) imaging of cell-based therapies provides a direct readout of therapeutic efficacy by assessing the fate of implanted cells. To permit long-term cellular imaging, RGs are traditionally required to be integrated into the cellular genome. This poses a potential safety risk and regulatory bottleneck for clinical translation as integration can lead to cellular transformation. To address this issue, we have developed non-integrative, replicating minicircles (MCs) as an alternative platform for safer monitoring of cells in living subjects. We developed both plasmids and minicircles containing the scaffold/matrix attachment regions (S/MAR) of the human interferon-beta gene, driven by the CMV promoter, and expressing the bioluminescence RG firefly luciferase. Constructs were transfected into breast cancer cells, and expanded S/MAR minicircle clones showed luciferase signal for greater than 3 months in culture and minicircles remained as episomes. Importantly, luciferase activity in clonal populations was slowly lost over time and this corresponded to a loss of episome, providing a way to reversibly label cells. To monitor cell proliferation in vivo, 1.5 × 10(6) cells carrying the S/MAR minicircle were implanted subcutaneously into mice (n = 5) and as tumors developed significantly more bioluminescence signal was noted at day 35 and 43 compared to day 7 post-implant (p<0.05). To our knowledge, this is the first work examining the use of episomal, self-limited, replicating minicircles to track the proliferation of cells using non-invasive imaging in living subjects. Continued development of S/MAR minicircles will provide a broadly applicable vector platform amenable with any of the numerous RG technologies available to allow therapeutic cell fate to be assessed in individual patients, and to achieve this without the need to manipulate the cell's genome so that safety concerns are minimized. This will lead to safe tools to assess treatment response at earlier time points and improve the precision of cell-based therapies.

Show MeSH
Related in: MedlinePlus