(F-209) Modifying lipid microbubble composition to enhance the delivery of gene-editing cargo into transfection-resistant cell types via microbubble-mediated sonoporation

Introduction:: Successful gene therapies require the effective cytosolic delivery of gene-editing cargo into cells. As the field of gene therapy advances, genetic cargos have become larger and more complex , such as those used in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems, consequently creating barriers to achieving efficient cytosolic delivery without perturbing cell viability. This is particularly concerning for sensitive cell types, for which current well-established transfection techniques (for example lipofection and electroporation) often cause significant cell death when eliciting high transfection efficiencies. Thus, there is a need for more robust methods of transfection that enable the intracellular delivery of large cargos into transfection-resistant cell types while minimizing the trade-off between transfection efficiency and cell viability.

Sonoporation is a transfection technique that harnesses the bioeffects of gas-filled microbubble oscillation induced by ultrasound exposure. Under the presence of an acoustic field, microbubbles can safely facilitate transient membrane permeabilization of neighbouring cells for intracellular delivery of surrounding cargo. As such, microbubble-enabled sonoporation represents a promising pathway towards enabling safe and successful transfection of sensitive cells. However, this potential has yet to be realized, whereby transfection efficiencies with this method using conventional microbubbles are not competitive with other well-established transfection techniques. To address this challenge, this project aims to modify the microbubble lipid shell composition to improve the transfection efficiency of sonoporation for a variety of difficult-to-transfect cell types while preserving cell viability.


Materials and Methods:: Rationally designed microbubble formulations with varying lipid shell chemistries and mechanical properties were generated to promote transient membrane destabilization and increased cell membrane sensitivity to sonoporation. The stability, average size, and population distribution of the formulations in the microbubble library were characterized using resistive pulse sensing. An acoustically compatible sonoporation setup for suspension and adherent cell types was developed in-house. Ultrasound parameters, incubation times, cell concentrations, and microbubble to cell ratios were explored to optimize the sonoporation parameter space. Double negative T cells and keratinocytes were used as examples of transfection-resistant primary human cell types for sonoporation experiments. Cells were sonoporated with fluorescent messenger ribonucleic acid cargo and different microbubble formulations and subsequently quantified using flow cytometry for transfection efficiency and cell viability.


Results, Conclusions, and Discussions:: A novel library of 16 microbubbles was developed, designed intentionally to improve sonoporation utility. It included microbubbles formulated with porphyrin-lipid, lyso-lipid and EDTA, all of which have established membrane intercalating properties. Using a centrifugal isolation technique, microbubbles were produced in high yield (1-4 x109 microbubbles/mL) and comparable size (1.5-2 um peak diameter size), important for ensuring un-confounded comparability between conventional and novel microbubbles. This library was applied systematically to double negative T cells and keratinocytes . In T cells, modest (10-40%) transfection was achieved with sonoporation while maintaining cell viabilities >80%. The variability within the untreated patient-derived T cells yielded challenges in further systematic sonoporation optimization. As such, optimization was continued in immortalized keratinocytes.

This project develops new microbubbles intended to enhance the capabilities of sonoporation. By developing different formulations designed to change microbubble properties in a specific way, strategies to further improve cell membrane permeabilization via sonoporation are elucidated. Improvements to the transfection efficiency of sonoporation can transform this transfection technique into a modality that can efficiently deliver large cargos into therapeutically important but transfection-resistant cell lines while conserving their viability. This would ultimately enable broader development of more safe and effective gene therapies for a wide range of sensitive primary cell types and cargos.


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