A diphtheria toxin resistance marker for in vitro and in vivo selection of stably transduced human cells.

Gabriele Picco,Consalvo Petti,Livio Trusolino,Andrea Bertotti,Enzo Medico

doi:10.1038/srep14721

Abstract

We developed a selectable marker rendering human cells resistant to Diphtheria Toxin (DT). The marker (DTR) consists of a primary microRNA sequence engineered to downregulate the ubiquitous DPH2 gene, a key enzyme for the biosynthesis of the DT target diphthamide. DTR expression in human cells invariably rendered them resistant to DT in vitro, without altering basal cell growth. DTR-based selection efficiency and stability were comparable to those of established drug-resistance markers. As mice are insensitive to DT, DTR-based selection can be also applied in vivo. Direct injection of a GFP-DTR lentiviral vector into human cancer cell-line xenografts and patient-derived tumorgrafts implanted in mice, followed by systemic DT administration, yielded tumors entirely composed of permanently transduced cells and detectable by imaging systems. This approach enabled high-efficiency in vivo selection of xenografted human tumor tissues expressing ectopic transgenes, a hitherto unmet need for functional and morphological studies in laboratory animals.

Highlights

To kill human cells, diphtheria toxin requires the presence on the cell surface of its receptor, heparin-binding EGF-like growth factor (HBEGF), and of diphthamide biosintesis proteins, in particular of Diphthamide Biosynthesis Protein 2 (DPH2)
We evaluated HBEGF and DPH2 expression in a series of gene expression datasets: (i) 151 colorectal cancer (CRC) cancer cell lines[21], (ii) 515 CRC patient-derived cancer xenografts (PDXs), and (iii) expression data for colorectal, glioblastoma, head and neck and pancreatic cancer obtained from The Cancer Genome Atlas via the CBioPortal[22]
To assess if silencing of the genes involved in diphthamide biosynthesis confers resistance to diphtheria toxin (DT), HCT116 colorectal cancer (CRC) cells, expressing intermediate levels of HBEGF, were chosen as a model and transduced with 18 different shRNAmir constructs, targeting the DPH2, DPH5, DPH6 and DPH7 transcripts (Supplementary Table 1)

Summary

Introduction

Additional genes, including DPH5, DPH6, and DPH7, have been shown to encode proteins essential for diphthamide formation and DT-mediated toxicity in human cells[16,17,18]. To develop a selectable marker conferring resistance to DT, we considered an RNA interference approach, using short hairpin sequences inserted into a primary microRNA transcript backbone (shRNAmirs). This design adds a Drosha processing site to the hairpin construct that has been shown to greatly increase knockdown efficiency[20]. Four to six different shRNAmir sequences were tested for each of the key diphthamide biosynthesis genes

Methods

Results

Conclusion