Maximal Lentivirus-Mediated Gene Transfer and Sustained Transgene Expression in Human Hematopoietic Primitive Cells and Their Progeny

Abstract

Gene therapy using permanent modifications of hematopoietic stem cells (HSC) has increasing potential applications for both genetic and acquired diseases. Considerable progress has been made recently in gene transfer to HSC by the use of lentivirus-derived vectors, which have the capacity to transduce noncycling cells. However, overall efficiency of HSC transduction reported so far is still not sufficient for numerous applications. We describe here an improved HSC transduction protocol, using the previously described lentiviral vector, that leads to more than 90% transduction of human CD34+ cells from cord blood, including NOD-LtSz-scid/scid repopulating cells. Moreover, under the same conditions, we transduce more than 75% and 80% of CD34+ cells mobilized in peripheral blood and from bone marrow, respectively. We further show that transgene expression is stable through time and hematopoietic cell differentiation in vitro as well as in vivo. Such a high HSC transduction efficiency opens new opportunities for both gene therapy applications and functional studies of regulator proteins of hematopoiesis.

Human CD34+cells differentiate into microglia and express recombinant therapeutic protein

“…Eighteen weeks after transplantation, EGFP was expressed in 90-98% of human bone marrow (CD45 ϩ ) cells (Fig. 5A), indicating efficient and stable gene transfer and expression, as already shown with this lentiviral vector (18). We analyzed the brain of four mice with 50-68% engraftment.…”

“…Human UCB and MPB CD34 ϩ cells were transduced according to previously described procedures (17,18) with a self-inactivating lentiviral͞HIV vector (TRIP⌬U3) carrying expression cassettes for the human ALD protein or the enhanced GFP (EGFP) under the control of the elongation factor 1␣ promoter. Briefly, CD34 ϩ cells were incubated with lentiviral vector in the presence of four recombinant human cytokines (stem cell factor, Flt3-ligand, IL-3, pegylatedmegakaryocyte growth and differentiation factor; UCB ϭ 100͞ 10͞100͞60 ng͞ml, MPB ϭ all 10 ng͞ml).…”

High-level β-globin expression and preferred intragenic integration after lentiviral transduction of human cord blood stem cells

“…We focused on normal cord blood as a target population for these initial studies because of the high frequency of NOD/SCID mouse-repopulating cells in cord blood (45,46) and their reported ease of transduction after brief in vitro exposure to virus (25,28,29,32,(47)(48)(49). The demonstrated levels of anti-sickling β A-T87Q -globin protein in these cells constitute a significant advance and now underscore the need to extend these studies to adult HSCs.…”

NACA is a positive regulator of human erythroid-cell differentiation

“…Transduction was performed with either NACA or EGFP lentiviral Journal of Cell Science 118 (8) vector particles added at a concentration corresponding to 2500 ng p24 per ml (Amsellem et al, 2002). After 24 hours at 37°C, a second set of particles was added to the cultures.…”

“…For that purpose, we engineered the lentiviral TRIP∆U3-EF1α-Stagged-NACA vector (Sirven et al, 2001;Amsellem et al, 2002), which contains the coding sequence of NACA tagged at its N-terminus with an S-tag sequence. CD34 + cells were transduced by this lentiviral vector or the EGFP control vector and then cultured under conditions that trigger either erythroidor granulocytic-cell differentiation.…”