High Efficiency Retroviral-Mediated Gene Transduction into CD34⁺Cells Purified from Peripheral Blood of Breast Cancer Patients Primed with Chemotherapy and Granulocyte-Macrophage Colony-Stimulating Factor

Abstract

The hematopoietic system has been one of the major targets for designing human gene therapy protocols. In the present system, we have transduced LNL6, one of the most commonly used retroviral vectors in gene therapy, into purified CD34+ cells from peripheral blood of patients primed with chemotherapy and granulocyte-macrophage colony-stimulating factor (GM-CSF). Purification of CD34+ cells was achieved by incubation with a murine anti-CD34 monoclonal antibody (9C5), and subsequently with paramagnetic microspheres (Dynal) coated with sheep anti-mouse IgG1 (Fc). The CD34+ cells were released from the beads by treatment with chymopapain. Flow cytometry analysis using the anti-CD34 antibody HPCA-2-FITC targeted at another epitope of CD34 showed that 78-97.5% of the cells thus purified were CD34+. After retroviral-mediated gene transfer, polymerase chain reaction (PCR) analysis revealed that 67-100% of the hematopoietic colonies contained the marker gene neo, indicating that CD34+ cells purified by immunomagnetic microsphere method from peripheral mononuclear cells primed with hematopoietic growth factors are highly susceptible to retroviral-mediated gene transfer. The expression of neo as determined by reverse transcription (RT)-PCR appeared to be unstable and not persistent. Taken together, our data suggest that LNL6 is a suitable vector for gene marking of hematopoietic progenitors but not for gene therapy protocols based on persistent gene expression.

Retroviral vector design studies toward hematopoietic stem cell gene therapy for mucopolysaccharidosis type I

“…transplant recipients 8,9 which may be related to methylation of proviral LTR 10,12 and cytokine-regulated inhibition of viral promoters. 13 However, modifications of LTR, such as replacing the U3 enhancer of the 3Ј LTR with the erythroid-specific GATA-1 autoregulatory enhancer, have demonstrated enhanced and targeted transgene expression in erythroblastic progeny of transduced stem cells.…”

“…8,9 This promoter inactivation is often observed in association with increased proviral methylation. activity per 1% relative transgene frequency in F MPS .…”

Intracellular immunization of human fetal cord blood stem/progenitor cells with a ribozyme against human immunodeficiency virus type 1.

“…In this study, we largely followed the previous protocols for enrichment of CD34+ cells and for retroviral transduction (7). We report here studies using retrovirus vectors carrying the ribozyme gene driven by two different polymerase III promoters to transduce CD34+ hematopoietic stem/progenitor cells isolated from fetal cord blood and then challenging the progeny cells with a macrophage tropic strain of HIV-1.…”

“…Ho and colleagues (7) used immunomagnetic beads to enrich for CD34+ cells from bone marrow, mobilized peripheral blood, and fetal cord blood. A purity of 85%-95% of CD34+ cells was routinely obtained by using this procedure (7). Moreover, the isolated CD34+ cells were highly susceptible to retroviral vector-mediated gene transfer: up to 100% transduction was reported (7) with LNL6, one of the most commonly used retroviral vectors.…”

Gene therapy targeting cord blood-derived CD34+ cells from HIV-exposed infants: preclinical studies

“…Each colony was individually processed for DNA extraction using the method described previously. 21 PCR reactions using neo-specific primers were performed in 50 l solution containing reagents described previously 13 except that neo-sequence specific primers were used: neo3 (5′-GATCAAGAGACAGGAT-GAGG-3′) and neo4 (5′-CAGCCATGATGGATACTTTC-3′). The PCR program was as follows: 94°C for 3 min followed by 35 cycles of 94°C for 30 s, 54°C for 30 s and 72°C for 30 s, and then 72°C for 10 min.…”