Article

Transient expression of genes delivered to newborn rat liver using recombinant adeno-associated virus 2/8 vectors.

INSERM U948, Biothérapies Hépatiques, CHU Hotel-Dieu, Nantes, France.
The Journal of Gene Medicine (impact factor: 2.48). 06/2009; 11(8):689-96. DOI:10.1002/jgm.1343 pp.689-96
Source: PubMed

ABSTRACT In vivo adeno-associated virus (AAV) delivery to adult liver results in sustained expression of the transgene. However, it has been suggested that AAV delivery to the newborn liver may result in transient expression. In the present study, we analysed transgene expression after AAV8 delivery of a therapeutic or a marker gene to newborn rat liver.
Recombinant AAV 8 vectors carrying either the human UGT1A1 cDNA or the lacZ gene were injected intravenously in 2-day-old Gunn or Wistar rats. Serum bilirubin level was recorded in Gunn rats and beta-galactosidase expression was monitored by immunohistochemistry or enzyme activity. The molecular forms of AAV genome were analysed by the polymerase chain reaction and Southern blotting in whole liver and by the quantitative polymerase chain reaction in macroscopically dissected beta-galactosidase clusters.
In Gunn rat, complete serum bilirubin normalization occurred after AAV delivery but hyperbilirubinemia resumed thereafter. Similarly, beta-galactosidase expression was maximum at day 7, but only a few (less than 1%) beta-galactosidase positive cells were recorded at 1 or 3 months. These cells gathered in small clusters and the AAV copy number was 75-fold higher in positive cell clusters than in the surrounding parenchyma.
The results obtained in the present study show that in vivo AAV delivery to newborn rats results in transient expression in most hepatocytes. Expression of the trangene was persistent in small clusters of cells and preliminary data support the hypothesis that integration of the viral genome occurs in these clusters. Altogether, our data confirm the low efficiency of AAV vectors for gene therapy of liver diseases when delivered in the newborn period.

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Keywords

AAV copy number
 
AAV genome
 
AAV8 delivery
 
beta-galactosidase expression
 
complete serum bilirubin normalization
 
day 7
 
enzyme activity
 
human UGT1A1 cDNA
 
liver diseases
 
low efficiency
 
macroscopically dissected beta-galactosidase clusters
 
newborn liver
 
newborn rat liver
 
Recombinant AAV 8 vectors
 
Serum bilirubin level
 
transient expression
 
viral genome
 
vivo AAV delivery
 
vivo adeno-associated virus
 
whole liver