Transduction of Human Hematopoietic Stem Cells by Lentiviral Vectors Pseudotyped with the RD114-TR Chimeric Envelope Glycoprotein

Abstract

Lentiviral vectors are efficiently pseudotyped with RD114-TR, a chimeric envelope glycoprotein made of the extracellular and transmembrane domains of the feline leukemia virus RD114 and the cytoplasmic tail of the murine leukemia virus amphotropic envelope. RD114-TR-pseudotyped vectors may be concentrated by centrifugation, are resistant to complement inactivation, and are suitable for both ex vivo and in vivo gene therapy applications. We analyzed RD114-TR-pseudotyped, HIV-1-derived lentiviral vectors for their ability to transduce human cord blood, bone marrow, and peripheral blood mobilized CD34(+) hematopoietic stem/progenitor cells. Transduction efficiency was analyzed in CD34(+) cells in liquid culture, in CD34(+) clonogenic progenitors in semisolid culture, and in CD34(+) repopulating stem cells after xenotransplantation in NOD-SCID mice. Compared with a standard VSV-G-based packaging system, RD114-TR-pseudotyped particles transduced hematopoietic stem/progenitor cells at lower multiplicity of infection, with lower toxicity and less pseudo-transduction at comparable vector copy number per genome. Potential changes in the CD34(+) cell transcription profile and phenotype on transduction with RD114-TR-pseudotyped vectors was comparatively investigated by microarray analysis. Our study shows that the biology of repopulating hematopoietic stem cells and their progeny is not affected by transduction with RD114-TR-pseudotyped lentiviral vectors. RD114-TR is compatible with the development of lentiviral stable packaging cell lines, and may become the envelope of choice for clinical studies aiming at safe and efficient genetic modification of human hematopoietic stem cells.

RD2-MolPack-Chim3,a Packaging Cell Line for Stable Production of Lentiviral Vectors for Anti-HIV Gene Therapy

“…RD2-MolPack-Chim3 has been designed to produce LV for targeting HSC since it carries the RD114-TR envelope, which has been previously (Sandrin et al, 2002;Neff et al, 2004;Di Nunzio et al, 2007) and in this study shown to have a preferential tropism for CD34…”

A novel lentiviral vector targets gene transfer into human hematopoietic stem cells in marrow from patients with bone marrow failure syndrome and in vivo in humanized mice

“…We coexpressed RD114 glycoprotein as an "entry/fusion" glycoprotein because its receptor is present at high levels on hCD34 ϩ cells and because RD114 glycoprotein LVs efficiently transduce HSCs. 18,19 Insertion of a ligand such as SCF in the influenza HA allowed excellent functional presentation on the LV particles, but completely compromised the vector-cell fusion capacity of the HA glycoprotein. Therefore, SCFHA can bind to the c-Kit receptor but does not efficiently fuse with the target cell.…”

“…To achieve this, the VSVG glycoprotein was exchanged for another fusion partner, a mutant RD114 glycoprotein of an endogenous feline virus, RD114 ( Figure 1A). RD114 is an attractive candidate for in vivo use because: (1) RD114 glycoprotein is resistant to degradation by human complement 17 ; (2) RD114-pseudotyped LVs efficiently transduce CD34 ϩ HSCs [17][18][19] ; and (3) a RD114 glycoprotein mutant (RDTR) allowing incorporation onto LVs has been engineered. 17 We report here that RDTR/SCFHA-displaying vectors achieve a highly selective transduction of hCD34 ϩ cells in unfractionated total cord blood (CB) and BM.…”

Pseudotyped Lentiviral Vectors: One Vector, Many Guises

“…Indeed, numerous studies have demonstrated pseudotyping of LVs with envelopes from other retroviruses such as MLV, [22][23][24] Gibbon Ape Leukemia Virus (GALV), 25,26 Feline Leukemia Virus (RD114), [26][27][28][29] Amphotropic Retrovirus (Ampho), [30][31][32] 10A1 MLV (10A1), 33,34 and Ecotropic retrovirus (Eco). 31,34,35 Retroviruses are generally conserved in their genome and virion structure.…”