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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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Proliferation of S/MAR MC labeled cells can be monitored over time in living subjects.A) S/MAR MC labeled breast cancer cells were implanted into the right flank of Nu/Nu mice and bioluminescence imaging (BLI) was performed over time. As tumors developed more luminescent signal was noted. B) This observation was confirmed by performing region of interest analysis over the tumor and measuring average radiance (p/s/cm2/sr) at days 7, 20, 28, 35 and 43 post-implantation. Significantly higher BLI signal (n = 5; * p<0.05) was noted at days 35 and 43 post-implantation compared to day 7. Error bars are S.E.M..
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pone-0073138-g004: Proliferation of S/MAR MC labeled cells can be monitored over time in living subjects.A) S/MAR MC labeled breast cancer cells were implanted into the right flank of Nu/Nu mice and bioluminescence imaging (BLI) was performed over time. As tumors developed more luminescent signal was noted. B) This observation was confirmed by performing region of interest analysis over the tumor and measuring average radiance (p/s/cm2/sr) at days 7, 20, 28, 35 and 43 post-implantation. Significantly higher BLI signal (n = 5; * p<0.05) was noted at days 35 and 43 post-implantation compared to day 7. Error bars are S.E.M..

Mentions: To show that S/MAR MCs can be used as a platform for tracking proliferating cells in vivo we implanted 1.5×106 MDA-MB-231 Luc2-S/MAR MC labeled cells (clone 3-7; day 61 post-transfection) into the right flank of 6 week old female Nu/Nu mice and performed BLI over time (Figure 4 and Figure S1). As shown in Figure 4A, as tumors developed we saw an increase in the bioluminescent signal in the flank of these animals. We confirmed this observation by quantifying the amount of bioluminescent signal emanating from tumors. To do this, we performed ROI analysis over tumor sites and detected significant increases in average radiance at days 35 and 43 post-implantation compared to day 7. (Figure 4B). Therefore, as cells divided in vivo the S/MAR MCs continued to replicate, verifying that these constructs can be used to perform RG imaging of dividing cells in living subjects.


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)

Proliferation of S/MAR MC labeled cells can be monitored over time in living subjects.A) S/MAR MC labeled breast cancer cells were implanted into the right flank of Nu/Nu mice and bioluminescence imaging (BLI) was performed over time. As tumors developed more luminescent signal was noted. B) This observation was confirmed by performing region of interest analysis over the tumor and measuring average radiance (p/s/cm2/sr) at days 7, 20, 28, 35 and 43 post-implantation. Significantly higher BLI signal (n = 5; * p<0.05) was noted at days 35 and 43 post-implantation compared to day 7. Error bars are S.E.M..
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073138-g004: Proliferation of S/MAR MC labeled cells can be monitored over time in living subjects.A) S/MAR MC labeled breast cancer cells were implanted into the right flank of Nu/Nu mice and bioluminescence imaging (BLI) was performed over time. As tumors developed more luminescent signal was noted. B) This observation was confirmed by performing region of interest analysis over the tumor and measuring average radiance (p/s/cm2/sr) at days 7, 20, 28, 35 and 43 post-implantation. Significantly higher BLI signal (n = 5; * p<0.05) was noted at days 35 and 43 post-implantation compared to day 7. Error bars are S.E.M..
Mentions: To show that S/MAR MCs can be used as a platform for tracking proliferating cells in vivo we implanted 1.5×106 MDA-MB-231 Luc2-S/MAR MC labeled cells (clone 3-7; day 61 post-transfection) into the right flank of 6 week old female Nu/Nu mice and performed BLI over time (Figure 4 and Figure S1). As shown in Figure 4A, as tumors developed we saw an increase in the bioluminescent signal in the flank of these animals. We confirmed this observation by quantifying the amount of bioluminescent signal emanating from tumors. To do this, we performed ROI analysis over tumor sites and detected significant increases in average radiance at days 35 and 43 post-implantation compared to day 7. (Figure 4B). Therefore, as cells divided in vivo the S/MAR MCs continued to replicate, verifying that these constructs can be used to perform RG imaging of dividing cells in living subjects.

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