Development of a universal anti-polyethylene glycol reporter gene for noninvasive imaging of PEGylated probes.
A reporter gene can provide important information regarding the specificity and efficacy of gene or cell therapies. Although reporter genes are increasingly used in experimental and clinical studies, a highly specific yet nonimmunogenic reporter that can track genes and cells in vivo by multiple imaging technologies still awaits development. In this study, we constructed a versatile and nonimmunogenic reporter gene to noninvasively image gene expression or cell delivery by optical imaging, MRI, and small-animal PET.


METHODS
We cloned and expressed a membrane-anchored anti-polyethylene glycol (PEG) reporter that consists of the Fab fragment of a mouse anti-PEG monoclonal antibody, AGP3, fused to the C-like extracellular-transmembrane-cytosolic domains of the mouse B7-1 receptor. Binding of PEGylated probes (PEG-NIR797 for optical imaging, PEG-superparamagnetic iron oxide for MRI, and (124)I-PEG for small-animal PET) were examined in vitro and in vivo. In addition, we compared the specificity, immunogenicity, and probe toxicity of the anti-PEG reporter with the gold standard reporter gene, type 1 herpes simplex virus thymidine kinase (HSV-tk). Finally, we derived a humanized anti-PEG reporter and evaluated its imaging function in vivo with subcutaneous and metastatic tumor models in mice.


RESULTS
The cells or tumors that stably expressed anti-PEG reporters selectively accumulated various PEGylated imaging probes and could be detected by optical imaging, MRI, and small-animal PET. Importantly, the anti-PEG reporter displayed an imaging specificity comparable to the HSV-tk reporter but did not provoke immune responses or cause toxicity to the host. Furthermore, the humanized anti-PEG reporter retained high imaging specificity in vivo.


CONCLUSIONS
The highly specific and nonimmunogenic anti-PEG reporter may be paired with PEGylated probes to provide a valuable system to image gene expression or cell delivery in experimental and clinical studies.
Chuang
Kuo-Hsiang
KH
Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Wang
Hsin-Ell
HE
Cheng
Ta-Chun
TC
Tzou
Shey-Cherng
SC
Tseng
Wei-Lung
WL
Hung
Wen-Chun
WC
Tai
Ming-Hong
MH
Chang
Tien-Kuei
TK
Roffler
Steve R
SR
Cheng
Tian-Lu
TL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology* / Q000633:toxicity; D003001:Cloning, Molecular; D005260:Female; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D006367:HeLa Cells; D018259:Herpesvirus 1, Human / Q000201:enzymology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology / Q000633:toxicity; D008279:Magnetic Resonance Imaging; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D015335:Molecular Probes / Q000235:genetics* / Q000276:immunology* / Q000633:toxicity; D041681:NIH 3T3 Cells; D009369:Neoplasms / Q000000981:diagnostic imaging / Q000378:metabolism; D055620:Optical Phenomena; D011092:Polyethylene Glycols / Q000378:metabolism*; D049268:Positron-Emission Tomography; D013937:Thymidine Kinase / Q000235:genetics
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D015335:Molecular Probes; D011092:Polyethylene Glycols; D013937:Thymidine Kinase