Design of Retinoid-Responsive Suicide Gene Systems For Neuroblastoma Treatment

Background:
Neuroblastoma (NB) is a paediatric disease of the sympathetic nervous system, accounting for 15% of childhood cancer deaths. High grade tumours remain difficult to treat, with tumour relapse being almost invariably fatal (ref.1). During residual disease therapy patients receive high dose retinoic acid (RA) as a cell differentiation treatment. This improves 5-year overall survival, but only modestly (ref.2). RA induces tumour cell differentiation in culture, but again not completely (ref.3). Thus, RA’s maximal potential as a clinical agent remains unclear. We therefore propose an alternative way of harnessing the clinical potential of RA signalling, by generating a “suicide vector” system (ref.5) which converts the differentiation response to a cytotoxic response in tumour cells. RA generates 100-1000-fold increases in transcription of target genes and we hypothesise that linking toxin genes to RA-responsive promoters in plasmids should generate a suitable suicide vector for NB. Our pilot vector indeed generates strong, inducible cytotoxicity even at very low RA doses (Figure 1). We then propose to deliver vectors to cells in culture and in vivo using our liposome-based nanotechnology that has been show to target NB tumours in a pre-clinical model (ref.5). The student would therefore design and optimised the vectors, examine ways in which to maximise their delivery to tumour cells using nanotechnology and then pilot this suicide therapy approach pre-clinically.

Aims/Objectives:
The long-term aim is to re-purpose RA as a directly cytotoxic NB therapy.

Objective 1 (Month 1-24):
To develop an optimised, DNA minicircle-based suicide vector encoding highly RA-responsive toxin genes in cultured tumour cells. The student will use recombinant DNA approaches to construct plasmids encoding luciferase and toxin genes driven by retinoid-responsive promoters. These will be optimised for maximal, inducible expression in transfected tumour cell lines, using luciferase and cell survival assays. To minimise expression of toxins in non-tumour tissues, the student will also investigate the effectiveness of miRNA target sequences in vectors. Lastly, the vectors will be modified for use as minicircle DNAs and tested for their expression level and longevity in transfected cultured cells.
Objective 2 (Month 12-36):
To optimise our peptide-based liposome technology for targeting a range of NB cells including PDX cells. The student will test the cell targeting ability of existing nanoparticles in NB cell lines and PDX lines and will design and test novel, peptide-based targeting strategies for these liposomes to improve targeting efficiency and range.
Objective 3 (Month 24-36):
The third objective it to demonstrate a complete suicide vector system capable of impeding xenografted NB tumours. Once a minicircle vector and peptide targetting strategy is optimised in cultured tumour cells, the student will complete the study by adapting the liposomes for optimal use in vivo, largely by incorporation of PEGylated lipids (ref.5). These liposomes will then be tested in a NB xenograft model. Animals treated with vehicle or RA and the effectiveness of the suicide system in reducing tumour growth will be assessed.

References:
1          Matthay et al. Nature reviews. Disease primers 2, 16078, (2016).
2          Matthay et al. J Clin Oncol 27, 1007-1013, (2009).
3          Clark, O., Daga, S. & Stoker, A. W. Cancer Lett 328, 44-54, (2013).
4          Karjoo et al. Advanced Drug Delivery Reviews 99, 113-128, (2016).
5          Grosse et al. The FASEB journal 24, 2301-2313, (2010).