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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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Luciferase activity and S/MAR MCs are slowly lost over time in labeled cell populations.A) Normalized luciferase expression was measured at both day 64 and 121 post-transfection in three S/MAR MC clonal populations. All clones showed a trend (p = 0.18) towards decreased normalized luciferase expression over long periods of time in culture. S/MAR clone 3-7 was cultured up to day 178 after transfection and continued to show a slow loss of luciferase activity. B) The decrease in luciferase expression corresponded to a decrease in Luc2-S/MAR MC as shown via Southern blot analysis. A single band was seen in both control DNA spiked with 100 pg of S/MAR MC and DNA from S/MAR MC clone 3-7 at day 64 post-transfection. However, a band is barely discernable at day 121 from S/MAR MC clone 3-7, indicating a slow loss of S/MAR MC over time.
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pone-0073138-g003: Luciferase activity and S/MAR MCs are slowly lost over time in labeled cell populations.A) Normalized luciferase expression was measured at both day 64 and 121 post-transfection in three S/MAR MC clonal populations. All clones showed a trend (p = 0.18) towards decreased normalized luciferase expression over long periods of time in culture. S/MAR clone 3-7 was cultured up to day 178 after transfection and continued to show a slow loss of luciferase activity. B) The decrease in luciferase expression corresponded to a decrease in Luc2-S/MAR MC as shown via Southern blot analysis. A single band was seen in both control DNA spiked with 100 pg of S/MAR MC and DNA from S/MAR MC clone 3-7 at day 64 post-transfection. However, a band is barely discernable at day 121 from S/MAR MC clone 3-7, indicating a slow loss of S/MAR MC over time.

Mentions: Both RG expression and episomes are slowly lost in S/MAR MC labeled cells While we detected luminescent signal in the culture dish over time, the results in Figure 2B do not reflect differences in the number of cells in each dish at the time of imaging. Therefore, we performed a luminometer assay and protein assay on lysates from S/MAR MC clonal populations to measure Fluc activity (relative light units; RLU) normalized to protein content (µg) (Figure 3A). Comparing results at days 64 and 121 post-transfection across the three S/MAR MC clones we noted a trend (p = 0.18) towards decreased normalized Fluc activity (64% decrease for clone 3-7, 86% decrease for clone 3-5, and 87% decrease for clone 2-1), signifying a slow loss of Fluc activity over time. For S/MAR clone 3-7, which showed the highest Fluc activity of all clones and was cultured for the longest period of time, Fluc activity continued to decline up to day 178 post-transfection (97% decrease compared to day 64).


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)

Luciferase activity and S/MAR MCs are slowly lost over time in labeled cell populations.A) Normalized luciferase expression was measured at both day 64 and 121 post-transfection in three S/MAR MC clonal populations. All clones showed a trend (p = 0.18) towards decreased normalized luciferase expression over long periods of time in culture. S/MAR clone 3-7 was cultured up to day 178 after transfection and continued to show a slow loss of luciferase activity. B) The decrease in luciferase expression corresponded to a decrease in Luc2-S/MAR MC as shown via Southern blot analysis. A single band was seen in both control DNA spiked with 100 pg of S/MAR MC and DNA from S/MAR MC clone 3-7 at day 64 post-transfection. However, a band is barely discernable at day 121 from S/MAR MC clone 3-7, indicating a slow loss of S/MAR MC over time.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073138-g003: Luciferase activity and S/MAR MCs are slowly lost over time in labeled cell populations.A) Normalized luciferase expression was measured at both day 64 and 121 post-transfection in three S/MAR MC clonal populations. All clones showed a trend (p = 0.18) towards decreased normalized luciferase expression over long periods of time in culture. S/MAR clone 3-7 was cultured up to day 178 after transfection and continued to show a slow loss of luciferase activity. B) The decrease in luciferase expression corresponded to a decrease in Luc2-S/MAR MC as shown via Southern blot analysis. A single band was seen in both control DNA spiked with 100 pg of S/MAR MC and DNA from S/MAR MC clone 3-7 at day 64 post-transfection. However, a band is barely discernable at day 121 from S/MAR MC clone 3-7, indicating a slow loss of S/MAR MC over time.
Mentions: Both RG expression and episomes are slowly lost in S/MAR MC labeled cells While we detected luminescent signal in the culture dish over time, the results in Figure 2B do not reflect differences in the number of cells in each dish at the time of imaging. Therefore, we performed a luminometer assay and protein assay on lysates from S/MAR MC clonal populations to measure Fluc activity (relative light units; RLU) normalized to protein content (µg) (Figure 3A). Comparing results at days 64 and 121 post-transfection across the three S/MAR MC clones we noted a trend (p = 0.18) towards decreased normalized Fluc activity (64% decrease for clone 3-7, 86% decrease for clone 3-5, and 87% decrease for clone 2-1), signifying a slow loss of Fluc activity over time. For S/MAR clone 3-7, which showed the highest Fluc activity of all clones and was cultured for the longest period of time, Fluc activity continued to decline up to day 178 post-transfection (97% decrease compared to day 64).

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