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

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 regula...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	John A Ronald, Lorena Cusso, Hui-Yen Chuang, Xinrui Yan, Anca Dragulescu-Andrasi, Sanjiv Sam Gambhir
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/7c355f60dfc54862924813edb17c3918
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:7c355f60dfc54862924813edb17c3918
record_format	dspace
spelling	oai:doaj.org-article:7c355f60dfc54862924813edb17c39182021-11-18T08:57:50ZDevelopment and validation of non-integrative, self-limited, and replicating minicircles for safe reporter gene imaging of cell-based therapies.1932-620310.1371/journal.pone.0073138https://doaj.org/article/7c355f60dfc54862924813edb17c39182013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24015294/?tool=EBIhttps://doaj.org/toc/1932-6203Reporter 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.John A RonaldLorena CussoHui-Yen ChuangXinrui YanAnca Dragulescu-AndrasiSanjiv Sam GambhirPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 8, p e73138 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q John A Ronald Lorena Cusso Hui-Yen Chuang Xinrui Yan Anca Dragulescu-Andrasi Sanjiv Sam Gambhir Development and validation of non-integrative, self-limited, and replicating minicircles for safe reporter gene imaging of cell-based therapies.
description	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.
format	article
author	John A Ronald Lorena Cusso Hui-Yen Chuang Xinrui Yan Anca Dragulescu-Andrasi Sanjiv Sam Gambhir
author_facet	John A Ronald Lorena Cusso Hui-Yen Chuang Xinrui Yan Anca Dragulescu-Andrasi Sanjiv Sam Gambhir
author_sort	John A Ronald
title	Development and validation of non-integrative, self-limited, and replicating minicircles for safe reporter gene imaging of cell-based therapies.
title_short	Development and validation of non-integrative, self-limited, and replicating minicircles for safe reporter gene imaging of cell-based therapies.
title_full	Development and validation of non-integrative, self-limited, and replicating minicircles for safe reporter gene imaging of cell-based therapies.
title_fullStr	Development and validation of non-integrative, self-limited, and replicating minicircles for safe reporter gene imaging of cell-based therapies.
title_full_unstemmed	Development and validation of non-integrative, self-limited, and replicating minicircles for safe reporter gene imaging of cell-based therapies.
title_sort	development and validation of non-integrative, self-limited, and replicating minicircles for safe reporter gene imaging of cell-based therapies.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/7c355f60dfc54862924813edb17c3918
work_keys_str_mv	AT johnaronald developmentandvalidationofnonintegrativeselflimitedandreplicatingminicirclesforsafereportergeneimagingofcellbasedtherapies AT lorenacusso developmentandvalidationofnonintegrativeselflimitedandreplicatingminicirclesforsafereportergeneimagingofcellbasedtherapies AT huiyenchuang developmentandvalidationofnonintegrativeselflimitedandreplicatingminicirclesforsafereportergeneimagingofcellbasedtherapies AT xinruiyan developmentandvalidationofnonintegrativeselflimitedandreplicatingminicirclesforsafereportergeneimagingofcellbasedtherapies AT ancadragulescuandrasi developmentandvalidationofnonintegrativeselflimitedandreplicatingminicirclesforsafereportergeneimagingofcellbasedtherapies AT sanjivsamgambhir developmentandvalidationofnonintegrativeselflimitedandreplicatingminicirclesforsafereportergeneimagingofcellbasedtherapies
_version_	1718421092043849728

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

Ejemplares similares