1. No category

Targeted Gene Delivery to Vascular Tissue In Vivo by Tropism-Modified
Adeno-Associated Virus Vectors
Stephen J. White, Stuart A. Nicklin, Hildegard Büning, M. Julia Brosnan, Kristen Leike,
Emmanuel D. Papadakis, Michael Hallek and Andrew H. Baker
Circulation. 2004;109:513-519; originally published online January 19, 2004;
doi: 10.1161/01.CIR.0000109697.68832.5D
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2004 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7322. Online ISSN: 1524-4539
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circ.ahajournals.org/content/109/4/513
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial
Office. Once the online version of the published article for which permission is being requested is located,
click Request Permissions in the middle column of the Web page under Services. Further information about
this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation is online at:
http://circ.ahajournals.org//subscriptions/
Downloaded from http://circ.ahajournals.org/ by guest on June 9, 2014
Targeted Gene Delivery to Vascular Tissue In Vivo by
Tropism-Modified Adeno-Associated Virus Vectors
Stephen J. White, DPhil*; Stuart A. Nicklin, PhD*; Hildegard Büning, PhD; M. Julia Brosnan, PhD;
Kristen Leike; Emmanuel D. Papadakis, BSc; Michael Hallek, MD; Andrew H. Baker, PhD
Background—Gene therapy offers an unprecedented opportunity to treat diverse pathologies. Adeno-associated virus
(AAV) is a promising gene delivery vector for cardiovascular disease. However, AAV transduces the liver after systemic
administration, reducing its usefulness for therapies targeted at other sites. Because vascular endothelial cells (ECs) are
in contact with the bloodstream and are heterogeneous between organs, they represent an ideal target for site-specific
delivery of biological agents.
Methods and Results—We isolated human venous EC-targeting peptides by phage display and genetically incorporated
them into AAV capsids after amino acid 587. Peptide-modified AAVs transduced venous (but not arterial) ECs in vitro,
whereas hepatocyte transduction was significantly lower than with native AAV. Intravenous infusion of engineered
AAVs into mice produced reduced vector accumulation in liver measured 1 hour and 28 days after injection and delayed
blood clearance rates compared with native AAV. Peptide-modified AAVs produced enhanced uptake of virions in the
vena cava with selective transgene expression. Retargeting was dose dependent, and coinfusion of either heparin or free
competing peptides indicated that uptake was principally independent of native AAV tropism and mediated via the
peptide.
Conclusions—AAV tropism can be genetically engineered by use of phage display– derived peptides to generate vectors
that are selective for the vasculature. (Circulation. 2004;109:513-519.)
Key Words: gene therapy 䡲 endothelium 䡲 viruses 䡲 peptides
C
ardiovascular gene therapy is becoming a realistic clinical goal, as results in clinical trials for vein graft failure1
and stable angina2 prove. A critical feature of efficient gene
delivery is target cell accessibility in vivo. Although certain
surgical procedures (eg, bypass grafting) allow access, many
sites are inaccessible (eg, pulmonary or renal tissue) but
would be amenable to systemic delivery if appropriate vectors were available. Endothelial cells (ECs), which are in
continuous contact with the bloodstream and integrally involved in cardiovascular abnormalities, are appropriate targets. The concept of EC targeting originates from studies that
defined in vivo heterogeneity, such that many EC surface
receptors seem to be unique to individual organs.3,4 Uptake of
basic gene transfer vectors, including adenovirus, AAV, and
lentivirus, by ECs after systemic delivery is poor, because the
liver rapidly sequesters virus5–7; thus, only therapies that take
advantage of this natural tropism are feasible.
Adeno-associated virus (AAV) is a promising gene transfer
vector for cardiovascular therapies. AAV tropism is dictated
by distribution of its primary cell-tethering receptor heparan
sulfate proteoglycan (HSPG)8 and internalizing the coreceptors fibroblast growth factor receptor 19 and ␣v␤5 integrin.10
Although AAV transduces ECs, the efficiency is lower
relative to hepatocytes.11 Modifying AAV to remove native
tropism in combination with provision of target cell–specific
tropism (retargeting) is a prerequisite for development of
systemically injectable vector platforms. New tropism may be
supplied through antibodies, ligands, or small targeting peptides (reviewed by Nicklin and Baker12). A defined site in the
capsid of AAV serotype 2 (AAV-2)13 has been identified as
an efficient site for insertion of targeting peptides. We have
recently demonstrated that AAV-2 can be efficiently and
selectively targeted to human ECs in vitro.11 Here, we have
isolated 2 novel venous EC-selective targeting peptides that
redefine AAV infectivity profiles in vitro and in vivo.
Methods
Cell Culture
Human umbilical vein ECs (HUVECs), human coronary artery ECs,
and primary human hepatocytes were purchased from TCS (Botolph
Received May 9, 2003; de novo received August 12, 2003; revision received September 23, 2003; accepted September 25, 2003.
From the Bristol Heart Institute, Bristol Royal Infirmary, Bristol (S.J.W., E.D.P.), and the Division of Cardiovascular and Medical Sciences, University
of Glasgow (S.A.N., M.J.B., A.H.B.), UK; and the Laboratorium für Molekulare Biologie, Genzentrum, Ludwig-Maximilians Universität München (H.B.,
K.L., M.H.), the Medizinische Klinik III, Klinikum der Universität München (M.H.), and Gesellshaft für Strahelforschung (M.H.), Munich, Germany.
*The first 2 authors contributed equally to this work.
Correspondence to Dr A.H. Baker, Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, G11 6NT UK. E-mail
ab11f@clinmed.gla.ac.uk
© 2004 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/01.CIR.0000109697.68832.5D
513
Downloaded from http://circ.ahajournals.org/
by guest on June 9, 2014
514
Circulation
February 3, 2004
Peptide Sequences From Phage Display
Sequence
MSNNPIRPPTSG
Frequency
Sequence
Frequency
2
MQPRPQTLTPAS
1
MSTPPIREQAAH
1
MSLTTPPAVARP
10
MSQTPYARPQYV
1
MSSPTVSSAPQY
1
LQPPSMITHPST
2
MTRIQDSPYDLR
1
MLMPQPAHHNNS
1
MTPIQSTQYPHS
1
after position 587 in the AAV-2 capsid, and produced as described
previously.11 Viruses express lacZ from the cytomegalovirus promoter. Genomic particle (GP) titers were quantified by dot blot and
real-time polymerase chain reaction (PCR). For heparin column
assays, AAV was loaded onto columns with heparin immobilized on
cross-linked 4% agarose beads (Sigma). The flow-through, 3 column
washes (PBS, 1 mmol/L MgCl2, 2.5 mmol/L KCl), and elution
fractions (PBS containing 1 mmol/L MgCl2, 2.5 mmol/L KCl, and 1
mol/L NaCl) were analyzed by dot blot.
MAMPQPADHNNS
1
THAMSHLDKAH
1
In Vitro Cell Transduction Assays
VSGMSVPVQLAR
1
MTQTPRTTPWPD
1
MGMTVPENLIVQ
2
AFPMVGGPDHFR
1
MTNLPTVTQFPP
1
MTSNPHLNPGPR
1
SMRGLPELNPRI
1
MTGSQQTLHPPP
1
LTSPFSTPLNPR
2
AQAMANPLGSHI
1
AAV transduction was quantified at 5000, 10 000, or 20 000
GPs/cell in the presence of helper Ad (multiplicity of infection, 10).
Cells were exposed to virus for 16 hours, the media were changed,
and the cells were subsequently incubated until 72 hours. Cells were
lysed, and ␤-galactosidase expression was quantified by use of
Galactolight Plus (Tropix).
MNMTPPPHSPPK
1
AMSMTTMPHSPN
1
MTPFPTSNEANL
6
MTQVYTPPPTST
1
MMPPPTSLPSPS
1
MATQPLSGSRLSG
1
MAGQPKDSSKTL
1
MAVQPPNTSTSN
1
AAVPPPYVMSRP
1
LSTPLPYDMRRS
1
LTPPNQVLNPLY
1
MTQQPPLPHPAK
1
MTPIATSIPPQM
1
LAKPLPTTSNTG
1
MAINDTYPPPRP
1
LSKPIPHIPSSIG
1
MTSPHPQTPNLT
1
CICRGVGCCLLL
1
VLSMQTPPTPLL
1
MTPTTPIPSLPQ
1
MGHNINIPRTPL
1
LAQNPIYRAHPH
1
LTVPVPVSFAVH
1
ANTPPHTILSTE
1
MSDLLIEYPPYI
1
MTLPHELRDGAL
1
SSRIPGFPDPLH
1
Virus Distribution Studies In Vivo
A total of 1.5⫻1010 or 3⫻1011 GPs of AAVwt, AAVmtp, or AAVmsl
were infused intravenously into male 5-week-old BALB/C mice
before euthanasia. Organs were removed and snap frozen, and DNA
was extracted (Qiagen) and quantified by use of PicoGreen (Cambridge Biosciences). A lacZ quantification standard curve was
generated from serial dilutions of each AAV preparation by use of
FastStart DNA Master SYBR Green I (Roche) (4 mmol/L MgCl2)
with 0.5 mmol/L forward (5⬘-ATCTGACCACCAGCGAAATGG3⬘) and reverse (5⬘-CATCAGCAGGTGTATCTGCCG-3⬘) lacZ
primers. Total DNA (20 ng) from a defined tissue mass was
amplified, and products were quantified by melting curve analysis at
crossover points using the Lightcycler Data Analysis software
(Roche). Results were expressed as genomic AAV particles/mg
All peptides and their frequencies of isolation are indicated. Candidate
consensus motifs are indicated by underlining, and peptides in bold represent
the most frequently identified peptides.
Claydon). Human saphenous vein ECs were prepared as described
previously.14 ECs were cultured in complete endothelium media
(TCS), 20% (vol/vol) FCS, and antibiotics. HepG2 hepatocytes and
the murine venous EC lines IP-1B and SVEC-4-1015 were maintained in minimal essential medium, 10% (vol/vol) FCS, and
antibiotics. Human saphenous vein smooth muscle cells (VSMCs)
were isolated by explant preparation and cultured in DMEM (4500
mg/L glucose), 20% (vol/vol) FCS, and antibiotics. Peripheral blood
mononuclear cells were isolated from whole blood by use of Ficoll
Paque (Pharmacia Biotech).
Phage Display
Libraries (12-mer, New England BioLabs) were panned on 2
successive cultures of VSMCs, HepG2, and peripheral blood mononuclear cells (preclearing steps), then on HUVECs. Weakly associated phages were removed, high-affinity phages isolated by lysis in
30 mmol/L Tris/10 mmol/L EDTA (pH 8.0), and individual plaques
sequenced after 5 rounds. Peptide homology was performed via
BLAST (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Recovery
studies for homogenous phages were as described previously.16
Briefly, 107 phages were incubated with cells for 1 hour at 4°C,
washed 5 times, lysed in hypertonic buffer (30 mmol/L Tris/
10 mmol/L EDTA), and quantified by titration.
Production of AAV Vectors
pRCmtp and pRCmsl were constructed with oligonucleotides encoding SMTPFPTSNEANLGGGS and SMSLTTPPAVARPGGGS (peptide sequence italicized), annealed into the MluI-AscI site of pRC⬘99
Figure 1. Analysis of phage binding in vitro. A total of 107
phages displaying MSLTTPPAVARP or MTPFPTSNEANL peptides were biopanned and washed extensively, and tightly
bound phages were recovered from (A) HUVECs, VSMCs, or
HepG2 hepatocytes. *P⬍0.001 vs binding to VSMCs and hepatocytes. B, Binding to mouse EC lines IP-1B and SVEC-4 to 10.
*P⬍0.05 vs insertless phage control.
Downloaded from http://circ.ahajournals.org/ by guest on June 9, 2014
White et al
Targeted Gene Delivery In Vivo
515
Figure 2. Analysis of AAV transduction in
vitro. A, HUVECs were infected with
10 000 and 20 000 GPs/cell of either
AAVwt, AAVmsl, or AAVmtp, and
␤-galactosidase was quantified at 72
hours. B, Transduction of HepG2 hepatocytes at 5000 and 10 000 GPs/cell with
analysis as for ECs. C, Transduction of
HUVECs at 10 000 GPs/cell by AAVwt,
AAVmtp, and AAVmsl in presence of
competing heparin (1 IU/5⫻105 GPs
AAV). D, Heparin column binding assay.
See Methods for details. Flow-through,
column washes, and elution fractions (1
mol/L NaCl) were analyzed by dot blot.
tissue. The following reaction conditions were used: denaturation,
95°C, 600 seconds; amplification, 95°C, 3 seconds; annealing, 60°C,
10 seconds; elongation, 72°C, 20 seconds; and data collection, 89°C,
1 second (30 to 50 cycles). Products were analyzed by melting-curve
analysis after initial denaturation at 95°C with cooling at 20°C/s to
45°C, incubation for 15 seconds, and termination at 99°C. For
competition experiments, AAVs were coinfused with either 100 IU
heparin or 0.5 mg free C-terminally amidated peptide (MSLTTPPAVARPGGGS or MTPFPTSNEANLGGGS). DNA was extracted,
quantified, and subjected to quantitative PCR as described above.
Analysis of Transgene Expression
␤-Galactosidase activity was measured in tissue homogenates with
the Galactolight Plus kit (Tropix). Protein concentration was quantified with the microBCA assay (Pierce). Detection of
␤-galactosidase by immunohistochemistry was performed on paraformaldehyde-fixed, wax-embedded sections with rabbit anti–␤galactosidase antibody (ICN Biomedicals) 1/1200 or matched rabbit
IgG nonimmune control, followed by detection with biotinylated
goat anti-rabbit secondary antibody (1/200), ABC kit (Vector Laboratories) and Sigma Fast diaminobenzidine staining tablets (Sigma).
Sections were counterstained with hematoxylin for 5 seconds.
Statistical Analysis
Data were analyzed by ANOVA for multiple comparisons or
unpaired Student’s t test and are shown as mean⫾SEM. Data were
considered significant at a value of P⬍0.05.
Results
Identification of Targeting Peptides
First, we sought to identify novel EC-targeting peptides by
phage display. Using a selective in vitro biopanning procedure (see Methods), we identified 49 individual peptides
(Table), many of which were isolated between 2 and 10 times.
Peptides MSLTTPPAVARP and MTPFPTSNEANL were
repeated 10 and 6 times, respectively. A number of consensus
motifs were identified (Table). No homology to human
sequences was identified by database searches. Phage recovery studies on HUVECs, VSMCs, and hepatocytes with 23 of
the selected peptides demonstrated EC-selectivity (not shown
except MSLTTPPAVARP and MTPFPTSNEANL [Figure
1A]). Both peptide-displaying phages demonstrated 5- to
20-fold enhanced binding to murine EC lines compared with
control phage (Figure 1B).
Characterization of Peptide-Modified AAV
Vectors In Vitro
We genetically inserted MSLTTPPAVARP or MTPFPTSNEANL peptides into AAV-2 after residue 587 and
created AAVmsl and AAVmtp with titers comparable to
those of wild-type AAV (AAVwt) (not shown). In HUVECs
(Figure 2A), transduction for each AAV was dose dependent,
and both AAVmsl and AAVmtp were equivalent to AAVwt.
In HepG2 hepatocytes, transduction was very different (Figure 2B). As expected, AAVwt produced very high transduction (⬇100 times the levels in HUVECs). AAVmsl produced
significantly higher transduction than AAVmtp. Both
AAVmsl and AAVmtp produced significantly lower transduction than AAVwt (Figure 2B). We compared heparin
dependence for each AAV in ECs (Figure 2C). Transduction
with AAVwt and AAVmsl showed a tendency to decrease,
but AAVmtp-mediated transduction of ECs was significantly
enhanced in the presence of heparin, as observed in previous
studies.11 AAVwt and AAVmsl transduction in HepG2 cells
Downloaded from http://circ.ahajournals.org/ by guest on June 9, 2014
516
Circulation
February 3, 2004
was significantly inhibited by heparin (not shown). AAVmtp
produced negligible HepG2 transduction that was inhibited
with heparin, demonstrating that it represented residual native
tropism (not shown). Infectivity profiles and heparin dependence for each virus in primary human hepatocytes was
essentially the same as in HepG2 cells, although the absolute
levels of transgene expression were lower (not shown). A
direct comparison of transduction levels in HepG2 hepatocytes and primary human hepatocytes at 10 000 GPs/cell
revealed that AAVwt produced 200 ␮g ␤-galactosidase/mg
protein in HepG2 hepatocytes but 8 ␮g ␤-galactosidase/mg
protein in primary hepatocytes. Both peptide modifications
significantly reduced the efficiency of AAV transduction into
primary hepatocytes, resulting in an improvement in the
primary hepatocyte–to–primary EC ratio of 4:1 for AAVwt to
1:1 for both AAVmsl and AAVmtp. Heparin dependence was
also assessed by use of heparin columns (Figure 2D). In
contrast to AAVwt (found exclusively in high-salt eluate),
substantial proportions of AAVmsl and AAVmtp were detected in the column wash, consistent with the celltransduction studies (Figure 2C). Importantly, both peptidemodified AAVs showed reduced transduction in primary
human coronary ECs, demonstrating their venous selectivity
(not shown). Because of the cross-species targeting we
observed (Figure 1B), we also assessed transduction of each
AAV in the murine venous EC line IP-1B. Each peptidemodified AAV produced efficient transduction, and AAVmtp
was not significantly different from AAVwt (not shown).
Assessment of the transduction of human VSMCs confirmed
that each peptide-modified vector transduced venous ECs
selectively and not other vascular cell types (not shown).
Characterization of Peptide-Modified AAV
Vectors In Vivo
We infused 1.5⫻1010 GPs of each AAV into BALB/c mice
and assessed particle biodistribution by real-time PCR 1 hour
after infusion. Real-time PCR allows tracking of virions to
specific sites in vivo without other confounding components
of AAV transduction, eg, trafficking and nuclear gene conversion, which vary widely in different tissues but occur
independently of cell-surface binding. AAVwt accumulated
predominantly in the liver (Figure 3A). Both peptide insertions significantly reduced liver virion accumulation (Figure
3A). Likewise, lung and kidney accumulation (Figure 3A)
was significantly lower for AAVmtp and AAVmsl compared
with AAVwt, whereas brain (Figure 3A) and heart (Figure
3A) were not different from AAVwt. Because reduced virion
accumulation was evident for both AAVmsl and AAVmtp,
we quantified virions present in blood 30, 60, and 180
minutes after infusion (Figure 3B). Interestingly, both mutants had delayed clearance from the blood at early time
points compared with AAVwt. No differences were observed
at 180 minutes. We next analyzed arterial and venous blood
vessels (Figure 4). Uptake in the aorta was significantly lower
for AAVmsl and AAVmtp than AAVwt (Figure 4A). In
contrast, AAVmsl levels in the vena cava were similar to
AAVwt (Figure 4B). Importantly, AAVmtp demonstrated
significantly higher accumulation than AAVwt (Figure 4B),
indicating that the venous selectivity observed by in vitro
Figure 3. Biodistribution in vivo using modified AAV vectors. A,
1.5⫻1010 GPs of AAVwt, AAVmtp, and AAVmsl or PBS were
infused into BALB/c mice, and particle distribution was quantified by real-time PCR in all major organs. *P⬍0.05 vs AAVwt
(n⫽6 mice/group). B, Circulating AAVwt, AAVmsl, and AAVmtp
were quantified at 30, 60, and 180 minutes after infusion of
1.5⫻1010 GPs using real-time PCR (n⫽3 mice/group).
studies was replicated in vivo. Next, we assessed the specificity
of the targeting to vena cava using competition assays at 1 hour
(Figure 4). Coinfusing heparin with AAVs significantly blocked
AAVwt but not AAVmsl or AAVmtp in the vena cava at 1 hour
(Figure 4C). For both AAVmsl and AAVmtp, coinfusion of the
respective free peptide in vivo significantly reduced accumulation in venous tissue (Figure 4D).
We next analyzed AAV distribution 28 days after infusion
(Figure 5). Reduced AAV accumulation in the liver for
AAVmsl and AAVmtp was confirmed by real-time PCR
(Figure 5A). Similar reductions were observed in spleen and
lung (Figure 5A). We analyzed AAVwt and AAVmtp further
at a high dose (3⫻1011 GPs/mouse) and observed that the
reduced virion accumulation was dose dependent (Figure
5B). At this dose, AAVmsl produced 29.7⫾9.7% and
AAVmtp, 2.9⫾0.45% (n⫽4 in all groups) of AAVwt values.
Importantly, in the vena cava at low dose (1.5⫻1010 GPs/
mouse), AAVmsl and AAVmtp were readily detected by
real-time PCR, but AAVwt was undetectable in all animals
tested (Figure 5C). Again, this was dose dependent when
AAVwt and AAVmtp were analyzed at the higher dose
(Figure 5C). We compared the mean vena cava–to-liver ratios
Downloaded from http://circ.ahajournals.org/ by guest on June 9, 2014
White et al
Targeted Gene Delivery In Vivo
517
Figure 4. Retargeting to vasculature in vivo
using modified AAV vectors. 1.5⫻1010 GPs of
AAVwt, AAVmtp, and AAVmsl were infused
into mice and accumulation in (A) aorta and (B)
vena cava quantified by real-time PCR.
*P⬍0.05 vs AAVwt (n⫽6 mice/group). C, Accumulation of AAVwt, AAVmsl, and AAVmtp in
vena cava was quantified in absence and
presence of competing heparin (*P⬍0.05 vs
AAVwt, n⫽4 mice/group) and (D) in presence
of 0.5 mg/mouse competing free peptide (P)
for AAVmsl and AAVmtp (*P⬍0.05 vs value in
absence of peptide, n⫽4 mice/group).
at 1 hour, and they were 0.1 for AAVwt and 1.3 for AAVmtp.
At 28 days after injection of 3⫻1011 GPs, AAVwt produced
a mean vena cava–to-liver ratio of 0.016, and for AAVmtp, it
was 6.5. Thus, there was a 400-fold improvement in vena
cava–to-liver gene delivery with AAVmtp. To further define
selectivity, we analyzed aortas at 28 days after infection, and
both AAVmsl and AAVmtp produced reduced accumulation
compared with AAVwt (not shown).
Ultimately, transgene expression is the most important
parameter. We therefore analyzed ␤-galactosidase transgene
expression at 28 days after infusion in both AAVwt- and
AAVmtp-infected animals at 3⫻1011 GPs/mouse. In accordance with real-time PCR data, transgene expression in liver
homogenates was significantly reduced for AAVmtp compared with AAVwt (Figure 6A). Transgene levels in tissue
homogenates from other organs were not above background
levels for both AAVwt and AAVmtp (not shown). Immunohistochemical analysis of the vena cava revealed selective
expression of transgene in cells at the luminal surface of
AAVmtp- but not AAVwt- or PBS-treated animals (Figure
6B). ␤-Galactosidase activity was not detected in the aortic
tissue from animals treated with either AAV (not shown).
Discussion
The concept of developing systemically injectable gene
transfer vectors is important for a number of potential
cardiovascular gene therapies. This work provides, for the
first time, proof of concept for this possibility through
isolation and characterization of venous EC-selective peptides and AAV vectors. First, we demonstrate reduced virion
accumulation in the major organs in which AAVwt is
detected, predominantly the liver, for both modified AAVs,
which translated to a significant reduction in hepatic transgene expression. Second, peptide-engineered AAVs had prolonged blood-circulating times compared with AAVwt, presumably because of the reduction in liver sequestration.
Third, we observed enhanced gene delivery for AAVmtp into
a specific vascular site in vivo at the level of virion accumulation and transgene expression. Retargeting was evaluated
by real-time PCR to track virions and transgene expression by
analysis of ␤-galactosidase to assess the ultimate aim, retargeting of transgene expression in vivo. Thus, these findings
represent the first description of significantly altered AAV
biodistribution in vivo after systemic administration. Further
analysis of the selectivity of the retargeting is interesting. At
Downloaded from http://circ.ahajournals.org/ by guest on June 9, 2014
518
Circulation
February 3, 2004
Figure 5. AAV organ distribution and vascular retargeting 28
days after infusion. A, AAVwt, AAVmsl, and AAVmtp virion quantification in liver, spleen, and lung 28 days after infusion of
1.5⫻1010 GPs/animal (*P⬍0.05 vs AAVwt, n⫽4 mice/group). B,
Real-time PCR quantification in liver 28 days after injection of
3⫻1011 GPs/mouse (*P⬍0.05 vs AAVwt, n⫽4 mice/group). C,
AAV quantification in vena cava 28 days after infusion of
1.5⫻1010 (n⫽4 mice/group; U.D., undetectable) or 3⫻1011 GPs/
mouse (*P⬍0.05 vs AAVwt, n⫽3 mice/group).
1 hour, both peptide-modified AAVs and AAVwt could be
detected in the vena cava, and neither AAVmsl nor AAVmtp
required heparin-dependent mechanisms. Importantly, at 28
days, with a low dose of each AAV, only AAVmsl and
AAVmtp could be detected in the vena cava, providing
evidence that in vitro venous selectivity was borne out in
vivo. This finding for AAVwt is supported by the work of
Seisenberger et al,17 who found that cell-surface docking can
take place without cell infection as virions escape into the
extracellular milieu. The in vitro transduction data also
suggest that AAVwt-mediated transduction into hepatocytes
is more dependent on HSPG binding than for ECs. It was
shown previously that ECs express high levels of HSPG;
however, they are present in the extracellular matrix, sequestering AAV away from the cell surface, preventing infection. 18 Because AAVmtp produced residual heparindependent transduction in hepatocytes, incubation of ECs
with heparin blocks its sequestration into HSPG in the
extracellular matrix, enabling transduction via interaction of
the peptide with the cell surface.
AAV-2 has a predominantly hepatic tropism when delivered systemically.5,19 Although primary and coreceptors have
been identified for AAV-2,8 –10 the mechanisms that dictate
tropism after systemic administration are not entirely elucidated. We previously reported that insertion of a small
targeting peptide at residue 587 conferred heparin-
Figure 6. Transgene expression in vivo. A, Transgene expression in liver homogenates 28 days after infusion of 3⫻1011 GPs/
mouse of AAVwt or AAVmtp. *P⬍0.05 vs AAVwt. B, Immunohistochemical detection of ␤-galactosidase expression in vena
cava at 28 days after infusion. Representative sections from 4
animals/group. Arrows indicate examples of transgene-positive
cells. Bar ⬇20 ␮m (applicable to all panels).
independent transduction and significantly reduced AAV-2
infection of nontarget cells.11 This dual phenotype is hypothesized to be a result of the close physical association between
the peptide insertion and the HSPG binding motif.20 This
raises the possibility of simultaneously detargeting and retargeting AAV-2 through single-step insertion of targeting
peptides.
This tropism modification was generated at the level of the
primary virus– cell interaction, an essential first step in the
virion infection. The tropism of AAV for efficient transgene
expression is also modulated through efficient internalization,
trafficking, and nuclear gene conversion.21–23 AAV targeting
may ultimately require refining with the use of ligands that
mediate selective cell-surface binding and virus trafficking.11
Enhanced gene transfer for cancer gene therapy has also
recently been demonstrated after direct intratumoral injection
of AAV targeted with the RGD-4-C peptide,24 which targets
␣v integrins.25
Therapeutic applications in the vasculature via systemic
delivery are varied and will require a repertoire of targeting
peptides unique for individual applications, such as has been
demonstrated by in vivo phage panning.3,4 Clearly, phage
display will afford the selection of peptides that target to
arterial endothelium and sites of disease in vivo based on
heterogeneity of target receptor expression. Incorporation of
such peptides is needed to realize the full potential of this
technology. In summary, this study demonstrates the ability
to rationally design and construct genetically engineered
“designer” gene therapy vectors for selective delivery of
genes to target vascular cells in vivo.
Downloaded from http://circ.ahajournals.org/ by guest on June 9, 2014
White et al
Acknowledgments
The authors thank the British Heart Foundation, the Medical Research Council, UK, and the Deutsche Forschungsgemeinschaft. We
thank Margaret Cunningham and Melanie Smith for technical
assistance.
References
1. Mann MJ, Whittemore AD, Donaldson MC, et al. Ex-vivo gene therapy
of human vascular bypass grafts with E2F decoy: the PREVENT singlecentre, randomised, controlled trial. Lancet. 1999;354:1493–1498.
2. Grines CL, Watkins MW, Helmer G, et al. Angiogenic gene therapy
(AGENT) trial in patients with stable angina pectoris. Circulation. 2002;
105:1291–1297.
3. Rajotte D, Arap W, Hagedorn M, et al. Molecular heterogeneity of the
vascular endothelium revealed by in vivo phage display. J Clin Invest.
1998;102:430 – 437.
4. Arap W, Kolonin MG, Trepel M, et al. Steps towards mapping the human
vasculature by phage display. Nat Med. 2002;8:121–127.
5. Koeberl DD, Alexander IE, Halbert CL, et al. Persistent expression of
human clotting factor IX from mouse liver after intravenous injection of
adeno-associated virus vectors. Proc Natl Acad Sci U S A. 1997;94:
1426 –1431.
6. Huard J, Lochmuller H, Acsadi G, et al. The route of administration is a
major determinant of the transduction efficiency of rat tissues by
adenoviral recombinants. Gene Ther. 1995;2:107–115.
7. Ohashi K, Park F, Kay MA. Role of hepatocyte derived hyperplasia in
lentivirus-mediated liver transduction in vivo. Hum Gene Ther. 2002;13:
653– 663.
8. Summerford C, Samulski RJ. Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. J Virol.
1998;72:1438 –1445.
9. Qing K, Mah C, Hansen J, et al. Human fibroblast growth factor receptor
1 is a co-receptor for infection by adeno-associated virus 2. Nat Med.
1999;5:71–77.
10. Summerford C, Bartlett JS, Samulski R-J. ␣V␤5 integrin: a co-receptor
for adeno-associated virus type 2 infection. Nat Med. 1999;5:78 – 82.
11. Nicklin SA, Buening H, Dishart KL, et al. Efficient and selective AAV-2
mediated gene transfer directed to human vascular endothelial cells. Mol
Ther. 2001;4:174 –181.
Targeted Gene Delivery In Vivo
519
12. Nicklin SA, Baker AH. Tropism-modified adenoviral and adenoassociated viral vectors for gene therapy. Curr Gene Ther. 2002;2:
273–293.
13. Girod A, Ried M, Wobus C, et al. Genetic capsid modifications allow
efficient re-targeting of adeno-associated virus type 2. Nat Med. 1999;5:
1052–1056.
14. Nicklin SA, Reynolds PN, Brosnan MJ, et al. Analysis of cell-specific
promoters for viral gene therapy targeted at the vascular endothelium.
Hypertension. 2001;38:65–70.
15. O’Connell KA, Edidin M. A mouse endothelial cell line immortalized by
simian virus 40 binds lymphocytes and retains functional characteristics
of normal endothelial cells. J Immunol. 1990;144:521–525.
16. Nicklin SA, White SJ, Watkins SJ, et al. Selective targeting of gene
transfer to vascular endothelial cells by use of peptides isolated by phage
display. Circulation. 2000;102:231–237.
17. Seisenberger G, Ried MU, Endreß T, et al. Real-time single-molecule
imaging of the infection pathway of an adeno-associated virus. Science.
2001;294:1929 –1932.
18. Pajusola K, Gruchala M, Joch H, et al. Cell-type-specific characteristics
modulate the transduction efficiency of adeno-associated virus type 2 and
restrain infection of endothelial cells. J Virol. 2002;76:11530 –11540.
19. Nathwani AC, Davidoff A, Hanawa H, et al. Factors influencing in vivo
transduction by recombinant adeno-associated viral vectors expressing
the human factor IX cDNA. Blood. 2001;97:1258 –1265.
20. Xie Q, Bu W, Bhatia S, et al. The atomic structure of adeno-associated
virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Sci
U S A. 2002;99:10405–10410.
21. Hansen J, Qing K, Kwon H-J, et al. Impaired intracellular trafficking of
adeno-associated virus type 2 vectors limits efficient transduction of
murine fibroblasts. J Virol. 2000;74:992–996.
22. Hansen J, Qing K, Srivastava A. Adeno-associated virus type 2-mediated
gene transfer: altered endocytic processing enhances transduction efficiency in murine fibroblasts. J Virol. 2001;75:4080 – 4090.
23. Duan D, Yue Y, Yan Z, et al. Endosomal processing limits gene transfer
to polarised airway epithelia by adeno-associated virus. J Clin Invest.
2000;105:1573–1587.
24. Shi W, Bartlett JS. RGD inclusion in VP3 provides adeno-associated
virus type 2 (AAV2)-based vectors with a heparan sulfate-independent
cell entry mechanism. Mol Ther. 2003;7:515–525.
25. Pasqualini R, Koivunen E, Ruoslahti E. ␣v integrins as receptors for
tumor targeting by circulating ligands. Nat Biotechnol. 1997;15:542–546.
Downloaded from http://circ.ahajournals.org/ by guest on June 9, 2014