Femtosecond Laser (FS) Poration for Nonviral Corneal Gene Transfer

Abstract

Purpose : Corneal gene therapy is currently limited by the ability to target DNA delivery to specific cells, particularly corneal keratocytes. The purpose of this study was to test the hypothesis that FS laser pulses below the energy threshold required for laser induced optical breakdown, can be used to open temporary membrane pores and facilitate gene transfection into stromal keratocytes.

Methods : Confluent cultures of SV40 transformed rabbit keratocytes (TRK) were FS-porated with a spot density of 100 pulses per mm² at 1 µJ/pulse from a 1030nm <200 FS laser in the presence of the fluorescent dye Carboxyfluorescein (CF), and immediately imaged. FS-porated TRK cells were also evaluated by live/dead assay, in the presence of Ethidium Homodimer (EH) and Calcein AM to detect dead and live cells respectively and assess the temporary nature of the pores. Cells were also FS-porated in the presence of 5 or 10 µg of eGFP T7 CMV plasmid construct with a spot density of 400 pulses per mm². For comparison, cells were also treated with FuGene at 3:1 or 4:1 transfection reagent to DNA. Cells treated with DNA were cultured for 48 hours to allow for transcription then fixed and DAPI stained to identify cells.

Results : FS-poration of CF treated cells showed a grid of fluorescent cells spaced 100 µm apart, while brightfield imaging showed no cellular damage within the porated region. Imaging of live/dead treated cells revealed regions of cells which stained with both dyes, indicating survival of FS-porated cells, while no dead, EH only labeled cells, were detected. FS-porated cells treated with 5 and 10 µg DNA produced GFP positive cells only within the treated region, resulting in 1.6 ± 0.6% and 6.2 ± 0.9% positive cells, respectively. Since each mm² region only contains 400 poration locations, these values represent 5.0 ± 1.7% and 16.1 ± 7.8% of the possible poration locations. By comparison cells treated with FuGene produced similar transfection efficiencies of 17.6 ± 4.5% and 15.6 ± 9.9%.

Conclusions : These findings indicate that FS-poration can provide transfection efficiencies comparable to that achieved by FuGene without damaging cells. We propose that this technique could be used for targeted, transepithelial keratocyte gene delivery with no limit to the size of genes used.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.