Targeted integration of adeno-associated virus (AAV) into human chromosome 19.

Abstract

A key feature in adeno‐associated virus (AAV) replication is efficient integration of the viral genome into host cell DNA to establish latency when helper virus is absent. The steps involved in this process remain largely uncharacterized, even though AAV integration was first documented 20 years ago. Using a protein‐‐DNA binding method we isolated AAV‐‐cellular junction DNA sequences. The cellular component hybridized to a single restriction fragment in the virus‐free parental cell line, and also co‐migrated with AAV‐specific sequences in numerous latently infected cell lines. Analysis of somatic cell hybrids indicated that this cellular sequence maps to the distal portion of the q arm of human chromosome 19. In situ hybridization of AAV DNA to chromosomes from latently infected cells confirms the physical location of AAV integrations to be q13.4‐ter of chromosome 19. Sequence analysis of several independent integration sites shows breakpoints occurring within a 100 bp cellular region. This non‐pathogenic parvovirus thus appears to establish viral latency by integrating its DNA specifically into one chromosomal region. Such specific integration is so far unique among the eukaryotic DNA viruses. The incorporation of site‐specific integration into AAV vector schemes should make this vector system attractive for human gene therapy approaches.

DsAAV8-mediated expression of glucagon-like peptide-1 in pancreatic beta-cells ameliorates streptozotocin-induced diabetes

“…Furthermore, basal secretion of GLP-1 from beta-cells, as occurs with insulin, This targeted approach of GLP-1 production in beta-cells using AAV8 vector technology could potentially reduce the frequency of repeat administration required with current GLP-1-based therapies. While wild-type AAV is capable of integration into the human genome at a specific site on chromosome-19, designated AAVS1, 38,39,58 the vector used in this study was engineered to lack the Rep protein required for mediating this integration. Pancreatic beta-cells proliferate at a slow rate 59 and long-term episomal maintenance of transgenes delivered through AAV vectors has been demonstrated in slowly dividing cells.…”

Gene transfer and expression in oligodendrocytes under the control of myelin basic protein transcriptional control region mediated by adeno-associated virus

“…Without helper viruses its genome integrates into host chromosome DNA, preferentially into the human chromosome 19. 8 Recombinant AAV vector is generated by removing the rep and cap genes and placing a 'transgene' between the ITRs.…”

Long-term in vivo cochlear transgene expression mediated by recombinant adeno-associated virus

“…Persistence of AAV within tissues may be due to either its integration within the genome of the transduced cell or its retention as an unintegrated episome. 4,7,8 The relative decline of transgene expression seen over a 6 month period suggests a possible loss of the AAV from the cell. Thus, the decline of transgene expression is consistent with an extrachromosomal presence of AAV genome.…”