Development of Renal-targeted Vectors Through Combined In Vivo Phage Display and Capsid Engineering of Adenoviral Fibers From Serotype 19p 1 British Heart Foundation Glasgow Cardiovascu
Trang 1Development of Renal-targeted Vectors Through
Combined In Vivo Phage Display and Capsid
Engineering of Adenoviral Fibers From Serotype 19p
1 British Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, UK; 2 Department of Immunology,
Scripps Research Institute, La Jolla, California, USA; 3 Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA;
4 Haemostasis and Thrombosis, Medical Research Council Clinical Sciences Centre, Imperial College London, London, UK
The potential efficacy of gene delivery is dictated by the
infectivity profile of existing vectors, which is often
restric-tive In order to target cells and organs for which no
effi-cient vector is currently available, a promising approach
would be to engineer vectors with novel transduction
profiles Applications that involve injecting adenovirus
(Ad) vectors into the bloodstream require that native
tropism for the liver be removed, and that targeting
moieties be engineered into the capsid We previously
reported that pseudotyping the Ad serotype 5 fiber for
that of Ad19p results in reduced hepatic transduction In
this study we show that this may be caused, at least in
part, by a reduction in the capacity of the Ad19p-based
virus to bind blood coagulation factors It is therefore
a potential candidate for vector retargeting, focusing
on the kidney as a therapeutic target We used in vivo
phage display in rats, and identified peptides HTTHREP
and HITSLLS that homed to the kidneys following
intra-venous injection We engineered the HI loop of Ad19p to
accommodate peptide insertions and clones Intravenous
delivery of each peptide-modified virus resulted in
selec-tive renal targeting, with HTTHREP and HITSLLS-targeted
viruses selectively transducing tubular epithelium and
glomeruli, respectively Our study has important
implica-tions for the use of genetic engineering of Ad fibers to
produce targeted gene delivery vectors
Received 15 December 2006; accepted 25 April 2007; published online
5 June 2007 doi: 10.1038/sj.mt.6300214
IntroductIon
Gene delivery is limited by the ability of available vector systems
(either viral or non-viral) to deliver sufficient levels of therapeutic
transgenes to target cells and tissues in vivo to provide an
effica-cious phenotype For strategies based on the commonly used
delivery that is restricted only to defined cell types such as liver
hepatocytes Access to local tissue can enable tissue transduction
to be carried out, but access to therapeutically relevant targets is often impossible or impractical In this context, there is a need to develop targeting strategies to enable gene delivery to these sites
in vivo Such strategies have evolved rapidly in recent years and
include utilization of alternate vector serotypes as well as meth-odologies to alter the infectivity of existing vectors, including peptides and antibody targeting systems In many of these strate-gies, however, the required route of delivery is via the bloodstream with “homing” of the vector to the target tissue Alternative sero-types of some commonly used vectors have been isolated, and many possess alternative tropism on account of differences in cell tethering and entry mechanisms, thereby enabling targeting
to defined tissues For example, adeno-associated virus serotypes
6 (AAV-6) and 9 (AAV-9) show significant levels of delivery of genes to skeletal and cardiac muscle following intravenous injec-tion, when compared with AAV-2, thereby potentially accelerat-ing the development of gene therapeutics for skeletal and cardiac
number of alternate serotypes are available, altered cell infectivity can be achieved For example, Ad vectors engineered with many subgroup B fibers target CD46, thereby altering infection at the
via the bloodstream The fiber protein exposed on the surface of the adenoviral capsid is the main determinant of tropism, and has been the predominant target of retargeting strategies (reviewed in Ref 9) In the case of Ad5, extensive mutagenesis of the fiber has resulted in reduced liver transgene expression but has not enabled
that there are still some uncertainties about the extent to which
receptors in the liver are utilized by Ad5 vectors in vivo Recently
there have been reports of the important role played by blood serum proteins, including coagulation factors IX (FIX) and X (FX), in driving hepatic delivery of Ad5 through heparan sulphate proteoglycans (HSPGs) and/or low-density lipoprotein
Ad vectors based on serotype 5 will likely require elimination of coagulation factor binding Because coagulation factor binding
Correspondence: Andrew H Baker, British Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow, 126 University Place,
Glasgow G12 8TA, UK E-mail: ab11f@clinmed.gla.ac.uk
Trang 2is probably mediated through the fiber,13 an alternative approach
would be to identify serotypes that show reduced coagulation
factor binding We previously reported that the fiber of serotype
19p, a subgroup D virus not associated with any known human
disease, exhibits severely reduced liver tropism when delivered
intravenously into rats, and this was in the absence of enhanced
potential platform on which to build a targeting system for
deliv-ery of genes to non-hepatic tissue
poten-tial for in vivo targeting of Ad following intravascular injection,
and appropriate targeting sequences can be identified by
tech-niques including in vivo phage display The flexibility of phage
display allows entire peptide repertoires to be screened in vitro,
ex vivo, or in vivo in order to isolate highly efficient and
that it is possible to identify small peptide motifs that target the
vasculature of defined tissues and organs following intravenous
tech-nology to the delivery of bioactive therapeutics, using peptides
isolated by phage display, has potential clinical utility, and this
has already been proven in a limited range of applications For
example, intravenous administration of peptides isolated for
spe-cifically targeting tumors or white fat, when coupled to drugs and
pro-apoptotic peptides, resulted in tumor regression or fat
and selective in vivo gene delivery by viral vector systems has been
somewhat slower to emerge, because this involves genetic
engi-neering of complex viral capsid proteins We have therefore
uti-lized Ad5 pseudotyped with the Ad19p fiber in order to develop
targeted viral gene therapy vectors by intravenous administration,
by combining in vivo phage display with the genetic engineering
of the HI loop of Ad serotype 19p fibers We have focused on the
kidney as a therapeutic target because it is fundamentally
impor-tant in a range of diseases and there is a clear absence of suitable
vector systems for non-invasive targeting
results
Binding of coagulation factors and resulting
cell infectivity is reduced with Ad19p as
compared to Ad5
We previously reported that replacing the Ad serotype 5 fiber with
has been found that an important mechanism for hepatic
seques-tration of Ad vectors is the binding of blood coagulation factors,
such as FIX and FX, to the capsid so as to “bridge” the virus to
HSPGs and/or low density lipoprotein receptor-related protein in
concen-trations of FIX or FX exerted any influence on Ad19p-mediated
transduction, and compared the results with Ad5 (using a
wild-type fiber, Ad5 and a coxsackievirus and Ad receptor
by both FIX and FX Ad19p-mediated transduction was also
significantly enhanced by FIX and FX, but to substantially lower
16
a
b
c
d
6 ) 14 12 10 8 6 4 2
160
3 )/mg protei
n 140 120 100 80 60 40 20 0 Ad5 Virus injection
�RU
�RU start
Time (s)
end
Ad5
Ad5
Ad19p-Eco47
Ad19p-Eco47
Ad19p-Eco47
+FIX +FX
CHO-KI CHO-pgsA745 Ad19p + FIX + FX + FIX + FX + FIX + FX
− +FIX +FX
−
+FIX +FX
− +FIX +FX
−
100 75 50 25 0 0.00 0.25 0.50 Virus titre (× 1011) 0.75 1.00 1.25
Figure 1 Assessment of coagulation factor binding by modified viruses (a) HepG2 cells were distributed in the plates 24 hours prior
to infection Plated cells were washed in phosphate-buffered saline (PBS) and 50 µl of serum free media with physiological levels (1 IU/ml)
of either factor IX (FIX) or FX added This was followed by infection with the appropriate Ad at 10,000 virus particles (VP)/cell Cells were incubated for 3 hours at 37 °C before being washed and the media replaced Cells were incubated for 72 hours before quantification of
transgene expression *P < 0.05 versus transduction in the absence
of clotting factors (b) CHO-K1 (wild-type) and CHO-pgsA745
the effects and mechanisms of coagulation factors on Ad19p-modi-fied vector transduction Plated cells were washed in PBS and 50 µl of serum free media with physiological levels (1 IU/ml) of either FIX or
FX added This was followed by infection with 20,000 VP/cell of the appropriate Ad Cells were incubated for 3 hours at 37 °C before being washed and the media replaced Cells were incubated for 48 hours
before quantification of transgene expression *P < 0.05 versus
trans-duction in the absence of clotting factors (c, d) Ad5 or Ad19p vectors
immo-bilised onto a CM-5 sensor chip in 50 mmol/l Tris pH 7.4; 150 mmol/l
25 °C Depicted are sensorgrams of c FX (showing specific binding) of
Ad5 and, to a lesser extent, Ad19p-Eco47 (d) The steady state change
(δRU) in respiratory unit (RU) on binding of the virus to the FX was
plotted against virus concentration CHO, Chinese hamster ovary; RLU, relative light units.
Trang 3levels than with AdKO1 and Ad5 (Figure 1a) This suggests that
Ad19p may show reduced binding to coagulation factors To
investigate this further we utilized two approaches, one
surface plasmon resonance (SPR) First, wild-type CHO-K1 cells
were transduced with Ad5 and Ad19p in the presence or absence
of FIX and FX Basal levels of transduction for both Ad5 and
Ad19p were low, likely reflective of lack of primary receptor
an effect that was ablated in CHO-pgsA745 cells, thereby
demon-strating that coagulation factor-mediated cell transduction relies
showed enhanced transduction in CHO-K1 cells but, in
in CHO-pgsA745, thereby demonstrating that binding of the
Ad19p:FX complex to the cell surface occurs through HSPG (Figure 1b) Next, in order to examine the direct interaction of the Ad5 and Ad19p viruses with FX, we carried out SPR analy-sis FX was covalently coupled to biosensor chips and the virus (at varying concentrations) was injected Both viruses showed specific binding to FX, but there was a substantially weaker
interac-tion was calcium-dependent, consistent with the characteristics
Ad19p showed substantially reduced binding to FX in
that viruses with fibers derived from Ad19p show lower levels of binding and sensitivity to the coagulation factor pathway than Ad5 does This, at least in part, may be the underlying cause of
suggests that Ad19p fibers have a potential role to play in vector targeting strategies with intravascular injection routes
Identification of renal homing peptides by in vivo
phage display
Using Wistar Kyoto (WKY) rats, we carried out three rounds of
in vivo phage display to identify peptides that had the ability to
Twelve- to thirteen-week-old WKY rats (n = 3 rats/phage display
were recovered, titered and normalized per gram of tissue By the third round, we observed selective enrichment of the phage display library for the kidney in comparison with non-target
analy-sis of phage in the kidneys (>200 individual phage for rounds 2
found in all tissues This highlights the fact that phage display has the potential to identify peptides on the basis of either ubiquitous
or tissue-selective markers From the sequencing analysis, the peptides HTTHREP (HTT) and HITSLLS (HIT) were selected for the experiments, as they were found to be exclusively selective
In order to assess the kidney targeting capacity of each pep-tide, we injected animals with a high titer stock of each individual phage We utilized a pre-dosing regimen wherein animals were injected with control (not expressing a peptide) phage 5 minutes prior to injection of the candidate peptide-expressing phage (Figure 3a), as we have described previously.25 Pre-dosing was
Phage IV infusion
5 min circulation,
saline perfusion
9
8 7 6 5 4 3 2
Kidney
1st RD 2nd RD 3rd RD
Lung Heart Brain
1
Kidney retrieval
Phage recovery
and amplification
Recycle
Sequence Titer restricted
libary
Figure 2 In vivo phage display (a) Schematic representation of in vivo
phage display in the rat, so as to identify renal targeting peptides
Recov-ered and amplified phage from the kidneys 5 minutes after injection were
subjected to a total of three rounds of in vivo phage display, followed by
sequence analysis after round 2 and 3 (b) Phage recoveries over
sequen-tial rounds of phage display *indicates P < 0.05, NS, nonsignificant
IV, intravenous.
table 1 consensus sequences identified for the kidney following three
rounds of in vivo phage display
linear 7mer library n/total phage sequenced round
A total of 214 phage were sequenced.
table 2 Phage isolated from non-target organs were sequenced
following the third round of in vivo phage display
APASLYN (4.88%) APASLYN (2.27%) APASLYN (2.27%) HAAIHIS (4.88%) LPKNWSS (4.55%) VLTAGPW (7.695) YLQAPVH (4.88%) LVSQPHP (4.55%)
A total of 111 phage were sequenced.
found only in one organ, while others such as APASLYN were the encoded peptides It was clear that there were some peptides
Trang 4carried out in order to pre-saturate non-specific phage binding
sites in the liver, so as to analyze peptide-mediated targeting
with-out the confounding factor of reticulo-endothelial entrapment
of the phage particle The biodistribution of each phage
elevated phage accumulation for HIT and HTT in the kidney as
compared to the control phage, with no significant changes seen
in the liver When the recovery of either HITSLLS, HTTHREP or
control phage from the kidney was expressed as a percentage of
the liver, it was approximately tenfold higher in HITSLLS infused
animals and approximately 100-fold higher in HTTHREP infused
In order to produce targeted gene therapy vectors based on
Ad19p, we genetically modified the fiber gene from Ad19p to
accommodate peptide insertions, and inserted oligonucleotides
encoding each peptide individually into the HI loop (a site that
Alignment of the Ad5 and 19p fiber nucleotide sequences
showed that the HI loop of 19p was considerably smaller than
Ad5 and did not contain a suitable restriction site for
inser-tion of oligonucleotides encoding each targeting peptide We
used a polymerase chain reaction (PCR) strategy (see Materials
site between amino acids 331 and 332 in order to facilitate the
insertion of oligonucleotides for renal-targeting peptides (see
Materials and Methods) The resulting virus (Ad19p-Eco47)
(Figure 4b) was compared with parental Ad19p (non-modified
fiber) to confirm there were no differences in in vitro infectivity
(Figure 4c) Subsequently we cloned oligonucleotides encoding
each candidate renal targeting peptide into the Ad19p-Eco47
fiber-expressing plasmid and produced peptide-modified
those observed with non-modified fibers, as assessed by Western
Having derived all peptides by in vivo phage display, and
given that the critical aspect of targeting is expression of the
encoded transgene in vivo, we proceeded to inject rats with each
virus We killed animals 5 days after injection of each virus (and
Ad5 as a further control) to determine the extent and selectiv-ity of renal targeting Immunohistochemical analysis of kidneys
peptide-modified vectors showed readily detectable levels of transgene
in the kidneys, to a significantly higher level than that seen in
differ-ent peptide-modified vectors produced differdiffer-ent cellular distri-bution patterns in the kidneys Ad19p-HIT showed extensive
2
−3 )
NS
NS NS
10 8
10 7
10 6
10 5
10 4
10 3
10 2
10 1
3
1
NS NS NS
7 × 10 10
7 × 10 11
2 × 10 11
Control
12 10
8 6 4
2 0.17 Control HIT HTT 1.17
10.84 HIT
HTT
Kidney Phage reco Kidney
Lung Heart
Figure 3 Analysis of selected peptide-expressing phage (a)
Demon-stration of saturation kinetics of M13 phage in the liver, kidney, lung,
and heart at increasing doses *indicates P < 0.05 versus control phage
(b) Phage recoveries from kidney and liver (plaque forming units (PFU)/g
control phage to saturate non-specific binding *P < 0.05 versus control
phage (c) Phage recoveries expressed as a percentage of phage in the
liver Individual values shown above each bar NS, non significant.
Ad19p
-Eco47 HTT HIT
Ad19p parental
5
5 10 15
20 20
20 25 30 35
3 )/mg protein 40
30 35 50 kd
Ad19p Ad19p-Eco47
Ad19p-Eco47
Ad19p
c a
e d
b
HI loop
Figure 4 engineering of Ad19p fibers for targeting peptide insertion
(a) Schematic illustrating the protocol followed for producing the modi-fied vectors (refer to Materials and Methods) (b) Model of the predicted
structure of the Ad19p fiber with and without insertion of the restriction
site to allow cloning (c) In vitro comparison of Ad19p and Ad19p-Eco47
Rat glomerular endothelial cells were infected with increasing doses of either virus for 3 hours at 37 ºC They were then washed and the media was replaced Seventy-two hours after infection, cells were harvested and
β-galactosidase was measured and normalized to protein (d) Model of
the predicted structure of the Ad19p fiber with each peptide inserted into
the HI loop (e) Western blot of fiber monomer Ten µg of viral protein
was loaded, and the membrane was probed with the anti-fiber antibody 4D2 (Neomarkers, Fremont, CA) at 1:1,000 Ad, adenovirus; PCR, poly-merase chain reaction; RLU, relative light units.
Trang 5localization within the glomerulus with little or no staining in
and selective staining within the epithelial cells of the tubules
(Figure 5) Immunohistochemical analysis revealed no transgene
Further analysis of non-target organs including spleen, heart
(Figure 6a), lungs, and brain (not shown) also failed to detect
transgene expression In our previous study on Ad19p liver
targeting following intravascular injection, we used a
pre-dos-ing regimen to assess hepatocyte transduction in the absence
assessed targeting of Ad5, Ad19p-Eco47, and both
peptide-modified vectors Levels of β-galactosidase-positive hepatocytes
were: for Ad5, (83 ± 1%); for Eco47, (9 ± 1%); for
is, liver targeting of modified Ad19p vectors was not altered as
compared to Ad19p control virus, whether in the presence or
absence of Kupffer cell clearance Hence, each peptide-modified
vector produced efficient and selective transgene expression in
the kidneys following intravenous administration
Finally, we harvested organs 1 hour after injection of the same
dose of virus in order to assess early virion accumulation in the
liver and kidneys As expected, virion levels in the liver were
relatively high but essentially equivalent for Eco47,
Ad19p-HIT and Ad19p-HTT, likely as a result of profound Kupffer cell interactions However, although levels in the kidney at 1 hour after injection were far lower than in the liver, significant increases were nevertheless observed with both peptide-modified Ad19p vectors (Figure 7) This highlights the potential of the engineered Ad19p
Low power
Ad5
Ad19p-Eco47
Ad19p-HIT
Ad19p-HTT
Ad19p-HIT
Ad19p-HTT
Ad19p-Eco47
Ad5
High power
60
50
40
30
10
0
PBS Ad5 Ad19p-Eco47Ad19p-HIT Ad19p-HTT
a
b
Figure 5 Analysis of kidney targeting in vivo Eight-week old male
of each modified vector or phosphate-buffered saline (PBS) and killed
5 days later (a) Immunohistochemistry performed in kidney sections
Black staining indicates β-galactosidase activity Representative sections
are shown (n = 6 rats per group) Scale bar = 100 µm (b) Quantitative
analysis of transgene-expressing cells *P < 0.05 versus
Ad19p-Eco47-injected rats and Ad5-Ad19p-Eco47-injected rats NS, not significant Ad, adenovirus.
Liver
a
b
Spleen
Heart
90 80 70 60 50
40 30 20 10 Ad5 Ad19p-Eco47 Ad19p-HIT Ad19p-HTT
Figure 6 Analysis of non-renal tissue for reporter gene activity
virus particles per rat of each modified vector or phosphate-buffered saline and killed 5 days later Immunohistochemistry was performed on
the liver, spleen, and heart Black staining indicates β-galactosidase activ-ity Representative sections are shown (n = 6 rats per group) Scale bar =
100 µm (b) Quantitative analysis of transgene-expressing cells from liver
sections of animals infused using a pre-dose strategy Ad, adenovirus.
10,000
1,000
100
10
0.1 Kidney
Liver
Ad19p Eco47
Ad19p HIT Ad19p HTT
1
Figure 7 Assessment of early particle delivery to liver and kidney
particles per rat of each modified vector or phosphate-buffered saline and killed following perfusion at 1 hour after injection Virion quanti-fication was carried out by means of Taqman Data Analysis software (Applied Biosystems), using SYBR green DNA was extracted from the
kidney and 200 ng total DNA was amplified using LacZ primers, and the products were quantified using Taqman *P < 0.05 versus Ad19p-Eco47
Ad, adenovirus.
Trang 6system for mediating selective kidney transduction even in the
presence of rapid Kupffer cell uptake It also illustrates that
strate-gies to ablate the Kupffer cell interaction of Ad19p-based vectors
may further enhance targeting potential
dIscussIon
In this study we rationally combined in vivo phage display with
engineering of the Ad19p fiber to accommodate incorporated
peptides within the constraints of the fiber’s minimal HI loop We
showed that Ad19p-based vectors have lower coagulation factor
binding and sensitivity to this pathway when compared with Ad5,
thus contributing to the reduced hepatic tropism of the vectors
Furthermore, we developed in vivo phage display to identify renal
targeting peptides and demonstrated the incorporation of these
peptides into Ad19p pseudotyped vectors In vivo we showed
selective targeting to the kidney Of particular importance is the
finding that specific cell types in the kidney are targeted by
differ-ent peptides, because this suggests a broadly applicable approach
to targeting not only selected organs but also individual cell types
within each organ
Retargeting of Ad vectors for selective gene therapy via the
intravenous route has been widely attempted but has been
ham-pered by a number of issues including complex interplay in vivo
may contribute to the tropism of Ad and also to the host innate
immune responses Although a variety of capsid modifications
and peptide insertions have been utilized to generate convincing
data showing enhanced and/or selective gene delivery in vitro or
convinc-ingly retargeted genetically-engineered Ads following
intrave-nous injection We used in vitro cell gene transfer experiments
and SPR to assess direct FX:virus binding as well as to show that
viruses based on Ad19p showed reduced sensitivity to
coagula-tion factor binding when compared with Ad5 The binding of FIX
view of the fact that Ad5 infection has been shown to be heparin
sensitive, the KKTK motif in the fiber shaft has been suggested
mutation of the KKTK motif reduces liver transduction of Ad5
in vivo.10–12 However, an Ad5 vector with a mutated KKTK motif
binds coagulation FX as effectively as does non-modified virus,
thereby demonstrating that this is not the site of direct
to clarify and interpret the lowered binding capacity observed
with Ad19p fibers In parallel with this, we have recently shown
that many other Ad5-based vectors with fibers from subgroup
of cells in vitro In future studies, therefore, it will be critically
important to ascertain affinity constants for different
interac-tions between Ad vectors with alternative fibers
Although Ad5 viruses have been targeted using different
sites within capsid proteins, including the fiber HI loop, penton,
HI loop, because SWISS-MODEL analysis suggested that peptide
exposure at the fiber surface would be optimal Although the HI
loop of Ad19 is much smaller than that of Ad5, we were able to
document efficient virus production with each of the peptide-modified viruses created The incorporation of relatively small peptides into this site was shown not to hinder virus assembly propagation The principal thrust of our strategy was to modify a virus fiber that naturally shows poor liver tropism and combine this with peptide engineering Depending on the extent of modi-fication in the Ad19p fiber HI loop that can be tolerated without hindering virus production, it may be possible to use engineered peptides in tandem or, indeed, direct larger targeting peptides into this locale However, targeting with 7-mer peptides isolated
by phage display has been shown to be efficient for renal gene delivery It may be possible to improve targeting even further by strategies to block Kupffer cell interactions This would (poten-tially) enhance the bioavailability of peptide-modified Ad for the kidney While the receptor(s) for Ad19p remains unknown, we have shown previously that the Ad19p fiber supports the
cells (data not shown), thereby suggesting that the receptor(s) utilized by Ad19p is present on rat tissue This allows the sys-tematic analysis of vector retargeting strategies under conditions
in which the primary receptor for the parental fiber is present Ad19p can also infect human saphenous vein endothelial cells
would suggest that the primary receptor targeted by Ad19p has a similar pattern of expression in rats and humans, although fur-ther work is required to elucidate these similarities fully
To date, renal gene therapy has been achieved by applying the
using an alternative, easily accessible tissue or organ such as liver,
useful for analyzing individual transgenes in pre-clinical settings, its use is limited to secreted transgene products only The vectors
we used in our study will allow high-level transgene expression locally, but via the non-invasive intravenous route Additionally, strategies for targeting individual cells within the kidney can
be adopted, with specific peptide-modified vectors to target the glomerulus or tubular epithelium, as required Furthermore, these vectors are especially important in studies on rats, and represent unique molecular tools, given that transgenic strategies are by no means routine
In the process of our study, with all aspects taken together, we have rationally designed a new generation of adenoviral vectors that achieve efficient transgene expression in the kidneys follow-ing intravenous delivery We further demonstrate targetfollow-ing of defined cell types within this organ This has important implica-tions for the future design of genetically engineered Ad vectors
as well as for the development of renal gene therapy, an area of research hitherto hampered by a lack of suitable vectors
MAterIAls And MetHods
All in vivo work was carried out under the UK Home Office regulation
Animals (Scientific Procedures) Act 1986.
Virus preparation and purification Stocks of recombinant pseudotyped Ads expressing the modified fibers were generated by transfection of 293T cells (American Type Culture Collection, Manassas, VA) with the modi-fied plasmids followed by superinfection with a fiber-deleted Ad5 vector 44,45 Briefly, 293T cells were transfected with the appropriate fiber-expressing
Trang 7plasmid Sixteen hours later cells were superinfected with an E1, E3,
fiber-deleted rAd5 (Ad5ΔF) (β-galactosidase) at 2,000 virus particles (VP)/cell
Virus particles were purified by CsCl ultracentrifugation and dialyzed into
10 mmol/l Tris (pH 8.1), 150 mmol/l NaCl and 10% glycerol Virus particles
were quantified by protein assay against bovine serum albumin standards
according to the conversion: 1 μg protein = 4 × 10 9 VP 44 Fiber expression was
checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis and
Western blot assay using monoclonal anti-fiber antibody 4D2 (Neomarkers,
Fremont, CA).
were distributed in a 96-well plate 24 hours prior to infection, incubated
with serum-free media containing 20,000 VP/cell and 1 IU/ml of either
human Factor IX or human Factor X (Haemotological Technologies,
VT), left for 3 hours at 37 °C, washed, and maintained until harvesting
Forty-eight hours after infection β-galactosidase activity was quantified
by plate assay using a Wallac Victor luminometer and recombinant
β-galactosidase as a standard A bicinchoninic acid assay on the cell lysates
was performed to determine protein concentration, and the results were
expressed as relative light units/mg of protein Similarily, for transduction
experiments, cells were distributed in a 96-well plate 24 hours prior to
infection, incubated with fresh media containing the required multiplicity
of infection (as indicated), left for 3 hours, washed, and maintained until
harvesting Seventy-two hours after infection β-galactosidase activity was
quantified.
SPR SPR experiments were carried out with a Biacore X instrument
(Biacore, Stevenage, UK) Blood coagulation FX was immobilized onto a
CM5 biosensor chip according to the manufacturer’s instructions (1,926
respiratory unit of FX was immobilized) Virus in 50 mmol/l Tris pH 7.4;
150 mmol/l NaCl; 5 mmol/l CaCl2; 0.005% Tween 20 was passed over the
chip at a flow rate of 20 μl/min Sensor chips were regenerated by injection
of 10 mmol/l HEPES pH 7.4; 150 mmol/l NaCl; 3 mmol/l EDTA; 0.005%
Tween 20.
purchased from New England Biolabs (Hertfordshire, UK) Phage display
was carried out as previously described 46 Briefly, 12–13-week-old WKY
rats (n = 3 rats/phage display round) were anesthetized (halothane/O2
mix-ture) For round 1, 2 × 10 11 PFU of PhD 7 library were infused into the
fem-oral vein, and for rounds ×2 and 3, 2 × 10 11 PFU of amplified phage from
the kidney were infused (Figure 2) Five minutes after infusion the animals
were perfused through the heart at physiological pressure (120 mm Hg),
and the organs were removed and snap frozen Phage were recovered and
titered and normalized/gram of tissue.
Peptide sequencing Peptide-encoding DNA of phage were sequenced after
rounds 2 and 3 Up to 96 individual plaques/rat were selected and
ampli-fied PCR was utilized for amplifying the region of the peptide insertion
using sense GCA ATT CCT TTA GTG GTA CC-3′ and antisense
5′-CCC TCA TAG TTA GCG TAA-3′ primers PCR products were sequenced
using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems,
Warrington, UK) and analyzed on an ABI 377 automated sequencer.
Analysis of selected peptide-expressing phage Animals were infused with 2
× 10 11 PFU/rat of a control M13 phage lacking the LacZ α-complementation
Control phage was administered to block non-specific binding Five
min-utes after infusion 2 × 10 10 PFU of each individual phage (HITSLLS and
HTTHREP) were infused Five minutes after infusion animals were
per-fused through the heart at physiological pressure (120 mm Hg), and organs
removed and snap frozen 25 Phage in target and non-target organs were
recovered and titered and normalized/gram of tissue.
Generation of peptide-modified Ad19p fibers The Ad19p fiber
comple-mentary DNA in the plasmid pDV145 was constructed as previously
described 15 The HI loop fragment of Ad19p was excised on the Eco065I
and XbaI sites The HI loop was modified using primers to insert a unique cloning site Eco47III Using PCR, overlapping fragments were amplified
using two sets of primers The initial set of primers (i) 5′-GGA GTC GCG CAG CTT GTT GAC CAG CTC GGC-3′ and (ii) 5′-CCA ACT AAA GTC AAA TGT GAT AGA GTA TTC ACA TCC AGC GCT AGT TTC TTG
GTT AAA GG-3′ (Eco47III site underlined) amplified a 1,316-basepair
fragment containing the HI loop Similarly, a second set of primers, (iii) 5′-C CAA GAA ACT AGC GCT GGA TGT GAA TAC TCT ATC ACA TTT GAC TTT AGT TGG-3′, and (iv) 5′-GGC TGG CAA CTA GAA GGC
ACA GTC GAG GCT GAT CAG C-3′ (Eco47III site underlined) amplified
a 190-basepair fragment The two fragments were then subjected to PCR using the flanking primers 1 and 4 to produce a HI loop fragment (1,435
basepairs) with an additional Eco47III site to create a HI loop sequence
of NQETSAGCE (inserted amino acids underlined) The mutated HI
loop fragment was excised using the Eco065I and XbaI sites and ligated into pDV145 to create pDV145-Eco47III Oligonucleotides encoding the
selected peptides HTTHREP and HITSLLS were purchased from MWG
(Milton Keynes, UK) and ligated into Eco47III-digested pDV145-Eco47III
and sequenced to ensure correct orientation The plasmids pDV145-HITSLLS and pDV145-HTTHREP were produced and used for generating Ad19p-HIT and Ad19p-HTT viruses, respectively.
Peptide:fiber modelling Ad19p fiber knob protein sequences with selected insertions in the HI loop were submitted to SWISS-MODEL (http://swissmodel.expasy.org/) and modeled using ExPDB templates derived from 1uxb.pdb, 47 a crystal structure of Ad19p fiber knob in com-plex with sialic acid The bound sialic acid does not change the conforma-tion of the fiber knob 47 and was therefore ignored during modeling The models generated were visualized through Swiss-PdbViewer (http://www expasy.org/spdbv/).
In vivo virus administration and analysis of virion levels and gene delivery
In order to assess transgene expression, 8-week-old male WKY rats were infused with 3.5 × 10 11 VP each Five days after infusion, the animals were killed and the organs were removed For assessing transgene expression in the absence of Kupffer cells, a pre-dose protocol was followed 15 Briefly, 8-week-old male WKY rats were infused with 3 × 10 11 VP Ad null (no trans-gene) followed 4 hours later by 5 × 10 10 VP of peptide-modified Ad or control virus Five days after infusion, the animals were killed and the organs were removed In order to detect transgene expression, the organs were fixed
in formalin and wax-embedded Immunohistochemistry was performed
on 6 μm sections, using a rabbit polyclonal anti-β-galactosidase antibody
diluted 1/1,000 or matched rabbit IgG non-immune control (DAKO, Ely, UK) Detection was with biotinylated anti-rabbit secondary antibody, ABC kit and diaminobenzidine staining supplemented with nickel (all Vector Laboratories) Sections were counterstained with haematoxylin For the purpose of assessing particle delivery at an early time point after injection
(1 hour) animals (n = 4 per group) were perfused at physiological blood
pressure with saline, and the organs were removed and snap frozen DNA was extracted from a defined tissue mass (Qiagen, Crawley, UK) and
quan-tified using the Nanodrop ND-1000 (Wilmington, NC) A LacZ
quantifi-cation standard curve was constructed from serial dilutions of each Ad by use of SYBR green (Applied Biosystems, Warrington, UK) with 200 nmol/l sense (5′-TAC TGT CGT CGT CCC CTC AAA-3′) and antisense (5′-TAA
CAA CCC GTC GGA TTC TCC-3′) LacZ primers The total DNA was
amplified, and the products were quantified using Taqman Data Analysis software (Applied Biosystems, Warrington, UK) The following reaction conditions were in use: denaturation, 10 minutes at 95 °C; amplifica-tion, 15 seconds at 95 °C and 1 minute at 60 °C (45 cycles); dissociaamplifica-tion,
15 seconds at 95 °C, 15 seconds at 60 °C, and 15 seconds at 95 °C.
Statistical analysis Phage display was performed in groups of three animals per round The results represent mean values and SEM of the
data In vitro studies were carried out in triplicate on three independent
occasions In vivo studies were performed in groups of three animals each
Trang 8The results represent mean values and SEM of the data Students t-test was
performed, and statistical significance was defined as P < 0.05.
AcknowledgMents
The authors thank Nicola Britton and Crawford Halliday (BHF GCRC,
University of Glasgow, UK) for technical assistance This work was
funded by the National Kidney Research Fund, the Biotechnology and
Biological Research Council, UK, and the British Heart Foundation.
reFerences
1 Huard, J, Lochmuller, H, Ascadi, G, Jani, A, Massie, B and Karpati, G (1995) The route
of administration is a major determinant of the transduction efficiency of rat tissues by
adenoviral recombinants Gene Ther 2: 107–115.
2 Inagaki, K, Fuess, S, Storm, TA, Gibson, GA, Mctiernan, CF, Kay, MA et al (2006)
Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene
transfer superior to that of AAV8 Mol Ther 14: 45–53.
3 Gregorevic, P, Blankinship, MJ, Allen, JM, Crawford, RW, Meuse, L, Miller, DG et al
(2004) Systemic delivery of genes to striated muscles using adeno-associated viral
vectors Nat Med 10: 828–834.
4 Blankinship, MJ, Gregorevic, P, Allen, JM, Harper, SQ, Harper, H, Halbert, CL et al
(2004) Efficient transduction of skeletal muscle using vectors based on
adeno-associated virus serotype 6 Mol Ther 10: 617–678.
5 Pacak, CA, Mah, CS, Thattaliyath, BD, Conlon, TJ, Lewis, MA, Cloutier, DE et al
(2006) Recombinant adeno-associated virus serotype 9 leads to preferential cardiac
transduction in vivo Circ Res 99: e3–e9.
6 Gaggar, A, Shayakhmetov, DM and Lieber, A (2003) CD46 is a cellular receptor for
group B adenoviruses Nat Med 9: 1408–1412.
7 Tuve, S, Wang, H, Ware, C, Liu, Y, Gaggar, A, Bernt, K et al (2006) A new group
B adenovirus receptor is expressed at high levels on human stem and tumor cells
J Virol 80: 12109–12120.
8 Havenga, MJ, Lemckert, AA, Grimbergen, JM, Vogels, R, Huisman, LG, Valerio, D et al
(2001) Improved adenovirus vectors for infection of cardiovascular tissues J Virol
75: 3335–3342.
9 Nicklin, SA and Baker, AH (2002) Tropism-modified adenovirus and adeno-associated
viral vectors for gene therapy Curr Gene Ther 2: 273–293.
10 Nicol, CG, Graham, D, Miller, WH, White, SJ, Smith, TA, Nicklin, SA et al (2004)
Effect of adenovirus serotype 5 fiber and penton modifications on in vivo tropism in
rats Mol Ther 10: 344–354.
11 Smith, TA, Idamakanti, N, Rollence, ML, Marshall-Neff, J, Kim, J, Mulgrew, K et al
(2003) Adenovirus serotype 5 fiber shaft influences in vivo gene transfer in mice
Hum Gene Ther 14: 777–787.
12 Smith, TA, Idamakanti, N, Marshall-Neff, J, Rollence, ML, Wright, P, Kaloss, M et al
(2003) Receptor interactions involved in adenoviral-mediated gene delivery after
systemic administration in non-human primates Hum Gene Ther 14: 1595–1604.
13 Shayakhmetov, DM, Gaggar, A, Ni, S, Li, ZY and Lieber, A (2005) Adenovirus
binding to blood factors results in liver cell infection and hepatotoxicity J Virol
79: 7478–7491.
14 Parker, AL, Waddington, SN, Nicol, CG, Shayakhmetov, DM, Buckley, SM, Denby, L
et al (2006) Multiple vitamin K-dependent coagulation zymogens promote
adenovirus-mediated gene delivery to hepatocytes in vitro and in vivo Blood
108: 2554–2561.
15 Denby, L, Work, LM, Graham, D, Hsu, C, Von Seggern, DJ, Nicklin, SA et al (2004)
Adenoviral serotype 5 vectors pseudotyped with fibers from subgroup D show
modified tropism in vitro and in vivo Hum Gene Ther 15: 1054–1064.
16 Reynolds, PN, Nicklin, SA, Kaliberova, L, Boatman, BG, Grizzle, WE, Balyasnikova, IV
et al (2001) Combined transductional and transriptional targeting improves the
specificty of transgene expression in vivo Nat Biotechnol 19: 838–842.
17 Pasqualini, R and Ruoslahti, E (1996) Organ targeting in vivo using phage display
peptide libraries Nature 380: 364–366.
18 Kolonin, MG, Saha, PK, Chan, L, Pasqualini, R and Arap, W (2004) Reversal of obesity
by targeted ablation of adipose tissue Nat Med 10: 625–632.
19 Rajotte, D, Arap, W, Hagedorn, M, Koivunen, E, Pasqualini, R and Ruoslahti, E (1998)
Molecular heterogeneity of the vascular endothelium revealed by in vivo phage
display J Clin Invest 102: 430–437.
20 Arap, W, Kolonin, MG, Trepel, M, Lahdenranta, J, Cardo-Vila, M, Giordano, RJ et al
(2002) Steps towards mapping the human vasculature by phage display Nat Med
8: 121–127.
21 Ellerby, HM, Arap, W, Ellerby, LM, Kain, R, Andrusiak, R, Rio, GD et al (1999)
Anti-cancer activity of targeted pro-apoptosis peptides Nat Med 5: 1032–1038.
22 Arap, W, Pasqualini, R and Ruoslahti, E (1998) Cancer treatment by targeted drug
delivery to tumor vasculature in a mouse model Science 279: 377–380.
23 Nicklin, SA, Von Seggern, DJ, Work, LM, Pek, DC, Dominiczak, AF, Nemerow, GR
et al (2001) Ablating adenovirus type 5 fiber-CAR binding and HI loop insertion of
the SIGYPLP peptide generate an endothelial cell-selective adenovirus Mol Ther
4: 534–542.
24 Esko, JD, Stewart, TE and Taylor, WH (1985) Animal cell mutants defective in
glycosaminoglycan biosynthesis Proc Natl Acad Sci USA 82: 3197–3201.
25 Work, LM, Buning, H, Hunt, E, Nicklin, SA, Denby, L, Britton, N et al (2006) Vascular bed-targeted in vivo gene delivery using tropism-modified adeno-associated viruses
Mol Ther 13: 683–693.
26 Dmitriev, I, Krasnykh, V, Miller, CR, Wang, MH, Kashentseva, E, Mikheeva, G et al
(1998) An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell
entry mechanism J Virol 72: 9706–9713.
27 Nicklin, SA, Von Seggern, DJ, Work, LM, Pek, DC, Dominiczak, AF, Nemerow, GR
et al (2001) Ablating adenovirus type 5 fiber-CAR binding and HI loop insertion of
the SIGYPLP peptide generate an endothelial cell-selective adenovirus Mol Ther
4: 534–542.
28 Lyons, M, Onion, D, Green, NK, Aslan, K, Rajaratnam, R, Bazan-Peregrino, M et al
(2006) Adenovirus type 5 interactions with human blood cells may compromise
systemic delivery Mol Ther 14: 118–128.
29 Stone, D, Ni, S, Li, ZY, Shayakhmetov, D and Lieber, A (2006) Intravascular delivery of
adenovirus vectors rapidly targets platelets to the reticuloendothelial system Mol Ther
13: S143.
30 Kiang, A, Hartman, ZC, Everett, RS, Serra, D, Jiang, H, Frank, MM et al (2006)
Multiple innate inflammatory responses induced after systemic adenovirus vector
delivery depend on a functional complement system Mol Ther 14: 588–598.
31 Zinn, KR, Szalai, AJ, Stargel, A, Krasnykh, V and Chaudhuri, TR (2004)
Bioluminescence imaging reveals a significant role for complement in liver
transduction following intravenous delivery of adenovirus Gene Ther
11: 1482–1486.
32 Shayakhmetov, DM, Li, ZY, Ni, S and Lieber, A (2004) Analysis of adenovirus sequestion in the liver, transduction of hepatic cells, and innate toxicity after injection
of fiber-modified vectors Virology 78: 5368–5381.
33 Dechecchi, MC, Tamanini, A, Bonizzato, A and Cabrini, G (2000) Heparan sulphate glycosaminoglycans are involved in adenovirus type 5 and 2-host cell interactions
Virology 268: 382–390.
34 Dechecchi, MC, Melotti, P, Bonizzato, A, Santacatterina, M, Chilosi, M and Cabrini, G (2001) Heparan sulfate glycosaminoglycans are receptors sufficient to mediate the
initial binding of adenovirus types 2 and 5 J Virol 75: 8772–8780.
35 Kritz, AB, Nicol, CG, Dishart, KL, Nelson, R, Holbeck, S, Von Seggern, DJ et al (2007)
Adenovirus 5 fibers mutated at the KKTK putative HSPG binding site show restricted retargeting capacity when engineered with targeting peptides in the HI loop
Mol Ther 15: 741–749.
36 Parker, AL, McVey, JH, Doctor, JH, Lopez-Franco, O, Waddington, SN, Havenga, MJ (2007) Influence of coagulation factor zymogens on the infectivity of adenoviruses
pseudotyped with fibers from subgroup D J Virol 81: 3627–3631.
37 Arnberg, N, Kidd, AH, Edlund, K, Olfat, F and Wadell, G (2000) Initial interactions
of subgroup D adenoviruses with A549 cellular receptors: sialic acid versus alpha(v)
integrins J Virol 74: 7691–7693.
38 Choi, YK, Kim, YJ, Park, HS, Choi, K, Paik, SG, Lee, YI et al (2003) Suppression of glomerulosclerosis by adenovirus-mediated IL-10 expression in the kidney Gene Ther
10: 559–568.
39 Lan, HY, Mu, W, Tomita, N, Huang, XR, Li, JH, Zhu, HJ et al (2003) Inhibition of renal
fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in
rat UUO model J Am Soc Nephrol 14: 1535–1548.
40 Chao, J, Zhang, JJ, Lin, KF and Chao, L (1998) Adenovirus-mediated kallikrein gene
delivery reverses salt-induced renal injury in Dahl salt-sensitive rats Kidney Int
54: 1250–1260.
41 Dai, C, Yang, J and Liu, Y (2002) Single injection of naked plasmid encoding hepatocyte growth factor prevents cell death and ameliorates acute renal failure in
mice J Am Soc Nephrol 13: 411–422.
42 Wang, C, Dobrzynski, E, Chao, J and Chao, L (2001) Adrenomedullin gene delivery attenuates renal damage and cardiac hypertrophy in Goldblatt hypertensive rats
Am J Physiol Renal Physiol 280: F964–F971.
43 Isaka, Y, Brees, DK, Ikegaya, K, Kaneda, Y, Imai, E, Noble, NA et al (1996) Gene
therapy by skeletal muscle expression of decorin prevents fibrotic disease in rat kidney
Nat Med 2: 418–423.
44 Von Seggern, DJ, Huang, S, Fleck, SK, Stevenson, SC and Nemerow, GR (2000) Adenovirus vector pseudotyping in fiber-expressing cell lines: improved transduction
of Epstein-Barr virus-transformed B cells J Virol 74: 354–362.
45 Jakubczak, JL, Rollence, ML, Stewart, DA, Jafari, JD, Von Seggern, DJ, Nemerow, GR
et al (2001) Adenovirus type 5 viral particles pseudotyped with mutagenized fiber
proteins show diminished infectivity of coxsackie B-adenovirus receptor-bearing cells
J Virol 75: 2972–2981.
46 Work, LM, Nicol, CG, Denby, L and Baker, AH (2004) In vivo biopanning: a
methodological approach to identifying novel targeting ligands for delivery of
biological agents to the vasculature Methods Mol Med 108: 395–413.
47 Burmeister, WP, Guilligay, D, Cusack, S, Wadell, G and Arnberg, N (2004) Crystal structure of species D adenovirus fiber knobs and their sialic acid binding sites
J Virol 78: 7727–7736.