In situ transduction of cells in human corneal limbus using adeno-associated viruses: an ex vivo study
Abstract 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...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-12-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-26926-0 |
| _version_ | 1797973642241376256 |
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| author | Hyeck-Soo Son Albert S. Jun James W. Foster Wei Wang Yassine Daoud Gerd U. Auffarth Madhuparna Roy |
| author_facet | Hyeck-Soo Son Albert S. Jun James W. Foster Wei Wang Yassine Daoud Gerd U. Auffarth Madhuparna Roy |
| author_sort | Hyeck-Soo Son |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-04-11T04:07:31Z |
| format | Article |
| id | doaj.art-8a67638419a64c2da2b58066ec2f0519 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-11T04:07:31Z |
| publishDate | 2022-12-01 |
| publisher | Nature Portfolio |
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| series | Scientific Reports |
| spelling | doaj.art-8a67638419a64c2da2b58066ec2f05192023-01-01T12:17:17ZengNature PortfolioScientific Reports2045-23222022-12-011211810.1038/s41598-022-26926-0In situ transduction of cells in human corneal limbus using adeno-associated viruses: an ex vivo studyHyeck-Soo Son0Albert S. Jun1James W. Foster2Wei Wang3Yassine Daoud4Gerd U. Auffarth5Madhuparna Roy6Wilmer Eye Institute, Johns Hopkins Medical InstitutionsWilmer Eye Institute, Johns Hopkins Medical InstitutionsWilmer Eye Institute, Johns Hopkins Medical InstitutionsWilmer Eye Institute, Johns Hopkins Medical InstitutionsWilmer Eye Institute, Johns Hopkins Medical InstitutionsUniversity Hospital HeidelbergWilmer Eye Institute, Johns Hopkins Medical InstitutionsAbstract 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.https://doi.org/10.1038/s41598-022-26926-0 |
| spellingShingle | Hyeck-Soo Son Albert S. Jun James W. Foster Wei Wang Yassine Daoud Gerd U. Auffarth Madhuparna Roy In situ transduction of cells in human corneal limbus using adeno-associated viruses: an ex vivo study Scientific Reports |
| title | In situ transduction of cells in human corneal limbus using adeno-associated viruses: an ex vivo study |
| title_full | In situ transduction of cells in human corneal limbus using adeno-associated viruses: an ex vivo study |
| title_fullStr | In situ transduction of cells in human corneal limbus using adeno-associated viruses: an ex vivo study |
| title_full_unstemmed | In situ transduction of cells in human corneal limbus using adeno-associated viruses: an ex vivo study |
| title_short | In situ transduction of cells in human corneal limbus using adeno-associated viruses: an ex vivo study |
| title_sort | in situ transduction of cells in human corneal limbus using adeno associated viruses an ex vivo study |
| url | https://doi.org/10.1038/s41598-022-26926-0 |
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