PRECLINICAL ASSESSMENT OF HUMAN HEMATOPOIETIC PROGENITOR-CELL TRANSDUCTION IN LONG-TERM MARROW CULTURES

Long-term marrow cultures (LTMCs) were established from 27 human marro ws. Hematopoietic cells were subjected to multiple rounds of exposure to retroviral vectors during 3 weeks of culture. Seven different retro viral vectors were evaluated. LTMCs were assessed for viability, repli cation-competent retrovirus, progenitors capable of proliferating in i mmune-deficient mice, and gene transfer. The average number of adheren t cells and committed granulocyte-macrophage progenitors (CFU-GM) reco vered from LTMCs was 28% and 11% of the input totals, respectively. Th ere was no evidence by marker rescue assay or polymerase chain reactio n (PCR) of replication-competent virus production during LTMC. No toxi city to cellular proliferation due to the transduction procedure was o bserved. The adherent layers of LTMCs exposed to retroviral vectors we re positive for proviral DNA by PCR and by Southern blot analysis. Fif ty-three percent of 1,427 individual CFU-GM from transduced LTMC adher ent layers were positive for vector-derived DNA, For neo(r)-containing vectors, the average G418 resistance was 28% of 1,393 LTMC-derived CF U-GM. Forty percent of 187 tissues from 30 immune-deficient mice injec ted with human LTMC cells were positive for human DNA 4-5 weeks after adoptive transfer. These studies indicate that multiple exposures of h uman LTMCs to retroviral vectors result in consistent and reproducible LTMC viability and gene transfer into committed progenitors. Our resu lts further support the use of transduced LTMC cells in clinical trial s of hematopoietic stem cell gene transfer.