DIRECTED EVOLUTION OF AN ONCOLYTIC VESICULAR STOMATITIS VIRUS ADAPTED TO HUMAN MALIGNANT MENINGIOMA CELLS

Yıl 2024, Cilt: 7 Sayı: 1, 24 - 31, 15.02.2024

Burak Gizem Göleş , Hülya Yazıcı , John N. Davıs

https://doi.org/10.26650/JARHS2024-1300891

Öz

Objectives: Recombinantly-engineered versions of the oncolytic virus VSV are currently under clinical investigation for the treatment of several different types of cancer. Here we aim to enhance the cancer-killing oncolytic phenotype of VSV-1’GFP toward human malignant meningioma cells using a directed evolution approach.

Material and Methods: Two independent trials of repeated growth of VSV-1’GFP on cultures of meningioma IOMM-Lee cells were performed. This adaptation procedure allows for the selection of viral mutants that display an enhanced oncolytic phenotype. A fluorescent viral plaque assay was used to measure changes in plaque size indicative of enhanced viral growth on these cancer cells. Sanger sequencing was used to identify the viral mutations responsible.

Results: Adapted VSV-1’GFP from each of the growth trials yielded larger fluorescent plaques than control virus, indicating the emergence of viral mutants with increased growth on these meningioma cells. Plaques from adapted virus were 184%±9% (Trial 1) and 166%±7% (Trial 2) larger than control (n=60; p<0.001; ANOVA). Sequencing determined that adapted virus from Trial 1 harbored 3 mutations: a silent mutation Y178Y in the M gene, an E92K mutation in the G gene, and a K152R mutation in the L gene. Trial 2 yielded 3 mutations in the G gene: N36T, E92K, and E254K.

Conclusion: The E92K mutation of the viral G-protein emerged independently in both growth trials, suggesting that this change may play a role in producing the enlarged-plaque phenotype and enhanced oncolytic propagation in IOMM-Lee cells. Further investigations of the prospect for treating malignant meningiomas using VSV-based oncolytic virotherapy appear warranted and, to the best of our knowledge, the present study appears to be the first directed evolution experiment involving an oncolytic virus adapted to human meningioma cells.

Anahtar Kelimeler

Vesicular stomatitis virus, oncolytic virus, meningioma, mutagenesis

Teşekkür

I would like to express my sincere gratitude and appreciation for Dr. Murat GUNEL and his invaluable contribution to this article.

İNSAN MALIGANT MENİNJİOM HÜCRELERİNE UYUMLANMIŞ BİR ONKOLİTİK VEZİKÜLER STOMATİT VİRÜSÜNÜN YÖNLENDİRİLMİŞ EVRİMİ

Öz

Amaç: Onkolitik virüs VSV’nin rekombinant olarak tasarlanmış çeşitleri birkaç farklı kanser türünün tedavisi için klinik olarak araştırılmaktadır. Bu çalışmada, yönlendirilmiş evrim yaklaşımı kullanarak VSV-1’GFP’ nin insan malign meninjiyom hücrelerine yönelik kanser öldürücü onkolitik fenotipini geliştirmek hedeflenmektedir.

Gereç ve Yöntemler: Meninjiyom IOMM-Lee hücrelerinin kültürleri üzerinde VSV-1’GFP’nin tekrarlanan büyümesine ilişkin iki bağımsız deneme gerçekleştirildi. Bu adaptasyon prosedürü, gelişmiş bir onkolitik fenotip gösteren viral mutantların seçimine olanak tanır. Bu kanser hücrelerinde viral büyümenin arttığını gösteren plak boyutundaki değişiklikleri ölçmek için bir floresan viral plak tahlili kullanıldı. Sorumlu viral mutasyonları tanımlamak için Sanger dizilimi kullanıldı.

Bulgular: Büyüme denemelerinin her birinden uyarlanmış VSV-1’GFP, kontrol virüsünden daha büyük floresan plaklar verdi; bu, meninjiyom hücrelerinde artan büyüme ile viral mutantların ortaya çıktığını gösterir. Uyarlanmış virüsten alınan plaklar kontrolden (n=60; p<0,001; ANOVA) %184±%9 (Deneme 1) ve %166±%7 (Deneme 2) daha büyüktü. Dizileme, Deneme 1’den uyarlanan virüsün 3 mutasyon barındırdığını belirledi: M geninde sessiz bir Y178Y mutasyonu, G geninde bir E92K mutasyonu ve L geninde bir K152R mutasyonu. Deneme 2, G geninde 3 mutasyon ortaya çıkardı: N36T, E92K ve E254K.

Sonuç: Viral G-proteininin E92K mutasyonu, her iki büyüme denemesinde de bağımsız olarak ortaya çıktı; bu değişiklik, IOMM-Lee hücrelerinde genişlemiş plak fenotipinin ve gelişmiş onkolitik yayılımın üretilmesinde rol oynayabileceğini düşündürmektedir. Malign meninjiyomların VSV bazlı onkolitik viroterapi kullanılarak tedavi edilmesi olasılığına ilişkin daha fazla araştırma gerekli görülmektedir ve bilgimiz dâhilinde, mevcut çalışma, insan meninjiyom hücrelerine uyarlanmış bir onkolitik virüsü içeren ilk yönlendirilmiş evrim deneyi olarak literatüre katkı sunmaktadır.

Anahtar Kelimeler

Veziküler stomatit virüs, onkolitik virus, meninjiom, mutagenez

Kaynakça

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