| With the development of molecular biology, genetics and immunology, gene therapy especially gene therapy in tumor have made extraordinary progress and take steps to the clinical stage from experimental and fundamental studies. The concept of gene therapy is to introduce the functional gene into the target cells or host cells by suitable vectors, become the fraction of host genetic material by gene integration, and obtain the therapeutic object by correcting inherit defect or assigning new function to cells. Gene therapy usually includes gene substitution, gene correction, gene modification, gene inactivation and gene inactivation, and so on.The key point of successful gene therapy is that the objective gene can be delivered to the target cells and expressed effectively, but can't emerge in nontarget cells. So it is important to monitor the location and magnitude of the transgene. 2-[18F]fluoro-2-deoxy-D-glucose(18F-FDG), a glucose analog that accumulates within metabolically active cells when phosphorylated by hexokinase, has been commonly used as a diagnostic tool in clinical practice to assess neural and cardiac function and tumor progression. Moreover, PET reporter gene imaging can also provide repeated, noninvasive and quantitative assessment of the expression of reporter genes in tissues and organs. The basic concept underlying the imaging strategy is that a reporter transgene (PET reporter gene) capable of trapping or binding a suitable positron-emitting radionuclide-labeled tracer (PET reporter probe) is introduced into a target tissue via a suitable vector. After PET reporter probe administration, a PET imaging signal is generated as the PET reporter probe accumulates over time within tissues expressing the PET reporter transgene. Reporter genes can be used to image vector targeting and the level of repoter gene expression, to image the regulation of endogenous genes and signal-transduction pathways, and to monitor and quantitatively assess the expression of a second transgene.Herpes simplex virus type-1 thymidine kinase (HSV1-tk, Note: HSV1-tk refers to the genes while HSV1-TK refers to the corresponding enzymes.) is a commonly studied suicide gene for cancer gene therapy. The use of HSV1-tk gene therapy in animal models has led to many different clinical trials, including trials for glioma, mesothelioma, prostate cancer, leukemia, and lymphoma. At the same time, HSV1-tk has been extensively used in the study of noninvasive PET reporter gene imaging as reporter gene. Noninvasive imaging the biodistribution, magnitude, and time variation of HSV1-tk gene expression or coexpression of the reporter gene HSV1-tk and a therapeutic gene will enhance progress in suicide gene therapy of cancer, and open up further opportunities for gene therapy.The uracil nucleoside derivative 5-124I-iodo-2'-fluoro-2'-deoxy-1-β-D- arabino-5-iodouracil (124I-FIAU) and the acycloguanosine derivatives 8-18F-fluoroacyclovir(18F-ACV) , 8-18F-fluoroganciclovir (18F-GCV), 8-18F- fluoropenciclovir (18F-PCV) , and 9-[4-18F-fluoro-3-(hydroxymethyl)butyl] guanine (18F-FHBG), 9-[(3-18F-fluoro-1-hydroxy-2-propoxy)methyl] guanine, (18F-FHPG) has often been used as reporter probes of HSV1-tk gene. When these nucleoside analogues labeled with radionuclide enter into the cells that express HSV1-tk gene, they will be phosphorylated to 5'-phospho- nucleoside by HSV1-TK. Then they will be trapped in the cells transfected with HSV1-tk gene. Mammalian TK can't phosphorylate these nucleoside analogues due to its strong substrate specificity. So the cellular radioactivity reflects the expression level of HSV1-tk gene or other gene tansfected with HSV1-tk gene.18F-FHBG is a side-chain 18F-labeled analog of penciclovir. Preliminary studies have shown that 18F-FHBG can be phosphorylated selectively by HSV1-TK, but there is less efficient phosphorylation of 18F-FHBG by mammalian TK. So the uptake of 18F-FHBG in the cells that don't express HSV1-tk gene is very low. This characteristic improves 18F-FHBG's signal-to-noise ratio and may decrease the chance of toxicity in nontarget cells. The unique advantage shows more and more potential in radionuclider reporter gene imaging.In our studies, we constructed breast carcinoma T47D-tk cell that stably express HSV1-tk gene using retrovirus infection, and synthesize the reporter gene probe of HSV1-tk gene-- 18F-FHBG. PET-CT reporter gene imaging with 18F-FHBG in subcutaneous xenografts of living nude mice showed that the accumulation of 18F-FHBG can detect the location of HSV1-tk gene. The study can establish the foundation for further 18F-FHBG reporter gene imaging in our country and offer monitor method and scientific base for further HSV1-tk gene therapy in tumor.1 Construct breast carcinoma cells that stably express HSV1-tk gene and product subcutaneous xenografts in nude mice1.1 Construction of retrovirus vector that express HSV1-tk gene and production of retrovirus.The HSV1-tk was amplified by PCR using pHSV106 as a template. A pair of primer of HSV1-tk gene was designed and synthesized, and restrictive enzyme BamH I and Sal I sequences were added to upstream and downstream respectively. HSV1-tk gene cDNA was got using PCR and restrictive enzyme BamH I and Sal I sites were added to it. HSV1-tk gene was inserted into the MCS of pMD 18-T vector to gain pHSV1-tk (DON-AI)/18T, and the positive clone was confirmed by PCR and sequencing. The retrovirus expression vector pHSV1-tk-DON-AI was constructed by using gene recombination technique and confirmed by PCR and with restrictive enzyme BamH I and Sal I digestion.Then the recombinant plasmid was transfected into 293T cells with gag-pol and env expression vector by liposome-mediated gene transfer method to conduct retrovirus. After 48 hours, the retrovirus that contains HSV1-tk gene can be collected.1.2 Construction of breast carcinoma cell that express HSV1-tk geneT47D cells were infected with retrovirus that contain HSV1-tk gene for 6 hours, then were cultured with 10% fetal bovine serum in RPMI 1640 medium for 24hours. After that, change the culture to 10% fetal bovine serum (FBS) and 800μg/ml G418 in RPMI 1640 medium for 14 days. Anti-G418 clones were selected and amplified. Monoclone was selected by limiting dilution assay with 96 porus plask and the monoclonal T47D cell that express HSV1-tk stably was named as T47D-tk. The integration and expression of HSV1-tk gene in T47D-tk cells were identified by genome identification and RT-PCR. It shows that T47D-tk cells express HSV1-tk gene but T47D cells don't and the growth curve of T47D-tk and T47D cells have no significant difference. 1.3 Subcutaneous T47D and T47D-tk breast carcinoma xenografsTumors were grown in 4 or 5-wk-old female BALB/C nu/nu mice that weigh 16~20g by subcutaneous inoculation of 5×106 T47D-tk or T47D cells under the skin in the left and right shoulders. The tumor nodus was emerged at 10th day. It is about 3~4 weeks till the long diameter of tumor was approximately 1.0 cm, then animals were used for PET-CT imaging. The growth and volume of T47D and T47D-tk tumor has no difference (P>0.05).2 Radiosynthesis of tracer 18F-FHBGRapid, reproducible radiosynthesis and quality control of 18F-FHBG is the fundament of radionuclear reporter gene imaging. We synthesize 18F-FHBG with Tracelab FX-FN synthesizer manufactured by GE Company following a literature procedure with modifications.The tosylated precursors reacted with 18F-KF in the presence of kryptofix 2.2.2, followed by acidic hydrolysis to produce 18F-FHBG. 18F-FHBG was isolated by high performance liquid chromatography (HPLC) purification. Synthesis time was 110 min, including HPLC purification with radionuclide purity >99% and initial radioactive concentration was 3mCi/ml (111MBq/ml). The radiochemical yield was about 10% (uncorrected for decay).In our study, the 18F-FHBG solution is achromatic and transparent with pH 7.0. And 18F-FHBG is sterile and free from pyrogens. It complies with the standards of clinical medicine.3 Uptake of 18F-FHBG in vitro and biodistribution and PET-CT imaging 3.1 In vitro assay for 18F-FHBG uptake in T47D and T47D-tk cells T47D and T47D-tk cells that carry HSV1-tk gene were plated in triplicate to a 6 porus plask and cultured in 2mL of media for 24 h. Logarithmically growing cells were incubated with 10μCi (370kBq) of 18F-FHBG in RPMI 1640 that contains 10% FBS for 15, 30, 60, 120 and 240min. After incubation and removal of media, the cells were washed with ice-cold phosphate-buffered saline (PBS, 3×10 mL) and trypsinized. After neutralization with 10% fetal bovine serum in RPMI 1640 medium (1.5mL), cells were centrifuged for 5 min at 1000 rpm and supernatants were discarded. Cells were resuspended in cold PBS, and an aliquot was removed for cell counting. The cell suspensions were further centrifuged three times to exclude the ectosctivity and cellular radioactivity was measured in a Well-type Gamma Counter. The ratio of activity uptake in T47D and T47D-tk cells was obtained for each of 3 experiments per time point.In vitro studies revealed that the total accumulation of 18F-FHBG in T47D-tk cells was significantly higher than that in T47D cells. The uptake of 18F-FHBG increased rapidly in T47D-tk cells reaching a plateau by 60 min. Maximum incorporation of 18F-FHBG within a short time suggests that the rate of phosphorylation by the HSV1-tk was also very high. No significant increase in uptake in T47D cells was observed during the study. The ratio between T47D-tk and T47D cells decreased from 77 at 60min to 64 at 120min. A gradual but slow accumulation of 18F-FHBG in wild-type cells likely represents minimal baseline phosphorylation by host kinase.3.2 The biodistribution of 18F-FHBGThe biodistribution study was conducted on 15 mice. All the mice were divided into five groups randomly and injected with approximately 200μCi (7400KBq) 18F-FHBG each via tail vein. Choose any group of mice, collect the blood by removing the eyeball, and sacrifice them respectively at 15, 30, 60, 120, 240min after the injection of 18F-FHBG. The main organs was removed, weighed, and counted for 18F radioactivity. Radioactivity determinations were normalized by the weight of the tissue and amount of radioactivity injected, to obtain %ID/g.In vivo distribution studies show that the peak activity in blood within 15 minutes. The organs of higher activity were liver, gut, kidney and bladder. It is indicated that the tracer 18F-FHBG mainly excrete by digestive tract and urinary tract. There is no obvious activity in brain because 18F-FHBG can't cross normal blood-brain barrier. Based on these biodistribution, the diagnostic value of 18F-FHBG is limited to evaluate the expression of HSV1-tk gene in lower abdomen and central nervous system.3.3 PET-CT imaging with 18F-FHBG in nude miceMice with subcutaneous xenografts were injected via tail vein with approximately 200μCi (7400KBq) of 18F-FHBG synthesized as described previously. All the mice were placed in a prone position at the center of vision, and imaging were performed using PET scanner at 15, 30, 60, 120, 240 min after injection. The data collection uses 2D model with 3min per bed position. Images were reconstructed using an ordered-subset expectation maximization algorithm. At the end of imaging, all the mice were sacrificed and the main organs and tumors were excised and weighed. Activity of tissue samples were was measured with Well-type Gamma Counter and corrected decays. For each mouse, radioactivity uptake was expressed as the %ID/g of tissue. As the images show, 18F-FHBG accumulates significantly only in T47D-tk tumors on the left shoulder. Uptake in T47D tumor is as low as the background activity by 2 h. All other organs demonstrated low accumulation except the liver, gut, kidney and bladder, which is the same as biodistribution study. This again verifies the way of 18F-FHBG excretion. At 240min after the injection, the %ID/g of T47D and T47D-tk tumor is respectively 0.034±0.003%ID/g and 0.105±0.007 %ID/g.These results from cellular uptake, biodistribution and PET-CT images suggest that 18F-FHBG may be useful for localization of HSV1-tk gene expression in target tissue. A key premise underlying the use of PET imaging to monitor and quantify transgene expression is that, over a relevant physiologic range, the expression level of the reporter gene is directly related to the PET imaging signal, which in turn depends on the tissue accumulation of the PET reporter probe.In our studies, we successfully constructed retrovirus vector and package into retrovirus expressing HSV1-tk gene, constructed breast carcinoma T47D-tk expressing HSV1-tk gene stably, synthesized reporter probe 18F-FHBG and performed PET-CT imaging in nude mice with subcutaneous xenografts. The method is hoped to be the foundation of monitor of gene therapy in cancer by reporter gene imaging.In summary, radionuclide reporter gene imaging has the advantage of noninvasive and reproducible, and can be used to instruct the expression of animal or human gene, organ or cell transplantation of living animal or human and regulation of transgene expression. It can guide the gene therapy and predict the effect by monitoring the location, magnitude and variation of transgene during the gene therapy. Moreover, PET performed after coexpression with a reporter gene may not only enhance monitoring of clinical therapeutic gene delivery but also allow linkage of clinical observations and functional test results to transgene expression, thus refining understanding of therapeutic mechanisms. Further study about 18F-FHBG PET-CT reporter gene imaging is needed to promote the development of gene therapy.
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