Efficient Oncoretroviral Gene Transfer in Primary, Secondary and Tertiary NOD/SCID Mice under Serum-Free Conditions.
Authors: Gammaitoni Loretta , Lucchi Simona , Bruno Stefania , Tesio Melania , Gunetti Monica , Pignochino Ymera , Lazzari Lorenza , Aglietta Massimo , Rebulla Paolo , Piacibello Wanda 
Published: October 2019

Abstract
Stable oncoretroviral gene transfer into hematopoietic stem cells (HSC) provides permanent genetic disease correction. It is crucial to transplant enough transduced HSC to compete with and replace the defective host hemopoiesis. To increase the number of transduced cells the role of ex-vivo expansion was investigated. For a possible clinical application all experiments were done according to good anufacturing practice (GMP) guidelines. The combination of Flt3 ligand (FL), Stem Cell Factor (SCF), Thrombopoietin (TPO), and Interleukin-6 (IL6) has been shown to stimulate proliferation and self-renewal of very primitive (SCID-repopulating cells, SRC) hematopoietic cells. We asked whether it is possible to efficiently transduce HSC with oncoretroviral vector, to expand them and whether transduced cells retain their self-renewal potential, as demonstrated by their capacity to efficiently and serially engraft NOD/SCID mice A Gibbon ape leukemia virus (GALV)-pseudotyped vectors already approved for clinical application has been used to efficiently and durably deliver a defective, non functional form of the cell surface marker truncated low affinity nerve growth factor receptor (LNGFR) into primitive cord blood (CB) HSC. The transduction was performed, following an up-to 24 hour exposure to FL, TPO, IL-6 and SCF, in the presence of the growth factors in serum-free (SF) medium on retronectin (RT) coated plates. At day 3 post-transduction, total cells and CD34+ cells were expanded 24-fold and 8.5-fold respectively. More than 40% of the cells were CD34+. Transduction efficiency was >55%. Serial transplantation is the most reliable method to assess the stable expression of a gene in cells with high proliferative potential. Mice transplanted with transduced or mock-transduced, expanded cells showed higher levels of human engraftment than those transplanted with unmanipulated cells (56.7%, 55% and 39.9% respectively). LNGFR expression of CD45+ cells was 14.35± 4.27%. All secondary mice transplanted with cells from primary recipient BM resulted engrafted (21.7%, 13.5 and 2.8% respectively). LNGFR expression was 47.54± 3.1% respectively of human CD45+ cells. BM cells from secondary recipients were used for tertiary transplants. Only mice transplanted with expanded cells were positively engrafted. Two mice out of five transplanted with secondary recipient BM cells derived from mice transplanted with transduced and expanded cells, showed good levels of human engraftment (6.15% CD45+). LNGFR expression was 49.1± 4.4% of human CD45+ cells. FACS analysis of the different subpopulations showed LNGFR expression within the progenitor (CD34+), B (CD19+), myeloid (CD14+), erythroid (GpA+) and megakaryocyte cells (CD41+) in equivalent proportion. BM of the engrafted mice was placed in a human colony assay. Human colonies also were generated from the murine BM. In conclusion, we have validated a SF-protocol for efficient gene transfer into human CB HSC using a retroviral vector. Under these conditions, transduced and expanded cells repopulated NOD/SCID mice for 3 generations with a human multilineage graft stably expressing the transgene. In a view of future clinical applications, this protocol represents a major step towards the achievement of this goal.