Electroporation outperforms in vivo-jetPEI for intratumoral DNA-based reporter gene transfer

Abstract

Intratumoral delivery of drug-encoding plasmid DNA (pDNA) enables localised in vivo expression of biological drugs, offering an attractive alternative to conventional protein treatment. However, this requires physical or chemical methods to enhance the low transfection efficiency of naked pDNA. Electroporation and complexation with the polycation in vivo-jetPEI are both evaluated in the clinic for intratumoral pDNA delivery, but lack head-to-head comparison. This study therefore compared both methods for intratumoral DNA-based reporter gene transfer in a subcutaneous mouse tumour model. Intratumoral electroporation resulted in strong reporter expression that was restricted to the tumour area and persisted for at least ten days. Intratumoral expression after injection of pDNA-jetPEI complexes was two to three logs lower, did not exceed the background in most mice, and lasted less than five days even with repeated dosing. Remarkably, reporter expression was primarily detected in the lungs, presumably due to leakage of pDNA-jetPEI complexes into the systemic circulation. In conclusion, electroporation enabled more efficient, prolonged and tumour-specific reporter expression compared to intratumoral injection of pDNA complexed with in vivo-jetPEI. These results favour the use of electroporation for intratumoral DNA-based gene transfer, and suggest further optimisation of pDNA-jetPEI complexes is needed to improve their efficacy and biosafety.

Figure 1

Comparison of electroporation and in vivo-jetPEI for intratumoral DNA-based reporter gene transfer. C57BL/6J mice received a single intratumoral electrotransfer of 20 µg naked pFluc or a single intratumoral injection of 20 µg pFluc complexed with 3.2 µl in vivo-jetPEI, ten days after MC38 tumour cell injection. (A) Intratumoral fluc expression. Data were compared between both groups at different time points with Mann–Whitney tests (n = 5 mice per group, **p < 0.01). The dots indicate the individual fluc expression in each mouse, the solid lines the mean expression in each group, and the dotted line the mean bioluminescence background in untreated mice. (B) MC38 tumour growth. According to Mann–Whitney tests, the difference in tumour volume between both groups was not significant at all time points (n = 5 mice per group). The dots indicate the individual tumour volume for each mouse, the solid lines the mean volume for each group, and the arrow the time of intratumoral pFluc injection. p/sec: photons per second.

Figure 2

Single and repeated intratumoral administration of reporter pDNA complexed with in vivo-jetPEI. C57BL/6J mice received one or three intratumoral injections of pFluc-jetPEI complexes with the first dose ten days after MC38 tumour cell injection. (A) Intratumoral fluc expression over time. According to Mann–Whitney tests, the difference in expression between both groups was not significant at all time points (n = 3 mice per group). The dots indicate the individual fluc expression in each mouse, the solid lines the mean expression in each group, the dotted line the mean bioluminescence background in untreated mice, and the arrows the intratumoral pFluc-jetPEI injections. The injections on day 2 and 4 were performed shortly after bioluminescence imaging. (B) In vivo bioluminescence image of fluc expression in the repeated-dosing group, one day after the first pFluc-jetPEI injection. Next to the low fluc expression in the tumour area (indicated by the red circles), a clear signal was visible at the chest region. The third mouse showed aberrantly high off-target expression, interfering with the determination of the intratumoral fluc signal. This mouse is therefore not included in the graph in panel A, which is substantiated by a positive Grubbs’ test for outliers. (C) Ex vivo bioluminescence image of different organs of mice in the repeated-dosing group, ten days after the first pFluc-jetPEI injection. Red circles indicate the tumours, and white circles the lungs. The upper mouse, the lower mouse on the left and the lower mouse on the right correspond to the third, fourth and first mouse in panel B, respectively. p/sec: photons per second. p/sec/cm²/sr: photons per second that leave a square centimetre of tissue and radiate into a solid angle of one steradian.