title: Fluorescence-guided development of a tricistronic vector encoding bimodal optical and nuclear genetic reporters for in vivo cellular imaging.
abstract: BACKGROUND
  In vivo imaging using genetic reporters is a central supporting tool in the development of cell and gene therapies affording us the ability to selectively track the therapeutic indefinitely. Previous studies have demonstrated the utility of the human norepinephrine transporter (hNET) as a positron emission tomography/single photon emission computed tomography (PET/SPECT) genetic reporter for in vivo cellular imaging. Here, our aim was to extend on this work and construct a tricistronic vector with dual optical (firefly luciferase) and nuclear (hNET) in vivo imaging and ex vivo histochemical capabilities. Guiding this development, we describe how a fluorescent substrate for hNET, 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)), can be used to optimise vector design and serve as an in vitro functional screen.
  
  
  METHODS
  Vectors were designed to co-express a bright red-shifted firefly luciferase (FLuc), hNET and a small marker gene RQR8. Genes were co-expressed using 2A peptide linkage, and vectors were transduced into a T cell line, SupT1. Two vectors were constructed with different gene orientations; FLuc.2A.RQR8.2A.hNET and hNET.2A.FLuc.2A.RQR8. hNET function was assessed using ASP(+)-guided flow cytometry. In vivo cellular conspicuity was confirmed using sequential bioluminescence imaging (BLI) and SPECT imaging of transduced SupT1 cells injected into the flanks of mice.
  
  
  RESULTS
  SupT1/FLuc.2A.RQR8.2A.hNET cells resulted in >4-fold higher ASP(+) uptake compared to SupT1/hNET.2A.FLuc.2A.RQR8, suggesting that 2A orientation effected hNET function. SupT1/FLuc.2A.RQR8.2A.hNET cells were readily visualised with both BLI and SPECT, demonstrating high signal to noise at 24 h post (123)I-meta-iodobenzylguanidine (MIBG) administration.
  
  
  CONCLUSIONS
  In this study, a pre-clinical tricistronic vector with flow cytometry, BLI, SPECT and histochemical capabilities was constructed, which can be widely applied in cell tracking studies supporting the development of cell therapies. The study further demonstrates that hNET function in engineered cells can be assessed using ASP(+)-guided flow cytometry in place of costly radiosubstrate methodologies. This fluorogenic approach is unique to the hNET PET/SPECT reporter and may prove valuable when screening large numbers of cell lines or vector/mutant constructs.
authors: Division of Medicine, Centre for Advanced Biomedical Imaging (CABI), University College London, 72 Huntley Street, London, WC1E 6DD UK.
authors: Adam
authors: A
authors: Badar
authors: Division of Medicine, Centre for Advanced Biomedical Imaging (CABI), University College London, 72 Huntley Street, London, WC1E 6DD UK ; UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD UK.
authors: Louise
authors: L
authors: Kiru
authors: Division of Medicine, Centre for Advanced Biomedical Imaging (CABI), University College London, 72 Huntley Street, London, WC1E 6DD UK.
authors: Tammy L
authors: TL
authors: Kalber
authors: UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD UK.
authors: Amit
authors: A
authors: Jathoul
authors: UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD UK.
authors: Karin
authors: K
authors: Straathof
authors: Department of Chemistry and Institute of Nuclear Medicine, University College London, 235 Euston Road (T-5), London, NW1 2BU UK.
authors: Erik
authors: E
authors: Årstad
authors: Division of Medicine, Centre for Advanced Biomedical Imaging (CABI), University College London, 72 Huntley Street, London, WC1E 6DD UK.
authors: Mark F
authors: MF
authors: Lythgoe
authors: UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD UK.
authors: Martin
authors: M
authors: Pule
keywords: ASP+; BLI; Cell imaging; Multimodality imaging; Norepinephrine transporter; PET; Reporter genes; SPECT