The Influence of Patient-Related Factors on Ex Vivo Retroviral-Mediated Gene Transfer into Mobilized Peripheral Blood Myeloid Progenitors

Abstract

Mobilized human peripheral blood progenitor cells are potential alternatives to bone marrow cells as targets for ex vivo gene transfer. We report the transduction efficiency of retroviral-mediated gene transfer into myeloid progenitors of peripheral blood progenitor cell (PBPC) harvests, mobilized by high-dose cyclophosphamide and GM-CSF, compared with nonmobilized bone marrow (BM). Eleven PBPC samples were enriched for CD34⁺ cells, preincubated with IL-3 (10 ng/ml), IL-6 (50 ng/ml), and 10% autologous plasma for 42 hr, and transduced over a 6-hr incubation with IL-3 + IL-6 and a retroviral vector carrying the Neo^R gene. Neo^R -specific sequences were detected by polymerase chain reaction in 10 cell pellets (91%). Gene expression in CFU-GM colonies was found in nine transduced samples (82%), with a mean transduction efficiency of 5.2% (95% CI, 1.3–11.8%) CFU-GM per PBPC sample. In univariate analysis, a higher transduction efficiency into CFU-GM correlated significantly with a higher CFU-GM concentration in the CD34⁺-enriched sample (p = 0.009), a shorter interval from diagnosis (p < 0.001), and fewer months of prior cytotoxic treatment (p = 0.001); correlation with younger age was of borderline statistical significance (p = 0.077). In multivariate analysis a shorter interval from diagnosis and, to a lesser degree, a higher CFU-GM concentration in the CD34⁺-enriched sample were independent predictors of higher transduction efficiency. Twelve BM samples were similarly transduced; 11 pellets were PCR positive. CFU-GM Neo^R gene expression was 4.2% (95% CI, 2.0–7.2%) CFU-GM per BM sample, which was not significantly different from the transduction efficiency into PBPC cells. No correlation was found between the transduction efficiency of CFU-GM in BM samples and CFU-GM concentration in the CD34⁺-enriched sample, time from diagnosis, months of prior cytotoxic treatment, and/or patient age. Our data suggest that the transduction efficiency ex vivo may be influenced by time from diagnosis, CFU-GM concentration in the sample, and possibly by the extent of prior cytotoxic administration.

Overview summary

In this study, we measured the efficiency of transduction, using a retroviral vector, of the Neo^R gene into human myeloid progenitor cells, enriched for CD34⁺ and mobilized from cancer patient PBPC harvests. In a multivariant analysis, we demonstrated that a higher ex vivo transduction efficiency was independently related to the number of months since diagnosis, and CFU-GM in the sample. No such effect was seen with bone marrow progenitor cells.