Virale Vektoren für den Gentransfer in korneale Endothelzellen

 

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Zusammenfassung

Hintergrund: Das Hornhautendothel ist aufgrund der einfachen Erreichbarkeit der Zellen und der Beschaffenheit als Monolayers als Angriffspunkt für Gentherapien sehr gut geeignet. Während Adenoviren aufgrund ihrer Immunogenität für eine In-vitro-Anwendung problematisch sind, erweisen sich Lentiviren und adenoassoziierte Viren (AAV) als gute Carrier (Vektoren) für den Transfer genetischer DNS in Endothelzellen (EC). Lentiviren, entwickelt auf der Basis von HI-Viren, können allerdings die übertragenen Gene in die DNS der Empfängerzelle integrieren und führen zu einer dauerhaften Überexpression. Zur Minimierung der Risiken nach einer Alternative zu HIV-basierten Lentiviren suchend, ist Ziel dieser Studie der Vergleich eines lentiviralen Vektoren mit einem für Menschen apathogenen, nicht integrierenden AAV. Material und Methoden: Durchflusszytometrischer Vergleich der Proteinexpression nach Verwendung eines lentiviralen Vektors oder von AAV 2 / 2 zur Transduktion eines grün fluoreszierenden Proteins in murinen EC (Balb/C) und in humanen EC (Zelllinie und Primärzellen). Proof-of-Principle-Experiment zur Bestätigung der Funktionalität des lentiviralen Gentransfers durch Übertragung des anti-apoptotischen Proteins Bcl-xL. Ergebnisse: Die Kinetik der Proteinexpression nach Transduktion von EC mit lentiviralen Vektoren unterscheidet sich grundlegend von der nach Gentransfer mittels AAV. Im Gegensatz zum Gentransfer mittels AAV erfolgt eine Überexpression des Reportergens nach lentiviralem Transfer sehr schnell. Weiterhin konnten deutlich Unterschiede hinsichtlich der Transduktionsrate zwischen humaner und muriner EC-Linie sowie zwischen humaner EC-Linie und humane Primärzellen nachgewiesen werden. Die Funktionalität des lentiviralen Gentransfers konnte durch eine signifikante Reduktion der Apoptose in humanen und murinen EC bestätigt werden. Schlussfolgerung: AAV-Vektoren stellen eine Alternative zu lentiviralen Vektoren für den Gentransfer in korneale EC dar. Angesichts der Kultivierung von Spenderhornhäuten in Organkultur über 4 Wochen vor Transplantation durch Hornhautbanken ist ein Einsatz i. R. der Hornhautkultivierung, z. B. zur Senkung der Apoptoserate in Spenderhornhäuten, denkbar.

Abstract

Background: Corneal endothelium is an interesting target for in vitro gene transfer strategies as it is readily accessible thanks to its anatomic structure as a monolayer and its direct contact to culture medium. Whereas the use of adenoviruses as viral vectors (carriers) to endothelial cells (EC) has been described as problematic as to its immunogenicity, lentiviruses and adeno-associated viruses (AAV) are potent vectors for the transfer of genetic DNA into EC. Lentiviral vectors, developed on the basis of HI-viruses, can integrate the transferred gene into the host DNA and thus lead to a permanent protein expression. Evaluating apathogen alternatives to lentiviral vectors for humans, we herein compared non-integrating AAV to lentiviral gene transfer. Materials and Methods: A comparison was made of the kinetics of expression of a green fluorescent protein after transduction using a lentiviral vector and AAV 2 / 2 in a murine EC line, human EC line and human primary cells (flow cytometry). A proof of principle experiment was conducted to demonstrate the function after lentiviral gene transfer of the anti-apoptotic gene Bcl-xL. Results: The kinetics of protein expression after transduction of EC using a lentiviral or an AAV vector show fundamental differences. Contrary to gene transfer using AAV, a high expression of the reporter protein was readily detectable only hours after transduction using the lentiral vector. In addition, we could demonstrate distinct differences in protein expression characteristics between human and murine EC as well as human EC line and primary human EC. Function could be demonstrated by showing a significant reduction in apoptosis in both murine and human EC. Conclusion: AAV vectors are an alternative to lentiviral vectors for gene transfer to corneal EC. Given a cultivation time of donor corneas of up to 4 weeks before transplantation, translation to eye banking, e. g., to decrease apoptosis in corneal allografts, is conceivable.

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Dr. Thomas Armin Fuchsluger

Zentrum für Augenheilkunde, Universitätsklinikum Essen

Hufelandstraße 55

45122 Essen

Phone: ++ 49/2 01/7 23 29 00

Fax: ++ 49/2 01/7 23 59 17

Email: thomas.fuchsluger@uk-essen.de