Abstract
Simple SummaryReasoning that multiple DNA breaks will trigger programmed cell death, we generated lentiviral CRISPR-to-kill (C2K) vectors targeting highly repetitive SINE sequences for cancer gene therapy. In proof-of-concept experiments, C2K-Alu-vectors selectively killed human, but not murine cell lines, and efficiently inhibited the growth of patient-derived glioblastoma cell lines resistant to high-dose irradiation. In combination with tumor-targeting approaches, the C2K system might represent a promising tool for cancer gene therapy.CRISPR/Cas9 was described as a bacterial immune system that uses targeted introduction of DNA double-strand breaks (DSBs) to destroy invaders. We hypothesized that we can analogously employ CRISPR/Cas9 nucleases to kill cancer cells by inducing maximal numbers of DSBs in their genome and thus triggering programmed cell death. To do so, we generated CRISPR-to-kill (C2K) lentiviral particles targeting highly repetitive Short Interspersed Nuclear Element-Alu sequences. Our Alu-specific sgRNA has more than 15,000 perfectly matched target sites within the human genome. C2K-Alu-vectors selectively killed human, but not murine cell lines. More importantly, they efficiently inhibited the growth of cancer cells including patient-derived glioblastoma cell lines resistant to high-dose irradiation. Our data provide proof-of-concept for the potential of C2K as a novel treatment strategy overcoming common resistance mechanisms. In combination with tumor-targeting approaches, the C2K system might therefore represent a promising tool for cancer gene therapy.
Highlights
Suicide gene therapy (SGT) was proposed as a new therapy option for difficult-to-treat cancer more than 40 years ago
Current concepts in oncology mainly rely on the genetic modification of cancer cells to express prodrug-converting enzymes generating toxic metabolites in the presence of the respective prodrug, the most popular one being the Herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir system
Glioblastoma patient-derived “stem-like” cell lines GS-8 [21], NCH644 [22], and BT112 [23] were routinely cultured as neurospheres in serum-free Neurobasal Medium (NBM, Invitrogen, Carlsbad, CA, USA) supplemented with 1% Glutamine (Invitrogen), 2% B27 (Invitrogen), and 20 ng/mL of each epidermal growth factor and fibroblast growth factor–2 (PeproTech, Rocky Hill, NJ, USA)
Summary
Suicide gene therapy (SGT) was proposed as a new therapy option for difficult-to-treat cancer more than 40 years ago (reviewed, e.g., in [1,2,3]). Current concepts in oncology mainly rely on the genetic modification of cancer cells to express prodrug-converting enzymes generating toxic metabolites in the presence of the respective prodrug, the most popular one being the Herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir system. Several clinical studies provided promising results for SGT [4,5], but the use of prodrug-converting enzymes was associated with inherent technical limitations. For. HSV-TK, the latter included the presence of inactive splicing variants, cell-cycle dependence, slow killing kinetics and limited tissue penetration as well as myelotoxicity of the prodrug [6]. In order to address tumor- and potentially recurrence-initiating cells [10], completely novel, cell-cycle independent SGT approaches with low probability for resistance development might be necessary
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