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The occurrence of clonal perturbations and leukemia in patients transplanted with gamma-retroviral (RV) vector-transduced autologous hematopoietic stem and progenitor cells (HSPCs) has stimulated extensive investigation, demonstrating that proviral insertions may perturb adjacent proto-oncogene expression. Although enhancer-deleted lentiviruses are less likely to result in insertional oncogenesis, there is evidence that they may perturb transcript splicing, and one patient with a benign clonal expansion of lentivirally transduced HPSC has been reported. The rhesus macaque model provides an opportunity for informative long-term analysis to ask whether transduction impacts on long-term HSPC properties. We used two techniques to examine whether lentivirally transduced HSPCs from eight rhesus macaques transplanted 1-13.5 years previously are perturbed at a population level, comparing telomere length as a measure of replicative history and gene expression profile of vector positive versus vector negative cells. There were no differences in telomere lengths between sorted GFP+ and GFP- blood cells, suggesting that lentiviral (LV) transduction did not globally disrupt replicative patterns. Bone marrow GFP+ and GF- CD34+ cells showed no differences in gene expression using unsupervised and principal component analysis. These studies did not uncover any global long-term perturbation of proliferation, differentiation, or other important functional parameters of transduced HSPCs in the rhesus macaque model.

Activation of proto-oncogenes by retroviral insertion is an important issue delaying clinical development of gene therapy. We have reported the nonrandom persistence of hematopoietic clones with vector insertions within the MDS1/EVI1 locus following transplantation of rhesus macaques. We now ask whether prolonged culture of transduced CD34+ cells before transplantation selects for clones with insertions in the MDS1/EVI11 or other proto-oncogene loci. CD34+ cells were transduced with standard retroviral vectors for 4 days and then continued in culture for an additional 6 days before transplantation. A 15% of insertions identified in granulocytes 6 months post-transplant were in MDS1/EVI11, significantly increased compared to the frequency in animals transplanted with cells immediately following transduction. MDS1/EVI1 clones became more dominant over time post-transplantation in one animal that was followed long term, accompanied by an increased overall copy number of vector-containing granulocytes, with one MDS1/EVI1 clone eventually accounting for 100% of transduced granulocytes and marrow colony-forming unit (CFU). This vector insertion increased the expression of Evi1 mRNA. There was no overrepresentation of MDS1/EVI1 insertions contributing to lymphoid lineages. Strategies involving prolonged ex vivo expansion of transduced cells may increase the risk of genotoxicity. [ABSTRACT FROM AUTHOR]