Transduction Of Human CD34 Research Articles

Retroviral transduction of human CD34+ umbilical cord blood progenitor cells with a mutated dihydrofolate reductase cDNA.

Umbilical cord blood cells (UCB) have become a major target population for experimental and clinical studies using transfer of genes involved in inborn enzymatic diseases. Cord blood contains hematopoietic progenitor cells at a high frequency, and expanding these cells ex vivo generates sufficient numbers of hematopoietic precursors for transplantation into adults, e.g., as supportive treatment. As clinical reports about retroviral transduction into UCB cells have not been as encouraging as the first preclinical data, we have established a retroviral transduction system that allows expansion and selection of hematopoietic progenitor cells from UCB. CD34-enriched UCB cells were transduced with a retroviral vector encoding a mutated dihydrofolate reductase cDNA that confers MTX resistance. We observed increased resistance to MTX in transduced granulocyte macrophage-colony forming units (CFU-GM) after co-culture of CD34+ UCB cells with the virus-producing cell line, or after incubation with virus-containing supernatant. The supernatant-based transduction protocol included a prestimulation with recombinant interleukin-1 (rhIL-1), rhkit-ligand, and rhIL-3 to increase the percentage of cells in S phase to greater than 50%. Using this protocol we measured a 72-fold expansion of CFU-GM and a 2.5-fold selective advantage of transduced versus nontransduced progenitor cells after exposure to low-dose methotrexate in liquid culture. Polymerase chain reaction analysis revealed integration of proviral DNA into the majority of transduced colonies before and after ex vivo expansion. The retroviral vector and transduction protocol reported here provides an experimental system for selection and expansion of retrovirally transduced progenitor/stem cells from UCB that may help improve the efficiency of current clinical gene therapy strategies.

Transduction of human CD34+ hematopoietic progenitor cells by a retroviral vector expressing an RRE decoy inhibits human immunodeficiency virus type 1 replication in myelomonocytic cells produced in long-term culture

Genetic modification of hematopoietic stem cells with a synthetic "anti-human immunodeficiency virus type 1 (HIV-1) gene" which inhibits replication of HIV-1 may allow production of mature lymphoid and monocytic cells resistant to HIV-1 growth after autologous transplantation. Because productive HIV-1 replication requires binding of the Rev protein to the Rev-responsive element (RRE) within the viral transcripts for the HIV-1 structural proteins, anti-HIV-1 gene products which interfere with Rev-RRE interactions may inhibit HIV-1 replication. One such strategy involves overexpression of the RRE sequences in transcripts derived from retroviral vectors to act as decoys to sequester Rev protein and prevent its binding to the RRE element in HIV-1 transcripts. We developed an in vitro model to test the efficacy of this gene therapy approach in primary human hematopoietic cells. Human CD34+ hematopoietic progenitor cells from normal bone marrow or umbilical cord blood were transduced with retroviral vectors carrying RRE decoy sequences as part of a long terminal repeat-directed transcript expressing the neo gene (L-RRE-neo) or with a control vector expressing only the neo gene (LN). The transduced progenitors were allowed to differentiate into mature myelomonocytic cells which were able to support vigorous growth of the monocytotropic isolate of HIV-1, JR-FL. HIV-1 replication was measured in unselected cell populations and following G418 selection to obtain uniformly transduced cell populations. Inhibition of HIV-1 replication in the unselected cell cultures was between 50.2 and 76.7% and was highly effective (99.4 to 99.9%) in the G418-selected cultures. Progenitors transduced by either the L-RRE-neo vector or the control LN vector were identical with respect to hematopoietic growth and differentiation. These findings demonstrate the ability of an RRE decoy strategy to inhibit HIV-1 replication in primary human myelomonocytic cells after transduction of CD34+ progenitor cells, without adverse effects on hematopoietic cell function.

Transduction of human CD34+ haemopoietic progenitors of various origin using an original retrovirus vector derived from Fr-MuLV and clinically relevant procedures.

Transduction of human CD34+ haemopoietic progenitors of various origin using an original retrovirus vector derived from Fr-MuLV and clinically relevant procedures.