Antitumor Activity of an Oncolytic Adenovirus-Delivered Oncogene Small Interfering RNA

Abstract

Despite successes in animal models, cancer gene therapy with small interfering RNAs (siRNA) is hindered by the lack of an optimal delivery platform. We examined the applicability of the replication-competent, oncolytic adenovirus, ONYX-411, to deliver a mutant K-ras siRNA transgene to human cancer cells. Proof-of-principle studies showed an additive tumor growth-inhibitory response through siRNA-mediated K-ras knockdown and ONYX-411-mediated cancer cell lysis. A novel construct, termed Internavec ( for interfering RNA vector), was generated by cloning a K-ras v12 -specific siRNA ras-4 hairpin construct under the control of the human H1 promoter into the deleted E3b region of ONYX-411. Internavec acquired an increase in potency of f10-fold in human cancer cells expressing the relevant K-ras v12 mutation (H79, H441, and SW480), as defined by a reduction in the effective dose needed to achieve 50% growth inhibition (ED 50 ). Internavec remained attenuated in nonmalignant epithelial cells. Daily intratumoral injections of Internavec ( five daily injections of 1 Â 10 8 plaque-forming units) significantly reduced the growth of s.c. H79 pancreatic cancer xenografts in nu/nu mice by 85.5%, including complete growth suppression in three of five mice. Parental ONYX-411 or ONYX-411-siRNA GFP was markedly less effective (47.8% growth reduction, P = 0.03; and 44.1% growth reduction, P = 0.03, respectively). siRNA ras transgene activity contributed to cell cycle blockage, increased apoptosis, and marked down-regulation of Ras signaling-related gene expression (AKT2, GSK3b, E2F2, and MAP4K5 ). These findings indicate that Internavec can generate a two-pronged attack on tumor cells through oncogene knockdown and viral oncolysis, resulting in a significantly enhanced antitumor outcome.

Clinical development directions in oncolytic viral therapy

“…Improving development of tumor-specific promoters to limit viral replication in malignant tissue may enable greater confidence in the utilization of replication aggressive viruses. 14,[188][189][190][191][192][193][194][195][196][197] Ultimately these modifications will likely need to be built into viral constructs that deliver molecular targeted therapeutics [198][199][200][201][202] and can be utilized in combination with traditional therapeutics, [203][204][205][206][207][208] thereby creating a 'super' virus.…”

Targeting of P53-transcriptional dysfunction by conditionally replicating adenovirus is not limited by P53-homologues

“…Viral DNA quantification was performed by real-time PCR (qPCR MasterMix Plus; Eurogentec, Seraing, Belgium) using 100 ng of liver DNA and adenoviral hexonspecific primer probes as described before. 44 18S Genomic Control Kit (Eurogentec) served as internal control. For the investigation of liver damage, explanted livers were examined macroscopically, and ALT/AST activities were determined in serum samples from treated mice using the AST/ALT enzymatic assay kit (Bioo Scientific) according to the manufacturer's protocols.…”

Gene Therapy of Pancreatic Cancer

“…RNAi directed against mutated KRAS showed anti-tumor activity [15], limited the aggressive phenotype of the tumor cells [16] and potentiated gemcitabine antitumor activity [17]. Preclinical studies showed encouraging results with viral delivery systems such as oncolytic adenovirus [18]. However, this strategy has not been further tested in phase I clinical trials.…”