This study reports a general method of labeling enveloped viruses with semiconductor quantum dots (QDs) for use in single virus trafficking studies. Retroviruses, including human immunodeficiency virus (HIV), could be successfully tagged with QDs through the membrane incorporation of a short acceptor peptide (AP) that is susceptible to site-specific biotinylation and attachment of streptavidin-conjugated QDs. It was found that this AP tag-based QD labeling had little effect on the viral infectivity and allowed for the study of the kinetics of the internalization of the recombinant lentivirus enveloped with vesicular stomatitis virus glycoprotein (VSVG) into the early endosomes. It also allows for the live cell imaging of the trafficking of labeled virus to the Rab5(+) endosomal compartments. This study further demonstrated by direct visualization of QD-labeled virus that VSVG-pseudotyped lentivirus enters cells independent of clatherin- and caveolin-pathways, while the entry of VSVG-pseudotyped retrovirus occurs via the clathrin pathway. The studies monitoring HIV particles using QD-labeling showed that we could detect single virions on the surface of target cells expressing either CD4/CCR5 or DC-SIGN. Further internalization studies of QD-HIV evidently showed that the clathrin pathway is the major route for DC-SIGN-mediated uptake of viruses. Taken together, our data demonstrate the potential of this QD-labeling for visualizing the dynamic interactions between viruses and target cell structures.
Human embryonic stem (hES) cells are renewable cell sources that have potential applications in regenerative medicine. The development of technologies to produce permanent and site-specific genome modifications is in demand to achieve future medical implementation of hES cells. We report herein that a baculoviral vector (BV) system carrying zinc-finger nucleases (ZFNs) can successfully modify the hES cell genome. BV-mediated transient expression of ZFNs specifically disrupted the CCR5 locus in transduced cells and the modified cells exhibited resistance to HIV-1 transduction. To convert the BV to a gene targeting vector, a DNA donor template and ZFNs were incorporated into the vector. These hybrid vectors yielded permanent site-specific gene addition in both immortalized human cell lines (10%) and hES cells (5%). Modified hES cells were both karyotypically normal and pluripotent. These results suggest that this baculoviral delivery system can be engineered for site-specific genetic manipulation in hES cells.
BackgroundLentiviral vectors with broad tropism are one of the most promising gene delivery systems capable of efficiently delivering genes of interest into both dividing and non-dividing cells while maintaining long-term transgene expression. However, there are needs for developing lentiviral vectors with the capability to deliver genes to specific cell types, thus reducing the "off-target" effect of gene therapy. In the present study, we investigated the possibility of engineering the fusion-active domain of a fusogenic molecule (FM) with the aim to improve targeted transduction of lentiviral vectors co-displaying an anti-CD20 antibody (αCD20) and a FM.ResultsSpecific mutations were introduced into the fusion domain of a binding-deficient Sindbis virus glycoprotein to generate several mutant FMs. Lentiviral vectors incorporated with αCD20 and one of the engineered FMs were successfully produced and demonstrated to be able to preferentially deliver genes to CD-20-expressing cells. Lentiviral vectors bearing engineered FMs exhibited 8 to 17-fold enhanced transduction towards target cells as compared to the parental FM. Different levels of enhancement were observed for the different engineered FMs. A pH-dependent study of vector transduction showed that the broader pH range of the engineered FM is a possible mechanism for the resulted increase in transduction efficiency.ConclusionThe fusion domain of Sindbis virus glycoprotein is amenable for engineering and the engineered proteins provide elevated capacity to mediate lentiviral vectors for targeted transduction. Our data suggests that application of such an engineering strategy can optimize the two-molecular targeting method of lentiviral vectors for gene delivery to predetermined cells.
Development of methods to engineer gamma-retroviral vectors capable of transducing target cells in a cell-specific manner could impact the future of the clinical application of gene therapy as well as the understanding of the biology of transfer gene vectors. Two molecular events are critical for controlling the entry of gamma-retroviral vectors to target cells: binding to cell-surface receptors and the subsequent fusion of viral vector membrane and cellular membrane. In this report, we evaluated a method to incorporate a membrane-bound antibody and a fusogenic molecule to provide binding and fusion functions respectively, into gamma-retroviral vectors for targeted gene delivery. An anti-CD20 antibody and a fusogenic protein derived from Sindbis virus glycoprotein could be efficiently co-displayed on the surface of viral vectors. Vectors bearing anti-CD20 antibody conferred their binding specificity to cells expressing CD20. Enhanced in vitro transduction towards CD20-expressing cells was observed for gamma-retroviral vectors displaying both an antibody and a fusogen. We found that the biological activity of the fusogen played an important role on the efficiency of such a targeting strategy and were able to engineer several mutant forms of the fusogen exhibiting elevated fusion function to improve the overall efficiency of targeted transduction. We devised an animal model to show that subcutaneous injection of such engineered vectors to the areas xenografted with target cells could achieve targeted gene delivery in vivo. Taken together, we demonstrated as proof-of-principle a flexible and modular two-molecule strategy for engineering targeting gamma-retroviral vectors.
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.
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