Quantitative and mechanism-based investigation of post-nuclear delivery events between adenovirus and lipoplex

Abstract

Quantitative and mechanism-based information on differences in transfection efficiency between viral and non-viral vectors would be highly useful for improving the effectiveness of non-viral vectors. A previous quantitative comparison of intracellular trafficking between adenovirus and LipofectAMINE PLUS (LFN) revealed that the three orders of magnitude lower transfection efficiency of LFN was dominantly rate limited by the post-nuclear delivery process. In the present study, the contribution of transcription and translation processes to the overall differences in the transgene expression efficiency of nucleus-delivered DNA was independently evaluated by quantifying mRNA. As a result, transcription efficiency (E(transcript)) of LFN, denoted as transgene expression divided by the amount of nuclear pDNA was about 16 times less than that for adenovirus. Furthermore, translation efficiency (E(translate)), denoted as transfection activity divided by mRNA expression was approximately 460 times less in LFN. Imaging of the decondensed form of DNA by in situ hybridization revealed that poor decondensation efficiency of LFN is involved in the inferior E(transcript). Moreover, the inferior translation efficiency (E(translate)) of LFN was mainly due to electrostatic interactions between LFN and mRNA. Collectively, an improvement in nuclear decondensation and the diminution of the interaction between vector and mRNA is essential for the development of new generations of non-viral vectors.

Figure 1.

Summary of the contribution of the transcription and translation processes to the overall transgene expression efficiency of nuclear DNA (TE_nuc). The TE_nuc for adenovirus was approximately 7000 times higher compared to that for LFN. By measuring the amount of cellular mRNA, an intermediate component of the central dogma, contributions of the differences in the transcription process and the translation process to the overall difference in TE_nuc were quantitatively evaluated. In the transcription process, LFN was 16 times less efficient. In post-transcriptional processes, the translation efficiency of adenovirus was 460 times more efficient.

Figure 2.

Effect of the pre-loading of Ad core protein on LFN-mediated transfection activity. Cells were pre-infected by adenovirus encoding EGFP to load the adenovirus core proteins into the nucleus for 1, 3 and 6 h and at the indicated dose. pDNA encoding luciferase DNA was then transfected with LFN. At 6 h post-transfection with LFN, transgene expression was compared between non-infected control (open bar) and pre-infected cells. The vertical axis represents luciferase activity expressed as relative light units (RLUs)/mg protein. These data represent the mean values and standard deviation for triplicate experiments.

Figure 3.

Comparison of transgene expression after the nuclear microinjection of adenoviral genomic DNA and pDNA. Adenovirus genomic DNA was purified by a guanidine treatment, followed by sucrose density gradient centrifugation. After quantification of the concentration of genome DNA and pDNA by RealTime PCR, genomic DNA and pDNA was microinjected into the nucleus at a dose of 10 copies/nucleus with rhodamine-labeled dextran (Rho-Dex) as an injection marker. At 24 h post-microinjection, EGFP expression efficiency was evaluated.

Figure 4.

Detection of the free form of adenoviral genomic DNA and pDNA by in situ hybridization. After 10 h post-transfection with adenovirus (A) or LFN (B) at a dose of 20 copies/cell and 3.4 × 10⁶ copies/cell, the decondensed form of DNA was detected by in situ hybridization with TSA system. (C) Quantitative comparison of nuclear DNA between adenovirus and LFN. After 10 h post-transfection with adenovirus (20 copies/cell) or LFN (3.4 × 10⁶ copies/cell), nuclear DNA was quantified by RealTime PCR. Data are represented as copies/cell.

Figure 5.

Comparison of nuclear sublocalization between the adenovirus genome and pDNA. At 10 h post-transfection with Ad (20 copies/cell) or LFN (3.4 × 10⁶ copies/cell), nuclear adenoviral genomic (A) or pDNA (B) was detected by in situ hybridization (red). Cell nuclei were stained by DAPI (blue).

Figure 6.

Effect of adenovirus and LFN on the post-transcription process. (A, B) Interaction between mRNA and vectors. Adenovirus (A) or LFN (B), encoding EGFP was applied to a reverse transcription reaction using a luciferase mRNA (8.5 × 10⁹ copies/reaction). The cDNA product was quantified by RealTime PCR. (C, D) The influence of adenovirus and LFN on an in vitro translation system. About 4.3 × 10⁸ copies/reaction of mRNA encoding luciferase was subjected to the in vitro translation with or without adenovirus (C) or LFN (D), at a dose of 6.8 × 10⁹ copies/reaction. Protein synthesis was quantified by a luciferase assay. (E) Influence of adenovirus and LFN on the luciferase expression stably expressed in the HeLa cells. At 10 h post-transfection with Ad or LFN at the indicated doses, transgene expression was determined by a luciferase assay. Data are represented as the percent of untreated cells. These data represent the mean values and standard deviation for triplicate experiments.