Ultrasound-directed, microbubble-assisted Crispr/Cas9 delivery in RPE cells

Abstract

Purpose : Crispr/Cas9 is a popular gene editing tool whose therapeutic potential in ophthalmology is being actively explored. Our goal is to develop a microbubble (MB)-assisted sonoporation method to deliver the Crispr/Cas9 constructs into the eye for the treatment of hereditary retinal diseases with minimal invasiveness and high spatial precision. This study investigates the feasibility of sonoporating Crispr/Cas9 plasmids into cultured human retinal pigment epithelial (RPE) cells, a cell type that is heavily involved in the pathology of the outer retinal degeneration.

Methods : Sonoporation was tested in ARPE-19 cells and HEK cells. The GFP plasmid and the RPE65 Crispr/Cas9 knockout plasmid with a GFP tag were employed to fluorescently label the transfected cells. The GTS Sonoporation System equipped with a plane wave transducer (center frequency of 1 MHz) was used as the ultrasound (US) source. The cell monolayer cultured on a cover slip was placed in a customized polydimethylsiloxane (PDMS) well supplied with the MBs and the DNA plasmids. US was delivered from the transducer placed ~35 mm below and the entire system was submerged in heated distilled water. Transfection under variations of sonoporation parameters was examined by fluorescence microscopy. The trypan blue assay was employed to test cell viability.

Results : Our in vitro experimental setup successfully introduced the GFP plasmid and the commercial Crispr/Cas9 plasmid into the HEK and the ARPE-19 cells. Among the sonoporation parameters examined, increase of the acoustic intensity from 2 to 4 W/cm² enhanced the GFP delivery by 1.6 fold and increase of the MB concentration from 0.4 to 4% (V/V) resulted in a 6.8 fold enhancement. However, the transfection efficiency of the Crispr/Cas9 plasmid was substantially lower than that of the GFP under similar sonoporation conditions, either at 100 Hz or 1000 Hz pulse repetition frequency (PRF). All sonoporation experiments did not lead to increased cell death.

Conclusions : This study shows the possibility of sonoporating an all-in-one Crispr/Cas9 plasmid into cultured human HEK cells and RPE cells at 1 MHz acoustic frequency. The lower delivery efficiency of Crispr than GFP may stem from the bulky size of the all-in-one Crispr plasmid that encodes both the Cas9 nuclease and the guide RNA (gRNA). Further studies are underway to investigate the microbubble-assisted sonoporation of more versatile Cas9 and gRNA constructs.

This is a 2021 ARVO Annual Meeting abstract.