Pharmacogenetic heterogeneity of transgene expression in muscle and tumours

Abstract

Background: Recombinant adenoviruses are employed to deliver a therapeutic transgene in the liver, muscle or tumour tissue. However, to rationalise this delivery approach, the factors of variation between individuals need to be identified. It is assumed that differences between inbred strains of laboratory animals are considered to reflect differences between patients. Previously we showed that transgene expression in the liver of different rat strains was dependent on the transcription efficiency of the transgene. In the present paper we investigated if transfection of muscle and tumour tissue were also subject to such variations.

Methods: Variation, in transgene expression, after intramuscular gene delivery was determined in different rodent strains and gene expression in tumours was investigated in different human and rodent cell lines as well as in subcutaneously implanted rodent tumours. The molecular mechanisms involved in transgene expression were dissected using an adenovirus encoding luciferase. The luciferase activity, the viral DNA copies and the luciferase transcripts were assessed in cultured cells as well as in the tissues.

Results: Large differences of luciferase activity, up to 2 logs, were observed between different rodent strains after intramuscular injection of Ad Luciferase. This inter-strain variation of transgene expression was due to a difference in transcription efficiency. The transgene expression level in tumour cell lines of different tissue origin could be explained largely by the difference of infectibility to the adenovirus. In contrast, the main step responsible for luciferase activity variation, between six human breast cancer cell lines with similar phenotype, was at the transcriptional level.

Conclusion: Difference in transcriptional efficiency in muscles as observed between different inbred strains and between human breast cancer cell lines may be expected to occur between individual patients. This might have important consequences for clinical gene therapy. The variation between tumour types and tissues within a species are mainly at the levels of infectivity.

Figure 1

Transgene expression in the plasma after intra-muscular gene delivery. Plasma levels of endostatin (yellow bars), mhATF-BPTI (blue bars) and ATF (red bars) in Wag/Rij rats (A) and in Brown Norway rats (B) after intra muscular injection of 10¹⁰ iu Ad5 Adapt encoding the respective transgenes. Ten animals per group, data are expressed as mean ± SD. The higher levels of endostatin compared to the ATF and ATF-BPTI is due to the pharmacokinetic characteristics of these molecules.

Figure 2

Luciferase expression in the gastrocnemius muscle of 3 rat strains after intra-muscular delivery. Luciferase activity was determined 48 hours after intra-muscular injection of 10⁹ iu (white bars, n = 6), 3 10⁹ iu (striped bars, n = 4), and 10¹⁰ iu (black bars, n = 9) Ad5 Adapt Luc. The mass of the gastrocnemius is similar in the 3 strains. Data are expressed as mean ± SD. The value of the mean is shown above the bars.

Figure 3

Infectibility of the gastrocnemius muscle to the adenoviral vector in the 3 rat strains. The same muscles were used as in Fig. 2. The number of Adenoviral DNA copies per muscle was determined by duplex real time PCR. The number of DNA copies was determined in a sample of a homogenate of the whole gastrocnemius muscle and then extrapolated to the total mass of the gastrocnemius. Data are expressed as mean ± SD. The value of the mean is shown above the bars.

Figure 4

Variation among mouse strains in transgene expression and infectibility to adenovirus after intra-muscular injection. The animals were injected with 3 10⁹ iu Ad5 Adapt Luc in the right gastrocnemius. A: Luciferase activity. B: Number of Adenoviral DNA copies per muscle determined by duplex real time PCR. The number of DNA copies was determined from a sample of a homogenate of the whole gastrocnemius muscle and then extrapolated to the total muscle mass. Data are expressed as mean ± SD. The value of the mean is shown above the bars.

Figure 5

Luciferase transcripts in mouse muscles after intra-muscular injection. Quantification of the luciferase mRNA in the muscle of the mice injected with 3 10⁹ iu Ad Adapt Luc by Northern blotting. These data refer to the muscles as depicted in Fig. 4. The RNA was extracted from a homogenate of the whole gastrocnemius and a northern blot was performed. The density of the bands was determined with a phosphorimager. Mean values ± SD, depicted on the figure, are expressed in arbitrary units. The mouse strain is indicated on the figure. Lane 1–6, 9–13, 16–20, 23–26, 29–33, 36–39: animals injected with Ad5 Adapt Luc. Lane 7–8, 14–15, 21–22, 27–28, 34–35, 40–41: animals injected with 3 10⁹ iu Ad5 Adapt Empty. Lane 42: negative control plasmid. The amplicon product is 310 base pair. The density of the β-Actin transcripts bands was also quantified and proved to be similar in all the animals (825 ± 74).

Figure 6

Comparison of in vitro and in vivo gene transfer in tumours. A: Luciferase activity in rats and mouse tumours 48 hours after intratumoral injection of 10¹⁰ iu Ad5 Adapt Luc (n = 5). The tumours are growing subcutaneously, and were about 8 × 8 mm at the time of injection. Values indicate means ± SD. B: Luciferase activity measured in lysate of cell cultures 48 hours after in vitro infection with Ad Adapt Luc at different MOI. Values are mean of a triplicate ± SD.

Figure 7

Luciferase activity, viral DNA and luciferase mRNA in different human cancer cell lines after adenoviral transfection. A: Luciferase activity measured in cell lysate 48 hours after infection with different MOI. B: Number of Adenoviral DNA copies per cell determined by duplex real time PCR. The number of cells was determined by microscopic counting and the number of genome by real time PCR of the GAPDH gene. All cell lines had similar ploidy. C: Quantification of the luciferase mRNA in the cell lines after infection at different MOI by Northern blotting. The density of the bands was determined with a phosphorimager. The β-actin blotting was also performed afterwards to control the quality of the RNA extraction and the quantity of RNA loaded. The density of the β-actin bands was similar in all the cell lines (data not shown). Values indicate mean of a triplicate ± SD.

Figure 8

Gene transfer in two rat cancer cell lines. A: Luciferase activity measured in cell lysate 48 hours after infection at different MOI. B: Number of Adenoviral DNA copies per cell determined by duplex real time PCR. The number of cells was determined by microscopic counting. C: Quantification of the luciferase mRNA by Northern blotting. The densities of the luciferase and β-actin blots were determined with a phosphorimager. Values show mean of a triplicate ± SD.

Figure 9

Cytology of the different human breast cancer cell lines. The photographs show cell cultures of the different breast cancer cell lines (magnification × 200). All cell lines develop a glandular phenotype: cuboid cell shape and clusters of few cell layers around a canalicule formation (black arrows).

Figure 10

Luciferase activity and viral DNA in human breast cancer cell lines. A: Luciferase activity measured in cell lysate 48 hours after infection at different MOI. B: Number of Adenoviral DNA copies per cell determined by duplex real time PCR. The number of cells was determined by microscopic counting and the number of genome by real time PCR of the GAPDH gene. All the cell lines had similar ploidy. C: Quantification of the luciferase mRNA in the cell lines after infection at different MOI by Northern blotting. The density of the bands was determined with a phosphorimager. The β-actin blotting was also performed afterwards to control the quality of the RNA extraction and the quantity of RNA loaded. The density of the β-actin bands was similar in all the cell lines (data not shown). Values are means of a triplicate ± SD.