Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology.
Ether-linked glycerophospholipids (ether lipids, EL) are membrane-interactive drugs selectively cytotoxic toward neoplastic cells compared with normal cells. No conclusive explanation has yet been provided for this selectivity. We now present data indicating that the drug 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OMe) induces apoptosis, or programmed cell death, in human leukemic cells. Apoptotic death is induced selectively by ET-18-OMe in HL60 cells, which are sensitive to the drug's cytotoxic action, but not in the resistant K562 cell line. Enrichment of HL60 cells with cholesterol (HL60-CHOL cells) significantly protects the cells from the cytotoxic effect and from the induction of apoptosis by ET-18-OMe; the percentage of fragmented DNA is only 17% for HL60-CHOL, compared with 50% in native HL60 cells after exposure to 20 microns ET-18-OMe for 24 hr. Our study provides a possible explanation for differences in sensitivity to EL among different cell types and illustrates an indirect interaction of EL with cellular DNA.
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.
The ability of 2 recent ether-lipid derivatives, aza-phospholipids BN52205 and BN52211, to induce apoptosis in different leukemia cell lines was investigated using I-octadecyl-2-methyl-rac-glycero-3- phosphocholine (ET-18-OCH3) as a positive control. HL60, K562, Molt-4 and U937 cells were exposed for 24 hr to 20 microM of drug. The 2 aza-derivatives were as cytotoxic as ET-18-OCH3: BN52205 and BN52211 selectively induced apoptotic death in HL60, Molt-4 and U937 cells, but not in the K562-resistant cell line. Around 50% of DNA was fragmented in HL60 cells after exposure to the aza-derivatives, and 34% and 20% of DNA was fragmented in Molt-4 and U937 cells respectively. Similar results were obtained when cells were exposed to ET-18-OCH3. Our data confirm that ether lipids induce apoptosis in a variety of human leukemic cells, providing a possible explanation for their selectivity and mechanism of action.
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