ADENOASSOCIATED VIRUS 2-MEDIATED HIGH-EFFICIENCY GENE-TRANSFER INTO IMMATURE AND MATURE SUBSETS OF HEMATOPOIETIC PROGENITOR CELLS IN HUMAN UMBILICAL-CORD BLOOD

Recombinant adeno-associated virus 2 (AAV) virions were constructed co ntaining a gene for resistance to neomycin (neo(R)), under the control of either the herpesvirus thymidine kinase (TK) gene promoter (vTK-Ne o), or the human parvovirus B19 p6 promoter (vB19-Neo), as well as tho se containing an upstream erythroid cell-specific enhancer (HS-2) from the locus control region of the human beta-globin gene cluster (vHS2- TK-Neo; vHS2-B19-Neo). These recombinant virions were used to infect e ither low density or highly enriched populations of CD34(+) cells isol ated from human umbilical cord blood. In clonogenic assays initiated w ith cells infected with the different recombinant AAV-Neo virions, equ ivalent high frequency transduction of the neo(R) gene into slow-cycli ng multipotential, erythroid, and granulocyte/macrophage (GM) progenit or cells, including those with high proliferative potential, was obtai ned without prestimulation with growth factors, indicating that these immature and mature hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions. Successful transduction did not require and was not enhanced by prestimulation of these cell popu lations with cytokines. The functional activity of the transduced neo gene was evident by the development of resistance to the drug G418, a neomycin analogue. Individual high and low proliferative colony-formin g unit (CFU)-GM, burst-forming unit-erythroid, and CFU-granulocyte ery throid macrophage megakaryocyte colonies from mock-infected, or the re combinant virus-infected cultures were subjected to polymerase chain r eaction analysis using a neo-specific synthetic oligonucleotide primer pair. A 276-bp DNA fragment that hybridized with a neo-specific DNA p robe on Southern blots was only detected in those colonies cloned from the recombinant virus-infected cells, indicating stable integration o f the transduced neo gene. These studies suggest that parvovirus-based vectors may prove to be a useful alternative to the more commonly use d retroviral vectors for high efficiency gene transfer into slow or no ncycling primitive hematopoietic progenitor cells, without the need fo r growth factor stimulation, which could potentially lead to different iation of these cells before transplantation.