Amplified femtosecond laser permeabilization and poration for nonviral corneal gene delivery

Abstract
In the cornea, topical application is the most desired method for administration of gene therapy, but the
effective delivery to the corneal stroma is limited by two major factors. First, tight junctions between epithelial
cells create a highly effective barrier to large molecule diffusion. Second, the delivery agent carrying the
therapeutic DNA must also be capable of entering the cells of the targeted tissue without posing a safety hazard
to the patient or medical personnel. Circumventing the epithelial barrier currently requires the removal of the
corneal epithelium to deliver therapeutic agents into the corneal stroma. This causes patient discomfort, delayed
visual recovery, and increased risk of bacterial infection and corneal scarring. To tackle this first roadblock, we
have developed a novel, FS laser-based, corneal epithelial micromachining approach capable of creating
microchannels through the epithelium which greatly enhance transepithelial diffusion without resulting in long
term damage. Next, delivery of the therapeutic DNA into stromal cells is often effectively accomplished using
viral vectors such as adeno-associated (AAV) or lentiviral vectors for transport, both of which are capable of
infecting all three major corneal cell types, epithelium, keratocyte, and endothelium. While both are relatively
safe, they both struggle with limited carrying capacity and immunogenicity. We propose that subthreshold LIOB
FS laser pulses could be used to open membrane pores in stromal keratocytes without damage to cells and
surrounding tissue depending on pulse energies. If successful, FS-poration could be used to replace viral vectors
with plasmid delivery and provide for safer gene delivery with no limit to gene size. Combined with our epithelial
microchannels, this technique could result in a topical transepithelial gene delivery technique with no limit to the
size of genes used.

Grant Number: 1R21EY036176-01A1
NIH Institute/Center: NIH
Principal Investigator: Samantha Bradford