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Fluorescence-guided development of a tricistronic vector encoding bimodal optical and nuclear genetic reporters for in vivo cellular imaging.

Badar A, Kiru L, Kalber TL, Jathoul A, Straathof K, Årstad E, Lythgoe MF, Pule M - EJNMMI Res (2015)

Bottom Line: In vivo cellular conspicuity was confirmed using sequential bioluminescence imaging (BLI) and SPECT imaging of transduced SupT1 cells injected into the flanks of mice.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Medicine, Centre for Advanced Biomedical Imaging (CABI), University College London, 72 Huntley Street, London, WC1E 6DD UK.

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.

No MeSH data available.


Related in: MedlinePlus

Fluorescence and radionuclide cell uptake studies. (a) ASP+-guided flow cytometry assessing hNET function in SupT1/NT (white), SupT1/FLuc.2A.RQR8.2A.hNET (beige), SupT1/hNET.2A.FLuc.2A.RQR8 (light grey) and SupT1/hNET.l.dCD34 (dark grey) sorted cells. Mean fluorescence intensities under the curves are presented. (b) hNET function in SupT1/hNET.2A.FLuc.2A.RQR8 cells was further characterised with an 125I-MIBG radiosubstrate uptake assay. Percent uptake was determined for SupT1/NT (white), SupT1/hNET.2A.FLuc.2A.RQR8 (light grey) and SupT1/hNET.l.dCD34 (dark grey) cells via gamma counting. Error bars are the mean ± SD for n = 3. Stats tests performed were ANOVA and Tukey’s HSD post hoc.
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Fig3: Fluorescence and radionuclide cell uptake studies. (a) ASP+-guided flow cytometry assessing hNET function in SupT1/NT (white), SupT1/FLuc.2A.RQR8.2A.hNET (beige), SupT1/hNET.2A.FLuc.2A.RQR8 (light grey) and SupT1/hNET.l.dCD34 (dark grey) sorted cells. Mean fluorescence intensities under the curves are presented. (b) hNET function in SupT1/hNET.2A.FLuc.2A.RQR8 cells was further characterised with an 125I-MIBG radiosubstrate uptake assay. Percent uptake was determined for SupT1/NT (white), SupT1/hNET.2A.FLuc.2A.RQR8 (light grey) and SupT1/hNET.l.dCD34 (dark grey) cells via gamma counting. Error bars are the mean ± SD for n = 3. Stats tests performed were ANOVA and Tukey’s HSD post hoc.

Mentions: Interestingly, using ASP+, we found that 2A orientation within the tricistronic vector affected hNET function. Sorted SupT1/FLuc.2A.RQR8.2A.hNET and SupT1/hNET.2A.FLuc.2A.RQR8 cells were subject to ASP+-guided flow cytometry with mean fluorescence intensities measured as from the AUC. Despite resulting in a 1.7-fold lower MFI compared to the bicistronic reference cells (SupT1/hNET.l.dCD34), cells encoding the tricistronic vector with 2A at the N-terminus of hNET (SupT1/FLuc.2A.RQR8.2A.hNET) gave rise to a >4-fold higher MFI (2292.33 ± 80.39 MFI) compared to cells encoding 2A at the C-terminus of hNET (SupT1/hNET.2A.FLuc.2A.RQR8) (549 ± 20.43 MFI) (Figure 3a). hNET function in SupT1/FLuc.2A.RQR8.2A.hNET cells was further characterised via radiosubstrate uptake assay. These cells demonstrated >18-fold higher 125I-MIBG uptake (60.25% ± 1.34%) compared to non-hNET expressing control cells (SupT1/NT) (3.18% ± 0.37%). Similar to ASP+ accumulation profiles, 125I-MIBG uptake in SupT1/FLuc.2A.RQR8.2A.hNET was 1.2-fold lower than in SupT1/hNET.l.dCD34 cells, suggesting reduced hNET pumping capabilities due to higher genetic load on tricistronic compared to bicistronic vector.Figure 3


Fluorescence-guided development of a tricistronic vector encoding bimodal optical and nuclear genetic reporters for in vivo cellular imaging.

Badar A, Kiru L, Kalber TL, Jathoul A, Straathof K, Årstad E, Lythgoe MF, Pule M - EJNMMI Res (2015)

Fluorescence and radionuclide cell uptake studies. (a) ASP+-guided flow cytometry assessing hNET function in SupT1/NT (white), SupT1/FLuc.2A.RQR8.2A.hNET (beige), SupT1/hNET.2A.FLuc.2A.RQR8 (light grey) and SupT1/hNET.l.dCD34 (dark grey) sorted cells. Mean fluorescence intensities under the curves are presented. (b) hNET function in SupT1/hNET.2A.FLuc.2A.RQR8 cells was further characterised with an 125I-MIBG radiosubstrate uptake assay. Percent uptake was determined for SupT1/NT (white), SupT1/hNET.2A.FLuc.2A.RQR8 (light grey) and SupT1/hNET.l.dCD34 (dark grey) cells via gamma counting. Error bars are the mean ± SD for n = 3. Stats tests performed were ANOVA and Tukey’s HSD post hoc.
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Related In: Results  -  Collection

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Fig3: Fluorescence and radionuclide cell uptake studies. (a) ASP+-guided flow cytometry assessing hNET function in SupT1/NT (white), SupT1/FLuc.2A.RQR8.2A.hNET (beige), SupT1/hNET.2A.FLuc.2A.RQR8 (light grey) and SupT1/hNET.l.dCD34 (dark grey) sorted cells. Mean fluorescence intensities under the curves are presented. (b) hNET function in SupT1/hNET.2A.FLuc.2A.RQR8 cells was further characterised with an 125I-MIBG radiosubstrate uptake assay. Percent uptake was determined for SupT1/NT (white), SupT1/hNET.2A.FLuc.2A.RQR8 (light grey) and SupT1/hNET.l.dCD34 (dark grey) cells via gamma counting. Error bars are the mean ± SD for n = 3. Stats tests performed were ANOVA and Tukey’s HSD post hoc.
Mentions: Interestingly, using ASP+, we found that 2A orientation within the tricistronic vector affected hNET function. Sorted SupT1/FLuc.2A.RQR8.2A.hNET and SupT1/hNET.2A.FLuc.2A.RQR8 cells were subject to ASP+-guided flow cytometry with mean fluorescence intensities measured as from the AUC. Despite resulting in a 1.7-fold lower MFI compared to the bicistronic reference cells (SupT1/hNET.l.dCD34), cells encoding the tricistronic vector with 2A at the N-terminus of hNET (SupT1/FLuc.2A.RQR8.2A.hNET) gave rise to a >4-fold higher MFI (2292.33 ± 80.39 MFI) compared to cells encoding 2A at the C-terminus of hNET (SupT1/hNET.2A.FLuc.2A.RQR8) (549 ± 20.43 MFI) (Figure 3a). hNET function in SupT1/FLuc.2A.RQR8.2A.hNET cells was further characterised via radiosubstrate uptake assay. These cells demonstrated >18-fold higher 125I-MIBG uptake (60.25% ± 1.34%) compared to non-hNET expressing control cells (SupT1/NT) (3.18% ± 0.37%). Similar to ASP+ accumulation profiles, 125I-MIBG uptake in SupT1/FLuc.2A.RQR8.2A.hNET was 1.2-fold lower than in SupT1/hNET.l.dCD34 cells, suggesting reduced hNET pumping capabilities due to higher genetic load on tricistronic compared to bicistronic vector.Figure 3

Bottom Line: In vivo cellular conspicuity was confirmed using sequential bioluminescence imaging (BLI) and SPECT imaging of transduced SupT1 cells injected into the flanks of mice.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Medicine, Centre for Advanced Biomedical Imaging (CABI), University College London, 72 Huntley Street, London, WC1E 6DD UK.

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.

No MeSH data available.


Related in: MedlinePlus