Image-guided Control of Transgene Expression Based on Local Hyperthermia

Abstract

Spatial and temporal control of transgene expression is one of the major prerequisites of efficient gene therapy. Recently, a noninvasive, physical approach has been presented based on local heat in combination with a heat-sensitive promoter. This strategy requires tight temperature control in vivo. Here, we use MRI-guided focused ultrasound (MRI-FUS) with real-time feedback control on a whole-body clinical MRI system for a completely automatic execution of a predefined temperaturetime trajectory in the focal point. Feasibility studies on expression control were carried out on subcutaneously implanted rat tumors. A stable modified C6 glioma cell line was used carrying a fused gene coding for thymidine kinase (TK) and green fluorescent protein (GFP) under control of the human heat-shock protein 70 (HSP70) promoter. In vitro studies showed strong induction of the TK -GFP gene expression upon heat shock under various conditions and localization of the protein product in the nucleus. In vivo tumors were subjected to a 3-min temperature elevation using MRI-FUS with a constant temperature, and were analysed 24 hr after the heat shock with respect to GFP fluorescence. Preliminary results showed strong local induction in regions heated above 40°C, and a good correspondence between temperature maps at the end of the heating period and elevated expression of TK -GFP. Mol Imaging (2003) 2, 11 -17.

Evaluation of a synthetic CArG promoter for nitric oxide synthase gene therapy of cancer

“…3 Specific characteristics of tumour cells 4,5 or of the tumour microenvironment 6 can also be targeted, but it is also preferable in a therapeutic strategy to include a level of exogenous control over the magnitude and duration of gene expression to enhance targeting specificity. To this end much research has focused on externally inducible promoters including those activated by heat, 7,8 radiation 9 and drugs. 10 Radiation is one of the most important modalities used to treat solid tumours, so it is logical to incorporate radiation-inducible elements into transgene constructs to be used in gene therapy protocols in combination with radiotherapy.…”

Microglia used as vehicles for both inducible thymidine kinase gene therapy and MRI contrast agents for glioma therapy

“…Wild-type microglia were transduced by lentiviral constructs to express the fusion protein TK-GFP in an inducible manner, as described by Guilhon et al 28 when studying C6 glioma sensitivity for thermotherapy induced by FUS. As a first step, the thermoinduction and efficiency of the expressed transgene were studied through the cell viability, mitochondrial potential and engagement of the apoptotic pathway.…”

“…The fusion gene TK-GFP under the HSP70 promoter has been constructed as described by Guilhon et al 28 Briefly, the TK-GFP gene was extracted from the plasmid ETLGB 29 and inserted in the pcDNA3-HS. 30 The construct with the TK-GFP gene under the control of the HSP70 promoter was isolated after bglII and SalI digestions.…”

“…Wild-type human microglial cell line (CHME-5) transduced with a plasmid containing the thymidine kinase-GFP (TK-GFP) fusion gene placed under the minimal human HSP70 promoter 28 is termed 'PHT cells'. After the heat shock (441C for 20 min), the transduced microglia (PHT cells) expressed the fusion protein TK-GFP (Figure 1a and b).…”

Real‐time adaptive methods for treatment of mobile organs by MRI‐controlled high‐intensity focused ultrasound

“…The combination of HIFU and MRI (MR-HIFU) is especially promising because MRI enables highly precise target localization and is the only noninvasive imaging technique that has been shown to allow accurate temperature mapping in vivo (8 -11). A large range of applications of MR-HIFU has been demonstrated in the clinical field, e.g., in ablation of breast tumors (4,12,13) and uterine fibroids (3,14,15), in preclinical studies on gene therapy (16), and for local drug delivery (17). MR-HIFU has shown great potential for improving therapeutic precision and reducing costs.…”