Retrovirus-Mediated Gene Transfer into Human CD34⁺38^lowPrimitive Cells Capable of Reconstituting Long-Term CulturesIn Vitroand Nonobese Diabetic–Severe Combined Immunodeficiency MiceIn Vivo

Abstract

Factors that may improve retroviral transduction of primitive human hematopoietic cells were studied using MFG-based vectors containing a LacZ gene and produced either by a murine (psi-Crip) or a human (Tasaf) cell line. Cord blood (CB) or bone marrow (BM) CD34+ cells were stimulated and transduced in the presence of three cytokines (interleukin 3 [IL-3], IL-6, and stem cell factor [SCF; c-Kit Ligand]). In the supernatant infection protocol, hematopoietic progenitor cells as measured by X-Gal staining of colony-forming unit cells (CFU-Cs) were transduced more effectively with Tasaf (20%) than with psi-Crip (8%). In contrast, there was no difference between these two cell lines in a coculture protocol. However, gene transfer into more primitive CD34+CD38- subsets and in LTC-IC-derived colonies was low. The use of a large number of cytokines including FLT3-L and PEG-rhMGDF increased the transduction efficiency into CD34+CD38(-)-derived CFU-Cs (35% by PCR) or LTC-ICs (10%). A virus pseudotyped with gibbon ape leukemia virus (GALV) envelope further improved gene transfer to 60 and 48% for LacZ+ CFU-C- and LTC-IC-derived colonies, respectively. These conditions of transduction allowed multilineage engraftment of primitive cord blood cells in NOD-SCID mice. Moreover, 10% (at least) of the human hematopoietic cells recovered from the marrow of these immunodeficient animals were transduced. These data suggest that the efficiency of transduction of human hematopoietic primitive cells can be significantly improved by judicious combinations of recombinant cytokines and high retroviral titers.

Characterization of novel safe lentiviral vectors derived from simian immunodeficiency virus (SIVmac251) that efficiently transduce mature human dendritic cells

“…Since the transduction efficiency of target cells is dependent on the type of glycoprotein used to coat retroviral vectors, [49][50] we next determined the infectivity of SIV vector particles generated with envelope glycoproteins derived from 10A1-MLV (murine leukemia virus), GALV (gibbon ape leukemia virus) and with an influenza virus H7-HA Gene Therapy hemagglutinin. To minimize variations in expression, all four glycoproteins types were produced using an expression backbone identical to that used in previous experiments for VSV-G-pseudotype formation.…”

Development of a suspension packaging cell line for production of high titre, serum-resistant murine leukemia virus vectors

“…To compare their infectivity, MFGnlsLacZ, a retroviral vector expressing the reporter gene LacZ fused with a nuclear localisation signal, 22 was introduced into the four clones by transduction with helper-free MFGnlsLacZ pseudotypes bearing gibbon ape leukemia virus (GALV) Env. 23 The LacZ titre produced by the four different clones was evaluated on TE671 cells. All four clones produced a titre which was up to 10 times higher than the titre produced by the bulk CC31A population ( Table 1).…”

“…TELCeB6, TELCeB6/AF-7 and TELCeB6/GAF are TE671-derived packaging cells producing helper free, MLV-based retro-virus vector particles which encode the MFGnlslacZ genome and bear no MLV-A and GALV envelope glycoproteins, respectively. 7,23,42 A HT1080 based packaging cell line, FLYA4 lacZ 3 was also used. 7 Antibodies Goat polyclonal anti-MLV RT, anti-RLV P30 and anti-RLV gp69/71 (Quality Biotech Inc, Camden, USA) were used to detect MLV Gag and Env proteins.…”

Surface-engineering of lentiviral vectors

“…Lentiviral vectors pseudotyped with the glycoproteins from Ross River virus show superiority over vectors pseudotyped with VSV-G for in vivo gene delivery to the liver of mice [41]. Likewise, screening of a large panel of pseudotyped vectors established the superiority of the gibbon ape leukemia virus (GALV) and the cat endogenous retroviral glycoproteins (RD114) for transduction of progenitor and differentiated hematopoietic cells [22,34,[42][43][44][45]. Finally, owing to the capacity of the influenza virus, Sindis virus, MLV and GALV glycoproteins, among others, to pseudotype MLV and lentiviral vectors [22,25,46], several Engineering Lentiviral Vectors S85 approaches can be undertaken to modify their tropism in order to modify the host range of vectors (see below).…”