Comparison of HIV- and EIAV-Based Vectors on Their Efficiency in Transducing Murine and Human Hematopoietic Repopulating Cells

Abstract

The use of lentiviral vectors for gene transfer into hematopoietic stem cells has raised considerable interest as these vectors can permanently integrate their genome into quiescent cells. Vectors based on alternative lentiviruses would theoretically be safer than HIV-1-based vectors and could also be used in HIV-positive patients, minimizing the risk of generating replication-competent virus. Here we report the use of third-generation equine infectious anemia virus (EIAV)- and HIV-1-based vectors with minimal viral sequences and absence of accessory proteins. We have compared their efficiency in transducing mouse and human hematopoietic stem cells both in vitro and in vivo to that of a previously documented second-generation HIV-1 vector. The third-generation EIAV- and HIV-based vectors gave comparable levels of transduction and transgene expression in both mouse and human NOD/SCID repopulating cells but were less efficient than the second-generation HIV-1 vector in human HSCs. For the EIAV vector this is possibly a reflection of the lower protein expression levels achieved in human cells, as vector copy number analysis revealed that this vector exhibited a trend to integrate equally efficiently compared to the third-generation HIV-1 vector in both mouse and human HSCs. Interestingly, the presence or absence of Tat in viral preparations did not influence the transduction efficiency of HIV-1 vectors in human HSCs.

Permanent partial phenotypic correction and tolerance in a mouse model of hemophilia B by stem cell gene delivery of human factor IX

“…This is in agreement with ours and others data using lentiviral-eGFP for the transduction of human SCID repopulating cells 15,18 and suggests that we have targeted at least short-term repopulating multilineage hematopoietic progenitor cells.…”

“…18 The enriched hematopoietic stem cell fraction (Lin À ) was obtained using the StemSep (StemCell Technologies) immunomagnetic cell selection system according to manufacturer's recommendations. Purity was standardly 495%.…”

“Microenvironmental contaminations” induced by fluorescent lipophilic dyes used for noninvasive in vitro and in vivo cell tracking

“…Transduction of HL-60 cells was performed with an HIV-1-based self-inactivating (SIN) lentiviral vector (pHRЈSINcPPT-SEW-SffV-IRES-eGFP lentivirus), which carries the enhanced green fluorescent protein (eGFP) reporter gene under the control of the spleen focus-forming virus LTR (a kind gift from Prof A. Thrasher, Institute of Child Health, London, United Kingdom) as previously described. 24 The human osteosarcoma cell line Cal-72 (purchased from German Collection of Microorganisms and Cell Cultures [DSMZ], http:// www.dsmz.de) and maintained in Dulbecco modified Eagle medium with 10% FCS ϩ 2mM L-glutamine ϩ 1ϫ insulin-transferrin-sodium selenite. Human umbilical vein endothelial cells (HUVECs; Clonetics) were cultured in endothelial growth medium 2 (Clonetics), and GP293T cells (kind gift from Prof Eric So, Institute of Cancer Research, London, United Kingdom) were maintained in Dulbecco modified Eagle medium supplemented with 10% FCS and 1% antibiotics.…”

“…Lineage (lin)-negative cells from ␤-actin-Luciferase mice bone marrow cells were obtained using the mouse hematopoietic progenitor cell enrichment kit (StemCell Technologies) following the manufacturer's instructions. After fluorescent staining of the cells, they were plated in serum-free medium supplemented with optimized cytokine cocktail 24 either in 24-well plates for flow cytometric (FC) analysis of viability and fluorescence intensity on day 1 or in triplicates in a black 96-well plate for bioluminescence imaging on day 7.…”

New Vectors for Stable and Safe Gene Modification

“…Although EIAV-based vectors are about 10-fold less efficient in several human cell lines than HIV-1 based vectors (Ikeda et al, 2002), they have demonstrated good efficiency for treatment of neuromuscular pathologies in animal models (Azzouz et al, 2004;Yip et al, 2006). In addition, they are able to transduce human HSCs (Siapati et al, 2005) and are very efficient in vivo. Several groups have demonstrated their potential applicability for gene therapy applications using animal models of neurological disorders, macular dystrophy and choroidal neovascularisation.…”