title: Development and validation of non-integrative, self-limited, and replicating minicircles for safe reporter gene imaging of cell-based therapies.
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.
authors.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.
authors.forename: John A
authors.initials: JA
authors.lastname: Ronald
authors.forename: Lorena
authors.initials: L
authors.lastname: Cusso
authors.forename: Hui-Yen
authors.initials: HY
authors.lastname: Chuang
authors.forename: Xinrui
authors.initials: X
authors.lastname: Yan
authors.forename: Anca
authors.initials: A
authors.lastname: Dragulescu-Andrasi
authors.forename: Sanjiv Sam
authors.initials: SS
authors.lastname: Gambhir
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000473:pathology / Q000628:therapy; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D064987:Cell- and Tissue-Based Therapy / Q000379:methods*; D003952:Diagnostic Imaging / Q000379:methods*; D005260:Female; D017930:Genes, Reporter / Q000235:genetics*; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D010957:Plasmids / Q000235:genetics*; D012093:Replicon / Q000235:genetics*