Targeting lentiviral vector to specific cell types through surface displayed single chain antibody and fusogenic molecule

Abstract

BackgroundViral delivery remains one of the most commonly used techniques today in the field of gene therapy. However, one of the remaining hurdles is the off-targeting effect of viral delivery. To overcome this obstacle, we recently developed a method to incorporate an antibody and a fusogenic molecule (FM) as two distinct molecules into the lentiviral surface. In this report, we expand this strategy to utilize a single chain antibody (SCAb) for targeted transduction.ResultsTwo versions of the SCAb were generated to pair with our various engineered FMs by linking the heavy chain and the light chain variable domains of the anti-CD20 antibody (αCD20) via a GS linker and fusing them to the hinge-CH2-CH3 region of human IgG. The resulting protein was fused to either a HLA-A2 transmembrane domain or a VSVG transmembrane domain for anchoring purpose. Lentiviral vectors generated with either version of the SCAb and a selected FM were then characterized for binding and fusion activities in CD20-expressing cells.ConclusionCertain combinations of the SCAb with various FMs could result in an increase in viral transduction. This two-molecule lentiviral vector system design allows for parallel optimization of the SCAb and FMs to improve targeted gene delivery.

Engineered lentiviral vectors pseudotyped with a CD4 receptor and a fusogenic protein can target cells expressing HIV-1 envelope proteins

“…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.…”

Recombinase Polymerase Amplification (RPA) of CaMV-35S Promoter and nos Terminator for Rapid Detection of Genetically Modified Crops

“…In the RPA platform, the phage-derived recombinase initially aggregates with the primers and forms nucleoprotein filaments. Then, the filaments scan the template DNA for homologous sequences and catalyze strand exchange at cognate sites [5]. The displaced strand is bound by a single-stranded DNA binding protein, and the primers then are extended by Bsu DNA polymerase.…”

Lentiviruses allow widespread and conditional manipulation of gene expression in the developing mouse brain

“…For example, we used VSVG pseudotyped HIV-1 viruses characterized by a high tropism but the use of different envelope proteins, including those fused to singlechain antibodies (Lei et al, 2010), may allow the targeting of specific tissues and cell types. Whereas the use of loxP-containing lentiviruses in available Cre-expressing lines allows the use of our approach in applications as diverse as the Cre/loxP (or similar) system can provide (Fig.…”