72 research outputs found
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 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, 1.5Ă10(6) cells carrying the S/MAR minicircle were implanted subcutaneously into mice (nâ=â5) 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.The authors would like to acknowledge the imaging support provided by the Stanford Small Animal Imaging FacilityPublicad
Inhibition of Multidrug Resistance by SV40 Pseudovirion Delivery of an Antigene Peptide Nucleic Acid (PNA) in Cultured Cells
Peptide nucleic acid (PNA) is known to bind with extraordinarily high affinity and sequence-specificity to complementary nucleic acid sequences and can be used to suppress gene expression. However, effective delivery into cells is a major obstacle to the development of PNA for gene therapy applications. Here, we present a novel method for the in vitro delivery of antigene PNA to cells. By using a nucleocapsid protein derived from Simian virus 40, we have been able to package PNA into pseudovirions, facilitating the delivery of the packaged PNA into cells. We demonstrate that this system can be used effectively to suppress gene expression associated with multidrug resistance in cancer cells, as shown by RT-PCR, flow cytometry, Western blotting, and cell viability under chemotherapy. The combination of PNA with the SV40-based delivery system is a method for suppressing a gene of interest that could be broadly applied to numerous targets
Evaluation of flight efficiency for Stockholm Arlanda Airport arrivals
Analysis of punctuality of airport arrivals, as well as identification of causes of the delays within transition airspace, is an important step in evaluating performance of the Terminal Maneuvering Area (TMA) Air Navigation Services: without knowing the current performance levels, it is difficult to identify which areas could be improved. Deviations from the flight plans is one of the major reasons for arrival delays. In this work, we quantified the impact of the deviations from the flight plans on the fuel burn. One of the main reasons of fuel waste is non- optimal vertical profiles during the descent phase. We calculated how much extra fuel is wasted due to vertical flight inefficiency within Stockholm TMA.Peer ReviewedPostprint (published version
Activatable Oligomerizable Imaging Agents for Photoacoustic Imaging of Furin-Like Activity in Living Subjects
Photoacoustic (PA) imaging is continuing
to be applied for physiological
imaging and more recently for molecular imaging of living subjects.
Owing to its high spatial resolution in deep tissues, PA imaging holds
great potential for biomedical applications and molecular diagnostics.
There is however a lack of probes for targeted PA imaging, especially
in the area of enzyme-activatable probes. Here we introduce a molecular
probe, which upon proteolytic processing is retained at the site of
enzyme activity and provides PA contrast. The probe oligomerizes via
a condensation reaction and accumulates in cells and tumors that express
the protease. We demonstrate that this probe reports furin and furin-like
activity in cells and tumor models by generating a significantly higher
PA signal relative to furin-deficient and nontarget controls. This
probe could report enzyme activity in living subjects at depths significantly
greater than fluorescence imaging probes and has potential for molecular
imaging in deep tumors
Activatable Oligomerizable Imaging Agents for Photoacoustic Imaging of Furin-Like Activity in Living Subjects
Photoacoustic (PA) imaging is continuing
to be applied for physiological
imaging and more recently for molecular imaging of living subjects.
Owing to its high spatial resolution in deep tissues, PA imaging holds
great potential for biomedical applications and molecular diagnostics.
There is however a lack of probes for targeted PA imaging, especially
in the area of enzyme-activatable probes. Here we introduce a molecular
probe, which upon proteolytic processing is retained at the site of
enzyme activity and provides PA contrast. The probe oligomerizes via
a condensation reaction and accumulates in cells and tumors that express
the protease. We demonstrate that this probe reports furin and furin-like
activity in cells and tumor models by generating a significantly higher
PA signal relative to furin-deficient and nontarget controls. This
probe could report enzyme activity in living subjects at depths significantly
greater than fluorescence imaging probes and has potential for molecular
imaging in deep tumors
Activatable Oligomerizable Imaging Agents for Photoacoustic Imaging of Furin-Like Activity in Living Subjects
Photoacoustic (PA) imaging is continuing
to be applied for physiological
imaging and more recently for molecular imaging of living subjects.
Owing to its high spatial resolution in deep tissues, PA imaging holds
great potential for biomedical applications and molecular diagnostics.
There is however a lack of probes for targeted PA imaging, especially
in the area of enzyme-activatable probes. Here we introduce a molecular
probe, which upon proteolytic processing is retained at the site of
enzyme activity and provides PA contrast. The probe oligomerizes via
a condensation reaction and accumulates in cells and tumors that express
the protease. We demonstrate that this probe reports furin and furin-like
activity in cells and tumor models by generating a significantly higher
PA signal relative to furin-deficient and nontarget controls. This
probe could report enzyme activity in living subjects at depths significantly
greater than fluorescence imaging probes and has potential for molecular
imaging in deep tumors
Activatable Oligomerizable Imaging Agents for Photoacoustic Imaging of Furin-Like Activity in Living Subjects
Photoacoustic (PA) imaging is continuing
to be applied for physiological
imaging and more recently for molecular imaging of living subjects.
Owing to its high spatial resolution in deep tissues, PA imaging holds
great potential for biomedical applications and molecular diagnostics.
There is however a lack of probes for targeted PA imaging, especially
in the area of enzyme-activatable probes. Here we introduce a molecular
probe, which upon proteolytic processing is retained at the site of
enzyme activity and provides PA contrast. The probe oligomerizes via
a condensation reaction and accumulates in cells and tumors that express
the protease. We demonstrate that this probe reports furin and furin-like
activity in cells and tumor models by generating a significantly higher
PA signal relative to furin-deficient and nontarget controls. This
probe could report enzyme activity in living subjects at depths significantly
greater than fluorescence imaging probes and has potential for molecular
imaging in deep tumors
S/MAR MCs can label cells in culture for extended periods of time and remain episomal.
<p>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 (3â5 and 3â7) 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 ”g) 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.</p
Detecting cancers through tumor-activatable minicircles that lead to a detectable blood biomarker
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