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(GFP)-expressing reporter virus in three types of cell that are
found in the human blood system, namely, lymphocytes,
macrophages, and endothelial cells (Figure 4b). The CAR
mechanism in GalH-AV and ManH-AV was removed by
modification. Endothelial cells[38] were not transfected by
either, whereas AV remained active. Lymphocytes, which are
not transfected by AV,[39] were used as a negative control. A
small amount of AV transfection was seen in the lymphocyte
sample owing to the presence of contaminating mono-
cytes.[40,41] Finally, transfection of macrophages by means of
retargeting through the mannose receptor[18,20] was examined
by using ManH-AV. Excitingly, significant transduction was
observed with ManH-AV.[42] AV also showed transfection of
macrophages possibly through integrin binding.[8] GalH-AV
showed no transduction of macrophages which suggests that
ManH-AV transduction is
a
specific, sugar-mediated
uptake.[43]
For the first time, by the use of controlled and precise
glycosylation chemistry we have successfully modified the
fragile structure of AV with carbohydrates and modulated its
function. AV transfection can now be adapted to carbohy-
drate–protein receptor interactions as putative lysine glyco-
sylation “switches off” normal receptor pathways and
“switches on” specific sugar-mediated pathways; the clear
potential in therapy is under investigation.
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[29] Yields determined from the total DNA present in the sample by
PicoGreen analysis.
[30] Adenoviral capsid proteins exist as homodimers or homotrimers
that can be disrupted easily by the addition of a denaturant such
as sodium dodecylsulphate (SDS) or guanidinium chloride. The
level of destruction of the adenovirus was analyzed by the
PicoGreen method (see Supporting Information), whereas spin
column purification would have removed degraded viral par-
ticles. PicoGreen analysis of DNA solution levels is routinely
used to indirectly calculate the number of viral particles present
in solution and has the advantage of high sensitivity, which is
useful when protein concentrations are too low for accurate
analysis as is often the case with adenoviral titrations. PicoGreen
analysis performed on intact viral particles showed only back-
ground fluorescence. By comparison of viral samples after
glycosylation with the unglycosylated batch, we are able to
determine the effect of glycosylation on the structure of
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[31] See Supporting Information.
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[33] Purchased from Molecular Probes (www.probes.com).
[34] Purchased from Galab.
[35] The retention time for the galactosylated particles was slightly
longer than that for the unmodified particles owing to a
nonspecific interaction.
Received: August 31, 2004
Revised: November 23, 2004
Keywords: carbohydrates · cell recognition · drug delivery ·
.
glycosylation · viruses
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107, ManH-AV 0; macrophage: AV 1.1 ꢁ 109, ManH-AV 4.5 ꢁ 108).
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Angew. Chem. Int. Ed. 2005, 44, 1057 –1061