Detection of adeno-associated virus type 2 in sorted human bone marrow progenitor cells

Wild-type adeno-associated virus (wtAAV) is a helper-dependent human parvovirus which has the ability to integrate into the genome of a wide variety of human cells, including those of the hemopoietic lineages. Recombinant adeno-associated virus (rAAV) is becoming a good candidate for virally mediated gene therapy. rAAV is likely to be a safe vector in clinical gene transfer, as it has never been associated with any disease despite previous studies showing that up to 70% of adults are seropositive for wtAAV. Seroconversion appears to occur early in life. wtAAV is an upper respiratory tract virus that is gut secreted, but little is known about the integration of latent wtAAV in hemopoietic lineages. Unlike retroviruses, which have been the most common vehicles for gene transfer to date, wtAAV appears to have a preferred integration site in the target cell which has been termed AAVS1. Several studies have shown that wtAAV can only integrate into only one of the pair of chromosome 19 in a cell. This may have implications for the use of rAAV in gene transfer because patients with latent virus would be refractory to further infection with rAAV. We used a polymerase chain reaction (PCR) assay to detect the presence of wtAAV in the bone marrow samples from 106 patients who presented at our institution. We were able to detect the presence of integrated virus in 18 whole marrow samples. Subsequently CD34(+) and CD3(+) cell subsets were sorted from the cryopreserved marrow of three PCR-positive individuals to assess integration of virus in these cell lineages. In all three samples tested, we were unable to detect wtAAV virus in the CD34(+) hematological precursor cells, but a detectable level of integrated viral DNA was demonstrated in the CD3(+) cell fraction. Our findings therefore suggest that CD34(+) cells might remain a good target for rAAV-mediated gene transfer despite previous wtAAV infection.