1. Measles Virus Glycoprotein Pseudotyped Lentiviral Vectors Transduce Cytokine Stimulated and Resting Hematopoietic Stem Cells at an Efficiency Without Precedent

Abstract

Hematopoietic stem cell (HSC) based gene therapy holds promise for the cure of many inherited and acquired diseases. The field is now moving towards the use of lentiviral vectors (LVs) evidenced by recent clinical trials. In these trials, VSV-G-LVs were applied at high doses in combination with strong cytokine-cocktails, which might compromise the ‘HSC’ character of the cells. Thus, a challenge in the field is to be able to transduce HSCs with high efficiency without compromising their stemness.Previously, we have shown that measles virus glycoprotein displaying LVs (MV-LVs) were able to transduce efficiently resting T, and B cells and dendritic cells. Now, we evaluated these MV-LVs for hCD34+-cell transduction under mild cytokine prestimulation in order to better preserve the ‘HSC’ characteristics. After a single application, MV-LVs transduced 100% of TPO+SCF stimulated hCD34+-cells. Even more striking was that these MV-LVs allowed efficient transduction of up to 70% of quiescent hCD34+-cells, while VSV-G-LVs reached 5% of transduction at the most. This indeed coincided with the fact that resting hCD34+ early progenitor cells express barely the low density lipid receptor, recognized by VSVG-LVs for cell entry (Frecha et al., Blood 2014). Importantly, reconstitution of primary and secondary recipient NSG mice with MV-LV transduced pre-stimulated or resting hCD34+-cells demonstrated that this high transduction levels in all analyzed myeloid and lymphoid engrafted lineages in all hematopoietic tissues. Notably, we found increased transduction levels (up to 100%) in all human cells in these secondary recipients for the resting MV-LV transduced hCD34+ cells, emphasizing that these vectors preferentially gene-marked HSCs.Solely the MV receptor, CD46, was found responsible for high level MV-LV entry into hCD34+-cells as shown by antibody blocking. Interestingly, a significantly different integration pattern for MV-LVs was revealed in resting and cytokine-pre-stimulated hCD34+-cells.All together, these MV-LVs paves the way towards HSC-based gene therapy of multiple diseases including diseases characterized by low numbers of HSCs such as Fanconi Anemia, for which high level HSC correction is needed combined with minimal cell activation to be successful in the clinic.