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Published on: May 27, 2011
Potency-enhancing mutations in E3-19K and i-leader increase the cytolytic activity of the PH20/SPAM1-armed oncolytic
Aleksei A Stepanenko1,2, Anastasiia O Sosnovtseva1,3,4, Anastasiia A Vasiukova1
1Department of Fundamental and Applied Neurobiology, V.P. Serbsky National Medical Research Center of Psychiatry and Narcology of the Ministry of Health of the Russian Federation, 119034 Moscow, Russia.
Abstract:
The oncolytic adenovirus Ad5-delta-24-RGD (Ad5Δ24RGD) exhibits suboptimal therapeutic efficacy in high-grade glioma. Here, we show that combining two specific potency-enhancing mutations in E3-19K and i-leader (designated 19KSS-iLQ125Ter) markedly increases the cytolytic efficiency and the size of vital dye-stained plaques. Unexpectedly, these mutations do not enhance actual spread efficiency, measured as the size of fluorescent plaques, in most tested cell lines. In contrast, the type of fiber modification (F5RGD4C, F5/3, or F5/35) strongly influences actual spread efficiency, with F5RGD4C generally conferring the greatest enhancement. In some cell lines, the fiber modification F5RGD10(2C) or verapamil treatment further improves actual spread efficiency. Nelfinavir inhibits spread and plaque formation of oncolytic adenoviruses with the 19KSS-iLQ125Ter modifications, irrespective of adenovirus death protein (ADP) expression. Expression of the reporter transgenes EGFP and Fluc or the human hyaluronidase PH20/SPAM1 from the E1B-55K region or downstream of the L3-23K or L5-Fiber region differentially affects the oncolytic potency of Ad5-delta-24-RGD-19KSS-iLQ125Ter. We identified an insertion site downstream of the L3-23K region that supports relatively high hPH20 activity while preserving the enhanced oncolytic potency of the virus. Combining 19KSS-iLQ125Ter with hPH20 expression may potentially improve therapeutic benefit in glioma.
Insights
New mutations in oncolytic adenovirus (Ad5Δ24RGD) enhance tumor cell killing but not spread. Fiber modifications and hyaluronidase expression show promise for improving glioma treatment efficacy.
Area of Science:
- Oncolytic virotherapy
- Gene therapy
- Cancer research
Background:
- Oncolytic adenovirus Ad5-delta-24-RGD (Ad5Δ24RGD) has limited efficacy in high-grade glioma.
- Enhancing viral spread and cytolytic efficiency is crucial for improving oncolytic adenovirus therapy.
Purpose of the Study:
- To investigate the impact of specific mutations and modifications on the therapeutic efficacy of Ad5Δ24RGD in glioma.
- To identify strategies for enhancing oncolytic adenovirus potency and spread.
Main Methods:
- Introduction of potency-enhancing mutations (19KSS-iLQ125Ter) into Ad5Δ24RGD.
- Evaluation of viral spread efficiency using fluorescent plaques and cytolytic efficiency via vital dye staining.
- Assessment of different fiber modifications (e.g., F5RGD4C) and transgene expression (e.g., hPH20).
- Inhibition studies using nelfinavir and analysis of adenovirus death protein (ADP) expression.
Main Results:
- The 19KSS-iLQ125Ter mutations significantly increased cytolytic efficiency and vital dye-stained plaque size.
- These mutations did not enhance actual spread efficiency in most cell lines.
- Fiber modification type, particularly F5RGD4C, strongly influenced spread efficiency.
- Nelfinavir inhibited viral spread and plaque formation.
- Expression of hPH20 from a specific insertion site preserved enhanced oncolytic potency.
Conclusions:
- The 19KSS-iLQ125Ter mutations improve the cytolytic capacity of Ad5Δ24RGD.
- Fiber modifications are critical for enhancing viral spread.
- Combining 19KSS-iLQ125Ter mutations with hPH20 expression presents a potential strategy to improve therapeutic outcomes in glioma.
