Amplified femtosecond laser permeabilization and poration for nonviral corneal gene delivery

University Of California-Irvine, Irvine CA

Investigators

Abstract

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.