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In situ transduction of cells in human corneal limbus using adeno-associated viruses: an ex vivo study

This study aimed to evaluate the efficacy of in situ adeno-associated virus (AAV)-mediated gene delivery into the human corneal limbal region via targeted sub-limbal injection technique. Human cadaveric corneal tissues were fixed on an artificial anterior chamber. Feasibility of sub-limbal injection...

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Main Authors: Son, Hyeck Soo (Author) , Jun, Albert S. (Author) , Foster, James W. (Author) , Wang, Wei (Author) , Daoud, Yassine (Author) , Auffarth, Gerd U. (Author) , Roy, Madhuparna (Author)
Format: Article (Journal)
Language:English
Published: 28 December 2022
In: Scientific reports
Year: 2022, Volume: 12, Pages: 1-8
ISSN:2045-2322
DOI:10.1038/s41598-022-26926-0
Online Access:Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1038/s41598-022-26926-0
Verlag, lizenzpflichtig, Volltext: https://www.nature.com/articles/s41598-022-26926-0
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Author Notes:Hyeck-Soo Son, Albert S. Jun, James W. Foster, Wei Wang, Yassine Daoud, Gerd U. Auffarth & Madhuparna Roy

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520 |a This study aimed to evaluate the efficacy of in situ adeno-associated virus (AAV)-mediated gene delivery into the human corneal limbal region via targeted sub-limbal injection technique. Human cadaveric corneal tissues were fixed on an artificial anterior chamber. Feasibility of sub-limbal injection technique was tested using trypan blue and black India ink. An enhanced green fluorescent protein (eGFP) encoding AAV DJ was injected into sub-limbal region. After AAV injection, corneal tissues were incubated in air-lift culture and prepared for immunohistochemical analysis. Cell survivial and expression of eGFP, stem cell markers (p63α and cytokeratin 19 (KRT19)), and differentiation marker cytokeratin 3 (KRT3) were evaluated using confocal microscopy. Both trypan blue and black India ink stained and were retained sub-limbally establishing specificity of the injection technique. Immunohistochemical analysis of corneas injected with AAV DJ-eGFP indicated that AAV-transduced cells in the limbal region co-express eGFP, p63α, and KRT19 and that these transduced cells were capable of differentiating to KRT3 postitive corneal epithelial cells. Our sub-limbal injection technique can target cells in the human limbus in a reproducible and efficient manner. Thus, we demonstrate that in situ injection of corneal limbus may provide a feasible mode of genetic therapy for corneal disorders with an epithelial etiology. 
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