Lack of Site-Specific Integration of the Recombinant Adeno-Associated Virus 2 Genomes in Human Cells

Abstract

The adeno-associated virus 2 (AAV)-based vector system has been suggested for its potential use in human gene therapy because the wild-type (wt) AAV genome appears to integrate into the human chromosomal DNA in a site-specific manner. We systematically investigated the integration patterns of the recombinant AAV genomes lacking one or both the viral coding sequences. Four recombinant AAV genomes were constructed containing the genes for resistance to tetracycline (TcR) and the herpesvirus thymidine kinase (TK) promoter-driven gene for resistance to neomycin (neoR; vTc.Neo), the genes for resistance to ampicillin (ApR) and TK-neoR (vAp.Neo), the genes for AAV replication (rep) genes and TK-neoR (vRep.Neo), and the AAV capsid (cap) genes and TK-neoR (vCap.Neo). The integration pattern of each of the recombinant AAV genomes in individual clonal isolates of the human nasopharyngeal carcinoma cell line (KB) analyzed on Southern blots using a neo-specific DNA probe was distinctly different. In addition, in none of the clones examined was the proviral genome covalently linked to the previously described AAV right-junction (Rt.Jn.) human chromosomal DNA fragment, the putative specific-site of integration for the wt AAV genome. Furthermore, whereas a 276-bp DNA fragment could be readily amplified from each of these clones, using a neo-specific primer-pair by polymerase chain reaction (PCR), no amplified DNA product was obtained using the neo- and the Rt.Jn. primer-pair under identical conditions. Fluorescence in situ hybridization (FISH) analyses further revealed the lack of integration of the recombinant AAV into human chromosome 19, even in the presence of a functional rep gene as determined by rescue of the recombinant AAV genome in the presence of adenovirus. These data suggest that the recombinant AAV genomes integrate at sites that are different from that characterized for the wt AAV genome. These studies may have implications in the development of the AAV-based vector system for its potential use in human gene therapy.

Enhanced expression and stable transmission of transgenes flanked by inverted terminal repeats from adeno‐associated virus in zebrafish

“…Southern blot, dot blot, and progeny inheritance tests, however, strongly indicated that several copies of transgenes flanked by AAV-ITRs were randomly integrated into the zebrafish genome at a single locus and that this construct was stably transmitted. The integration pattern of transgenes flanked by AAV-ITRs is consistent with the random integration of recombinant AAV in vitro in the absence of Rep and Cap proteins (Ponnazhagan et al, 1997;Miao et al, 1998).…”

Absence of ocular malignant transformation after sub-retinal delivery of rAAV2/2 or integrating lentiviral vectors in p53-deficient mice

“…Although the wild-type virus efficiently integrates in a specific location in the human genome (AAVS1 on chromosome 19), 29 integration of rAAV genomes is typically passive, of low frequency and largely random. [30][31][32] The vast majority of rAAV vector genomes remain as episomes in the host cell nucleus, with only an estimated 0.5% of genomes integrating into the host genome, 33 probably at pre-existing chromosomal breaks. 34 However, as doses ranging from 10 11 to 10 12 virus particles may be administered to the human eye, 15,35 this small fraction of integrating vector genomes potentially equates to a substantial number of integration events.…”

Transfer of activation-dependent gene expression into T cell lines by recombinant adeno-associated virus

“…14 However, rAAV is limited by the size of DNA that can be packaged to between 3.5 kb and 5 kb and, unlike wild-type AAV, rAAV integrates randomly in the host chromosomes. 15 X-linked hyper IgM (X-HIM) is a primary immune deficiency disease that results from defects in the gene for CD40 ligand. 16 CD40 ligand is a 39 kDa glycoprotein expressed transiently on the surface of activated CD4 + T cells 17 and delivers contact-dependent help to B cells and monocytes.…”