Publications (12) View all
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Dataset: 1-Nonintegrating Lentiviral Vectors can Effectively Deliver Ovalbumin Antigen for Induction of Antitumor Immunity
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Dataset: SuppInfo-032112011
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Article: Enhanced real-time monitoring of adeno-associated virus trafficking by virus-quantum dot conjugates
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ABSTRACT: The unique spectral properties of semiconductor quantum dots (QDs) enable long-term live-cell imaging and ultrasensitive detection of viral particles, which in turn can potentially provide a practical means for detailed analysis of the underlying molecular mechanisms of virus entry. In this study, we report a general method of labeling adeno-associated virus serotype 2 (AAV2) with QDs for enhanced visualization of the intracellular behavior of viruses in living target cells. It was found that the mild conditions required for this QD conjugation reaction allowed for the retention of viral infectivity of AAV2. Furthermore, quantitative analysis of viral motility in living cells suggested that QD-labeling had no significant effect on the intracellular transport properties of AAV2 particles compared to those of conventional organic dye-labeled AAV2. Our imaging study demonstrated that QD-AAV2 was internalized mainly through a clathrin-dependent pathway and then trafficked through various endosomes. It was also observed that QD-AAV2 particles exploit the cytoskeleton network to facilitate their transport within cells, and the labeling study provided evidence that the ubiquitin–proteasome system was likely involved in the intracellular trafficking of AAV2, at least at the level of nuclear transport. Taken together, our findings reveal the potential of this QD-labeling method for monitoring the intracellular dynamics of virus–host cell interactions and interrogating the molecular mechanisms of viral infection in greater detail.ACS Nano 04/2011; 5(5):3523-3535. · 10.77 Impact Factor -
Article: Pseudotyping lentiviral vectors with aura virus envelope glycoproteins for DC-SIGN-mediated transduction of dendritic cells.
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ABSTRACT: Lentiviral vectors (LVs) pseudotyped with envelope proteins of alphaviruses have recently attracted considerable interest for their potential as gene delivery tools. We report the production of human immunodeficiency virus type 1 (HIV-1)-derived LVs pseudotyped with envelope glycoproteins derived from the Aura virus (AURA). We found that the AURA-glycoprotein-pseudotyped LVs use C-type lectins (DC-SIGN and L-SIGN) as attachment factors. These interactions with DC-SIGN are specific as determined by inhibition assays and appear to facilitate transduction through a pH-dependent pathway. AURA-pseudotyped LVs were used to transduce monocyte-derived dendritic cells (DCs) and the transduction was shown to be DC-SIGN mediated, as illustrated by competitive inhibition with DC-SIGN and L-SIGN antibodies and yeast mannan. Comparisons with LVs enveloped with glycoproteins derived from vesicular stomatitis virus and Sindbis virus suggest that AURA-glycoprotein-bearing LVs might be useful to genetically modify DCs for the study of DC biology and DC-based immunotherapy.Human gene therapy 03/2011; 22(10):1281-91. · 4.20 Impact Factor -
Article: Production of lentiviral vectors with enhanced efficiency to target dendritic cells by attenuating mannosidase activity of mammalian cells.
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ABSTRACT: Dendritic cells (DCs) are antigen-presenting immune cells that interact with T cells and have been widely studied for vaccine applications. To achieve this, DCs can be manipulated by lentiviral vectors (LVs) to express antigens to stimulate the desired antigen-specific T cell response, which gives this approach great potential to fight diseases such as cancers, HIV, and autoimmune diseases. Previously we showed that LVs enveloped with an engineered Sindbis virus glycoprotein (SVGmu) could target DCs through a specific interaction with DC-SIGN, a surface molecule predominantly expressed by DCs. We hypothesized that SVGmu interacts with DC-SIGN in a mannose-dependent manner, and that an increase in high-mannose structures on the glycoprotein surface could result in higher targeting efficiencies of LVs towards DCs. It is known that 1-deoxymannojirimycin (DMJ) can inhibit mannosidase, which is an enzyme that removes high-mannose structures during the glycosylation process. Thus, we investigated the possibility of generating LVs with enhanced capability to modify DCs by supplying DMJ during vector production. Through western blot analysis and binding tests, we were able to infer that binding of SVGmu to DC-SIGN is directly related to amount of high-mannose structures on SVGmu. We also found that the titer for the LV (FUGW/SVGmu) produced with DMJ against 293T.DCSIGN, a human cell line expressing the human DC-SIGN atnibody, was over four times higher than that of vector produced without DMJ. In addition, transduction of a human DC cell line, MUTZ-3, yielded a higher transduction efficiency for the LV produced with DMJ. We conclude that LVs produced under conditions with inhibited mannosidase activity can effectively modify cells displaying the DC-specific marker DC-SIGN. This study offers evidence to support the utilization of DMJ in producing LVs that are enhanced carriers for the development of DC-directed vaccines.Journal of Biological Engineering 01/2011; 5(1):1.
