Abstract
As glioblastomas are mostly localized infiltrative lesions, gene therapy based on the retroviral replicating vector (RRV) system is considered an attractive strategy. Combinations of multiple suicide genes can circumvent the limitations associated with each gene, achieving direct and synergistic cytotoxic effects, along with bystander cell killing. In this study, we constructed a semi-and pseudotyped-RRV (sp-RRV) system harboring two suicide genes—herpes simplex virus type 1 thymidine kinase (TK) and yeast cytosine deaminase (CD)—to verify the dissemination and antitumor efficacy of our sp-RRV system (spRRVe-sEF1α-TK/sRRVgp-sEF1α-CD) in seven patient-derived glioblastoma stem-like cells (GSCs). Flow cytometry and high-content analysis revealed a wide range of transduction efficiency and good correlation between the delivery of therapeutic genes and susceptibility to the prodrugs ganciclovir and 5-fluorocytosine in patient-derived GSCs in vitro. Intra-tumoral delivery of spRRVe-sEF1α-TK/sRRVgp-sEF1α-CD, combined with prodrug treatment, synergistically inhibited cell proliferation and angiogenesis while increasing apoptosis and the depletion of tumor-associated macrophages in orthotopic glioblastoma xenografts. Genomic profiling of patient-derived GSCs revealed that the key genes preventing sp-RRV infection and transmission were associated with cell adhesion, migration, development, differentiation, and proliferation. This is the first report demonstrating that a novel sp-RRV-mediated TK/CD double suicide gene transfer system has high oncolytic power against extremely heterogeneous and treatment-refractory glioblastomas.
Highlights
Glioblastoma is highly infiltrative and extremely heterogeneous at the genetic and molecular level, and harbors glioblastoma stem-like cells (GSCs) with unique features including self-renewal, cellular quiescence, and undifferentiated phenotype distinct from their differentiated, proliferative progeny that comprise the bulk tumor mass [1,2]
We hypothesized that important green fluorescent (GFP)or red fluorescent (RFP)-only positive cells are as the protein dual-positive cells in protein assessing sp-retroviral replicating vector (RRV)
Dissemination, as GSCs transduced with therapeutic gene (TK cells or cytosine deaminase (CD))
Summary
Glioblastoma is highly infiltrative and extremely heterogeneous at the genetic and molecular level, and harbors glioblastoma stem-like cells (GSCs) with unique features including self-renewal, cellular quiescence, and undifferentiated phenotype distinct from their differentiated, proliferative progeny that comprise the bulk tumor mass [1,2]. All these characteristics contribute to therapeutic failure, disease recurrence, tumor evolution, and poor prognosis [1,2,3,4], indicating the importance of targeting. CD deaminates the prodrug 5-FC to form 5-FU that inhibits thymidylate synthase, resulting in depletion of deoxythymidine triphosphate (dTTP)
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