技术资料

Human immunodeficiency virus type 1 (HIV-1) vectors transduce rhesus blood cells poorly due to a species-specific block by TRIM5alpha and APOBEC3G,which target HIV-1 capsid and viral infectivity factor (Vif),respectively. We sought to develop a lentiviral vector capable of transducing both human and rhesus blood cells by combining components of both HIV-1 and simian immunodeficiency virus (SIV),including SIV capsid (sCA) and SIV Vif. A chimeric HIV-1 vector including sCA (chiHIV) was superior to the conventional SIV in transducing a human blood cell line and superior to the conventional HIV-1 vector in transducing a rhesus blood cell line. Among human CD34(+) hematopoietic stem cells (HSCs),the chiHIV and HIV-1 vectors showed similar transduction efficiencies; in rhesus CD34(+) HSCs,the chiHIV vector yielded superior transduction rates. In in vivo competitive repopulation experiments with two rhesus macaques,the chiHIV vector demonstrated superior marking levels over the conventional HIV-1 vector in all blood lineages (first rhesus,15 to 30% versus 1 to 5%; second rhesus,7 to 15% versus 0.5 to 2%,respectively) 3 to 7 months postinfusion. In summary,we have developed an HIV-1-based lentiviral vector system that should allow comprehensive preclinical testing of HIV-1-based therapeutic vectors in the rhesus macaque model with eventual clinical application. View Publication

The therapeutic potential of hematopoietic stem cell (HSC) gene therapy can be fully exploited only by reaching efficient gene transfer into HSCs without compromising their biologic properties. Although HSCs can be transduced by HIV-derived lentiviral vectors (LVs) in short ex vivo culture,they display low permissivity to the vector,requiring cytokine stimulation to reach high-frequency transduction. Using stringent assays of competitive xenograft repopulation,we show that early-acting cytokines synergistically enhanced human HSC gene transfer by LVs without impairing engraftment and repopulation capacity. Using S-phase suicide assays,we show that transduction enhancement by cytokines was not dependent on cell cycle progression and that LVs can transduce quiescent HSCs. Pharmacologic inhibition of the proteasome during transduction dramatically enhanced HSC gene transfer,allowing the reach of very high levels of vector integration in their progeny in vivo. Thus,LVs are effectively restricted at a postentry step by the activity of this proteolytic complex. Unexpectedly,cytokine stimulation rapidly and substantially down-regulated proteasome activity in hematopoietic progenitors,highlighting one mechanism by which cytokines may enhance permissiveness to LV gene transfer. These findings demonstrate that antiviral responses ultimately mediated by proteasomes strongly limit the efficiency of HSC transduction by LVs and establish improved conditions for HSC-based gene therapy. View Publication

Recent reports have indicated that human immunodeficiency virus (HIV) and murine leukemia virus (MLV) vectors preferentially integrate into active genes. Here,we used a novel approach based on genetic trapping to rapidly score several thousand integration sites and found that MLV vectors trapped cellular promoters more efficiently than HIV vectors. Remarkably,1 in 5 MLV integrations trapped an active promoter in different cell lines and primary hematopoietic cells. Such frequency was even higher in growth-stimulated lymphocytes. We show that the different behavior of MLV and HIV vectors was dependent on a different integration pattern within transcribed genes. Whereas MLV-based traps showed a strong bias for promoter-proximal integration leading to efficient reporter expression,HIV-based traps integrated throughout transcriptional units and were limited for expression by the distance from the promoter and the reading frame of the targeted gene. Our results indicate a strong propensity of MLV to establish transcriptional interactions with cellular promoters,a behavior that may have evolved to enhance proviral expression and may increase the insertional mutagenesis risk. Promoter trapping efficiency provides a convenient readout to assess transcriptional interactions between the vector and its flanking genes at the integration site and to compare integration site selection among different cell types and in different growth conditions. View Publication

CD4+ T cells are required for immunity against many viral infections,including HIV-1 where a positive correlation has been observed between strong recall responses and low HIV-1 viral loads. Some HIV-1-specific CD4+ T cells are preferentially infected with HIV-1,whereas others escape infection by unknown mechanisms. One possibility is that some CD4+ T cells are protected from infection by the secretion of soluble HIV-suppressive factors,although it is not known whether these factors are produced during primary antigen-specific responses. Here,we show that soluble suppressive factors are produced against CXCR4 and CCR5 isolates of HIV-1 during the primary immune response of human CD4+ T cells. This activity requires antigenic stimulation of naïve CD4+ T cells. One anti-CXCR4 factor is macrophage-derived chemokine (chemokine ligand 22,CCL22),and anti-CCR5 factors include macrophage inflammatory protein-1 alpha (CCL3),macrophage inflammatory protein-1 beta (CCL4),and RANTES (regulated upon activation of normal T cells expressed and secreted) (CCL5). Intracellular staining confirms that CD3+CD4+ T cells are the source of the prototype HIV-1-inhibiting chemokines CCL22 and CCL4. These results show that CD4+ T cells secrete an evolving HIV-1-suppressive activity during the primary immune response and that this activity is comprised primarily of CC chemokines. The data also suggest that production of such factors should be considered in the design of vaccines against HIV-1 and as a mechanism whereby the host can control infections with this virus. View Publication

HIV-1 replication in simian cells is restricted at an early postentry step because of the presence of an inhibitory cellular factor. This block reduces the usefulness of HIV-1-based lentiviral vectors in primate animal models. Here,we demonstrate that substitution of the cyclophilin A (CyPA) binding region in the capsid of an HIV-1-based lentiviral vector (LV) with that of the macrophage tropic HIV-1 Ba-L resulted in a vector that was resistant to the inhibitory effect and efficiently transduced simian cells. Notably,the chimeric gag LV efficiently transduced primary simian hematopoietic progenitor cells,a critical cellular target in gene therapy. The alterations in the CyPA binding region did not affect CyPA incorporation; however,transduction by the gag chimeric LV seemed to be relatively insensitive to cyclosporin A,indicating that it does not require CyPA for early postentry steps. In dual infection experiments,the gag chimeric LV failed to remove the block to transduction of the WT LV,suggesting that the gag chimeric LV did not saturate the inhibitory simian cellular factor. These data suggest that the CyPA binding region of capsid contains a viral determinant involved in the postentry restriction of HIV-1-based lentiviral vectors. Overall,the findings demonstrate that the host range of HIV-1-based LV can be altered by modifications in the packaging construct. View Publication

High-titer,HIV-1-based lentiviral vector particles were found to transduce cytokine-mobilized rhesus macaque CD34(+) cells and clonogenic progenitors very poorly (textless 1%),reflecting the postentry restriction in rhesus cells to HIV infection. To overcome this barrier,we developed a simian immunodeficiency virus (SIV)-based vector system. A single exposure to a low concentration of amphotropic pseudotyped SIV vector particles encoding the green fluorescent protein (GFP) resulted in gene transfer into 68% +/- 1% of rhesus bulk CD34(+) cells and 75% +/- 1% of clonogenic progenitors. Polymerase chain reaction (PCR) analysis of DNA from individual hematopoietic colonies confirmed these relative transduction efficiencies. To evaluate SIV vector-mediated stem cell gene transfer in vivo,3 rhesus macaques underwent transplantation with transduced,autologous cytokine-mobilized peripheral blood CD34(+) cells following myeloablative conditioning. Hematopoietic reconstitution was rapid,and an average of 18% +/- 8% and 15% +/- 7% GFP-positive granulocytes and monocytes,respectively,were observed 4 to 6 months after transplantation,consistent with the average vector copy number of 0.19 +/- 0.05 in peripheral blood leukocytes as determined by real-time PCR. Vector insertion site analysis demonstrated polyclonal reconstitution with vector-containing cells. SIV vectors appear promising for evaluating gene therapy approaches in nonhuman primate models. View Publication