Visualization of gene therapy with a liver cancer-targeted adeno-associated virus 3 vector

Abstract

Background: To evaluate the feasibility of a self-complementing recombinant adeno-associated virus 3 (scrAAV3) vector targeting liver cancer and non-invasively monitor gene therapy of liver cancer. Materials and methods: An scrAAV3-HSV1-TK-kallistatin (ATK) gene drug was constructed, which contained the herpes virus thymidine kinase (HSV1-TK) reporter gene and human endogenous angiogenesis inhibitor (kallistatin) gene for non-invasive imaging of gene expression. Subcutaneous xenografted tumors of hepatoma in nude mice were generated for positron emission tomography/computed tomography (PET/CT) imaging. The ATK group was injected with the ATK gene through the tail vein, and an imaging agent was injected 2 weeks later. PET/CT imaging was performed at 1 hour after injection of the imaging agent. The control group was injected with phosphate-buffered saline at the same volume as the ATK gene drug. HE staining is used for pathological observation of tumor sections. HSV1-TK and kallistatin expression was identified by immunofluorescence, real-time quantitative PCR, and western blotting. Results: Radioactivity on PET/CT images was significantly higher in the ATK group compared with the control group. 18F-FHBG uptake values of left forelegs in ATK and control groups were 0.591±0.151% and 0.017 ± 0.011% ID/g (n=5), respectively (P<0.05). After injection of the ATK gene drug, mRNA and protein expression of HSV1-TK and kallistatin in subcutaneous xenograft tumors was detected successfully. In vitro analysis demonstrated significant differences in the expression of HSV1-TK and kallistatin between ATK and control groups (P<0.05). Conclusions: The scrAAV3 vector has a strong liver cancer-targeting ability, and the ATK gene drug can be used for targeted and non-invasive monitoring of liver cancer gene therapy.

Keywords: Liver cancer; Molecular imaging; Self-complementary recombinant adeno-associated virus 3; Targeted therapy.

Figure 1

Monitoring of ATK by ¹⁸F-FHBG. We constructed an scrAAV3-HSV1-TK-kallistatin gene drug with a liver cancer-targeting ability. Studies have confirmed that scrAAV3 binds to HGFR and targets human hepatoma cells. The kallistatin gene inhibits neovascularization, which not only inhibits the growth of liver cancer xenografts, but also effectively inhibits metastasis and recurrence. The ATK gene drug was injected through the tail vein. Within tumor cells, HSV1-TK was transcribed and translated to produce the HSV1-TK enzyme. ¹⁸F-FHBG is a labeled analog of penciclovir and substrate for HSV1-TK. In the presence of HSV1-TK, the radiolabeled probe is phosphorylated and trapped within the cell. The magnitude of ¹⁸F-FHBG signals reflects the activity of the HSV1-TK enzyme and thus HSV1-TK gene expression.

Figure 2

Micro-PET/CT images of a representative animal. An ¹⁸F-FHBG PET/CT scan was performed to detect HSV1-TK expression in the left forearm of mice. Intense HSV1-TK uptake (arrows) was observed at the left forearm. (A) Coronal slices of an animal's PET imaging (left) and image of maximum intensity projection (right). (B) Transverse, coronal and sagittal images of PET/CT. (C) Transverse, coronal and sagittal images of PET.

Figure 3

Micro-PET/CT images of transplanted metastatic lymph nodes. An ¹⁸F-FHBG PET/CT scan was performed to detect HSV1-TK expression in the left forearm of mice. Two additional regions of high HSV1-TK uptake were found (red and yellow arrows), indicating lymph node metastasis. (A) Transverse sections of PET/CT of metastatic lymph nodes. (B and C) Transverse, coronal, and sagittal images of PET/CT of two metastatic lymph nodes.

Figure 4

Biodistribution data of ¹⁸F-FHBG in xenograft-bearing mice. Semi-quantitative analysis of ¹⁸F-FHBG uptake by various organs. A significant difference was found in the subcutaneous xenografted tumor between experimental mice injected with the ATK gene drug and control mice without injection of the ATK gene drug (n = 5, P<0.05). However, no significant difference was observed in other organs.

Figure 5

Protein and mRNA expression of HSV1-TK and kallistatin in vitro. The protein and mRNA expression of HSV1-TK and kallistatin in subcutaneous xenografted tumors were analyzed by immunofluorescence, qPCR, and western blotting in vitro. (A) HE staining results of HepG2 xenograft tumor tissues in nude mice. (B and C) Protein and mRNA expression of HSV1-TK and kallistatin of the ATK group was significantly different from that in the control group (P<0.05). (D) Immunofluorescence showed that the signals of HSV1-TK and kallistatin in the ATK group were higher than those in the control group.