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Abstract Retroviral gene delivery is widely used in T cell therapies for hematological cancers. However, viral vectors are expensive to manufacture, integrate genes in semirandom patterns, and their transduction efficiency varies between patients. In this study, several nonviral gene delivery vehicles, promoters, and additional variables were compared to optimize nonviral transgene delivery and expression in both Jurkat and primary T cells. Transfection of Jurkat cells was maximized to a high efficiency (63.0% ± 10.9% EGFP+ cells) by transfecting cells with Lipofectamine LTX in X‐VIVO 15 media. However, the same method yielded a much lower transfection efficiency in primary T cells (8.1% ± 0.8% EGFP+). Subsequent confocal microscopy revealed that a majority of the lipoplexes did not enter the primary T cells, which might be due to relatively low expression levels of heparan sulfate proteoglycans detected via messenger RNA‐sequencing. Pyrin and HIN (PYHIN) DNA sensors (e.g., AIM2 and IFI16) that can induce apoptosis or repress transcription after binding cytoplasmic DNA were also detected at high levels in primary T cells. Therefore, transfection of primary T cells appears to be limited at the level of cellular uptake or DNA sensing in the cytoplasm. Both of these factors should be considered in the development of future viral and nonviral T cell gene delivery methods.
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Modulation of acoustofluidic parameters to assess effect on molecular loading in human T cells.
T-cell therapies are rapidly emerging for treatment of cancer and other diseases but are limited by inefficient non-viral delivery methods. Acoustofluidic devices are in development to enhance non-viral delivery to cells. The effect of acoustofluidic parameters, such as channel geometry, on molecular loading in human T cells was assessed using 3D-printed acoustofluidic devices. Devices with rectilinear channels (1- and 2-mm diameters) were compared directly with concentric spiral channel geometries. Intracellular delivery of a fluorescent dye (calcein, 100 lg/ml) was evaluated in Jurkat T cells using flow cytometry after ultrasound treatment with cationic microbubbles (2.5% v/v). B-mode ultrasound pulses (2.5 MHz, 3.8 MPa output pressure) were generated by a P4-1 transducer on a Verasonics Vantage ultrasound system. Cell viability was assessed using propidum iodine staining (10 lg/ml). Intracellular molecular delivery was significantly enhanced with acoustofluidic treatment in each channel geometry, but treatment with the 1-mm concentric spiral geometry further enhanced delivery after acoustofluidic treatment compared to both 1- and 2-mm rectilinear channels (ANOVA p < 0.001, n ¼ 6/group). These results indicate that 3Dprinted acoustofluidic devices enhance molecular delivery to T cells, and channel geometry modulates intracellular loading efficiency. This approach may offer advantages to improve manufacturing of T cell therapies.
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- Award ID(s):
- 1950137
- PAR ID:
- 10322710
- Date Published:
- Journal Name:
- The journal of the Acoustical Society of America
- Volume:
- 150
- Issue:
- 4
- ISSN:
- 1520-9024
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1121/10.0007543
