ADENOASSOCIATED VIRUS 2-MEDIATED GENE-TRANSFER IN MURINE HEMATOPOIETIC PROGENITOR CELLS

Recombinant human adeno-associated virus 2 (AAV) virions were construc ted containing a gene for resistance to neomycin (neo(R)), under the c ontrol of either the herpesvirus thymidine kinase (TK) promoter (vTK-N eo), the murine colony-stimulating factor-1 (CSF-1) promoter (vCSF1-Ne o) or the CSF-1 promoter plus an upstream human erythroid cell-specifi c enhancer, HS-2 (vHS2-CSF1-Neo). Recombinant virions were used to inf ect low-density murine primary bone marrow cells. In hematopoietic pro genitor cell assays initiated with cells infected with these recombina nt virions, myeloid as well as erythroid cell colonies resistant to th e drug G418, a neomycin analogue, were readily obtained, indicating th at the murine hematopoietic progenitor cells were susceptible to infec tion by the recombinant AAV virions and that the transduced neo gene w as functionally active in these cells. Whereas only approximately 10% of the colony-forming unit-granulocyte/macrophage (CFU-GM) colonies cl oned from mock-infected cells survived the G418-selection at a final a ctive concentration of 250 mu g/mL of the drug, the extent of the CFU- GM colony formation initiated with the recombinant AAV-Neo virions was as follows: 15% with vTK-Neo, 22% with vCSF1-Neo and 49% with vHS2-CS F1-Neo. In addition, only 14% of the burst-forming unit-erythroid (BFU -E) colonies from mock-infected cells were resistant to G418, whereas 82% of the BFU-E colonies initiated with cells infected with vHS2-CSF1 -Neo virions survived the drug selection, suggesting that a human eryt hroid cell-specific enhancer was able to potentiate expression of the transduced neo(R) gene from a murine promoter. Individual CFU-GM and B FU-E colonies from mock-infected or recombinant AAV-Neo virus-infected cultures were subjected to polymerase chain reaction (PCR) analysis u sing a neo-specific synthetic oligonucleotide primer-pair. A 276 bp DN A fragment that hybridized with a neo-specific DNA probe on Southern b lots was detected only in colonies cloned from the recombinant virus-i nfected cells, indicating stable integration of the transduced neo gen e. These studies suggest the feasibility of using the AAV-based vector system in an animal model as a prelude to evaluating its safety and e fficacy in human gene therapy.