Adenovirus Gene Transfer to Amelogenesis Imperfecta Ameloblast-Like Cells
ABSTRACT To explore gene therapy strategies for amelogenesis imperfecta (AI), a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5) vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR) on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including "pK7" and/or "RGD" motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3) fiber "knob" domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold) of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both α(v)β3/α(v)β5 integrins and heparan sulfate proteoglycans (HSPGs) highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI.
- SourceAvailable from: sagepub.com[Show abstract] [Hide abstract]
ABSTRACT: Tooth enamel is a unique mineralized tissue in that it is acellular, is more highly mineralized, and is comprised of individual crystallites that are larger and more oriented than other mineralized tissues. Dental enamel forms by matrix-mediated biomineralization. Enamel crystallites precipitate from a supersaturated solution within a well-delineated biological compartment. Mature enamel crystallites are comprised of non-stoichiometric carbonated calcium hydroxyapatite. The earliest crystallites appear suddenly at the dentino-enamel junction (DEJ) as rapidly growing thin ribbons. The shape and growth patterns of these crystallites can be interpreted as evidence for a precursor phase of octacalcium phosphate (OCP). An OCP crystal displays on its (100) face a surface that may act as a template for hydroxyapatite (OHAp) precipitation. Octacalcium phosphate is less stable than hydroxyapatite and can hydrolyze to OHAp. During this process, one unit cell of octacalcium phosphate is converted into two unit cells of hydroxyapatite. During the precipitation of the mineral phase, the degree of saturation of the enamel fluid is regulated. Proteins in the enamel matrix may buffer calcium and hydrogen ion concentrations as a strategy to preclude the precipitation of competing calcium phosphate solid phases. Tuftelin is an acidic enamel protein that concentrates at the DEJ and may participate in the nucleation of enamel crystals. Other enamel proteins may regulate crystal habit by binding to specific faces of the mineral and inhibiting growth. Structural analyses of recombinant amelogenin are consistent with a functional role in establishing and maintaining the spacing between enamel crystallites.Critical Reviews in Oral Biology & Medicine 02/1995; 6(2):84-108.
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ABSTRACT: Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin alpha2beta1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.Matrix biology: journal of the International Society for Matrix Biology 06/2010; 29(5):411-9. · 3.56 Impact Factor
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ABSTRACT: A sequential dissociative extraction scheme is described in which tooth matrix proteins are extracted first in 4 M guanidine HCl, pH 7.4, and then in 4 M guanidine HCl, 0.5 M EDTA, pH 7.4, both with protease inhibitors present. The latter step dissolves the mineralized portion of the tissue and extracts noncollagenous proteins closely associated with hydroxyapatite crystallites in the mineralized matrix. In fetal bovine enamel, the initial dissociative extraction step completely removes proline-rich amelogenins from the tissue without dissolving the enamel apatite. The amelogenin proteins consist of several species on polyacrylamide gel electrophoresis with sodium dodecyl sulfate, but display anomalous migration behavior relative to conventional marker proteins in this technique. Subsequent extraction of fetal bovine enamel with guanidine HCl/EDTA removes matrix enamelins, acidic glycoproteins that are tightly bound to the enamel hydroxyapatite. This latter fetal protein type has not been isolated previously. The enamelins are adsorbed strongly by DEAE-cellulose in 7 M urea and totally adsorb to synthetic apatite, even in 4 M guanidine HCl. The enamelins display normal behavior on polyacrylamide gels and stain positively for sialic acid/phosphate and carbohydrate. With advancing tooth maturation, amelogenins disappear while enamelins are conserved. Gel filtration chromatography in 4 M guanidine HCl showed amelogenin components at apparent molecular weights of approximately 25,000, 15,000, 9,500, 7,500, and 6,000, while the enamelins eluted at Mr positions of approximately 72,000, 56,000, 42,000, 30,000, 21,000, 13,000, and 8,000. The gel filtration data showed a clear shift in molecular size population from higher to lower components for both amelogenins and enamelins with progressive enamel maturation.Journal of Biological Chemistry 11/1980; 255(20):9760-8. · 4.60 Impact Factor
Adenovirus Gene Transfer to Amelogenesis Imperfecta
Anton V. Borovjagin1,4, Juan Dong2,4., Michael J. Passineau7., Changchun Ren3,4, Ejvis Lamani3,4,
Olga A. Mamaeva4, Hongju Wu5,6, Enid Keyser5, Miho Murakami6, Shuo Chen8, Mary MacDougall3,4*.
1Department of Periodontics, University of Alabama at Birmingham School of Dentistry, Birmingham, Alabama, United States of America, 2Department of Orthodontics,
University of Alabama at Birmingham School of Dentistry, Birmingham, Alabama, United States of America, 3Department of Oral and Maxillofacial Surgery, University of
Alabama at Birmingham School of Dentistry, Birmingham, Alabama, United States of America, 4Institute of Oral Health Research, University of Alabama at Birmingham
School of Dentistry, Birmingham, Alabama, United States of America, 5Department of Obstetrics and Gynecology, University of Alabama at Birmingham, Birmingham,
Alabama, United States of America, 6Division of Human Gene Therapy, Department of Medicine, The Gene Therapy Center, University of Alabama at Birmingham,
Birmingham, Alabama, United States of America, 7Division of Cardiovascular Medicine and Allegheny-Singer Research Institute, West-Penn Allegheny Health System,
Pittsburgh, Pennsylvania, United States of America, 8Department of Pediatric Dentistry, Dental School University of Texas Health Science Center at San Antonio, San
Antonio, Texas, United States of America
To explore gene therapy strategies for amelogenesis imperfecta (AI), a human ameloblast-like cell population was
established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major
enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus
type 5 (Ad5) vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR) on those cells relative
to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic
capsid modifications including ‘‘pK7’’ and/or ‘‘RGD’’ motif-containing short peptides incorporated in the capsid protein fiber
as well as fiber chimera with the Ad serotype 3 (Ad3) fiber ‘‘knob’’ domain. All fiber modifications provided an augmented
transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-
fold) of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both avb3/
avb5 integrins and heparan sulfate proteoglycans (HSPGs) highly expressed by AI-ameloblasts as revealed by gene transfer
blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a
model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated
gene therapy for AI.
Citation: Borovjagin AV, Dong J, Passineau MJ, Ren C, Lamani E, et al. (2011) Adenovirus Gene Transfer to Amelogenesis Imperfecta Ameloblast-Like Cells. PLoS
ONE 6(10): e24281. doi:10.1371/journal.pone.0024281
Editor: Rory Edward Morty, University of Giessen Lung Center, Germany
Received November 20, 2010; Accepted August 9, 2011; Published October 7, 2011
Copyright: ? 2011 Borovjagin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Enamel is produced by highly specialized epithelial ameloblasts
that differentiate from the inner enamel epithelium originating
from the enamel organ epithelium (EOE) [1,2]. Secretory
ameloblasts synthesize and secrete a number of enamel proteins
that include: the most abundant enamel matrix protein amelo-
genin (Amel) , ameloblastin (Ambn) [4,5], the largest secreted
enamel glycoprotein enamelin (Enam) , amelotin (Amtn) ,
and odontogenic ameloblast-associated protein (ODAM, also
known as Apin) . Enamel matrix also contains two major
matrix proteinases: matrix metalloproteinase 20 (MMP-20, also
known as enamelysine)  and kallekrein 4 (KLK 4; also known as
EMSP 1) [10–12]. During the ameloblast secretory phase MMP-
20 and KLK 4 augment matrix mineralization through proteolytic
degradation of Amel and other matrix proteins. This leads to the
removal of enamel’s organic components, followed by apoptosis of
Non-syndromic genetic diseases affecting enamel formation
have been broadly classified as amelogenesis imperfecta (AI). The
most prevalent autosomal dominant (AD) form of AI is commonly
caused by the ENAM gene mutations, while the X-linked form of
AI is caused by alterations in the AMELX gene . Several
treatment options have been described to rehabilitate AI patients,
ranging from preventive intervention to interim composite
restorations, orthodontic treatment, orthognathic surgery and
placement of cast crowns .
The complex secretory pattern of ameloblasts and narrow
developmental window for tooth formation makes this process
highly susceptible to disruption resulting in irreparable tooth
malformation early in an affected individual’s life. The restricted
temporal pattern of amelogenesis suggests feasibility of ameloblast-
targeted gene replacement strategy aimed at rescuing the AI-
associated phenotype by transiently replacing defective gene
products in the ameloblast cellular layer within this time window.
A gene delivery vector, which could be injected into ameloblasts to
PLoS ONE | www.plosone.org1 October 2011 | Volume 6 | Issue 10 | e24281
accomplish this aim, offers a conceptually plausible approach to
restoring tooth formation in AI-afflicted individuals.
A gene therapy vector capable of efficient delivery and
expression of genes in ameloblasts is pivotal for such a strategy.
Human adenovirus type 5 (Ad5)-based vectors offer the strongest
in vivo transgene expression of limited duration without host DNA
integration [16,17], thereby minimizing human safety concerns.
Further, advanced-generation ‘‘gutless’’ Ad vectors with reduced
immunogenic properties [18,19] could potentially be employed to
minimize immune response in patients and prolong expression of
the therapeutic/replacement gene if necessary. Those factors
make Ad an attractive system for localized time-limited gene
expression in ameloblasts. Improving the efficiency of target cell
transduction to minimize viral load and the associated Ad immune
response is pivotal for clinical applications of the above gene
Tissues vary in their susceptibility to infection by Ad5, based
primarily upon their expression of the native Ad5 receptor,
Coxsackie-and-Adenovirus Receptor (CAR) . Susceptibility of
ameloblasts to Ad5 infection has not been addressed to date due to
the lack of a human AI-ameloblast culture as a model for exploring
gene delivery strategies. Our purpose, therefore, was to establish a
primary human AI ameloblast-like cell population and evaluate
the utility of tropism-modified Ad5 vectors for effective delivery to
these cells as a potential gene therapy strategy for AI. This study
represents a first step towards optimization of gene delivery
strategy for AI ameloblasts using Ad5-based vectors.
Materials and Methods
Human alveolar basal epithelial adenocarcinoma A549 cells as
well as rhabdomyosarcoma cell line (RD) were obtained from
American Tissue Culture Collection (ATCC). All cells were
cultured in 10% fetal bovine serum (FBS) (HyClone, Logan UT,
USA) supplemented Dulbecco’s Modified Eagle’s Medium and
Ham’s F-12 Nutrient (50:50) Mixture (DMEM/F-12) with 2 mM
L-glutamine and 100 mg/ml Penicillin/Streptomycin (Mediatech,
Herndon, VA USA).
Establishment of Human AI Ameloblast-like Cells
Extracted third molars from a 14 year-old female patient
presenting with a spontaneous case of AI (IRB approval from
University of Texas Health Science Center at San Antonio) were
obtained with written informed consent of the parent. Crown
EOE tissue was dissected and placed in explant cultures as
previously described . An ameloblast primary cell population
(AI-WAm) was cultured, expanded and frozen stocks prepared.
Cells (passage 4–6) were grown in 4-well chamber slides, fixed
with 4% formaldehyde (10 minutes at room temperature), and
permeabilized with 0.25% Triton X-100. After washes in PBS, the
samples were blocked with 10% BSA (Sigma, St. Louis, MO) and
incubated overnight at 4uC with one of the following primary
antibodies: rabbit polyclonal anti-Amel (Sigma, St. Louis, MO),
rabbit polyclonal anti-cytokeratin 14 (ab53115; Abcam, Cam-
bridge, MA); goat polyclonal anti-Enam (C-18; Santa Cruz
Biotechnology, Santa Cruz, CA), mouse anti-human syndecan 4
(Abcam, Cambridge, MA) at 1:50 dilutions in 3% BSA/PBS or
mouse monoclonal anti-hCAR antibody (clone RmcB, Millipore,
Billerica, MA), mouse monoclonal anti-human HSPG/GAG 10E4
antibody (F58-10E4, Seikagaku Biobusiness Corp), mouse anti-
human integrin avb3 (LM609 clone) or avb5 (P1F6 clone)
monoclonal antibodies (500 mg/ml) (Millipore, Billerica, MA) at
1:100 dilutions in 3% BSA/PBS at room temperature for 2 hrs.
The cells were washed with PBS followed by incubation with anti-
rabbit or anti-goat HRP polymer conjugate (SuperPicTureTM
Polymer Detection Kit) for Amel, Enam and cytokeratin 14 and
the color reaction developed according to instructions. For CAR,
HSPG or integrin staining, the cells were washed with PBS,
incubated for an hour with Alexa Fluor 488-conjugated goat anti-
mouse IgG (1:1000, Invitrogen Molecular Probes, Carlsbad, CA),
washed and counterstained at room temperature for 1 minute
with 300 nM DAPI (Invitrogen Molecular Probes, Carlsbad, CA)
for nuclear staining. All samples were mounted with Crystal/
Mount (Biomeda, Foster City, CA) and visualized using a Nikon
inverted microscope (Nikon Instruments Inc., Melville, NY). All
images were taken with Roper Scientific digital camera using the
same exposure time (300 ms for FITC and 30 ms for DAPI
images) using either 106, 406 or a 606 objectives and overlaid
using the NIS-Element ARTMsoftware.
In situ Alkaline Phosphatase (ALP) Histochemistry
ALP in situ histochemistry of AI-WAm cells and mouse 3-day
postnatal tooth sections were performed as described previously
All Ad5 vectors used in this study were replication-deficient (E1-
deleted). Construction of the capsid-modified vectors: Ad5 (G/L),
Ad5-RGD (G/L), Ad5-pK7 (G/L) and Ad5-pK7/RGD (G/L)
containing two reporter genes: Green Fluorescent Protein (GFP)
and firefly luciferase (Luc), each driven by a separate cytomega-
lovirus (CMV) promoter, was described previously . The
serotype chimera fiber modification was represented by two
different vectors: Ad5/3 (L)  and Ad5/3 (G), constructed by
homologous recombination in bacteria  using a shuttle vector
pKAN.F5/3  and a pVK900 backbone with deleted fiber gene
and CMV-GFP cassette in place of the E1 region (Krasnykh,
In vitro Gene Transfer Assay
Infectivity of the Ad vectors with native and modified fibers in
AI-WAm cells was determined in gene transfer experiments using
expression of two different reporters: GFP (G) and Luc (L).
TCID50 titers for CsCl-purified viral preparations were deter-
mined by the Ka ¨rber equation  in HEK293 cells.
Transduction of all cells with unmodified vector Ad5 (G/L) was
carried out at the infection dose of either 10 or 50 TCID50/cell,
whereas doses of fiber-modified Ads retaining CAR-tropism or
Ads re-targeted to Ad3 receptors (Ad5/3) were empirically
adjusted in our preliminary experiments by Luc expression levels
in CAR/CD46-positive A549 cells . Besides, comparable
levels of the reporter gene (Luc or GFP) expression from all the
vectors in A549 cells were verified in parallel for every gene
transfer experiment to control the accuracy of vector dosing. All
luciferase assays were performed at 20 hours post infection using a
luciferase assay kit (Promega, Madison, WI). Expression of GFP
reporter was analyzed by GFP fluorescence of infected cells
visualized using a Nikon inverted microscope with Nikon digital
camera (Nikon Instruments Inc., Melville, NY) and quantified
using multifunctional Synergy HT plate reader (Bio-Tek Instru-
ments, Winooski, VT, USA). Bioluminescence was quantified
using a luminometer (Femtomaster, Zylux, Germany).
The lack of replication competent adenovirus (RCA) contam-
ination in vector preparations was verified for each vector by
determining the E4 copy number in infected AI-WAm cells at 6,
Adenovirus Gene Transfer to AI-Ameloblasts
PLoS ONE | www.plosone.org2 October 2011 | Volume 6 | Issue 10 | e24281
20 and 36 hrs post infection. Extraction of total DNA from Ad-
infected cells was carried out using DNeasy Kit (Qiagen, Valencia,
CA) according to the manufacturer’s instructions. No statistically
significant increase in E4 copy number over time was considered
as an evidence for the lack of RCA contamination in viral
preparations that could potentially affect the results of our gene
transfer assays via amplification of the transgene expression.
A 1:1 mixture of purified recombinant avb3/avb5 integrins (2 mg)
or either 5 or 50 mg of heparin were used as competitors for cellular
integrins and HSPGs, respectively. Various fiber-modified Ad5
vectors were pre-incubated with the competitors in 50 ml of 2% FBS
medium for 30 minutes at room temperature prior to infection of
56103AI-WAm cells in 96-well plate at an MOI of 10 TCID50/cell.
The gene transfer (Luc) assay was then performed as described
RNA isolation and cDNA synthesis
Total RNA was isolated fromcells usingRNeasyMini Kit(Qiagen,
Valencia, CA) and treated with DNase1 (Qiagen) to eliminate DNA
contamination. First-strand cDNA was synthesized using 1 mg of
RNA and M-MLV reverse transcriptase kit (Applied Biosystems,
Foster City, CA) according to the manufacturer’s protocol.
Quantification of mRNA by Real Time PCR
Quantitative real-time PCR (qPCR) was performed with a 7500
Applied Biosystems Real-Time PCR Detection System (Foster City,
CA) using a SYBR Green Master mix (SABiosciences, Frederick,
MD). Relative gene expression was presented as DCT values
calculated for each gene’s cDNA by normalization to CTvalues for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a house-
in a given cell type relative to that in RD (control) cells using the
‘‘22DDCTmethod’’ . Primers for AMELX, ENAM, AMBN,
AMTN and ODAM genes were purchased from SABiosciences.
Primers used for quantification of hCAR by qPCR were described
previously . The following PCR primers were used for
quantification of hCD46: Forward: 59-CTTTCCTTCCTGG-
CGCTTTC-39; Reverse: 59-CGGAGAAGGAGTACAGCAGCA
-39 and GAPDH: Forward: 59-AGGTCGGTGTGAACGGATT-
TG-39; Reverse: 59-TGTAGACCATGTAGTTGAGGTCA-39.
Quantification of Adenoviral genomes
(Qiagen, Valencia, CA). Ad5 E4 gene copy number in each sample
was determined by q-PCR using a Taqman E4 probe (59–FAM-
TGGCATGACACTACGACCAACAC GATCT-TAMRA-39) and
E4-specific PCR primers (Forward: 59-GAGTGCGCCGAGA-
CAAC-39; Reverse: 59-TGGATGCCACAGGATTCCAT-39). Hu-
man b-actin primers were as described previously . E4 copy
number values were calculated from sample CTvalues based on a
standard curve obtained by serial dilutions of purified Ad5 genomic
DNA with known E4 copy number as described previously [31,32].
QPCR of the b-actin gene in the same samples was performed to
quantify total cellular DNA using a standard curve generated from
serial dilutions of known amount of total cellular DNA isolated from
AI-WAm cells. The E4 copy number values (measured in triplicate)
were normalized for total AI-WAm cellular DNA in each sample.
Expression of CAR, avb3/avb5 integrins, heparan sulfate
proteoglycans (HSPG), syndecan 4, or CD46 was analyzed by
flow cytometry (FACS) method using a Becton-Dickinson
FACSCalibur device (UAB FACS Core). Confluent cells were
collected by digestion with Versene, pelleted at 1000 rpm and
resuspended in 0.5 ml of 5% FBS/PBS. Cells (26105/sample)
were incubated at 4uC for 1 hr with one of the following
antibodies: human CAR-specific monoclonal antibody RmcB
(Millipore, Billerica, MA) at 1:100 dilution, anti-avb3 integrin
(LM609 clone) or anti-avb5 integrin (P1F6 clone) monoclonal
antibody (Millipore, Billerica, MA), each at 5 mg/ml final
concentration, anti-syndecan 4 antibody (Abcam, Cambridge,
MA) at 1:50 dilution, anti-HSPG 10E4 antibody (F58-10E4,
Seikagaku Biobusiness Corp) at 1:100 dilution or anti-human
CD46/isotype control R-phycoerythrin (PE)-conjugated antibod-
ies (eBioscience Inc., San Diego, CA), each at 1:25 dilution, and
washed 3 times with PBS. Incubation with RmcB, anti-integrin
avb3/avb5 antibodies, anti-syndecan 4, or 10E4 antibodies was
followed by 1 hr incubation with Alexa 488-conjugated secondary
antibody (1:1000 dilution) and triple wash with PBS.
Microsoft Excel statistical software was used for a two-tailed
Student’s t-Test to determine statistical significance of the
observed differences. The latter were considered significant at
Establishment of Ameloblast-like Cells from an AI Patient
Developing third molars (Fig. 1A) from a female hypoplastic AI
patient were obtained after elective surgery and used to establish
EOE explant cultures. A cell population termed AI-WAm (Fig. 1B)
was isolated and confirmed to be of epithelial origin by cytokeratin
14 staining (Fig. 1C). This population contained less than 0.1% of
ALP-positive larger cells, likely to represent stratum intermedium-
like cells (Fig. 1C and D). The AI-WAm population was enriched
for ameloblast-like cells, as shown by negative ALP staining
(Fig. 1D), showing homogeneous Amel (Fig. 1E and F) and Enam
(Fig. 1, G and H) expression at the protein level and mRNA level
(Fig. 1I). Amel localization was intracellular with a polarized
pattern (Fig. 1F, arrowheads). Furthermore, RT-qPCR analysis of
these cells showed expression of mRNAs for all tested enamel
proteins Ambn, Amel, Enam, Amtn and ODAM. As expected, all
the enamel matrix protein genes, except AMTN, were expressed
significantly higher in AI-WAm and normal EOE cell populations
relative to the dental pulp cells (P,0.05), representing a control
cell type of ectomesenchymal origin. While expression of the
enamel protein genes was detectable in the dental pulp cells, their
mRNA levels were extremely low (Fig. 1I).
Susceptibility of AI-WAm Cells to Ad5 Transduction is
Limited by hCAR Expression
Ad5-mediated gene transfer to AI-WAm cells was initially
investigated by a conventional gene transfer assay using a capsid-
unmodified Ad5 vector encoding firefly Luc as a reporter. While
susceptibility of AI-WAm cells to Ad5 transduction was on average
23-fold lower than that of A549 cells, frequently used as a CAR-
positive control , gene transfer to AI-ameloblasts was on
average 3-fold more efficient than to ‘‘CAR-negative’’ RD cells
 (Fig. 2A). In line with that, flow cytometry (FACS) analysis
suggested a substantially lower level of hCAR protein in AI-WAm
cells (MFI=9 versus 3.5 of control) as compared to A549
(MFI=22.7 versus 3.6 of control) or HEK293-T cells (MFI=55
versus 5 of control) (Fig. 2D). Notably, hCAR expression in AI-
WAm cells on mRNA level was only 6.3-fold lower than in A549
Adenovirus Gene Transfer to AI-Ameloblasts
PLoS ONE | www.plosone.org3October 2011 | Volume 6 | Issue 10 | e24281
cells as revealed by RT-qPCR analysis (Fig. 2B and C). While
immunohistochemistry (IHC) in AI-WAm cells demonstrated a
weaker hCAR staining as compared to A549 cells, it was clearly
positive relative to RD cells, frequently used as a ‘‘CAR-negative’’
control (Fig. 2E). Interestingly, in both A549 and AI-WAm cells
hCAR showed mostly diffuse cytoplasmic localization in contrast
to HEK293-T cells, used as another positive control for hCAR
staining, where the receptor showed a distinct localization to the
tight junctions of intercellular contacts (Fig. 2E arrows). In the
aggregate, our data were consistent with lower expression of
hCAR in AI-ameloblasts relative to A549 cells and especially to
HEK293-T cells. The latter cells are known to be highly
susceptible to transduction with unmodified Ad5 in agreement
with almost a 15-fold higher expression of CAR in those cells on
mRNA level (data not shown) and high level of CAR protein
evidenced by nearly 10-times more labeled cells (86.3% versus
0.14% of control) as compared with AI-WAm cell population
(Fig. 2D). Thus, limited susceptibility of AI-WAm cell population
to infection with unmodified Ad5 vector was consistent with
reduced expression and/or cell surface localization of the major
Ad5 receptor hCAR in these cells.
Capsid Modifications Improve Ad5 Gene Transfer to AI-
Since integrins avb3 and avb5 play a critical role in Ad5
internalization step [34,35], we also examined AI-WAm cells for
expression of these integrins by flow cytometry. The AI-WAm cells
displayed a robust expression of avb3 (78.3% of labeled cells,
MFI=196.3) as well as avb5 integrins (68.2% of labeled cells,
MFI=128.6) (Fig. 3A). Expression of both integrins was also
evident from IHC staining (Fig. 3B) that was consistent with the
flow cytometry data. Taking into account higher expression of
avb3 and avb5 integrins on AI-WAm cells, we reasoned that their
transduction by Ad5 could be improved by genetic capsid
modifications of the viral fiber protein with short integrin-binding
cyclic peptide CDCRGDCFC (RGD-4C) bearing an arginine-
Figure 1. Characterization of the human AI-ameloblast cell population by immunohistochemistry, ALP in situ histochemistry and
qRT-PCR analysis. A. Image of a tooth extracted from an AI patient that was used to establish an EOE primary cell culture. B. Phase contrast image
of AI-WAm cell monolayer. C. AI-WAm cells stained for ALP activity followed by immunostaining for the epithelial marker cytokeratin 14; a single,
highly ALP-positive cell is evident (arrow). D. Mouse molar stained for ALP activity showing ALP-negative secretory ameloblasts (Am) with highest
activity (dark purple) in the stratum intermedium (SI) followed by the stellate reticulum (SR). Low (E) and high (F) magnification of AI-WAm cells
positively stained for the major enamel protein Amel. Arrowheads on panel F indicate cells that appear polarized with unidirectional orientation of
the Golgi apparatus. Low (G) and high (H) magnification of AI-WAm cells positively stained for the largest enamel protein Enam. I. Quantitative
expression levels of the enamel matrix protein genes AMBN, AMELX, ENAM, AMTN and ODAM in AI-WAm cells relative to dental pulp and normal EOE
cells as determined by RT-qPCR following normalization to a housekeeping gene GAPDH and presented as DDCt values. Statistical analysis was
carried out as described in Materials and Methods. Scale bars are: 50 mm (B, F, H), 100 mm (C, D, E, G).
Adenovirus Gene Transfer to AI-Ameloblasts
PLoS ONE | www.plosone.org4 October 2011 | Volume 6 | Issue 10 | e24281
Figure 2. Ad5 gene transfer to AI-WAm cells is limited by deficiency in expression and/or cell surface localization of the Ad5
receptor CAR. A. Gene transfer efficiency of a human Ad5 vector expressing Luc reporter (Ad5 (L)) to an AI patient-derived ameloblast-like cells (AI-
WAm) at different multiplicities of infection (MOI) (MOI=10, 50 and 250 TCID50/cell) in comparison to CAR-positive A549 and CAR-negative RD cells
by conventional Luc assay at 20 hours post infection. Results are presented in Relative Luc Units (RLU) per cell with mean values shown above each
bar plus/minus standard deviation. All differences were statistically significant (P,0.05) B. Expression levels of hCAR mRNA in AI-WAm and A549 cells
Adenovirus Gene Transfer to AI-Ameloblasts
PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e24281
glycine-aspartate (RGD) motif [36–38] incorporated in the HI-
loop of the fiber knob domain (Fig. 4). This RGD-modification has
been previously shown to greatly enhance Ad5 infectivity for av
integrin-positive cells [39–41].
On the other hand, the abundance of negatively-charged
heparan sulfate proteoglycans (HSPG) on the surface of epithelial
cells known from the literature  suggested that a positively-
charged heptalysine (pK7) peptide as an HSPG-targeting ligand
 can be used for Ad5 fiber modification to enhance infectivity
of Ad5 vectors in the ameloblast cell population.
Furthermore, we hypothesized that a double modification pK7/
RGD (Fig. 4) could provide additional augmentation of Ad5
transduction in AI-WAm cells, based on the earlier finding that
simultaneous incorporation of both pK7 and RGD ligands in the
Ad5 fiber knob results in a synergistic effect in some CAR-deficient
cells such as RD . In order to confirm expression of HSPG on
the surface of AI-WAm cells we carried out both flow cytometry
and IHC staining using antibodies against syndecan 4, known to
be up-regulated in AI-ameloblasts along with syndecan 2 and 3
, and 10E4 antibodies that recognize N-sulfated glucosamine
residues of glycosaminoglycans (GAG) as HSPG epitope and
commonly used to trace HSPGs . As predicted, ameloblasts
showed a robust expression of HSPGs and in particular, syndecan
4 (MFI of 335.0 and 286.7 versus 8.9 of control, respectively),
which was also supported by an efficient IHC staining with the
same antibodies (Fig. 3C and D).
It is noteworthy that syndecan 4-specific staining pattern was
distinct from the pattern observed for total HSPG suggesting
polarized localization of syndecan 4 versus a more uniform
intracellular distribution of total HSPG. Syndecan 4 staining
was also observed in CAR-negative RD cells and CAR-positive
A549 cells showing even more conspicuous signal polarization
in A549 cells (Fig. 3D, 3D insert). Expression of syndecan 4 in
A549 cells was significantly (P=0.0001) lower relative to total
cellular HSPGs in contrast to AI-WAm cells (P=0.286) and RD
cells (P=0.67) (Fig. 3C and D). Specificity of HSPG detection
with 10E4 antibodies was evidenced by a profound drop in the
cell fluorescence intensity upon pre-treatment of A549 cells with
heparitinase (Fig. 3E, peak shift indicated by the green arrow),
an enzyme specifically cleaving both N-acetylated and N-
sulfated glucosaminido-glucuronic acid linkages of heparan
sulfates and removing GAG side chains from cell surface HSPG
Finally, an Ad5/3 serotype chimera vector with fiber protein C-
terminal knob domain genetically replaced with that of Ad3 (Fig. 4)
was included in the panel of tested vectors because our
experiments evidenced a substantial expression of tentative Ad3
co-receptors including HSPGs  and CD46  on AI-WAm
cells. Expression of CD46 in AI-ameloblasts was confirmed both
on mRNA and protein levels using RT-qPCR (Fig. 2C), and flow
cytometry (data not shown), respectively.
Taking into account that all the vectors with small peptide
modifications of the fiber used in this study (except for Ad5/3)
retain an intact CAR-binding site of the fiber protein and thus are
potential CAR-binders, we utilized CAR-positive A549 cell line
for dose normalization of all viral vectors using Luc reporter
expression as a measure of Ad ability to bind to and internalize in
those cells, i.e. as a surrogate ‘‘gene transfer equivalent’’. Infectious
doses of all vectors were empirically adjusted in A549 cells to
provide equal or comparable Luc expression levels and then
applied to CAR-negative or CAR-deficient RD and AI-WAm cells
respectively to reveal relative effect of various fiber modifications
on Ad5 transduction. In order to compare Ad5/3 with the panel of
CAR-binding vectors we likewise adjusted its infectious dose to
that of unmodified Ad5 and the other fiber-modified vectors by
Luc expression levels in A549 cells. We reasoned that this strategy
of Ad5/3 dose normalization would be valid provided comparable
levels of Ad5 and Ad3 receptors in A549 cells. Comparison of
CD46 versus CAR expression on mRNA level by RT-qPCR in
various cells types suggested comparable gene expression levels for
those genes in both A549 cells and AI-WAm cells but not in RD
cells, which expressed a substantially higher level of CD46 mRNA
relative to CAR mRNA (Fig. 2C). Although flow cytometry assay
showed that A549 cells express high levels of both CAR (Fig. 2D)
and CD46 (data not shown) proteins, precise quantitative
assessment of their relative expression levels in this control cell
line by FACS analysis was not feasible.
The use of two different reporters GFP and Luc, indepen-
dently expressed by each vector, allowed for two independent
readouts inthesame experiment.
differences between the GFP (Fig. 5A) and the Luc (Fig. 5B)
readouts, both reporters consistently revealed a dramatic
augmentation of Ad5 infectivity in AI-WAm cells by all
capsid-modified vectors relative to unmodified control with a
superior effect of Ad5-pK7/RGD that was ranging from 85- to
405-fold depending on the multiplicity of infection (Fig. 5C).
Notably, Ad5/3 showed the lowest augmentation of gene
transfer in both AI-WAm and RD control cells relative to the
other vectors, given its comparable transduction level in A549
cells following vector normalization (Fig. 5A and B).
relative to that in RD cells as determined by qRT-PCR and presented as ‘‘fold difference’’. All differences were statistically significant (P,0.05). P(A549/AI-
WAm)=0.027. C. Quantitative analysis of hCAR and hCD46 mRNA expression levels in RD, AI-WAm and A549 cells as determined by qRT-PCR and
normalized to the housekeeping gene GAPDH. The data are presented as DDCt values. For AI-WAm P(CAR/CD46)=0.44; for A549 P(CAR/CD46)=0.127; for
RD P(CAR/CD46)=0.007; for CAR P(AI-WAm/A549)=0.049; P(RD/AI-WAm)=0.033; for CD46 P(RD/AI-WAm)=0.0011; P(AI-WAm/A549)=0.0008. P-values for all other
differences were ,0.05. D. Flow cytometry analysis of Ad5 receptor (CAR) expression in AI-WAm cell population in comparison with CAR-positive
(A549) and CAR-negative (RD) control cells. Cells were incubated with primary anti-CAR (RmcB) monoclonal antibody (Ab) followed by labeling with
Alexa 488-conjugated secondary antibodies. No primary Ab was used in negative control samples. The extent of shift in the fluorescent peak
positions (color lines) relative to control peak(s) of unlabeled cells (black dotted lines) reflects the extent of cell labeling, corresponding to the
receptor expression on each cell type and is expressed as Mean Fluorescence Intensity (MFI). Numbers above each peak correspond to percentage
(%) of gated (M2) cells calculated using subjective gating. Fluorescence intensity (X-axis) is plotted as histograms on log scale (X-axis) using Flowjo
7.6.4 software (Tree Star Inc., Ashland OR). Y-axis depicts total events (cells) and expressed either as counts or % of maximal. P(AI-WAm/A549)=0.0038;
P(AI-WAm/RD)=0.39; P(A549/RD)=0.0018; P(A549/HEK293T)=0.018; CAR P(AI-WAm/HEK293)=0.0001; P(HEK293/RD)=0.001; E. Comparison of hCAR expression in AI-
WAm and control cells by IHC staining. CAR-specific (RmcB) primary antibody (same as used for FACS analysis, D) was used to stain AI-WAm cells.
A549 and HEK293-T cells were used as positive and RD cells as negative controls for CAR expression. All cells were counter-stained for 5 min. with
300 nM DAPI to visualize nuclear DNA (blue). No primary antibody was used with control samples. Negative control (RD) cells show efficient nuclear
staining but no discernible CAR staining (green), while A549 cells demonstrate a strong CAR-specific signal and AI-WAm cells display a moderate level
of hCAR signal with diffuse pattern of cytosolic localization (white arrows) similar to that in A549 cells. In sharp contrast, CAR-overexpressing HEK293-
T cells show a distinct localization of CAR protein in the cell membrane tight junctions (red arrows) with lesser cytosolic staining. Scale bars
correspond to 100 mm.
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Figure 3. Expression analyses of HSPG and integrin molecules as alternate receptors for fiber-modified Ads on AI-WAm cells.
Expression of avb3 and avb5 integrins (A and B) and heparin sulfate proteoglycans (C–E) in AI-WAm cell population and control cells was analyzed by
flow cytometry (A, C, E) and IHC staining (B and D). Cells were incubated with primary anti-avb3 or anti-avb5 monoclonal antibodies for detection of
corresponding integrin molecules or 10E4 antibody for detection of HSPG side chains (GAG) or anti-human syndecan 4 monoclonal antibody,
followed by Alexa 488-conjugated secondary antibody. A and C, top charts: AI-WAm cells; middle charts: RD cells; bottom charts: A549 cells. For AI-
WAm cells: P(avb3/avb5)=0.75; P(Synd4/HSPG)=0.29; For RD cells: P(Synd4/HSPG)=0.67; for avb3: P(RD/A549)=0.38; for avb5: P(AI-WAm/A549)=0.23; for syndecan 4:
P(RD/A549)=0.2; for all other differences P,0.05; E. HSPG Ab (10E4) specificity control sample: A549 cells were treated with heparitinase (10 U/ml) for
Adenovirus Gene Transfer to AI-Ameloblasts
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Importantly, AI-WAm cells transduced with capsid-modified
vectors, particularly Ad5-pK7/RGD, showed sustained expression
of GFP in culture during 4 weeks (data not shown).
Validity of our approach of reporter expression-based vector
dose normalization in A549 cells was supported by a direct
quantification of the internalized Ad5 genomic DNA (E4 copy
number) by qPCR in a parallel experiment using the same viral
doses as in the gene transfer experiments. As evidenced by Fig. 5D,
the relative levels of the reporter gene expression seen in AI-WAm
cells following infection with the fiber-modified vectors (Fig. 5B
and C) recapitulate the rates of Ad5 genomic DNA transfer
(estimated as E4 copy number) to those cells upon vector
To verify that the mechanism for the observed infectivity
enhancement is indeed mediated by binding of the modified Ads
to avb3/avb5 integrins and/or HSPG molecules on the surface of
1 hr at 37uC to remove GAG side chains. Green arrow shows shift of the fluorescence intensity peak resulting from reduction in cell labeling with 10E4
antibody (MFI decrease). Other details are as in Fig. 2D. B and D, scale bars correspond to: 100 mm in top image panels (integrins/AI-WAm, 106
objective), 10 mm (insert, 606objective) and 50 mm (406objective) in all other panels. Insert shows syndecan 4 staining image (606) of A549 cells,
clearly demonstrating a polarized intracellular localization of the protein.
Figure 4. Schematic representation of the Ad5 fiber proteins carrying intact and modified C-terminal knob domains. Fiber
modifications are indicated in the corresponding vector names: Ad5 (G/L) has unmodified fiber knob and possesses the native CAR tropism; Ad5-RGD
contains a peptide ligand with an ‘‘RGD motif’’ in the HI loop (red loop) of the fiber knob; Ad5-pK7 contains a stretch of seven lysine residues (green
oval) fused to the C-terminus of the Ad5 knob via a (GS)5linker (green hook); Ad5-pK7/RGD incorporates both modifications in the corresponding
locales of the same fiber molecule; Ad5/3 contains a chimera fiber with Ad5 fiber ‘‘knob’’ domain (gray) replaced with the Ad serotype 3 (Ad3) knob
(blue), which retargets the vector to Ad3 receptor(s).
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Figure 5. Augmentation of Ad5 gene transfer to AI-WAm cells by Ad5 vectors with various fiber modifications. A. Representative
fluorescent microscopy images of cells infected with the array of recombinant Ad5 vectors with genetically-modified fibers shown on Fig. 4. Relative
gene transfer efficiencies in the AI-WAm and A549 cells were assessed by fluorescent microscopy of GFP-expressing cells transduced with the array of
modified vectors 20 hrs post infection; (G/L) indicates the presence of two reporter genes (GFP and Luc) each under control of its own CMV promoter;
(G) denotes a single-reporter GFP cassette. Infection with Ad5 (G/L) was performed at an MOI of 50 TCID50/cell, while doses of other vectors were
adjusted by Luc expression to match that of Ad5 (G/L) in A549 cells (see Materials and Methods and Results). A 50 TCID50/cell infection dose was used
for Ad5/3 (G) vector for normalization in A549 cells as it could not be adjusted by Luc expression like the other (G/L) vectors. Fluorescent images were
Adenovirus Gene Transfer to AI-Ameloblasts
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AI-ameloblasts we carried out gene transfer blocking experiments
using recombinant integrins or heparin as competitors for the
respective targeted molecules on the surface of target cells. Pre-
incubation of modified Ad vectors with 1:1 mixture of purified
avb3 and avb5 integrin proteins resulted in a profound blocking of
AI-WAm gene transfer by Ad5-RGD (G/L) vector (17.6% of
control) in contrast to Ad5-pK7 (G/L) (71% of control).
Transduction by Ad5 RGD/pK7 (G/L) vector was also inhibited
by integrins, but to a lesser extent (48.1% of control), supporting
(Fig. 6A). Conversely, when the viruses were pre-incubated with
heparin as an HSPG competitor, the Ad5-pK7 G/L vector
demonstrated a dramatic dose-dependent reduction in gene
transfer, while neither Ad5-RGD (G/L) (Fig. 6B), nor Ad5/3 (L)
(data not shown) vectors showed any statistically significant
reduction in transduction of AI-WAm cells. The double-modified
vector Ad5-pK7/RGD (G/L) showed a significantly lower
sensitivity to gene transfer blocking with heparin, consistent with
involvement of additional interaction of the modified vector with
avb3/avb5 integrins via the RGD-4C ligand. Similar reciprocal
blocking effects were observed for transduction of RD cells with
the same panel of viruses (data not shown). Thus expression of
syndecan 4 and possibly other HSPGs on the surface of AI-
ameloblasts along with avb3/avb5 integrins allows circumventing
CAR deficiency of those cells by using an integrin-binding RGD
ligand in combination with a positively charged polylysine (pK7)
incorporated in the Ad5 fiber knob domain.
We have established for the first time a human ameloblast-like
cell population (AI-WAm) derived directly from an AI patient
primary EOE tissue. The AI-WAm cells express AMELX, ENAM,
AMBN, AMTN and ODAM genes at both mRNA and protein
levels, which is consistent with the gene expression profile known
for ameloblasts. Amel immunostaining showed cell polarization
and was more robust than Enam staining, in line with the relative
proportions of these proteins in the secretory ambeloblasts .
Since Amtn and ODAM are highly expressed in maturation- and
postmaturation-stage ameloblasts [7,8,48], the AI-WAm cells,
expressing relatively low levels of those gene transcripts (see
Fig. 1I), are likely to represent the early maturation stage
ameloblasts. Of note, both AMELX and ENAM genes have been
excluded from a causative role in the disease pathogenesis for this
AI patient (our laboratory unpublished data).
With this unique research tool in hand we next sought to
evaluate the utility of Ad-based vectors for gene delivery to AI-
ameloblasts as a potential strategy for gene replacement therapy of
Efficiency of gene transfer by an Ad5 vector depends mainly
upon the efficiency of the virus attachment to cellular receptors
[49–53] and viral internalization [34,35,41]. Our initial experi-
ments indicated that AI-WAm cells are about 23 times more
resistant to Ad5 infection than A549 cells (see Fig. 2A), which was
generally consistent with the lower levels of CAR expression
observed in AI-ameloblasts on both mRNA (see Fig. 2B and C)
and protein levels (see Fig. 2D and E). Although intracellular
localization of hCAR is typically confined to tight junctions at the
sites of intercellular contacts , the IHC staining for hCAR in
AI-WAm revealed mostly diffuse cytoplasmic distribution of this
protein (see Fig. 2E). Surprisingly, a similar hCAR staining pattern
was observed also for hCAR-positive A549 cells, whereas in
HEK293-T cells, used as another positive control in the same
experiments, the protein showed a distinct localization to tight
junctions (see Fig. 2E) in full agreement with the literature .
This difference between HEK293-T and A549 cells was most
likely due to a substantially higher expression of hCAR in
HEK293-T cells as suggested by a 15-fold higher expression of its
mRNA in these cells as compared to A549 cells (data not shown).
This agreed with the notion that A549 cells were more resistant to
transduction with unmodified Ad5 as compared to HEK293-T or
HEK293 cells (our unpublished observations). Other possible
reasons for the observed difference in hCAR staining patterns
between A549 and HEK293-T cells could be related to differences
in physiological state (confluence, passage number) or growing
conditions of those cells.
In addition to lower expression of hCAR in AI-WAm cells
relative to A549 or HEK293-T control cells evidenced by our RT-
qPCR and flow cytometry data, the IHC staining suggested
paucity in cell surface-localization of the CAR protein, which
could in part account for the observed resistance of AI-ameloblasts
to Ad5 transduction.
To overcome the intrinsic CAR deficiency of AI-WAm cells we
chose an approach of Ad5 infectivity enhancement using an array
of Ad5 vectors with various genetic modifications of the capsid
protein fiber that allow targeting of the vectors to alternate cell-
surface molecules. We hypothesized that incorporation of a small
cyclic peptide (RGD-4C) with affinity to cellular integrins (avb3
and avb5) [23,36,38,39] or/and heptalysine peptide (pK7),
targeting polysaccharide moieties of glycosylated cell surface
proteins (HSPGs) [23,43], could substantially increase Ad5
binding efficiency to cells of epithelial origin such as AI-WAm.
Expression of HSPG and avb3/avb5 integrins in AI-WAm and
RD cells was confirmed by IHC staining and flow cytometry
analysis (Fig. 3). The flow cytometry data were generally consistent
captured with 106objective and exposure time of 2 sec. for A549 cells and 400 msec. for AI-WAm cells. Each vector infection was carried out in
triplicates. Shown are images of representative samples; B. Luc assay of lysates prepared from A549, RD and AI-WAm cell samples (same as shown in
A) following vector dose normalization in A549 cells using 10 ml of each lysate (100 ml) for bioluminescence analysis. The data are presented as
percentage (%) of the Ad5 (G/L) gene transfer efficiency (total RLU) in each cell type taken as 100%; RLU - relative Luc units, (L) - a single firefly Luc
reporter expression cassette. Other details are as in Materials and Methods. C. The original Luc assay data of the AI-WAm samples (shown in A and B)
presented as RLU per cell. Two different MOIs of Ad5 (G/L) infection were used for dose normalization: 10 and 50 TCID50/cell to illustrate dose
dependence of the infectivity enhancement effects. All other details are as in B. D. Test for replication competent adenovirus (RCA) contamination in
modified Ad vector preparations by qPCR analysis of Ad genomic DNA. AI-WAm cells were infected with A549-normalized vectors at the doses
corresponding to MOI=50 of the Ad5 (G/L) (C) and harvested 6, 20 and 36 hrs post infection for total DNA isolation. Viral genomes were quantified
by qPCR and presented as E4 copy numbers normalized for total cellular DNA quantified by qPCR of the housekeeping (GAPDH) gene. No statistically
significant increase in internalized genomic DNA evidences the lack of RCA-induced genomic DNA replication at 20 hours post infection (time point
of Luc assay), suggesting that the reporter gene expression has not been affected for any vector. Color coding and other details are as in B and C.
Stars above bars with the corresponding colors indicate changes relative to the 6 hr time point values with no statistical significance. P values (.0.05)
are shown on the corresponding bars. Color brackets (with P values on the top) indicate value changes between 20 hr and 36 hr time points; black
brackets (with P values on the top) show sample differences of no statistical significance (P.0.05) within the same time point. All other differences
are statistically significant (P,0.05).
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with IHC staining of cells for HSPGs and integrins. The staining
patterns for both integrins (avb3 and avb5) were similar in all cell
types with both diffuse cytosolic distribution and cell membrane-
localized signals (Fig. 3B). In contrast, staining patterns for total
HSPGs based on detection of GAG moieties was very distinct from
that for syndecan 4, which showed a highly polarized localization
Figure 6. The infectivity enhancement effect of fiber-modified Ad5 vectors is mediated by avb3/avb5 integrins and/or HSPG
molecules on AI-ameloblasts. A. Differential blocking of gene transfer to AI-WAm cells by integrins. Ad5 RGD shows the highest sensitivity to
integrin blocking, while transduction with Ad5-pK7/RGD (G/L) is only partially inhibited. Ad5-pK7 (G/L) gene transfer shows no statistically significant
inhibition by integrins. B. Blocking of AI-WAm gene transfer by modified vectors with heparin. Heparin shows a profound dose-dependent blocking
effect on transduction with pK7-modified Ads, as opposed to RGD-modified vector. Gray bars (with % values on the top) show percentage of the
residual gene transfer level (RLU) resulting from blocking relative to that of unblocked controls (100%) shown by black bar for each fiber-modified
vector. All bars represent mean values with standard deviations. All differences were statistically significant except where indicated by asterisk and P
values (P.0.05) on the data bars.
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throughout all analyzed cell types (Fig. 3D), particularly
conspicuous in A549 cells (Fig. 3F). This observation was
consistent with specific intracellular localization of syndecan 4 to
complex cytoskeletal adhesion sites, i.e., focal adhesions . Of
note, expression of syndecan 4 in ameloblasts was higher than in
A549 cells relative to total HSPG in those cells (Fig. 3C and D).
Adequate comparison of Ad vectors with various tropisms in the
context of the established dental cell population was an important,
but challenging task due to the problem of proper dose
normalization of vectors carrying different tropism modifications.
Gene transfer assay with reporter gene expression as readout is
typically employed to compare transduction efficiencies of viral
vectors using either ‘‘physical’’ or ‘‘infectious’’ titers for vector dose
normalization. Vector dose normalization for gene transfer
experiments has been a subject of controversy in the field because
the level of transgene expression in infected cells may not be
reflective of the number of viral particles (VP) used for
transduction or infectious (pfu/TCID50) titer i.e. viral ability to
form infectious progeny and produce plaques on infected cell
monolayer. Moreover, Ad infectious titers are typically determined
in helper (HEK293 or 911) cell lines regardless of tropism of
analyzed vectors, which potentially leads to under- or overesti-
mation of vector infectivity in other cell types with different
repertoire of cell surface receptors.
To minimize potential errors in vector dose normalization due
to the aforementioned factors we sought to employ a different
approach based on empirical adjustment of reporter gene (Luc)
expression levels for each virus in CAR-positive A549 cells as an
equivalent of Ad5 infectious dose or ‘‘transgene expression dose’’.
We reasoned that the efficiency of vector-encoded transgene
expression in a cell line with high levels of the native Ad5 receptor
(CAR) represents an ‘‘equivalent’’ of gene transfer (cell binding
and internalization) capability of fiber-modified Ad5 vectors that
retain their natural ability to bind CAR (non CAR-ablated). Our
rationale was based on the assumption that in CAR-expressing
cells transduction by infectivity-enhanced vectors would occur
predominantly via the native cognate receptor (CAR) pathway
with relatively lesser contribution of other receptors provided small
ligand modifications of the fiber do not significantly compromise
CAR tropism of the vectors. On the contrary, in CAR-deficient or
CAR-negative cells lines the cell binding mechanisms mediated by
Ad5 fiber modifications would become predominant and deter-
mine transduction efficiency of the modified vectors.
The validity of our approach for viral dose normalization in
A549 cells has been supported by the results of direct
quantification of internalized genomic DNA (E4 copy number)
for each modified virus in A549 (data not shown) and AI-WAm
cells (see Fig. 5D). This analysis showed direct correlation between
the Luc expression readouts following vector dose normalization
and the actual Ad5 gene transfer levels assessed by intracellular
quantification of Ad5 genomic DNA using the E4 region-specific
qPCR (see Fig. 5D).
This study demonstrated that each tested fiber modification
drastically enhanced Ad5 infectivity in CAR-deficient cells such as
RD and AI-ameloblasts (see Fig. 5). The observed synergistic effect
of the two small ligand modifications in the Ad5-pK7/RGD (G/L)
vector was consistent with the original report  using RD as
CAR-negative cells for evaluation of pK7 and/or RGD modifi-
cation and suggested that pK7/RGD double modification
represents an optimal fiber modification also for gene transfer to
AI-ameloblasts at therapeutically relevant doses.
Our reciprocal blocking experiments with heparin or a mixture
of recombinant avb3 and avb5 integrins demonstrated that the
mechanism for the observed infectivity enhancement of Ad5-RGD
(G/L) and Ad5-pK7 (G/L) vectors indeed involves their binding to
avb3/avb5 integrins and HSPGs, respectively. Our experiments
also suggest transduction through both types of molecules for
double-modified Ad5-pK7/RGD (G/L) virus (Fig. 6). The same
mechanism apparently mediated augmentation of transduction of
control RD cells with negligible expression of CAR but substantial
expression of HSPGs, including syndecan 4, since transduction of
these cells was blocked by heparin with similar efficiency (data not
shown). Although our blocking data generally agreed with the
earlier study , we observed a substantially more robust
inhibition of gene transfer by both integrins and heparin (Fig. 6).
Despite higher doses of heparin used in our experiments, which
could account for stronger blocking effects (58-fold and 11.2-fold
for Ad5-pK7 and Ad5-pK7/RGD, respectively, versus 3.3-fold
and 1.5-fold inhibition reported for the respective vectors in RD
cells previously), it had no inhibitory effect on Ad5-RGD vector
transduction in AI-WAm cells (Fig. 6B), in contrast to the slight
effect in RD cells reported previously . The reason for this
discrepancy is unclear and could reflect subtle differences in the
cell-binding mechanism of the HSPG-targeted Ads in AI-WAm
versus RD cells.
Integrins avb3 and avb5 have been implicated in internalization
of the group C adenoviruses (Ad2/Ad5), which involves their
interaction with an RGD motif of the Ad capsid’s penton base [34–
38,41] following the fiber knob-mediated step of the viral
attachment to CAR [20,33,49–53]. The affinity of RGD interaction
with avb3 and avb5 integrins is relatively lower than that of the
fiber-CAR interaction, although it is essential for triggering
formation of endosomes as well as for Ad5 endosome escape. The
latter step requires avb5 (b5 cytolasmic tail) but not avb3 for
endosome membrane permeabilization critical for Ad5 entering the
cytoplasm. Of note, differential role of avb3 and avb5 has been
suggested more recently for transduction of RGD-modified Ad
(Ad5-RGD), implicating primarily avb3 in binding to linear RGD
peptide of the penton base as well as to the cyclic peptide RGD-4C
in the fiber knob . In this regard, robust expression of avb3 on
AI-WAm is consistent with strong augmentation of the Ad5-RGD
(G/L) vector transduction of those cells observed in this study,
whereas higher overall expression levels of HSPGs and integrins in
AI-ameloblasts relative to RD cells was consistent with more
efficient transduction of AI-WAm cell population with the RGD
and/or pK7-modified viruses.
Because both replicative and replication-deficient Ad5/3
vectors carrying a chimera fiber modification demonstrated
superior infectivity enhancement in different types of cancer cells
both in vitro and in vivo [57–62], it was of interest to evaluate
efficacy of this modification for transduction of non-cancer
epithelial cells such as AI-WAm relative to the small modification
ligands. However, Ad5/3 dose normalization to the panel of
CAR-binding vectors presented a problem due to ablated CAR
tropism of the virus resulting from the replacement of the entire
Ad5 fiber knob domain with that of the serotype 3 Ad (Ad3)
known to target a different set of cellular receptors [24,63]. Several
groups have identified the membrane cofactor CD46 as an
attachment receptor for human Ad group B serotypes, including
Ad11 , Ad35  and Ad3 . Furthermore, high-
throughput receptor screening approach identified HSPGs as
low-affinity Ad3 co-receptor interacting with its fiber knob domain
. It has thus been suggested that more than one receptor exists
for species B Ads [46,47,64–67]. It remains controversial whether
CD46 functions as attachment receptor for all species B serotypes.
Most recently, the major Ad3 receptor was identified with
desmoglein 2 (DSG2) , making the relevance of CD46 to
the mechanism of Ad3 transduction highly questionable.
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Our strategy of Ad5/3 infectious dose normalization to those of
the fiber-modified CAR-binding Ads was originally based on the
assumption that Ad5/3 and Ad5 possess similar abilities to
transduce A549 cells resulting from comparable levels of CAR and
Ad3 receptor(s). Due to the fact that this study was carried out
prior to the discovery of DSG2 as a primary Ad3 receptor, in this
study we analyzed expression of CD46 as a tentative Ad3 receptor
 on A549 cells and found comparable expression of this
molecule to CAR at least on mRNA level (Fig. 2C). We also found
high levels of CD46 and CAR proteins in A549 cells (Fig. 2D and
E), in line with earlier reports , but were unable to determine
the relative ratio of those molecules quantitatively using flow
cytometry approach since anti-CAR and anti-CD46 antibodies
could have different affinities and cell labeling with different
antibodies might not reflect the actual level of each receptor.
Furthermore, after DSG2 was identified as the major Ad3
receptor, the analysis of CD46 expression in A549 cells became
no longer relevant to the assessment of the bona fide Ad3 receptor
levels and justification of our Ad5/3 normalization approach. In
this regard, to validate our strategy for Ad5/3 normalization in
A549 cells we employed a novel quantitative approach developed
in a separate study to determining relative efficiency of A549 cell
transduction by Ad5 and Ad5/3 vectors. Briefly, we used Ad5 and
Ad5/3 viruses with EGFP-labeled capsids  as fluorescent tags
in flow cytometry assay to probe A549 cells for the corresponding
cognate receptors under conditions of receptor saturation and viral
internalization block (4uC). Considering that the viruses had
comparable infectious titers and capsid labeling efficiencies, the
difference in Mean Fluorescent Intensities (MFI) of the cells bound
to viral particles of each type (peak positions) relative to MFI of
unlabeled cells (background) was reflective of the difference in
A549 cell binding capacity of each vector (Ad5 or Ad5/3) under
receptor saturation conditions. The above-mentioned approach
revealed that A549 cells were capable of binding somewhat larger
number of Ad5/3 particles than Ad5 particles (MFI=109.4 versus
MFI=38.2, with 97% and 94% labeled cells, respectively). In light
of these findings transduction efficiency of Ad5/3 vector
(normalized to other vectors in A549 cells) observed for AI-
WAm cells is likely to be underestimated and could potentially be
higher than observed in our experiments (see Fig. 5). This could
account for the relatively lower gene transfer augmentation
demonstrated by the Ad5/3 vectors (L or G) in AI-ameloblasts
as compared to the other fiber-modified vectors (Fig. 5B and C).
Although expression of DSG2 in pre-ameloblasts was evidenced
by gene expression arrays (our unpublished observations),
expression of this protein in AI-WAm cell population may be
down-regulated relative to HSPG and integrins, which could also
account for the relatively lower augmentation of their transduction
by the chimera Ad5/3 vectors.
This initial study thus defines an optimal gene delivery strategy
to ameloblast-like cell population derived from a human patient
with AI. Extrapolating in vitro results to clinical situation is a
common challenge of the gene therapy field, although some
general principles that have been derived from studies in cell lines
have been supported by observations from clinical trials . Ad-
mediated gene transfer is dose-dependent, but increasing Ad doses
inevitably leads to enhanced host immune response to Ad vectors
and systemic toxicities. Therefore, the superior efficiency of gene
transfer to AI-ameloblast cell population demonstrated by some
capsid-modified vectors in this study along with the observed
longevity of transgene expression (up to 4 weeks) in the target cells
offers a potential utility of fiber-modified Ad5 vectors for local
administration in dental tissues.
Several reasons warrant hope for Ad gene therapy applications
in dentistry. Often, the typically limited duration of Ad5-delivered
transgene expression in vivo [70–72] is a crucial obstacle, but in
developing teeth relatively short-term changes (weeks or months)
in matrix protein expression can determine the properties of the
mineralized tissues. Accordingly, a localized Ad-mediated gene
delivery strategy, rescuing essential components of the developing
tooth in a temporal fashion, could restore the complex
choreography of mineralized matrix formation during a critical
time window. Moreover, localized administration of an Ad5 vector
allows minimization of its clearance by pre-existing anti-Ad 5
antibodies [71,72]. Successful induction of bone formation by
bone morphogenic protein (BMP), delivered via an Ad gene
therapy vector by its localized microsurgical infusion ,
supports feasibility of Ad gene therapy applications also for
morphogenesis of dental tissues during permanent tooth forma-
tion. These perspectives warrant further evaluation of the Ad5-
pK7/RGD as a potential AI-gene therapy vector in a suitable
animal model of amelogenesis as a logical next step in the above
We thank Martha Galindo, Jennifer Schulze McDaniel and Yixin Wu for
their technical assistance, Dr. J. Michael Mathis (Louisiana State
University Health Science Center, Shreveport LA) for kindly providing
the CD46/isotype control antibodies, Dr. Ralph Sanderson for kindly
providing syndecan 4 and 10E4 antibodies as well as Heparitinase and Dr.
David T. Curiel for providing the fiber-modified vectors.
Conceived and designed the experiments: AVB MJP JD OAM M.
MacDougall. Performed the experiments: AVB MJP JD OAM CR EL EK
SC M. MacDougall. Analyzed the data: AVB OAM JD MJP M.
MacDougall. Contributed reagents/materials/analysis tools: M. Murakami
HW EK OAM SC M. MacDougall. Wrote the paper: AVB MJP OAM JD
M. MacDougall. Obtaining informed consent from the patient/parent: JD
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