Chemoselective attachment of small molecule effector functionality to human adenoviruses facilitates gene delivery to cancer cells

Article abstract of DOI:10.1021/ja104547x

We demonstrate here a novel two-step "click" labeling process in which adenoviral particles are first metabolically labeled during production with unnatural azido sugars. Subsequent chemoselective modification allows access to viruses decorated with a broad array of effector functionality. Adenoviruses modified with folate, a known cancer-targeting motif, demonstrated a marked increase in gene delivery to a murine cancer cell line.

Full text of DOI:10.1021/ja104547x

Published on Web 09/10/2010
Chemoselective Attachment of Small Molecule Effector Functionality to
Human Adenoviruses Facilitates Gene Delivery to Cancer Cells
Partha Sarathi Banerjee,†,§ Philomena Ostapchuk,‡ Patrick Hearing,‡ and Isaac Carrico*,†,§
Department of Chemistry, Department of Molecular Genetics and Microbiology, and Institute of Chemical Biology
and Drug DiscoVery, State UniVersity of New York Stony Brook, New York 11790
Received May 25, 2010; E-mail: isaac.carrico@sunysb.edu
Abstract: We demonstrate here a novel two-step “click” labeling
process in which adenoviral particles are first metabolically labeled
during production with unnatural azido sugars. Subsequent
chemoselective modification allows access to viruses decorated
with a broad array of effector functionality. Adenoviruses modified
with folate, a known cancer-targeting motif, demonstrated a
marked increase in gene delivery to a murine cancer cell line.
Virus-mediated gene delivery has been limited by the inability
to easily access a wide array of surface displayed functionality.
Here we demonstrate the chemoselective attachment of a cancer
selective small molecule, folate, to the surface of human adenovirus
type 5 (hAd5). Modification of the viral surface is mediated by the
metabolic incorporation of an azido sugar, O-linked N-acetylglu-
cosamine (O-GlcNAz), into the fiber protein during virus produc-
tion. Folate-decorated hAd5 demonstrates a significant increase in
transgene delivery to murine breast cancer cells.
Figure 1. A cartoon illustrating incorporated O-GlcNAz residue on the
adenoviral fiber protein and its subsequent chemical modification with
ligands. Potential sites of O-GlcNAz incorporation are indicated with red
circles. Either “click” (A) or Staudinger ligation (B) chemistries were used
to decorate metabolically labeled adenovirus.
Viral surface engineering has significant potential to tune the
virus-host interface enabling gene replacement therapy, oncolytic
therapy, and vaccine development.1-3 Despite being a long-standing
objective, targeting therapeutically relevant tissues remains a
significant hurdle.2 A contributing factor is the lack of general,
effective methods to introduce targeting ligands onto viral surfaces
that do not compromise virus production or infectivity. Currently
the majority of targeting efforts rely upon genetic manipulation of
virus surface proteins. However, due to the delicate nature of viral
protein assembly and infection, such changes often offset particle
production and cellular transduction.4,5 In order to sidestep virion
assembly issues and the limitations in available functionality
inherent to genetic manipulation, chemical modification of full viral
particles has been explored.6 The bulk of chemical modification
approaches utilize mono- or bifunctional PEG molecules, which
have the potential to both de- and retarget vectors to specific tissues.
As these approaches are dependent upon naturally occurring
residues, they lack the precision and control of genetic manipula-
tion.7 Alternatively, cysteines, inserted into adenoviral capsid
proteins at genetically amenable sites, allow the selective modifica-
tion of viral particles.8 Here, we demonstrate the selective in-
corporation of an azido sugar, N-azidoacetylglucosamine (GlcNAz),
at Ser-109 of the adenoviral fiber protein. Proximal to the primary
natural targeting motif, the introduced azide allows chemoselective
modification of full, infective adenovirus particles with peptides,
fluorophores, and small molecule targeting elements. Folate modi-
fication enabled efficient gene delivery to breast cancer cells
normally refractive to adenoviral infection.
Unnatural amino acids have been introduced into simple capsid
protein assemblies derived from bacteriophage, plant viruses, or
hepatitis B.9-11 Advantageously, such noncovalent homopolymeric
structures are easy to produce from E. coli and, as a result,
straightforward to engineer. In addition, a number of papers have
described the modification of natural residues on the surfaces of viruses
or virus-like particles with azides or alkynes.12,13 While this approach
does not provide any of the selectivity advantages of unnatural amino
acid incorporation, it does allow access to “click” reactions and, as a
result, higher levels of modification. These chemically addressable
nanoparticles are excellent candidates for imaging and drug delivery
applications; however their simple architecture prohibits infection and
gene delivery into mammalian cells.
Metabolic incorporation of azide bearing sugars into mammalian
cell surface proteins has been used for identification, functional analysis,
and imaging purposes.14,15 As viruses used for therapeutic applications
are generated in eukaryotic cell lines, use of unnatural sugars during
the propagation cycle is expected to potentially substitute for monosac-
charides within viral glycoproteins. As a result of low pathogenic
potential, ease of production, and established safety protocols, human
adenovirus type 5 has been developed extensively as a vector for gene
replacement and oncolytic therapy.16 Human Ad5 is reported to have
a single O-GlcNAc residue on Ser-109 of the fiber protein (Figure 1),
which exists as a homotrimer on 12 vertices of the virus capsid.17,18
Replacing this sugar residue with an azido analog would allow for
specific placement of an azide on the viral coat. Advantageously, Ser-
109 is located proximal to the natural primary targeting motif on the
fiber knob. Thus chemoselective placement of targeting moieties at
this site is expected to be effective for redirecting viral particles. Toward
this goal, E1 deleted Ad5 particles were propagated on HEK 293 cells.
† Department of Chemistry.
‡ Department of Molecular Genetics and Microbiology.
§ ICB&DD.
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Figure 2. Qualitative analysis of azido sugar incorporation into the fiber
protein of hAd5. (A) Anti-FLAG Western of nondenatured viral particles
treated with alkyne-FLAG under CuAAC conditions (100 mM Tris pH )
8; 1 mM CuBr; 3 mM Bathophenanthroline disulphonic acid disodium salt;
400 µM alk-FLAG; 12 h; RT). Top: anti-FLAG Western. Bottom:
Coomassie. (B) Fluorescent analysis of denatured O-GlcNAz-enabled viral
particles treated with alkyne-TAMRA under CuAAC conditions (ex: 532;
em: 580 BP 30). (C) Hexosaminidase treatment (5 µg/µL Hex-C; 37 °C
overnight) and subsequent Staudinger modification of partially purified
O-GlcNAz-labeled fiber protein (100 mM Tris pH ) 8; 400 µM phos-
FLAG; 2 h; RT). Top: anti-FLAG Western. Bottom: Coomassie.
During the infection period, the culture media was supplemented with
50 µM Ac4GalNAz (Supplementary Scheme 1) or Ac4GlcNAz
(Supplementary Scheme 2). Both sugars are metabolic precursors of
UDP-GlcNAz, a known substrate for O-GlcNAc Transferase.19 Viruses
produced from the infected 293 cells were purified on double CsCl
gradients and probed for incorporation of azido sugar with an
engineered peptide epitope FLAG (DYKDDDDK), bearing either an
N-terminal alkyne or a modified triaryl phosphine motif (Figure 1).
Purified viruses were modified with FLAG epitope and tetramethyl-
rhodamine (TAMRA) probes using the copper accelerated azide-alkyne
cycloaddition (CuAAC),20,21 commonly known as the “click” chem-
istry, or the Staudinger ligation reaction.22,23 Initial characterization
of the O-GlcNAc site of hAd5 indicated that it was enzymatically
inaccessible in complete virion. In order to determine if an installed
O-GlcNAz was chemically accessible, whole viruses were chemically
treated, and not denatured, prior to analysis. While western analysis
of viruses grown with either Ac4GalNAz or Ac4GlcNaz sugar
demonstrated effective labeling of the fiber protein (MW 61.5 kD)
with O-GlcNAz (Figure 2A), Ac4GalNAz-mediated labeling was
markedly higher. In order to confirm both the identity and placement
of the installed azide, modification analysis of both denatured virus
and partially purified fiber was carried out. Modification of the
denatured virus with a fluorescent probe under click conditions
demonstrated exclusive labeling of a protein at 62 kD (Figure 2B).
This corresponds to either fiber or protein IIIa, which comigrate. Fiber
was partially purified from a complete virus and treated with a
hexosaminidase known to remove both O-GlcNAc and O-GlcNAz.19
Subsequent chemical modification demonstrated almost a complete
loss of azide-dependent signal (Figure 2C). Cumulatively, these results
strongly indicate the fiber bears an O-GlcNAz modification, which is
the exclusive source of virus labeling.
Figure 3. Effective gene transduction of 4T1 cells with retargeted Ad5.
(A) Structure of alkyne-folate ligand. See Synthesis Scheme 3 in Supporting
Information. (B) GFP fluorescence microscopy images of alkyne-folate
modified metabolically labeled Ad5 virus and metabolically unlabeled Ad5
infected 4T1 cells. Microscopy was carried out on Glass Bottom dishes
using a Zeiss LSM 510 fluorescence microscope. Cells were infected at an
MOI of 50pfu/cell, and images were taken 24 h postinfection (lane i: 4T1
cells infected with folate-modified Ac4GalNAz-labeled Ad5; lane ii: 4T1
cells infected with metabolically unlabeled Ad5 (no Ac4GalNAz); lane iii:
4T1 cells infected with folate-unmodified Ac4GalNAz-labeled Ad5 (no
alkyne-folate); lane iv: 4T1 cells infected with folate-modified Ac4GalNAz-
labeled Ad5 pretreated with 1 mg/L folic acid).
Fluorescently labeled particles were analyzed to determine the
number of chemically addressable azides on the viral surface.
Quantification of the number of fluorophores on each virus by
fluorescent gel scanning demonstrated exclusive fiber labeling in
agreement with Western analysis (Supplementary Figure S2) and
an average attachment of 21.9 ( 1.5 dyes per particle (Supple-
mentary Table 1), consistent with previous O-GlcNAc characteriza-
tion indicating ∼50% occupancy at Ser109.18
Low CAR expression is exhibited in a number of cancers, including
ovarian, pancreatic, and gastrointestinal cancers, generally limiting the
effectiveness of hAd5 for oncolysis.24-27 As a result of tumor
associated overexpression and limited abundance in normal tissues,
the folate receptor has become an appealing target for cancer
therapy.28,29 In addition, folate conjugates are easily produced and
generally have a minor impact on receptor affinity. In order to explore
O-GlcNAz-mediated folate modification we synthesized an alkyne probe
containing a folic acid residue tethered by a small linker (Figure 3A and
In contrast to genetic engineering, we expected the introduction
of O-GlcNAz to have little impact on adenoviral physiology. Virus
production in the presence of either azido sugar demonstrated no
difference on producer cell viability. In addition, no reduction in
particle production was apparent as a result of azide incorporation
(Supplementary Figure S1A). Similarly, infectivity of O-GlcNAz-
labeled virus particles, as assessed via standard plaque forming
assay, remained unaffected (Supplementary Figure S1B).
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Supplementary Scheme 3). O-GlcNAz enabled adenovirus, encoding
a GFP transgene in the E1 deleted region of the viral genome regulated
by a CMV promoter, was conjugated to alkyne-folate via CuAAC.
Mouse breast carcinoma cells (4T1), known to express moderate to
high levels of folate receptors30 and be naturally refractive to human
adenovirus, were cultured in folate-deficient media for 2 weeks. The
cells were infected with the folate-decorated virus at an MOI of 50,
24 h after replating. GFP expression was visualized 1 day after
infection. The results demonstrate a high level of GFP expression
within the 4T1 cells infected with the folate-labeled virus (Figure 3B).
Human Ad5 produced without azido sugar, but treated with the folate
reagent under CuAAC conditions, failed to produce significant
transgene expression. In addition, free folate completely abrogated
infection of folate-modified hAd5, indicating folate receptor meditated
gene delivery. Quantification of infection was assessed using a Synergy
2 fluoresence plate reader (excitation 485 ( 10 nm; emission 528 (
10 nm) which showed a 3-4-fold increase of GFP expression in cells
infected with metabolically and chemically modified virions versus
unmodified virus. Infection assayed in the presence of increasing free
folate concentration demonstrated dose-dependent transfection inhibi-
tion of 4T1 cells (Supplemental Figure S3).
culture condition, adenovirus production and purification as well as
experimental details of labeling, detection, characterization, and target-
ing of modified viruses. The material is available free of charge via
the Internet at http://pubs.acs.org.
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Acknowledgment. Support fort his work was provided by the
NSF (CBET-1080909, ISC) and the NIH (5R01AI041636, PH).
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Supporting Information Available: Details of azido sugar sub-
strates and targeting folate molecule synthesis, peptide synthesis, cell
JA104547X
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