Quantitation of pulmonary transgene expression with PET imaging.
PET imaging represents a promising approach for noninvasive monitoring of reporter gene expression in living subjects. We evaluated the relationship between various methods of quantifying the imaging signal and in vitro assays of the expression of a PET reporter gene (a mutant Herpes simplex virus-1 thymidine kinase (mHSV1-tk); 9-(4-(18)F-fluoro-3-hydroxymethylbutyl)guanine ((18)F-FHBG) was used as the PET reporter probe.


METHODS
In 14 rats, pulmonary gene transfer was performed by intratracheal administration of various amounts of an adenovector containing a fusion gene encoding for mHSV1-tk and an enhanced green fluorescent protein. Three days later, the animals were divided into 2 groups. One group (n = 7) did not receive any other interventions. The other group was treated with alpha-naphthylthiourea (ANTU) to increase pulmonary vascular permeability. All rats were injected intravenously with (18)F-FHBG. Two additional rats in both groups received a null adenovector and served as controls. In the normal rats, repetitive blood samples were obtained and PET imaging was performed simultaneously using a dynamic imaging protocol. Rate constants estimating (18)F-FHBG transport (K(1)) or trapping (k(3)) within target cells were generated by compartmental modeling. After euthanasia, pulmonary uptake of (18)F-FHBG was determined using a gamma-counter in all rats, and in vitro assays of transgene expression were performed on lung tissue.


RESULTS
In normal rats, pulmonary uptake of (18)F-FHBG increased as thymidine kinase (TK) activity increased only at low levels of mHSV1-tk expression and then plateaued as TK activity continued to increase. Compartmental modeling failed to improve the correlation with in vitro assays of transgene expression. However, a linear relationship was obtained between the pulmonary uptake of (18)F-FHBG and in vitro assays of TK activity in rats treated with ANTU.


CONCLUSIONS
In rodent lungs, (18)F-FHBG uptake appears to be a function of both transport into tissues expressing the transgene as well as the level of transgene expression itself.
Richard
Jean-Christophe
JC
Washington University School of Medicine, St. Louis, Missouri, USA.
Zhou
Zhaohui
Z
Chen
Delphine L
DL
Mintun
Mark A
MA
Piwnica-Worms
David
D
Factor
Phillip
P
Ponde
Datta E
DE
Schuster
Daniel P
DP
D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000502:physiology; D017930:Genes, Reporter; D006147:Guanine / Q000031:analogs & derivatives* / Q000097:blood / Q000493:pharmacokinetics*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods; D008168:Lung / Q000000981:diagnostic imaging* / Q000378:metabolism*; D008657:Metabolic Clearance Rate; D019275:Radiopharmaceuticals / Q000097:blood / Q000493:pharmacokinetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013223:Statistics as Topic; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism*; D014055:Tomography, Emission-Computed / Q000379:methods*; D019076:Transgenes / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
C113308:9-(4-fluoro-3-hydroxymethylbutyl)guanine; D019275:Radiopharmaceuticals; D011994:Recombinant Proteins; D014764:Viral Proteins; D006147:Guanine; C099241:thymidine kinase, Canid herpesvirus 1; D013937:Thymidine Kinase