2. Academic Validation
  3. Potency-enhancing mutations in E3-19K and i-leader increase the cytolytic activity of the PH20/ SPAM1-armed oncolytic adenovirus Ad5Δ24RGD

Potency-enhancing mutations in E3-19K and i-leader increase the cytolytic activity of the PH20/ SPAM1-armed oncolytic adenovirus Ad5Δ24RGD

  • Mol Ther Oncol. 2026 Jan 23;34(1):201137. doi: 10.1016/j.omton.2026.201137.
Aleksei A Stepanenko 1 2,  Anastasiia O Sosnovtseva 1 3 4,  Anastasiia A Vasiukova 1,  Marat P Valikhov 1 3,  Anastasia A Chernysheva 1,  Gaukhar M Yusubalieva 5 6 7,  Vladimir P Chekhonin 1 2
Affiliations

Affiliations

  • 1 Department 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.
  • 2 Department of Medical Nanobiotechnology, Institute of Translational Medicine, N.I. Pirogov Russian National Research Medical University of the Ministry of Health of the Russian Federation, 117513 Moscow, Russia.
  • 3 Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences, 119991 Moscow, Russia.
  • 4 Laboratory of Regulation of Intracellular Proteolysis, Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences, 119991 Moscow, Russia.
  • 5 Laboratory of Cell Technologies, Federal Scientific and Clinical Center for Specialized Types of Medical Care and Medical Technologies of the Federal Medical and Biological Agency of the Russian Federation, 115682 Moscow, Russia.
  • 6 Laboratory of Immunotherapy of Solid Tumors, Federal Center for Brain and Neurotechnology of the Federal Medical and Biological Agency of the Russian Federation, 117513 Moscow, Russia.
  • 7 Laboratory of Molecular Mechanisms of Regeneration, Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences, 119991 Moscow, Russia.
PMID: 41716467 DOI: 10.1016/j.omton.2026.201137
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.

Keywords

E3-19K; L3-23K; L5-fiber; MT: Regular Issue; adenovirus death protein; glioblastoma; hyaluronidase PH20; i-leader; nelfinavir; oncolytic adenovirus; verapamil.

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