Targeting lentiviral vectors to specific cell types in vivo

Abstract

We have developed an efficient method to target lentivirusmediated gene transduction to a desired cell type. It involves incorporation of antibody and fusogenic protein as two distinct molecules into the lentiviral surface. The fusogen is constructed by modifying viral envelope proteins, so that they lack the ability to bind to their cognate receptor but still retain the ability to trigger pH-dependent membrane fusion. Thus, the specificity of such a lentiviral vector is solely determined by the antibody, which is chosen to recognize a specific surface antigen of the desired cell type. This specific binding then induces endocytosis of the surface antigen, bringing the lentivirus into an endosome. There, the fusogen responds to the low pH environment and mediates membrane fusion, allowing the virus core to enter the cytosol. Using CD20 as a target antigen for human B cells, we have demonstrated that this targeting strategy is effective both in vitro and in intact animals. This methodology is flexible and can be extended to other forms of cell type-specific recognition to mediate targeting. The only requirement is that the antibody (or other binding protein) must be endocytosed after interaction with its cell surface-binding determinant.antibody ͉ gene therapy ͉ lentivirus ͉ retrovirus ͉ targeted gene delivery

Visualization of targeted transduction by engineered lentiviral vectors

“…10 Codisplay of an anti-CD20 antibody and a fusogenic protein on the same virion is thought to be essential for this engineered lentivirus to infect the target cell. To image the virus, we constructed lentiviral particles harboring green fluorescence protein (GFP) fused to the N-terminus of the HIV accessory protein viral protein R (designated GFP-Vpr; Figure 1a).…”

“…To image the virus, we constructed lentiviral particles harboring green fluorescence protein (GFP) fused to the N-terminus of the HIV accessory protein viral protein R (designated GFP-Vpr; Figure 1a). GFPVpr-labeled lentivirus enveloped with both anti-CD20 antibody and fusogenic protein, which were produced as described, 10 except with the use of lentiviral backbone plasmid FUW lacking the GFP transgene ( Figure 1a) instead of FUGW and cotransfection of an additional plasmid that expresses GFP-Vpr. During virus synthesis, GFP-Vpr provided in trans can be incorporated into the virion via interaction between Vpr and the P6 region of the gag protein.…”

“…[5][6][7] Sometimes, to achieve a desirable therapeutic effect, the viral vectors must be capable of precisely delivering a gene of interest to specific cells without influencing non-target cells. [8][9][10] Many efforts have been made to develop such targeting viral vector systems mostly by altering the viral envelope glycoprotein. [11][12][13][14][15][16] Although certain envelope glycoproteins are structurally plastic enough to allow insertion of a new molecular recognition unit (such as peptide, single chain antibody, growth factor and so on) for targeting, this manipulation can adversely affect the delicate coupling interactions of the binding and fusion domains of glycoproteins, resulting in enveloped vectors with decreased infectivity to the target cells.…”

“…8,15,[17][18][19] We have previously developed an efficient method to target lentivirus-mediated gene transduction to a desired cell type. 10 Our engineering approach involved the incorporation of a targeting antibody and pH-dependent fusogenic protein as two distinct molecules on the lentiviral surface. Our hypothesis for targeted transduction was that antibody binding induces endocytosis, and then the virus is brought into an endosomal compartment where the low pH environment causes the fusogenic molecule to trigger membrane fusion and release the viral core into the cytosol.…”

αCAR IGF‐1 vector targeting of motor neurons ameliorates disease progression in ALS mice

“…Our approach is based on antibody mediated cell‐specific targeting and involves modification of the viral vector surface to redirect target specificity 23. More specifically, a binding‐deficient version of the α ‐virus Sindbis glycoprotein is used to pseudotype lentiviral vectors and to mediate fusion of viral and endosomal membrane, while the specificity is determined by an antibody (here the α CAR) chosen to recognize a specific surface receptor of the desired cell type 23, 24…”