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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.

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.

Synthetic ether lipids (EL) exert their antiproliferative action on leukemic cells through localization in the plasma membrane with subsequent biochemical effects which are still being elucidated. In the present study, the modulation of membrane-linked ATPase activity was investigated in relation to changes in membrane fluidity of HL60 and K562 human leukemic cells. Incubation of HL60 and K562 cells with EL under non-cytotoxic conditions caused significant membrane fluidization which was related to the membrane cholesterol (CHOL) levels. HL60 cells, which are sensitive to the cytotoxic action of EL, had a lower basal CHOL content. When HL60 cells were loaded with CHOL, Na+, K(+)-ATPase activity was reduced significantly compared to that of untreated cells. In contrast, CHOL-deprived K562 cells had twice the Na+,K(+)-ATPase activity of unmodified K562 cells. Na+K(+)- and Mg(2+)-ATPase activities were stimulated significantly in both cell lines by EL at concentrations lower than 20 microM. This stimulation was greater in cells richer in CHOL, such as K562 cells and CHOL-enriched HL60 cells. In contrast, Na+,K(+)-ATPase in both cell lines was inhibited by EL above 20 microM regardless of the CHOL content. Mg(2+)-ATPase activity was not related to cell CHOL content and was not inhibited by EL above 20 microM.

Lentiviral vectors transduce at high efficiency human hematopoietic stem/progenitor cells (HSCs) without compromising their self-renewing and repopulation capacity. Last-generation helper constructs and self-inactivating (SIN) vectors have minimized the chances of generating recombinant, replication-competent HIV derivatives during packaging, and drastically reduced the potential risks of insertional activation of oncogenes. To allow the clinical use of lentiviral vectors, development of packaging cell lines and standardized procedures for vector manufacturing, downstream processing and quality/safety testing appears necessary. However, generation of stable packaging cell lines has been hampered so far by the high toxicity of VSV-G and of some of the HIV helper proteins used in the packaging steps. More recently, lentiviral vectors have been pseudotyped with alternative, non-toxic envelope proteins such as derivatives of the RD114 feline leukemia virus envelope glycoprotein, allowing the generation of a prototype, stable packaging cell line (Ikeda et al., Nature Biotech 21:569, 2003). The receptor for RD114 is abundant on human hematopoietic cells, allowing efficient transduction by RD114-pseudotyped lentiviral vectors. We report a comparison between lentiviral vector preparations pseudotyped with VSV-G and the RD114-TR fusion glycoprotein in transducing human cord blood-derived CD34+ HSCs and peripheral blood lymphocytes (PBLs). RD114-TR-pseudotyped vectors transduce both cell types at much lower m.o.i than VSV-G-pseudotyped vectors (5–10 vs. 50–200), resulting in lower toxicity, higher persistence and less vector copy number per genome. In addition, RD114-TR-pseudotyped vectors transduce at higher efficiency human clonogenic progenitors (BFU-E, CFU-GM, CFU-GEMM), as assayed in semi-solid cultures in vitro. Transduction efficiency and average vector copy number in human repopulating HSCs and their progeny was determined after xeno-transplantation in NOD-SCID mice. Our results indicate that pseudotyping with RD114-TR is a promising alternative to VSV-G for developing clinical-grade lentiviral vector preparations aimed at genetic modification of human HSCs.