Efficient Transduction of Hemopoietic CD34 + Progenitors of Human Origin Using an Original Retroviral Vector Derived from Fr-MuLV-FB29: In Vitro Assessment

Abstract

A novel retroviral vector has been designed based on a Friend-murine leukemia virus (Fr-MuLV) FB29 strain. The latter has been selected according to characteristics of pathogenicity in mice where it induces a disease of the haemopoietic system affecting all lineages. Higher infectivity has also been demonstrated as compared to other strains. In accordance with these findings, the amphotropic producer clone used in this study carrying along the neomycine resistance gene (FOCH-Neo), harbors viral titers over 10⁷ cfu/ml. To investigate the potential of genetically engineering hematopoietic precursors, CD34⁺ progenitors were selected from cord blood, bone marrow, and peripheral blood mobilized stem cells (patients + solid tumors) and transduced with FOCH-Neo. High transduction rates were achieved using virus supernatant and minimal doses of hematopoietic growth factors during pretransduction and transduction steps. A polymerase chain reaction (PCR) assay investigating the presence of both neomycin-encoding and viral vector sequences tested positive in 45–90% of granulocyte-macrophage colony-forming units (CFU-GM) generating cells (bone marrow and peripheral blood derived cells) following transduction. An average of 35% colonies showed resistance to G418. Such levels of transduction proved reproducible using only supernatants harboring over 10⁷ cfu/ml. In those experiments where long-term in vitro cultures could be maintained over 5 weeks (all cord blood and 5 among 23 PBSC), efficient transduction of long-term culture initiating cell (LTC-IC) hematopoietic progenitors was demonstrated 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% of late unselected CFU-colonies. This novel retroviral vector harbors interesting features toward genetic modification of hematopoietic progenitors.

Overview summary

We have investigated the potential of a novel retroviral vector based on the FB29 special strain of Friend-murine leukemia virus to engineer hematopoietic precursors genetically. CD34⁺ progenitors were selected from cord blood, bone marrow, and peripheral blood and transduced with FOCH-Neo. Minimal doses of hematopoietic growth factors were used during pretransduction and transduction steps. Transduction was evaluated on the basis of the percentage of colony-forming unit (CFU)-colonies resistant to G418 (35% on average) and of a polymerase chain reaction assay investigating the presence of both neomycin-encoding and viral vector sequences. This tested positive in 45–90% CFU-colonies that were not submitted to G418 selection. Such data proved reproducible using supernatants harboring over 10⁷ cfu/ml with a multiplicity of infection (moi) of 10. In those experiments where long-term cultures could be maintained over 5 weeks, efficient transduction of long-term culture initiating cells (LTC-IC) was demonstrated on the basis of both resistance to G418 and virus integration. This novel retroviral vector harbors interesting features toward genetic modification of hematopoietic progenitors.