EFFICIENT TRANSDUCTION OF HEMATOPOIETIC CD34(-ORIGIN USING AN ORIGINAL RETROVIRAL VECTOR DERIVED FROM F-MULV-FB29 - IN-VITRO ASSESSMENT() PROGENITORS OF HUMAN)

A novel retroviral vector has been designed based on a Friend-murine l eukemia virus (Fr-MuLV) FB29 strain. The latter has been selected acco rding to characteristics of pathogenicity in mice where it induces a d isease of the haemopoietic system affecting all lineages. Higher infec tivity has also been demonstrated as compared to other strains. In acc ordance with these findings, the amphotropic producer clone used in th is study carrying along the neomycine resistance gene (FOCH-Neo), harb ors viral titers over 10(7) cfu/ml. To investigate the potential of ge netically engineering hematopoietic precursors, CD34(+) progenitors we re selected from cord blood, bone marrow, and peripheral blood mobiliz ed stem cells (patients + solid tumors) and transduced with FOCH-Neo. High transduction rates were achieved using virus supernatant and mini mal doses of hematopoietic growth factors during pretransduction and t ransduction steps. A polymerase chain reaction (PCR) assay investigati ng the presence of both neomycin-encoding and viral vector sequences t ested positive in 45-90 % of granulocyte-macrophage colony-forming uni ts (CFU-GM) generating cells (bone marrow and peripheral blood derived cells) following transduction. An average of 35% colonies showed resi stance to G418. Such levels of transduction proved reproducible using only supernatants harboring over 10(7) cfu/ml. In those experiments wh ere long-term in vitro cultures could be maintained over 5 weeks (all cord blood and 5 among 23 PBSC), efficient transduction of long-term c ulture initiating cell (LTC-IC) hematopoietic progenitors was demonstr ated on the basis of both resistance to G418 and virus integration. In the latter case, the PCR assay tested positive in as much as 35-60% o f late unselected CFU-colonies. This novel retroviral vector harbors i nteresting features toward genetic modification of hematopoietic proge nitors.