Adrenal gland infection by serotype 5 adenovirus requires coagulation factors.

Recombinant, replication-deficient serotype 5 adenovirus infects the liver upon in vivo, systemic injection in rodents. This infection requires the binding of factor X to the capsid of this adenovirus. Another organ, the adrenal gland is also infected upon systemic administration of Ad, however, whether this infection is dependent on the cocksackie adenovirus receptor (CAR) or depends on the binding of factor X to the viral capsid remained to be determined. In the present work, we have used a pharmacological agent (warfarin) as well as recombinant adenoviruses lacking the binding site of Factor X to elucidate this mechanism in mice. We demonstrate that, as observed in the liver, adenovirus infection of the adrenal glands in vivo requires Factor X. Considering that the level of transduction of the adrenal glands is well-below that of the liver and that capsid-modified adenoviruses are unlikely to selectively infect the adrenal glands, we have used single-photon emission computed tomography (SPECT) imaging of gene expression to determine whether local virus administration (direct injection in the kidney) could increase gene transfer to the adrenal glands. We demonstrate that direct injection of the virus in the kidney increases gene transfer in the adrenal gland but liver transduction remains important. These observations strongly suggest that serotype 5 adenovirus uses a similar mechanism to infect liver and adrenal gland and that selective transgene expression in the latter is more likely to be achieved through transcriptional targeting

Influence of Factor X on adenovirus transduction in warfarin-treated mice.

<p>A replication-deficient recombinant adenovirus encoding the Lac-Z gene (6×10⁸ PFU) was injected in warfarin-pretreated animals (n = 7) or warfarin-pre-treated, factor X-complemented animals (n = 5). Forty-eight hours later, liver biopsies or adrenal glands were collected. A) Whole liver were stained for β-galactosidase expression. B: Total RNA were extracted from the adrenal glands (two glands from the same animal were pooled), reversed-transcribed and subjected to quantitative PCR to detect LacZ expression or 18S RNA. The ratio LacZ/18S of adrenal glands collected from warfarin-pre-treated, factor X-complemented animals injected with adenovirus was set at 100%. The data presented are means+SEM. Statistical analysis: Student t test (Prism, Graph-Pad softwares).</p

Figure 2

<p><b>Effect of warfarin pre-treatment on adenoviral transgene expression.</b> A replication-deficient recombinant adenovirus encoding the Lac-Z gene (6×10⁸ PFU) was injected in control- (Adenovirus) or warfarin-pretreated animals (Warfarin and Adenovirus). Twenty-four hours later, liver biopsies or adrenal glands were collected. A: Measurement of the β-galactosidase protein in the samples was performed. The data presented are percentages of mean +/− SEM from untreated animals (n = 2), adenovirus-injected animals (n = 10) and adenovirus-injected animals, pre-treated with warfarin (n = 10). 100% represents the average of β-galactosidase activity in the adrenal glands (100% = 7584 β-gal units/mg of protein) or liver (100% = 24805 β-gal units/mg of protein) of adenovirus-injected animals. Statistical test: ANOVA (Prism, Graph-Pad softwares). B) Total RNA were collected from individual adrenal glands or liver biopsies, reversed-transcribed and subjected to quantitative PCR to detect LacZ or 18S RNA. The ratio LacZ/18S of liver or adrenal glands collected from animals injected with adenovirus was set at 100%. Data presented are duplicate determinations from a single adrenal gland and liver biopsy and is representative of 4 independent experiments.</p

Localization of the kidneys using ^99mTc-DMSA.

<p>Mice were injected intra-peritoneally with 95 MBq of ^99mTc-DMSA. Five hours later, the animals were anaesthetized and SPECT/CT scans performed. A) Transverse, coronal and sagittal sections centered on the kidneys. B) Volume rendering of the whole animal in which the kidneys appear in yellow. Legend: K: kidney, L: left, post: posterior side of the animal.</p

A: CAR expression in the liver and the adrenal glands.

<p>Total RNAs were extracted from liver biopsies or total adrenal glands and subjected to reverse transcription and PCR amplification. The PCR products were separated by electrophoresis. B and C: Mice were injected systemically with 10⁹ PFU of either AdHwt (n = 7) or AdH[GA]24 (n = 7) or PBS (n = 4). Forty-eight hours later, the animals were culled and the livers and adrenal glands were collected. B: Whole liver were stained for β-galactosidase expression. C: Total RNA were extracted from the adrenal glands (two glands from the same animal were pooled), reversed-transcribed and subjected to quantitative PCR to detect LacZ expression or 18S RNA. The ratio LacZ/18S of adrenal glands collected from animals injected with AdHwt was used as 100%. The data presented are means+SEM. Statistical test: ANOVA (Prism, Graph-Pad softwares).</p

Visualization of gene transfer in the liver upon intra-renal injection: Effect of warfarin.

<p>Direct injection of 5×10⁸ PFU Ad-CMV-rNIS in the left kidney was performed on control (A) or warfarin-treated mice (B). Forty-eight hours later, the mice were anaesthetized and SPECT/CT scans performed. The transverse, coronal and sagittal sections presented are centered on the liver. Legend: L: liver, S: Stomach.</p

Effect of warfarin pre-treatment on adenoviral transgene expression following intra-renal administration.

<p>Ad-CMV-rNIS, a replication-deficient recombinant adenovirus encoding the NIS gene (5×10⁸ PFU) was injected into the left kidney of control- (Adenovirus) or warfarin-pretreated animals (Warfarin and Adenovirus). Fourty-eight hours later, liver biopsies and adrenal glands were collected. Total RNA were collected from individual adrenal glands (on the left kidney) or liver biopsies, reversed-transcribed and subjected to quantitative PCR to detect rNIS expression or GAPDH RNA. The ratio rNIS/GAPDH of liver or adrenal glands collected from animals injected with adenovirus was set at 100%. Data presented are triplicate determinations from three adrenal gland and liver biopsies. The data presented are means+SEM. Statistical analysis: Student t test (Prizm, Graph-Pad softwares). **p≤0,01.</p

Visualization of gene transfer in the adrenal gland upon intra-renal injection: Effect of warfarin.

<p>Direct injection of 5×10⁸ PFU Ad-CMV-rNIS in the left kidney was performed on control (A) or warfarin-treated mice (B). Forty-eight hours later, the mice were anaesthetized and SPECT/CT scans performed. The transverse, coronal and sagittal sections presented are centered on the adrenal glands. Legend: AG: adrenal glands, L: liver, K: kidneys, S: stomach, post: posterior side of the animal.</p