We have developed a novel LV that displays the HIV-1 receptor (CD4) and the FM molecule. This LV/CD4+FM vector is inefficient in transducing normal cells, but can enter cells expressing HIV-1 Env proteins and mediate gene delivery with reasonable efficiency. The vector can be produced by transient transfection of 293T cells with an appropriate combination of plasmids harboring the lentiviral backbone, packaging proteins, CD4 and FM. The flow cytometry analysis confirmed the amazing ability of the transfected 293T cells to co-express all of necessary proteins for making this designed LV. The producer cells then bud vector particles containing CD4 and FM in the vector membrane and release them into the medium supernatant. An in vitro transduction assay revealed that the harvested supernatant containing LV/CD4+FM could efficiently transduce HIV-1 Env-expressing cells and only a background level of transduction was seen when this same vector was exposed to cells lacking HIV-1 Env. Various control experiments verified that both CD4 and FM are necessary for the Env-targeted transduction, and only background transduction was observed when vectors bearing either CD4 alone or CD4 along with a non-relevant protein were used. This specific transduction can be inhibited by the soluble CD4 protein or an endosome neutralization reagent (ammonium chloride) added to the cells. In amixed population of cells of which half were Env-positive and half were Env-negative, LV/CD4+FM could selectively modify Env-expressing cells and no transduction was detected towards cells lacking Env.
Several groups have reported various vector systems targeting Env-expressing cells using a strategy termed inverse fusion, in which the HIV-1 receptor and co-receptor molecules, CD4 and CCR5/CXCR4, respectively, are introduced into the surface of individual vector particles, directing them to cells positive for HIV-1 Env (Bittner et al., 2002
; Endres et al., 1997
; Mebatsion et al., 1997
; Peretti et al., 2006
; Schnell et al., 1997
; Somia et al., 2000
; Ye et al., 2005
). Coordinated interactions between incorporated CD4 and CCR5/CXCR4 on the vector surface with cell surface Env are sufficient to trigger Env-mediated fusion and achieve targeted gene delivery. This mimics the natural infection of HIV-1 to target cells, but with a swapped configuration. Success of this inverse fusion method has been demonstrated in vector systems derived from HIV-1 (Endres et al., 1997
), rhabdovirus (Mebatsion et al., 1997
), vesicular stomatitis virus (Schnell et al., 1997
), and murine leukemia virus (Somia et al., 2000
). The designer vectors based on this method, however, are limited to target cells infected with one tropism of HIV-1. Vectors pseudotyped with CD4 and CCR5 can only target cells expressing Env derived from the macrophage-tropic HIV-1 strain (R5 strain), while vectors enveloped with CD4 and CXCR4 are restricted to target cells expressing the T-cell tropic Env derived from the X4 strain of HIV-1. As a control group in the current study, we confirmed this tropism-limited targeting using the inverse fusion method ( and ). In contrast, our system does not involve the HIV-1 co-receptor and thus can overcome this tropism restriction. We showed that the single LV/CD4+FM vector could target both the R5- and X4-tropsim of HIV-1 Env and mediate gene delivery to cells expressing either type of Env with similar efficiency.
We hypothesized that the entry of the targeting LV is initiated when the vector carrying CD4 binds to the HIV-1 Env. Our vector–cell binding assay confirmed that vector-incorporated CD4 remained functional and was able to direct LVs to bind to 293T.EnvR5 cells. When soluble CD4 protein was added to the cells at the initial stage of transduction, a dose-dependent reduction of overall transduction was obtained. These data suggest that the interaction between vector CD4 and cell surface Env play a pivotal role in initiating the targeting. We further postulated that the binding should induce receptor-mediated endocytosis to uptake LVs into endosomal compartments, where the low pH environment can prompt the conformation change of the vector-carrying FM molecule and induce fusion between endosome and vector membranes (Joo and Wang, 2008
). Using an endosomal neutralization assay, we found that the transduction efficiencies of the targeting vectors decreased with increasing concentrations of NH4
Cl. This trend indicates that the drop in pH is important for vector transduction. When the three FM molecules (AKN, AGM, and SGN) were employed to test their ability to mediate targeted transduction by LV, we obtained different transduction efficiencies. Among them, the SGN vector (FUGW/CD4+SGN) was the most efficient vector. Similar variations in targeting efficiencies were also observed in our previous studies (Yang et al., 2008
). These FM molecules are based on a binding-deficient and fusion-competent version of the Sindbis virus glycoprotein with additional mutations in the E1 glycoprotein domain. Although these additional mutations (AGM and SGN) were originally identified from Sindbis viruses propagated in cholesterol-depleted cells and viruses carrying these mutations were believed to be less dependent on cholesterol for fusion, we speculate that these FM molecules might also have different responsiveness to the endosomal pH, with SGN being the most responsive and fusion-active. Our reported vector–liposome fusion assay actually supports this speculation, and SGN was found to be the most pH-sensitive fusogen (Lei et al., 2010
). Nevertheless, the reduction in transduction efficiency by the endosomal neutralization assay and the variations in transduction efficiencies among the different FM molecules provide sufficient evidence that endocytosis and endosomal trafficking must also be involved in the transduction process. The inhibition study by various endocytosis-inhibitory drugs revealed that the targeting LVs enter HIV-1 Env-expressing cells through clathrin-mediated endocytosis. Cells transfected to express the dominant-negative dynamin mutant were more resistant to vector transduction, indicating that the dynamin-dependent endocytosis pathway plays a crucial role in the internalization of the engineered vector as well. When the dominant mutants of Rab5 and Rab7 were introduced into target cells to regulate the maturation of endosomes at early and late stages, respectively, the transduction efficiency was only affected by the Rab5 mutant, suggesting that the success of vector transduction is largely associated with the trafficking in the early endosomes.
The therapeutic relevance of our HIV-1 Env-specific LV system was demonstrated in a mixed population of T cells with some of them infected to express Env proteins. We showed that Env-positive Jurkat cells could be targeted by LV/CD4+FM to express the suicide gene HSV1-TK, which caused them to be susceptible to the prodrug treatment. Our preliminary in vitro
study showed that the targeting vector FUWSR39tk/CD4+FM could significantly reduce the surface marker (LNGFR)-positive subpopulation of human T cells cultivated in the presence of the prodrug GCV, indicative of the elimination of HIV-1 Env-expressing cells because of the biocistronic nature of the IRES-linked Env and LNGFR. Experiments are under way to further investigate the reason as to why the targeting vector, in conjunction with GCV treatment, was unable to completely eliminate LNGFR/Env-expressing cells under our current experimental condition. This also raises an interesting question that if this method fails to eradicate infected HIV-1 virus completely, what effects might the imposed selective pressure have on the remaining virus reservoir? One logical possibility is that the selective pressure may cause the virus to become a CD4-indpendent strain. Hoffman et al. has actually identified such a strain capable of directly interact with the chemokine receptor CXCR4 to achieve infection (Hoffman et al., 1999
); this CD4-independent strain was further found to be more sensitive to neutralizing antibody treatment. It will be interesting to investigate whether such CD4-independent strains can emerge from our suicide gene therapy.
In conclusion, we have shown that functional incorporation of CD4 and FM into the envelope of LVs can be accomplished with resulting vectors having a rather selective ability to target HIV-1 Env-expressing cells. Compared to the inverse fusion method, the LVs bearing CD4 and FM exhibited higher transduction efficiencies and were able to target a broader range of HIV-1 Env proteins irrespective of their CCR5 or CXCR4 tropism. Although our preliminary experiment in vitro showed the feasibility of such a vector to selectively eliminate Env-expressing T cells through suicide gene therapy, further investigations will reveal whether this gene delivery vector system has a practical utility to specifically eradicate HIV-1-infected cells in vivo, thereby reducing the virus load of HIV-1-infected patients.