Minicircles for a two-step blood biomarker and PET imaging early cancer detection strategy

Abstract

Early cancer detection can dramatically increase treatment options and survival rates for patients, yet detection of early-stage tumors remains difficult. Here, we demonstrate a two-step strategy to detect and locate cancerous lesions by delivering tumor-activatable minicircle (MC) plasmids encoding a combination of blood-based and imaging reporter genes to tumor cells. We genetically engineered the MCs, under the control of the pan-tumor-specific Survivin promoter, to encode: 1) Gaussia Luciferase (GLuc), a secreted biomarker that can be easily assayed in blood samples; and 2) Herpes Simplex Virus Type 1 Thymidine Kinase mutant (HSV-1 sr39TK), a PET reporter gene that can be used for highly sensitive and quantitative imaging of the tumor location. We evaluated two methods of MC delivery, complexing the MCs with the chemical transfection reagent jetPEI or encapsulating the MCs in extracellular vesicles (EVs) derived from a human cervical cancer HeLa cell line. MCs delivered by EVs or jetPEI yielded significant expression of the reporter genes in cell culture versus MCs delivered without a transfection reagent. Secreted GLuc correlated with HSV-1 sr39TK expression with R² = 0.9676. MC complexation with jetPEI delivered a larger mass of MC for enhanced transfection, which was crucial for in vivo animal studies, where delivery of MCs via jetPEI resulted in GLuc and HSV-1 sr39TK expression at significantly higher levels than controls. To the best of our knowledge, this is the first report of the PET reporter gene HSV-1 sr39TK delivered via a tumor-activatable MC to tumor cells for an early cancer detection strategy. This work explores solutions to endogenous blood-based biomarker and molecular imaging limitations of early cancer detection strategies and elucidates the delivery capabilities and limitations of EVs.

Keywords: Biomarkers; Cancer detection; Minicircles; Reporter genes.

Figure 1.. Schematic of Two-Step Blood and Imaging-Based Cancer Screening Approach.

(a) Tumor-activatable MCs are either complexed with a transfection agent or encapsulated in extracellular vesicles and delivered to cells. Under the tumor-specific Survivin promoter, MCs will only be transcribed and translated within tumor cells to produce (1) a secretable reporter protein and (2) a PET reporter gene (PRG) enzyme. (b) A mouse tumor transfected with the tumor-activatable MCs secretes reporter protein into the bloodstream, which can be detected in a blood test. (c) A mouse tumor transfected by the tumor-activatable MCs creates PRG enzymes that phosphorylate and trap the PET reporter probe (PRP) within tumor cells. These cells retain the PRP and reveal the tumor location on the PET image.

Figure 2.. Tumor-activatable MC Delivery in Cell Culture.

(a) Vector maps of Survivin-driven PP and MC. The MC lacks the prokaryotic backbone (blue elements). (b) Agarose gel electrophoresis confirming MC size. (c) HeLa cells transfected with MC-pSur and jetPEI secreted higher levels of GLuc compared to control groups (n=3, p<0.001, 72h). (d) HeLa cells transfected with MC-pSur resulted in greater retention of [³H]PCV compared to control groups, indicating expression of HSV-1 sr39TK (n=3, p<0.0001, 72h). (e) Expression of reporter genes GLuc and HSV-1 sr39TK, as shown in (c) and (d), is highly correlated in cell culture. The correlation coefficient is 0.9836 and the coefficient of determination is 0.9676 (p<0.0001). PP, parental plasmid; MC, minicircle; pSur, Survivin promoter; GLuc, Gaussia Luciferase; [³H]PCV, tritiated Penciclovir.

Figure 3.. Tumor-activatable MC Delivery and GLuc Secretion in Mice.

(a) Baseline measurements of the GLuc assay in whole blood, prior to any MC-pSur administration. (b) At day 3, MC-pSur delivered intratumorally to tumor-bearing mice produced GLuc at higher levels compared to healthy mice (p=0.0620) and compared to mock treatments (p<0.01). (c) At day 7, tumor-activatable MC-pSur delivered using jetPEI produced GLuc at levels that distinguished between tumor-bearing and healthy mice (p<0.05). RLU, Relative Light Units; MC, minicircle; pSur, Survivin promoter; GLuc, Gaussia Luciferase.

Figure 4.. Tumor-activatable MC Delivery & PET Reporter Gene Expression in Tumor Models.

(a) Representative coronal and axial PET/CT images nine days after initial MC-pSur or mock injection. On the day of imaging, [¹⁸F]FHBG was injected intravenously and 10 min static PET scans were acquired 30 min later. Red arrows point to tumor location. (b) Quantification of [¹⁸F]FHBG retention within PET ROIs (top 25% voxels) drawn at tumor site (n=6, day 9, p=0.0058). (c) Quantification of [¹⁸F]FHBG retention in excised tumors as measured on a gamma counter (n=6, day 9, p=0.0196). (d) Corresponding GLuc measurements for imaged mice (day 9, p=0.2378). (e) Correlation of reporter genes GLuc and HSV-1 sr39TK expressed after delivery of MC-pSur, as shown in (c) and (d). The correlation coefficient is 0.6922 and the coefficient of determination is 0.4791 (p=0.1276). MC, minicircle; pSur, Survivin promoter; GLuc, Gaussia Luciferase; [¹⁸F]FHBG, 9-4-[¹⁸F]fluoro-3-(hydroxymethyl)butyl]guanine; %ID/cc, percent injected dose per cubic centimeter; %ID/g, percent injected dose per gram; ROI, region of interest.