Quantification of lentiviral vector copy numbers in individual hematopoietic colony-forming cells shows vector dose-dependent effects on the frequency and level of transduction

Lentiviral vectors are effective tools for gene transfer and integrate variable numbers of proviral DNA copies in variable proportions of cells. The levels of transduction of a cellular population may therefore depend upon experimental parameters affecting the frequency and/or the distribution of vector integration events in this population. Such analysis would require measuring vector copy numbers (VCN) in individual cells. To evaluate the transduction of hematopoietic progenitor cells at the single-cell level, we measured VCN in individual colony-forming cell (CFC) units, using an adapted quantitative PCR (Q-PCR) method. The feasibility, reproducibility and sensitivity of this approach were tested with characterized cell lines carrying known numbers of vector integration. The method was validated by correlating data in CFC with gene expression or with calculated values, and was found to slightly underestimate VCN. In spite of this, such Q-PCR on CFC was useful to compare transduction levels with different infection protocols and different vectors. Increasing the vector concentration and re-iterating the infection were two different strategies that improved transduction by increasing the frequency of transduced progenitor cells. Repeated infection also augmented the number of integrated copies and the magnitude of this effect seemed to depend on the vector preparation. Thus, the distribution of VCN in hematopoietic colonies may depend upon experimental conditions including features of vectors. This should be carefully evaluated in the context of ex vivo hematopoietic gene therapy studies

Lineage- and stage-restricted lentiviral vectors for the gene therapy of chronic granulomatous disease

Insertional mutagenesis represents a serious adverse effect of gene therapy with integrating vectors. However, although uncontrolled activation of growth-promoting genes in stem cells can predictably lead to oncological processes, this is far less likely if vector transcriptional activity can be restricted to fully differentiated cells. Diseases requiring phenotypic correction only in mature cells offer such an opportunity, provided that lineage/stage-restricted systems can be properly tailored. In this study, we followed this reasoning to design lentiviral vectors for the gene therapy of chronic granulomatous disease (CGD), an immune deficiency due a loss of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes, most often secondary to mutations in gp91(phox). Using self-inactivating HIV1-derived vectors as background, we first expressed enhanced green fluorescent protein (eGFP) from a minimal gp91(phox) promoter, adding various natural or synthetic transcriptional regulatory elements to foster both specificity and potency. The resulting vectors were assessed either by transplantation or by lentiviral transgenesis, searching for combinations conferring strong and specific expression into mature phagocytic cells. The most promising vector was modified to express gp91(phox) and used to treat CGD mice. High-level restoration of NADPH activity was documented in granulocytes from the treated animals. We propose that this lineage-specific lentiviral vector is a suitable candidate for the gene therapy of CGD