Abstract
Purpose :
Inherited corneal dystrophies may cause severe visual impairment. While phototherapeutic keratectomy or corneal transplantations may provide symptomatic relief, the procedures are associated with high infection and recurrence rates. Gene therapies may provide an alternative and a potentially permanent treatment. Adeno-associated viruses (AAVs) have emerged as the vectors of choice for ocular gene therapy thanks to their established safety profile. This study aimed to investigate limbal injections into human cadaveric tissues using 1) trypan blue as a visual guide and 2) two AAV serotypes to test the in situ transduction efficiency of human limbal stem cells (LSCs).
Methods :
Human cadaveric corneal tissues were fixed on an artificial anterior chamber (Katena, Parsippany, NJ, USA). Trypan blue (50-µl) was injected into the limbal region using a beveld 34-gauge needle (JBP Nanoneedle, South Korea) and a 1-ml syringe (BD Syringe, Franklin Lakes, NJ, USA). In parallel, 50-µl of AAV serotypes 1 and DJ (1011 genome copies(GC)/mL diluted in phosphate-buffered saline) harboring enhanced green fluorescent protein (eGFP) gene were injected into separate human cadaveric corneal tissues using the same injection technique. Tissues injected with AAV-eGFP were cultured for 2 weeks using the “air-lift” method and evaluated for immunohistochemical staining. Expression of eGFP as well as recombinant stem cell marker p63 was analyzed using a confocal microscope.
Results :
Histological assessment showed trypan blue staining of the sub-limbal region, confirming that LSCs can be targeted using the injection technique. Immunohistochemical analysis of the corneal tissues injected with AAV serotypes 1 and DJ show that LSCs co-stain p63alpha, eGFP, and DAPI.
Conclusions :
The injection technique could successfully target human LSCs in a reproducible manner, and led to effective transduction of LSCs with AAV serotypes 1 and DJ. Thus, we demonstrate that in situ injection of limbal cells may provide a feasible mode for corneal genetic therapy.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.