Relative efficiencies of DNA delivery and subsequent expression
Ensuring an appropriate level and duration of expression is essential in achieving an efficient and safe gene therapy. The delivery of the therapeutic gene to target cells has to be sufficiently high to elicit a response, and minimum therapeutic thresholds may vary dramatically between therapeutic strategies. Delivery modalities can be broadly grouped into biological, chemical or physical methods. Biological modalities used in our laboratory include the viral vectors Adeno-Associated Virus and replication incompetent Adenovirus; physical modalities currently being studied include electroporation and sonoporation, while commercially available lipofection reagents are also widely used. Non-viral methods delivering plasmid DNA are argued to present a relatively safe alternative to viral vectors. They are less immunogenic, toxicity is generally very low, plasmid DNA has greater potential for delivery of larger genetic units and large-scale production is relatively easy. Physical methods such as electroporation have been utilised effectively for
However, all the described methods have associated problems; the transfer of naked DNA is typically an inefficient process, with cell & tissue damage caused by administration of physical and chemical modalities, while adenoviral-mediated gene transfer is complicated by a host immune response to both the vector and transduced target cells (Jooss et al. 1998; Heller et al. 2008). In addition, only transient transgene expression is typically achieved by these approaches. Proposed causes of transient expression include loss of DNA due to cell turnover, immune responses against transfected cells and/or expressed proteins, and inhibition of transcription through host cell methylation of microbial DNA sequences (Prosch et al. 1996; Scheule 2000). AAV vectors have been shown not to elicit strong immune responses in general (Jooss, Ertl et al. 1998) (although immune responses against AAV have been reported following liver administration (Manno et al. 2006)) and the levels of transgene expression following AAV mediated delivery have been shown to increase post delivery in heart, brain and muscle tissues
The optimal gene delivery method for a given therapy will be dependant on tissue location, and type, as well as therapeutic strategy. While certain studies have been reported comparing the efficiencies from different vectors (Wang, A. Y. et al. 2004; Kealy et al. 2009), given the paucity of information regards direct comparisons between various delivery techniques, especially in the cancer setting, this study assesses the level and duration of reporter gene expression within target murine tissues when delivered by a range of commonly used gene delivery techniques; electroporation, sonoporation, lipofection, Adenovirus and Adeno-Associated Virus.2 Materials and methods
2.1. DNA constructs
pCMV-luc plasmid, which expresses firefly luciferase under the transcriptional control of the cytomegalovirus (CMV) promoter, was purchased from Promega (Wisconsin, USA). pCMV-LacZ plasmid, which expresses -Galactosidase under the transcriptional control of the CMV promoter, was purchased from Plasmid Factory (Bielefeld, Germany). Plasmid concentration was determined using the Nanodrop spectrophotometer (ND-1000 Spectrophotometer, Labtech Int, East Sussex, UK). Replication incompetent recombinant Adenovirus 5 particles encoding the luciferase gene under the transcriptional control of the CMV promoter were a kind gift from Prof. Andrew Baker, University of Glasgow, they were generated and titrated as described previously (Waddington et al. 2008). Replication incompetent Adenovirus 5 particles encoding the -galactosidase gene under the transcriptional control of the CMV promoter were a kind gift from the Regenerative Medicine Institute, NUI Galway, they were generated and titrated as described previously (Sharif et al. 2006). An AAV plasmid expressing firefly luciferase under the transcriptional control of the cytomegalovirus (CMV) promoter was constructed by first excising the firefly
2.2. Cell lines and tissue culture
Murine JBS fibrosarcoma tumour cells (Collins, C. G., Tangney et al. 2006) and murine CT26 colonic adenocarcinoma cells (obtained from ATCC) were maintained in culture at 37 °C in a humidified atmosphere of 5 % CO2, in Dulbecco’s Modified Essential Medium (GIBCO, Invitrogen Corp., Paisley, Scotland) supplemented with 10 % iron-supplemented donor calf serum (Sigma Aldrich Ireland, Ireland), 300 g/ml L-glutamine, and 10 mM HEPES (1-Piperazineethane sulfonic acid, 4-(2-hydroxyethyl) monosodium salt), (Sigma Aldrich Ireland, Ireland) pH 7.4. The murine MGC8 gastric carcinoma cell line was kindly provided by Dr. Robert Kammerer, Ludwig-Maximilians-University, Germany (Nockel et al. 2006), and was maintained in RPMI (Roswell Park Memorial Institute- Gibco) medium supplemented with 10 % iron-supplemented donor calf serum (Sigma Aldrich Ireland, Ireland) and 1mM sodium pyruvate (Sigma Aldrich Ireland, Ireland). Cell densities were determined by visual count using a haemocytometer. Cells were at 80 % confluency on the day of transduction
In vitro gene delivery
The efficacies of the different delivery methods
In vitro luciferase assay
Treated cells were analysed for luciferase activity using the Luciferase Assay System (Promega MSC, Dublin) 24 hr post transfection. Treated cells were counted and resuspended to 104 cells in 50 μl DMEM medium. 50 μl 1X lysis buffer was added to each of the samples and incubated for 5 min at room temperature. 100 μl Luciferase assay reagent was then added to each sample and the luminescence was measured with a Junior LB 9509 luminometer (Berthold Technology, Promega MSC, Dublin).
In vitro -galactosidase assay
Treated cells were analysed for -Galactosidase activity using the Roche -Gal Staining set (Roche Diagnostics GmbH, Penzberg, Germany) as per the manufacturer’s protocol. Briefly, cells were washed with PBS, and incubated in fixative (2% formaldehyde, 0.2 % gluteraldehyde in PBS) for 15 min. Cells were incubated in staining solution o/n at 37 oC. Cells were analysed in PBS under a light microscope and transfection efficiency (% stained cells) was calculated from 10 random viewing fields per well.
2.6. DNA/RNA extraction
Transfections were carried out as previously outlined with the CMVLacZ constructs. At 24 hr post transfection, cells were harvested for simultaneous DNA/RNA extraction using the Qiagen Allprep DNA/RNA Kit (Qiagen Crawley, West Sussex). Briefly, treated cells were counted and resuspended in 350 µl of Buffer RLT containing -mercaptoethanol and vortexed. The DNA and RNA extraction was carried out as per the manufacturer’s protocol. 5 µg RNA was treated with DNase 1 (DNAfree, Ambion) to remove contaminating genomic DNA. cDNA synthesis was carried out using 500 ng of the DNase treated RNA with the Qiagen Omniscript RT kit, per manufacturer’s instructions. The resulting cDNA was brought to a 50 µl volume using nuclease free water.
2.7. Quantitative real-time PCR
PCR was performed using the Lightcycler FastStart DNA Master Sybr Green system (Roche). PCR was carried out in a final volume of 20 µl using 0.5 µl of each primer (0.25 µM), 3 mM MgCl2. PCR was performed in a lightcycler (Roche) with a 15 min pre-incubation at 95 °C followed by 40 cycles of 15 s at 95 oC, 5 s at 60 °C, 5 s at 72 oC. PCR products were subjected to melting curve analysis using the light cycler system to exclude the amplification of unspecific products. PCR products were analysed by conventional agarose gel electrophoresis. Primers were synthesized by MWG Biotech. The following primers were used to detect the
2.8. Animals and tumour induction
All murine experimentation was approved by the University College Cork Animal Ethics Committee. Mice were obtained from Harlan Laboratories (Oxfordshire, England). They were kept at a constant room temperature (22 C) with a natural day/night light cycle in a conventional animal colony. Standard laboratory food and water were provided
In vivo gene delivery
Mice were randomly divided into experimental groups and subjected to specific experimental protocols. For tumour experiments, mice were treated at a tumour volume of approximately 100 mm3 in volume (5-7 mm major diameter). For liver transfection, a 1 cm subcostal incision was made over the liver and the peritoneum opened. The proximal portion of the liver was exposed and DNA administered as described below. The wound was closed in two layers, peritoneal and skin, using 4/0 prolene sutures (Promed, Killorglin, Ireland). For muscle experiments, a single intramuscular injection was carried out into the right or left quadriceps muscle of the animal. Mice were anaesthetized during all treatments by intraperitoneal (IP) administration of 200 µg xylazine and 2 mg ketamine.
For plasmid delivery by electroporation, a custom-designed applicator with 2 needles 4 mm apart was used, with both needles placed central to the tissue. Tissue was injected between electrode needles with plasmid DNA in sterile injectable saline in an injection volume of 50 µl. Concentration of plasmid was adjusted to administer 4 x 1012 gene copy numbers. After 80 seconds, square-wave pulses (1200 V/cm 100 µsec x 1 and 120 V/cm 20 msec, 8 pulses) were administered in sequence using a custom designed pulse generator (Cliniporator (IGEA, Carpi, Italy).
For plasmid delivery by ultrasound, tissue was injected with plasmid DNA as above. The ultrasound probe was then applied to the tissue and ultrasonic waves delivered at 1.0 W/cm2, 20 % duty cycle for 5 min (Sonoporator, Sonidel, Dublin, Ireland).
For plasmid delivery using Lipofection, tissue was injected with plasmid DNA/Lipofectamine 2000 complex in an injection volume of 100 µl. Concentration of plasmid was adjusted to administer 4 x 1012 gene copies.
Viral vector particles were administered by direct intratumoural, intramuscular or intra-hepatic injection in a volume of 50 µl. 2 X 108 – 2 X 109 GC of replication incompetent recombinant AAV2 particles, or 1 x 109 VP of replication incompetent recombinant Adenovirus 5 particles were used per administration.
2.10. Whole body luciferase imaging
2.11. Statistical analysis
The primary outcome variable of the statistical analyses was luminescence per cell per gene copy administered in each cell line or luminescence per gene copy administered in each organ measured at each time point. The principal explanatory variables were the delivery modalites used.
3.1. Comparison of transgene expression levels
Reporter gene expression was analysed following
In order to assess consistency across cell lines, delivery to a range of tumour cell models was examined. Efficiencies arising from Lipofectamine, Ad and AAV delivery to JBS was compared with those from CT26 and MGC8 (Nockel, van den Engel et al. 2006). Cancer cell type-specificity was clearly observed for each vector (Figure 1b). AAV also achieved the highest levels of expression in CT26 cells as with JBS cells. However, AAV failed to transduce MGC8 cells. There were also considerable relative differences observed in Lipofectamine transfected cells (p < 0.05), while Ad delivery resulted in expression in all cell lines, albeit with statistically significant variation between each (p < 0.05).
3.2. Analysis of DNA delivery and transcription efficiencies
In order to assess and compare DNA entry efficiency and subsequent transcription efficiencies for each delivery method, LacZ reporter gene DNA and mRNA was quantified by PCR. Prior to DNA/RNA extraction at 24 h post delivery, the number of cells was determined using trypan blue exclusion. The total number of LacZ DNA copies was expressed per cell at 24 h (Figure 2b). The highest number of DNA copies per cell was observed with Lipo, followed by AAV, which were both significantly higher than US, EP and Ad methods (p<0.02). Lipofection delivery does not involve physical generation of pores required by the other plasmid methods for delivery, nor is it dependent on the presence of cell surface receptors (e.g. CAR), which may be poorly expressed in certain cancers, which may in turn explain the poor Ad uptake by JBS cells observed here. The efficiency of DNA entry to cells was also calculated by comparing the number of transgene copies in extracted DNA and expressing it as a percentage of the number of transgene copies initially administered (Figure 2c). Results correlated with the above % cell transfection data, with viral and chemical methods displaying the highest efficiencies of gene delivery to cells in comparison with the physical modalities (p<0.02). There was no significant difference between AAV, Ad and Lipo methods (p>0.05).
Transcription efficiency was determined using qPCR analyses on LacZ DNA and mRNA (Figure 2d). The number of copies of transgene mRNA 24 h post-delivery was expressed as a percentage of the number of internalised DNA copies. Ad resulted in significantly higher ratio of mRNA:DNA compared with all other delivery methods (p<0.01). There was no significant difference in transcription efficiencies between the remaining delivery methods. With US and Lipo, while these methods may efficiently mediate delivery of plasmid DNA to the cytoplasm, subsequent trafficking to the nucleus and transgene transcription is not ensured. For AAV, the low level observed can be attributed to the rate limiting step associated with AAV mediated expression, involving synthesis of double stranded DNA from the single stranded genome prior to transcription (Ferrari et al. 1996). EP transcription efficiency was also significantly higher than US, AAV and Lipo (p<0.02). The combination of high and low voltage pulses used for electroporation here is believed to create transient pores in both the cell and nuclear membranes enhancing DNA entry and subsequent nuclear localisation (Gothelf & Gehl ; Chang 1992).
3.3. Duration of transgene expression in tumour, liver and skeletal muscle
Luminescence from plasmid and Ad reduced dramatically within 48 h post delivery to tumour and liver (Figure 3), and both plasmid and Ad reduced to background levels from day 7 in tumour, and day 14 (EP) or day 21 (Ad) in liver. Day 5 was the earliest practical time point for imaging of AAV for reasons including safety guidelines for animal experimentation with this vector. AAV-related expression also decreased in tumour, to background levels by day 16-post administration. However, a different pattern of transgene expression was observed for AAV in liver and quadriceps muscle, with an overall increase in luminescence over time. When muscle related expression was examined with plasmid, prolonged sustained luminescence was observed, with equivalent expression seen at day 370 and day 18 post electroporation (data not shown). However, unlike plasmid, complete loss of Ad mediated luciferase activity was observed when muscle was examined, with Ad expression increasing up to day 7, before reducing to background levels by day 21.
3.4. Comparisons between transgene expression levels in tumour, liver and skeletal muscle
To directly compare
It can be seen that the intra-modality pattern of expression differed from that observed
The method employed to deliver genes of interest is the primary parameter related to expression in a target tissue, and consequently has important therapeutic implications. Our findings delineate the relative efficiencies of five well-described delivery modalities, and highlight target organ/tissue specific variations in transfection capability. Furthermore the kinetics of gene expression arising from each modality were compared.
In order for data to be generated
Given the mechanism of AAV single-stranded DNA virus transduction of cells, it is likely that at the time of measurement of AAV-transduced cells
Uniform conditions were used for all tissues in this study. In the absence of titrations to determine the precise optimal parameters for each method for each target cell or tissue, we cannot rule out that the relative differences reported between vectors
Considerable variation in transgene expression was observed between modalities and cell types. Lipofectamine consistently transfected all cell types examined
Efficient reporter gene expression requires cell and nuclear DNA molecule entry, followed by transcription, translation and enzymatic activity on substrate. We analysed various steps for each delivery modality
There were significant inter-vector and inter-tissue variations in the times at which highest luminescence values were observed
While the patterns of luciferase expression from Ad and plasmid were similar in tumour and liver, this was not the case for muscle. It has previously been demonstrated with Ad gene delivery to muscle, that associated Ad transduction of Dendritic Cells resulted in presentation of transgene as antigen and subsequent T cell elimination of transgene expressing muscle cells (Jooss, Ertl et al. 1998). When we examined Ad mediated expression in quadriceps of athymic mice, no reduction was evident for up to 2 months, unlike in immunocompetent Balb/C mice where luminescence was absent from day 21-post muscle transduction (figure 3d). This suggests the involvement of T-cell inactivation of adenoviral-transduced cells in immunocompetent mice. Plasmid electroporation, on the other hand, has been shown not to elicit such transgene silencing immune responses (Vicat et al. 2000), and presents an attractive option in achieving long-term gene expression, especially in light of recent improvements in plasmid vectors (Gill et al. 2009).
Although Ad provided the highest immediate gene expression in all tissues, the potential for high level expression from AAV is highlighted in this study, with AAV2 providing expression in the same order of magnitude as Ad within the first month post administration, increasing to higher levels over time. Sustained transgene expression is desirable for many therapies and there is also potential to overcome transient expression from plasmids by inclusion of integrating transposon or S/MAR elements (Gill, Pringle et al. 2009). EP yielded the highest transfection among the non-viral techniques, in all tissues, unlike
Overall, the data generated here clearly define the relative efficiencies of the various delivery systems in a wide range of tissues
The results clearly define the relative efficiencies of these delivery systems in a range of situations, providing researchers with valuable information to support vector choice in therapeutic strategy design.
We thank Prof. Andrew Baker of the University of Glasgow and Martina Harte of the Regenerative Medicine Institute Galway for providing rAd particles. This work was supported in part by grants from Cancer Research Ireland (CRI07TAN) and Science Foundation Ireland (06/RF/BIC055 and 07/RF/BIMF542).
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