225. Preserving CD4+ T-Cells Phenotype and Function Upon Ex Vivo Lentiviral Transduction

Ex vivo gene modification of T-cell is an appealing strategy for the treatment of both immunodeficiencies and neoplasia. Cell activation remains an essential prerequisite to genetic modification by lentiviral transduction. However, most current protocols for T-cell activation alter significantly the phenotype of transduced cells. This phenotypic change can constitute a drawback when the gene modification aims to correct a gene defect while preserving the T cell-subsets diversity. As we plan to conduct an open label phase I/II gene therapy trial for HIV-1 infected patients presenting with lymphoma where the patients will receive autologous hematopoietic stem cells transplantation with gene modified CD34+ cells and CD4+ T-cells, we investigate the use of the TransAct™ reagent (a CD3/CD28 nanoscale activator reagent from Miltenyi Biotec, Bergisch Gladbach, Germany) for the clinical grade transduction of CD4+ T-cells with the LVsh5/C46 lentiviral vector (Cal-1, Calimmune, Inc. Tucson, USA). LVsh5/C46 is a SIN lentiviral vector that inhibits two crucial steps of CD4+ T cell infection by the HIV virus: (i) attachment of the virus to its target by downregulation of CCR5 via a short hairpin RNA, (ii) fusion of the virus to the target cell through expression of the C46 inhibitor. Compared to previously published T-cells transduction protocols, the use of Miltenyi TransAct™ permits an efficient transduction - evaluated by measurement of vector copy number through quantitative PCR - without major phenotypic modification. Indeed, after activation with the TransAct™ reagent, ex vivo transduced CD4+ T-cells display very few changes in their surface markers with conservation of naive (CCR7+CD62L+CD45RA+), central memory (CCR7+CD62L+CD45RA-) and effector memory (CCR7-CD62L-CD45RA-) subsets in superimposable proportions as initially. Moreover, expression of CD25 remains below 50% of cells suggesting a more “gentle “ activation of the transduced CD4+ T-cells. Phenotypic analysis of transduced cells show no overt modification of cell subsets containing TH1, TH2 and TH17 memory T-cells. Furthermore, transduced T-cells retained the ability to be primed towards the TH1, TH2 and TH17 pathways. Cell sorting of ex vivo modified cells demonstrate a preferential transduction of naive CD31+CCR7+CD62L+CD45RA+ T-cells as compared to other subsets. Besides, we did not find any significant impact of the transduction process on the TCRβ repertoire diversity as evaluated by high-throughput sequencing and analysis of diversity through the Gini-Simpson index or the Shannon index. This optimized protocol could be of valuable use for clinical trials aiming to provide the patient with diversified gene-corrected T-cells.