Frequency and Spectrum of Genomic Integration of Recombinant Adeno-Associated Virus Serotype 8 Vector in Neonatal Mouse Liver

Abstract

Neonatal injection of recombinant adeno-associated virus serotype 8 (rAAV8) vectors results in widespread transduction in multiple organs and therefore holds promise in neonatal gene therapy. On the other hand, insertional mutagenesis causing liver cancer has been implicated in rAAV-mediated neonatal gene transfer. Here, to better understand rAAV integration in neonatal livers, we investigated the frequency and spectrum of genomic integration of rAAV8 vectors in the liver following intraperitoneal injection of 2.0 ؋ 10 11 vector genomes at birth. This dose was sufficient to transduce a majority of hepatocytes in the neonatal period. In the first approach, we injected mice with a ␤-galactosidase-expressing vector at birth and quantified rAAV integration events by taking advantage of liver regeneration in a chronic hepatitis animal model and following partial hepatectomy. In the second approach, we performed a new, quantitative rAAV vector genome rescue assay by which we identified rAAV integration sites and quantified integrations. As a result, we find that at least ϳ0.05% of hepatocytes contained rAAV integration, while the average copy number of integrated doublestranded vector genome per cell in the liver was ϳ0.2, suggesting concatemer integration. Twenty-three of 34 integrations (68%) occurred in genes, but none of them were near the mir-341 locus, the common rAAV integration site found in mouse hepatocellular carcinoma. Thus, rAAV8 vector integration occurs preferentially in genes at a frequency of 1 in approximately 10 3 hepatocytes when a majority of hepatocytes are once transduced in the neonatal period. Further studies are warranted to elucidate the relationship between vector dose and integration frequency or spectrum.

Rescue of Severe Infantile Hypophosphatasia Mice by AAV-Mediated Sustained Expression of Soluble Alkaline Phosphatase

“…The rapid decline of ALP activity after AAV injection is thought to be due to a reduction of ALP expression in the liver. It was reported that AAV vector transduces the neonatal liver with high efficiency, but the expression is rapidly decreased mainly because episomal vector genomes are degraded in the liver at this developmental stage (Cunningham et al, 2008;Inagaki et al, 2008). There was no significant difference in bone mineralization between lentiviral-and AAV-treated mice.…”

Long-term correction of biochemical and neurological abnormalities in MLD mice model by neonatal systemic injection of an AAV serotype 9 vector

“…It was reported that serotype 8 AAV (AAV8) or AAV9 vector efficiently transduced both neonatal and adult liver, but the gene expression is only transient in neonatal liver. 17 The rapid decline of gene expression in the neonatal liver could be due to dilution and degradation of episomal vector genomes during active cell mitosis. Post-mitotic tissues such as the muscle, heart and brain are important targets for AAVmediated neonatal gene therapy for long-term expression.…”

Large Virus for an Even Bigger Task: Can the Mimivirus Close the Gene-Therapy Vector Void?

“…Inclusion of both the positive and the negative strand in the rAAV vector to overcome this reduces the coding capacity by a factor of two and, thus, potentially limits the possible genes of interest even further [24]. Lastly, rAAV-induced tumorigenesis has been suggested when high incidences of hepatocellular carcinomas were found in one particular clinical trial [25], possibly resulting from the rAAV preference of integration near active genes; however, evidence is still very limited and unconfirmed by other studies [26]. In addition, correct gene targeting may become positively influenced by the depletion Ku70 -one half of a heterodimeric protein complex involved in the initial steps of homologous recombination, thereby, (partially) counter acting harmful side effects of rAAV vector systems [27], whereas the small coding capacity would not pose a problem for the delivery of constructs encoding miRNAs to frustrate the current replication of viruses without attenuated vaccine or modify the expression of miRNA-responsive genes [28,29].…”