ARTICLE
Positron emission tomography imaging of novel
AAV capsids maps rapid brain accumulation
Jai Woong Seo
1,2
, Elizabeth S. Ingham
2
, Lisa Mahakian
2
, Spencer Tumbale
1
,BoWu
1
, Sadaf Aghevlian
1
,
Shahin Shams
2
, Mo Baikoghli
3
, Poorva Jain
1
, Xiaozhe Ding
4
, Nick Goeden
4
, Tatyana Dobreva
4
,
Nicholas C. Flytzanis
4
, Michael Chavez
5
, Kratika Singhal
6
, Ryan Leib
6
, Michelle L. James
1
,
David J. Segal
7
, R. Holland Cheng
3
, Eduardo A. Silva
2
, Viviana Gradinaru
4
&
Katherine W. Ferrara
1
✉
Adeno-associated viruses (AAVs) are typically single-stranded deoxyribonucleic acid
(ssDNA) encapsulated within 25-nm protein capsids. Recently, tissue-speci
fi
c AAV capsids
(e.g. PHP.eB) have been shown to enhance brain delivery in rodents via the LY6A receptor on
brain endothelial cells. Here, we create a non-invasive positron emission tomography (PET)
methodology to track viruses. To provide the sensitivity required to track AAVs injected at
picomolar levels, a unique multichelator construct labeled with a positron emitter (Cu-64,
t
1/2
=
12.7 h) is coupled to the viral capsid. We
fi
nd that brain accumulation of the PHP.eB
capsid 1) exceeds that reported in any previous PET study of brain uptake of targeted
therapies and 2) is correlated with optical reporter gene transduction of the brain. The PHP.
eB capsid brain endothelial receptor af
fi
nity is nearly 20-fold greater than that of AAV9. The
results suggest that novel PET imaging techniques can be applied to inform and optimize
capsid design.
https://doi.org/10.1038/s41467-020-15818-4
OPEN
1
Molecular Imaging Program at Stanford (MIPS), Department of Radiology, School of Medicine, Stanford University, Stanford, CA, USA.
2
Department of
Biomedical Engineering, University of California, Davis, CA, USA.
3
Department of Molecular and Cellular Biology, University of California, Davis, CA, USA.
4
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.
5
Department of Bioengineering, Stanford University,
Stanford, CA, USA.
6
Stanford University Mass Spectrometry, Stanford, CA, USA.
7
Genome Center and Department of Biochemistry and Molecular
Medicine, University of California, Davis, CA, USA.
✉
email:
kwferrar@stanford.edu
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1
1234567890():,;
T
herapeutic delivery to the brain has traditionally been
limited in volume. The background level of protein/
nanotherapeutics reaching the brain is on the order of 0.1
percent injected dose per cubic centimeter (% ID/cc)
1
, necessi-
tating more ef
fi
cient methods of delivery. Engineered adeno-
associated viruses (AAVs), single-stranded deoxyribonucleic acid
(ssDNA) encapsulated within 25-nm protein capsids, have
recently shown potential to greatly increase transduction as
compared with previous therapeutics
2
–
4
. AAVs can infect divid-
ing and non-dividing cells and result in highly ef
fi
cient long-term
transduction in a broad range of tissues
5
,
6
. This is particularly
signi
fi
cant as AAV gene therapy has a solid safety pro
fi
le, was
fi
rst
approved by the FDA in December 2017
7
and more than 200
clinical trials have been conducted since 1989
8
. Recently, AAVs
have been shown capable of delivering CRISPR-Cas9 gene
silencing in vivo
9
, expanding their potential utility. Using a
directed evolution approach to viral capsid engineering and
selection, AAV-PHP.eB, containing a 2-mer substitution and 7-
mer peptide insertion in a surface exposed loop of the capsid,
enhanced neuronal transduction throughout the brain compared
to the conventionally used AAV serotype 9 (AAV9)
3
. This 40 to
90-fold increased ef
fi
ciency is believed to result from a novel
interaction between virus and the brain endothelial cell receptor
LY6A
10
,
11
.
In vivo imaging has great potential to contribute to the design
and optimization of AAVs. The biodistribution of viral vectors
has previously been evaluated by real-time PCR, Southern blot-
ting of the transduced gene, western blotting, immunohis-
tochemistry (IHC), and in vivo imaging of reporter proteins
12
.
Many of these methods are invasive, relying on small quantities of
tissue at a single site and/or time point
13
. In vivo imaging can
determine the reporter protein level expressed from a transduced
gene across an entire region of interest (ROI) over time; however,
the underlying mechanisms for differences in the reporter protein
cannot be directly identi
fi
ed with this approach. Development of
a labeling method for non-invasive pharmacokinetics (PK) stu-
dies is valuable for several reasons. First, in vivo imaging can
directly and non-invasively assess endothelial receptor binding at
multiple time points. Second, PK can be non-invasively assessed
even with repeated administration, the potential for which
increases since capsid engineering and cargo development also
address issues related to AAV neutralization and
immunogenicity
5
,
14
,
15
. Third, quantitative imaging techniques
facilitate comparisons across strains and species.
We therefore set out to develop an imaging method to track
therapeutic viral constructs and quantify their binding to endo-
thelial surface receptors. Positron emission tomography (PET)
provides an ideal non-invasive method to track viral constructs in
brain-related and other diseases
16
. In particular graphical analysis
of plasma and tissue radiotracer uptake at multiple time points
produces a linear plot, the slope of which is related to the number
of available tracer binding sites. PET facilitates the interpretation
of endothelial binding and the quanti
fi
cation of reversible
receptor binding
17
,
18
. This provides a unique noninvasive
assessment of AAV uptake.
PET imaging has not previously been applied for systemic
AAV tracking. Surface modi
fi
cation of AAVs has previously
focused on tagging
fl
uorophores
19
–
24
to PEG
25
,
26
, or adding
peptides
27
,
28
, antibodies
23
, or small molecules
29
to the capsid.
Given that most earlier generations of AAV and other viral
therapies were not designed for speci
fi
c organ targeting, imaging
studies labeled multiple lysines on the capsid with a lesser impact
on organ-speci
fi
c endothelial targeting and transduction
30
.
Recently, the AAV capsid was labeled with I-124, but the study
was limited to direct intracranial injection to the brain and
therefore did not focus on the receptor binding characteristics or
endothelial accumulation
31
. Alternatively, reporter gene imaging
has been used to measure transduction but cannot quantify PK
32
.
Thus, our study
fi
lls a void in PET imaging of the PK of novel
capsids.
The challenge in monitoring the PK of systemically injected
AAVs with PET (particularly with high time resolution) is to
achieve a trackable level of radioactivity while matching the half-
life of the positron emitter to AAV circulation half-life, which
ranges from minutes to days
33
. An additional challenge is to
minimize conjugation to key AAV surface features. High molar
activity (MA) positron emitters, such as F-18 and Ga-68, typically
have a short half-life (
t
1/2
of 110 and 68 min, respectively); thus,
limiting their utility (Fig.
1
a). The dose for systemic adminis-
tration of AAVs in mice is low; ~10
11
–
12
vector genomes (vg) are
injected, corresponding to 0.2
–
2 pmol of AAVs. Cu-64 has a half-
life of 12.7 h and is therefore well suited to the AAV half-life in
blood
34
; however, combining
64
CuCl
2
(MA, ~20
μ
Ci/pmol) and 2
pmol of AAVs yields ~40
μ
Ci of labeled AAVs when the labeling
ratio of Cu-64 to AAVs is 1:1. Real-time high resolution imaging
is impaired with this very low level of radioactivity. In order to
facilitate high signal-to-noise (SNR) PET imaging at a low AAV
dose, we have therefore synthesized a bifunctional multichelator
that increases the MA of
64
Cu/molecule up to 10 times compared
to a single chelator.
Our study highlights the potential to use PET imaging to track
viral capsids after systemic injection, facilitating noninvasive
quantitation of organ accumulation and clearance and endothelial
receptor binding. The multichelator approach developed here is
applied for optical microscopy, system-level PET imaging and
autoradiography. Based on these analyses, we
fi
nd that brain
accumulation of PHP.eB, a novel AAV9 derivative with high
brain tropism, exceeds that reported in previous PET studies of
brain uptake of targeted therapies. Further, the high signal-to-
noise ratio obtained with the multichelator approach can be
exploited to quantify endothelial receptor af
fi
nity over the
fi
rst 30
min after injection. Here, brain af
fi
nity of the PHP.eB capsid is
enhanced nearly 20-fold as compared with the well-established
AAV9 capsid. Most importantly, the labeling method retains the
transduction ef
fi
cacy of the AAV and can be applied in future
studies to inform and optimize the design of AAVs and other
viral capsids.
Results
Syntheses of multichelators
. We have developed a bio-
orthogonal approach for coupling a multichelator and AAV,
based on conjugation to AAV surface lysines and cysteines and
used this approach to compare the PK of AAV9-PHP.eB (AAV9-
PHP.eB is denoted as PHP.eB hereafter) with AAV9 and AAV9-
tetracysteine (AAV9-TC). Conjugation to surface lysines was
previously shown to be feasible in
fl
uorescence imaging where
AAV surface lysines were modi
fi
ed with a
fl
uorescent dye, which
facilitated AAV tracking without hampering transduction ef
fi
-
ciency
19
–
22
,
35
. Based on surface solvent accessibility in the X-ray
structure of the AAV9 capsid
30
, the estimated number of exposed
lysines on AAV9 and PHP.eB ranges from 420 to 480 out of 1185
and 1245 total lysines, respectively (Fig.
1
b). This includes 7
–
8
lysines per viral protein (VP), with one viral particle composed of
60 units of VP. We based the surface lysine labeling strategies on
inverse electron demand Diels
–
Alder reactions (IEDDA), which
offer a fast, quantitative (>50,000 M
−
1
S
−
1
) orthogonal reaction
36
.
We modi
fi
ed a small number of the surface lysines with Tz-NHS
ester, followed by conjugation of the
64
Cu-multichelator-
transcyclooctene (TCO), (NOTA)
8
-TCO (Fig.
1
c). Multi-
chelator-maleimide, (NOTA)
8
-MI, was employed (Fig.
1
d) to
label AAV9-TC. Notation describing the labeled AAVs (e.g.
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64
Cu-PHP.eB) is de
fi
ned in Fig.
1
c, d. In order to con
fi
rm that the
lysine attachment does not alter receptor binding, we compared
labeling with this approach to labeling of cysteines available on a
tetracysteine (TC) engineered version of AAV9 using a
maleimide-thiol reaction of the multichelator (Supplementary
Fig. 1). In this virus, an (HRWCCPGCCKTF) motif was inserted
at amino acid 139 (residue numbered by VP1 sequence) of the
AAV9 capsid proteins (AAV9-139). The insertion site is located
in the N-terminal disordered region of capsid proteins VP1 and
VP2. A similar modi
fi
cation was made in AAV9 and was shown
to be accessible by labeling reagents without compromising viral
integrity
24
.
The multichelators, (NOTA)
8
-TCO and (NOTA)
8
-MI, were
synthesized through a solid phase reaction. Multistep coupling of
Fmoc-Lys(Fmoc)-OH from polyethylene glycol(PEG)
27
-Lys(Boc)
on resin afforded eight branched amines, further coupled with
tert-
Bu-NOTA-OH. A PEG spacer was included to separate the
multichelator and reactive functional group. After cleavage of
(NOTA)
8
-NH
2
(Supplementary Fig. 2a, 1) from the resin, 1 was
further functionalized to (NOTA)
8
-TCO (Supplementary Fig. 2a,
2) and (NOTA)
8
-MI (Supplementary Fig. 2a, 3). In all, 2 and 3
were isolated by HPLC presented monoisotopic mass peaks at
5026.67 (calculated mass: 5026.85 Da) and 4937.66 (calculated
mass: 4937.74 Da) in MALDI mass analysis (Supplementary
Fig. 2a), respectively. For optical studies of PHP.eB and AAV9
conjugated with the multichelator, (NOTA)
8
-A555-TCO with a
cysteine introduced to conjugate A555-maleimide was synthe-
sized as shown in Supplementary Fig. 2b. The mass (M
+
H
+
)of
a
b
c
d
Fig. 1 Strategy for labeling AAVs with a positron emitter. a
Table presents the number of AAVs systemically injected and the molar activity of positron
emitters.
b
Solvent accessible surface of AAV9 capsid
30
(PDB ID:3UX1) displayed by PyMOL software. Insets highlight a trimer around a threefold axis.
Orange and green color represent lysine and cysteine residues, respectively. Solvent radius is set as 1.4 angstrom. AAV capsid is composed of
60 structurally identical viral protein subunits (VPs) with 1:1:10 ratio of VP1:VP2:VP3.
c
,
d
are the labeling schemes of AAV-PHP.eB, AAV9, and AAV9-TC.
c
Surface modi
fi
cation with multichelators (MC) on lysine residues in capsids. (NOTA)
8
-TCO (incorporating a PEG
27
spacer) is employed for the
radiolabeling of Tz-AAV9 or Tz-PHP.eB after reaction of Tz-NHS ester with AAV9 or PHP.eB. (i, tetrazine-PEG
5
-NHS (Tz-NHS) ester, 1x PBS (pH 8), 4 °C,
overnight dialysis in 20 kDa molecular weight cut-off (MWCO) membrane).
d
The site-speci
fi
c radiolabeling on cysteine residues in AAV9-TC was
employed with the multichelator-maleimide conjugate ((NOTA)
8
-MI) incorporating Cu-64 after the reduction of tetracysteine with TCEP (ii, TCEP in 1x
PBS (pH 7.0
–
7.5)). Asterisk indicates average molar radioactivity of Cu-64 from commercial vendor as used in this study.
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3
(NOTA)
8
-cys(SH)-NH
2
(4), (NOTA)
8
-A555-NH
2
(5) and
(NOTA)
8
-A555-TCO (6) veri
fi
ed with MALDI mass analysis
were detected at 4874.54 (calculated mass: 4875.70 Da), 5844.85
(calculated mass: 5843.94) and 6245.47 (calculated mass:
6243.16), respectively.
Ef
fi
ciency of Cu-64 incorporation on single and multichelator
.
To assess the incorporation of copper on the multichelator,
increasing amounts of (NOTA)
8
-TCO were reacted with a known
amount of nonradioactive Cu-63 spiked with the radioactive Cu-
64, and the incorporation ratio was then compared with that
achieved with the single chelator (NOTA-TCO). We con
fi
rmed
that more than two single chelators, NOTA-TCOs, are required
to incorporate one copper molecule (Supplementary Fig. 3a), and
one multichelator, (NOTA)
8
-TCO, incorporated 5
–
8 copper
molecules (Supplementary Fig. 3b). Thus, the multichelator
achieves ~10 times higher molar radioactivity than the single
chelator (Supplementary Fig. 3).
Capsid surface modi
fi
cation maintains transduction ef
fi
ciency
.
To determine the maximum molar ratio of Tz-NHS and
(NOTA)
8
-TCO that can be incubated with PHP.eB:
CAG-GFP
(and similarly (NOTA)
8
-MI with AAV9-TC:
CAG
-
mNeonGreen
)
without hampering its integrity, we monitored the AAV trans-
duction ef
fi
ciency in HEK293T cells before and after labeling
(Fig.
2
a, b, Supplementary Figs. 4 and 5). In previous studies, the
conjugation of NHS (succinimidyl ester) to AAVs typically pro-
ceeded under strong basic conditions (0.1 M Na
2
CO
3
, pH 9.3);
however, the reported procedures have been inconsistently
detailed, and the experimental conditions vary widely (summar-
ized in Supplementary Table 1). To avoid harsh conditions, the
reaction was performed at pH 8 by mixing PBS and Na
2
CO
3
(v:v,
8:2), as is routinely exploited in preparation of antibody con-
jugates. Under this reaction condition, PHP.eB (4.2 × 10
11
vg, 0.7
pmol particles) was incubated with Tz-NHS and followed an
IEDDA reaction with (NOTA)
8
-TCO (Supplementary Fig. 4a).
SDS-PAGE analysis of PHP.eBs labeled with (NOTA)
8
-TCO
clearly showed three VP bands with similar molecular weight to
the unmodi
fi
ed control PHP.eB (Supplementary Fig. 4b). Incu-
bation with a molar ratio of 500 and above resulted in additional
high molecular-weight protein bands (Supplementary Fig. 4b)
and a signi
fi
cant reduction in
fl
uorescent-protein expressing cells
(Supplementary Fig. 4c). Both assays suggest that keeping the
molar ratio of (NOTA)
8
-TCO/PHP.eB below 500-fold maintains
transduction ef
fi
ciency of HEK293T cells and prevents the
aggregation of capsid proteins after labeling of PHP.eB. Limits on
the concentration of the chelator were more restrictive with
AAV9-TC. AAV9-TC:
CAG-mNeonGreen
(5.8 × 10
12
vg, 9.6
pmol) after reduction to HS-AAV9-TC by TCEP was reacted
with (NOTA)
8
-MIs at 14, 70, and 140 pmol (Supplementary
Fig. 5a). Multiple bands of over-labeled VPs were found when the
incubated (NOTA)
8
-MI/AAV9-TC ratio was 70-fold or more
(Supplementary Fig. 5b). For AAV9-TC, multiple bands likely
result from the non-speci
fi
c maleimide conjugation with primary
amines as previously reported
37
. In this previous report, a similar
protein band shift occurred in SDS-PAGE at dye:protein ratios
>40:1. Irrespective of the multiple band formation, AAV9-TC
transduction ef
fi
ciency was preserved at all levels of modi
fi
cation
(Supplementary Fig. 5c).
Under the optimized conditions, AAV9 and PHP.eB were then
labeled with multichelators that have been conjugated with Cu-64
following the procedure detailed in the Methods section.
Transduction ef
fi
ciencies of Tz-AAV9 and -PHP.eB (modi
fi
ed
from AAV9:
CAG-mNeonGreen
and PHP.eB:
CAG-GFP
with Tz-
NHS, respectively) and HS-AAV9-TC (a reduced form of AAV9-
TC:
CAG-mNeonGreen
) were then compared with those of intact
AAVs in HEK293T cells in vitro (1 × 10
6
vg/cell). As assessed by
fl
uorescent green protein expression in HEK293T cell images,
fl
uorescent-protein transduction was similar at 24 h after
incubation with intermediates before and after modi
fi
cation
(Fig.
2
a). Flow cytometry at 48 h con
fi
rmed the comparable
transduction ef
fi
ciency of modi
fi
ed and unmodi
fi
ed AAVs for Tz-
AAV9 and -PHP.eB and HS-AAV9-TC (Fig.
2
b). Most
importantly, the transduction ef
fi
ciency of
63
Cu-labeled PHP.
eB:
CAG-GFP
and PHP.eB:
CAG-GFP
in C57BL/6 mice was
evaluated to determine whether the multichelator in
fl
uenced
viral delivery and GFP production in the brain at 3 weeks after
tail vein administration (1.5 × 10
10
vg). The GFP mean
fl
uores-
cence intensity (MFI) within the brain was similar (Fig.
2
c, d,
n
=
4, n.s.) following injection of the unmodi
fi
ed (PHP.eB) or the
labeled AAV (
63
Cu-PHP.eB) and undetectable after saline
injection. Taken together, the results demonstrate that our
optimized labeling condition preserved the AAV
’
s functional
properties.
Characterization of radiolabeled capsid on viral proteins
. The
viral protein (VP) bands were visualized via protein staining
(blue, 1st and 2nd lane), and the radiolabeled VP bands were
imaged with sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis (SDS-PAGE) and autoradiography (gray, 3rd lane)
(Fig.
2
e). The band location of the three VPs (blue bands)
between Tz-PHP.eB and
64
Cu-PHP.eB, Tz-AAV9 and
64
Cu-
AAV9 and HS-AAV9-TC and
64
Cu-AAV9-TC were similar. The
relative radiolabeling of VP3 was greater than VP1 or VP2 for
PHP.eB and AAV9 (Fig.
2
e), directly related to the ratio of
protein abundance for the three VPs, 1:1:10 (VP1, VP2, VP3).
AAV9-TC was generated by site-speci
fi
c insertion of the
HRWCCPGCCKTF peptide motif at the VP1/VP2 interface at
the 139
th
amino acid
24
. As a result, the gel image from auto-
radiography of
64
Cu-AAV9-TC showed the VP2 band as the
major radiolabeled VP whereas the protein staining (blue) of
64
Cu-AAV9-TC was similar to the ratio of each VP (VP1:VP2:
VP3, 1:1:10) (right column image of Fig.
2
e).
PHP.eB size was unchanged after
63
Cu-multichelator labeling
.
The size of
63
Cu-labeled and unlabeled PHP.eB was 27.9 ± 0.64
and 27.1 ± 0.68 nm (
n
=
3), respectively, and detected as a single
peak (Supplementary Fig. 6).
Proteomic analysis of modi
fi
ed lysines on the capsid protein
.
We
fi
rst determined the lysine sites modi
fi
ed with Tz-NHS by
proteomic analysis. Mass lists from the analyses of excised gel
bands of VP1, VP2, and VP3 after reaction of PHP.eB with Tz-
NHS showed that tetrazines were predominantly incorporated on
two lysines (K557 and K567) (Supplementary Table 2), which
exist in all VPs. Importantly, the lysine at the 595th amino acid
located within the engineered peptide sequences (DGTLAVPFK),
critical for the distribution of PHP.eB to the brain and trans-
duction of its transgene
3
, remained unreacted. Based on the
crystal structure of AAV9
30
, neither of the modi
fi
ed sites are
located in the core of the threefold-proximal spikes, the region
considered to be responsible for most virus-host interactions.
K567 is located in the valley between the spikes, while K557 is on
the distal shoulder (Fig.
2
f). To the best of our knowledge, the two
sites have not been reported to be involved in receptor binding for
AAV9 derivatives.
Determination of the number of AAV labels
. We compared
results from optical labeling and electron microscopy to deter-
mine the number of labels. We examined the number of labels per
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ab
c
e
fg
d
Fig. 2 Transduction and labeling ef
fi
ciency of surface modi
fi
ed AAVs. a
Fluorescence microscopy images of human embryonic kidney(HEK) 293T cells
after 24 h incubation with intact AAVs (upper row, PHP.eB, AAV9, and AAV9-TC) and corresponding modi
fi
ed AAVs (lower row, Tz-PHP.eB, Tz-AAV9,
and HS-AAV9-TC) at 1 × 10
6
AAV/cell.
b
Percentage of green
fl
uorescent positive (GF
+
) HEK293T cells 2 days after incubation with unmodi
fi
ed AAVs
(PHP.eB, AAV9, and AAV9-TC, white bar with black circles) and the corresponding modi
fi
ed AAVs (gray bar with black squares), assessed by
fl
ow
cytometry. The frequency of GF
+
cells treated with unmodi
fi
ed and modi
fi
ed AAVs was similar and distinct from non-treated (NT, black triangles) cells
(
n
=
4 per group).
c
Representative GFP images of sagittal brain sections from a C57BL/6 mouse at 3 weeks after tail vein administration of
63
Cu-PHP.eB,
PHP.eB (1.5 × 10
10
vg) or saline (negative control) and
d
mean
fl
uorescence intensity (MFI) of sagittal brain sections (
63
Cu-PHP.eB: gray bar with black
squares, PHP.eB: white bar with black circles, saline: black triangles,
n
=
4).
e
SDS-PAGE of modi
fi
ed AAVs (Tz-PHP.eB, Tz-AAV9, and HS-AAV9-TC; lane
1) and radiolabeled AAVs (
64
Cu-PHP.eB,
64
Cu-AAV9, and
64
Cu-AAV9-TC; lane 2 and 3). The three bands depict viral protein (VP) 1
–
3 (L: standard
protein ladder). Lane 1
–
3 illustrate blue-stained VPs (lanes 1 and 2) and radiolabeled VPs (lane 3), respectively.
f
Illustration of AAV9 capsid with modi
fi
ed
lysines. Left: full view of AAV9, middle and right: top and side views of trimer viral proteins, respectively. The K557 (yellow) and K567 (red) lysine r
esidues
are highlighted.
g
Field view of direct-electron cryoEM images of PEG(40 kDa)-AAV9 (left image) and enhanced projection images of selected
PEG(40 kDa)-AAV9 capsids (six right images). White arrows mark the 40 kDa PEG molecules extended from the selected AAV capsids. Data are shown
as mean ± SD. Brown-Forsythe and Welch ANOVA with Dunnett
’
s T3 multiple comparison test compares means (
b
,
d
). Signi
fi
cance is presented as n.s.
(not signi
fi
cant), *
P
≤
0.05, **
P
≤
0.01, and ***
P
≤
0.001. Whole gel and gel autoradiography images and
P
values are shown in the source data. Scale bars:
100
μ
m(
a
), 2 mm (
c
), 50 nm (
g
, left), 20 nm (
g
, right).
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5
capsid using the multi-armed
fl
uorescent label (Supplementary
Figs. 2b and 7a) combined with quanti
fi
cation of the AAV con-
centration through a titer. We applied this approach for both the
amine- and thiol-directed coupling approaches. Labeling with
200
–
350 equivalents of Tz-NHS and reaction with 10
–
15
equivalents of (NOTA)
8
-A555-TCO gave 5.4 ± 2.3 (
n
=
6) and
3.5 ± 3.0 (
n
=
8) labels per PHP.eB and AAV9 capsid, respectively
(Supplementary Table 3). AAV9-TC reduced by 100 equivalents
of TCEP and subsequently reacted with 20 equivalents of A555-
C2 maleimide yielded 0.5 ± 0.3 (
n
=
4) ea/vg of A555-AAV9-TC
(Supplementary Table 3).
Furthermore, since the 5 kDa size of the (NOTA)
8
-A555
conjugate on AAV9 was not reliably visualized on cryogenic
electron microscopy (cryoEM), we conjugated a larger label (PEG
(40 kDa)-TCO) (Supplementary Fig. 7b) to the capsid. This label
was conjugated to AAV9 (denoted PEG(40 kDa)-AAV9) using
the same conditions used for the in vivo imaging and was used to
visualize the number of labels per virus (Supplementary Fig. 7a).
PEG(40 kDa)-AAV9 was obtained from a reaction with Tz-AAV9
and 4 equivalents of PEG(40 kDa)-TCO and showed one to three
labels per capsid on cryoEM images (Fig.
2
g).
AAV radiolabeling was achieved with high radiochemical
purity
. The 20
–
35 pmol of (NOTA)
8
-TCO and (NOTA)
8
-MI
were enough to incorporate >99% of 1
–
2 mCi Cu-64. In situ
incubation of Tz-AAV9 and -PHP.eB and HS-AAV9-TC with
these multichelators yielded
64
Cu-AAV9, -AAV9-TC, and -PHP.
eB to 2.2% (1.78 MBq (48
μ
Ci)), 6.6% (4.88 MBq (132
μ
Ci) and
7.5 ± 6% (3.9 ± 1.7 MBq (106 ± 45
μ
Ci)), respectively (decay cor-
rected). Radiochemical purity of radiolabeled AAVs on instant
radio-thin layer chromatography was above 98%.
PET imaging quanti
fi
ed brain accumulation and receptor
binding
. The PK and biodistribution of the
64
Cu-AAV9, -AAV9-
TC, and -PHP.eB capsids (as de
fi
ned in Fig.
1
) were assessed in
C57BL/6 mice (
n
=
3/group) with PET/CT as illustrated in
Fig.
3
a. The projection images acquired of AAVs revealed two
remarkable distinctions: the high brain uptake of PHP.eB and the
extended blood circulation of AAV9 (Fig.
3
b, Supplementary
Movies 1
–
3). Blood circulation of AAV9 (
t
1/2
=
5.0 h) was longer
than that of AAV9-TC (
t
1/2
=
2.4 h) and PHP.eB (
t
1/2
=
3.1 h)
(Fig.
3
c, Supplementary Tables 4 and 5). The faster clearance of
PHP.eB from blood, as compared to AAV9, is expected due to
rapid uptake within the brain (Supplementary Movie 2a at 4 h).
The mechanism for the enhanced clearance of AAV9-TC has not
been fully characterized; however, the tetracysteine motif
(HRWCCPGCCKTF) on AAV9-TC can remain reactive after
reduction by TCEP, and S-thiolation by serum proteins
38
can
reduce stability, potentially resulting in a protein corona or
aggregation over time
39
. Thus, while the initial (30 min) receptor
binding is expected to be similar, clearance from blood through
the liver and intestine (over hours) is expected to be enhanced for
AAV9-TC (Supplementary Movie 3a at 4 h). Brain accumulation
of PHP.eB was greater at all time points than that of AAV9 (
n
=
3,
P
=
0.0096 at 0 h,
P
=
0.0004 at 4 h,
P
=
0.0007 at 21 h) and
AAV9-TC (
n
=
3,
P
=
0.0116 at 0 h,
P
=
0.0003 at 4 h,
P
=
0.0006
at 21 h) (Fig.
3
c, Supplementary Table 6). Maximum uptake (%
ID/cc) of
64
Cu-PHP.eB in the entire brain was 35% ID/cc, with
the spatial maximum observed in the midbrain (Fig.
3
d) and
strong midbrain uptake clearly visualized in the projected PET/
CT brain image (Fig.
3
e). In addition, in order to assess the PK of
multichelator-labeled PHP.eB compared to that of PHP.eB, we
performed classical qPCR. Un-labeled PHP.eB and (NOTA)
8
-
A555-PHP.eB cleared at a similar rate from the blood pool
over 21 h (
t
1/2
=
4.8 h vs 5.3 h, respectively) (Fig.
3
f, left,
Supplementary Tables 4 and 7) and the blood clearance was
similar to that observed by PET (Fig.
3
c). The biodistribution of
PHP.eB and (NOTA)
8
-A555-PHP.eB was similar in the major
organs such as the brain, heart, liver, spleen, kidney and blood
(Fig.
3
f, right, Supplementary Table 8).
Sliced sagittal brain images in the cerebral cortex, thalamus,
midbrain and cerebellum from PET/CT (after 21 h), autoradio-
graphy (after 21 h), and ex vivo GFP
fl
uorescence (after 3 weeks)
showed a consistent distribution of the viral capsid tag and
corresponding transduced GFP protein expression (Fig.
3
g,
Supplementary Fig. 8). PHP.eB accumulation in the brain was
then analyzed in dynamic 5-min intervals over the
fi
rst 30 min.
4% and 10% ID/cc of PHP.eB was bound at 5 and 30 min,
respectively, whereas AAV9 and AAV9-TC accumulation was
<1% ID/cc (Fig.
3
h, Supplementary Fig. 8). Furthermore, Logan
plots, based on a reversible accumulation model, demonstrated
that the 30 min accumulation in brain was greater for PHP.
eB»AAV9 ~ AAV9-TC, with a distribution volume of 0.210,
0.011, and 0.011, respectively (Fig.
3
h). Thus, the af
fi
nity of PHP.
eB for the brain endothelium is estimated to be 20-fold higher
than for AAV9 and AAV9-TC. The receptor af
fi
nity of AAV9
was identical for the two labeling methods as assessed by the
initial 30-min Logan plot (Fig.
3
h).
Multichelator does not alter PHP.eB endothelial accumulation
.
We employed two optical probes: a probe in which an optical dye
(A555-NHS ester) was attached to the native capsid lysines and a
second optical probe ((NOTA)
8
-A555-TCO) conjugated to the
multichelator construct in a manner similar to the (NOTA)
8
-
TCO conjugate (Supplementary Fig. 7a). The binding of A555-
PHP.eB, A555-AAV9 (Supplementary Fig. 9), (NOTA)
8
-A555-
PHP.eB (Fig.
3
i) or (NOTA)
8
-A555-AAV9 (Supplementary
Fig. 10) to the brain endothelium, observed by confocal micro-
scopy at 4, 24, and 48 h after injection (Z-stack images in Sup-
plementary Movie 4), showed that punctate clusters were
observed at 4 h after injection. The
fl
uorescence intensity gra-
dually diminished by 24 h and was similar to saline injection
(Supplementary Fig. 10) at 48 h. Taken together, the optical and
PET images suggest that the early-bound PHP.eB crossed the BBB
within 48 h and speci
fi
c and effective binding of PHP.eB to the
brain endothelium was con
fi
rmed.
PET imaging elucidates strain and treatment-dependent PK
.
Since mouse strain dependence of PHP.eB BBB transcytosis has
been reported
10
,
40
, the PK, brain uptake and biodistribution of
64
Cu-PHP.eB were assessed by comparing BALB/c and C57BL/6
mice (Fig.
4
a, Supplementary Table 9). Dramatically-reduced
brain uptake of
64
Cu-PHP.eB was con
fi
rmed in BALB/c with
respect to C57BL/6 mice from the time-activity curve over 21 h
(Fig.
4
a) (
n
=
3,
P
=
0.0048 at 0 h,
P
=
0.0002 at 4 h,
P
=
0.004 at
21 h) and PET/CT images at 0 h (Fig.
4
b). Similar results (% ID/g)
were observed in the brain radioactivity from the biodistribution
at 21 h (Fig.
4
c,
n
=
3,
P
=
0.0193). In addition, the uptake of
PHP.eB was greater in the liver of BALB/c than C57BL/6 mice
(
n
=
3,
P
=
0.0627 at 4 h) (Fig.
4
d, e and Supplementary
Table 10). While the enhanced liver accumulation is anticipated
given the lack of brain accumulation, a strain-speci
fi
c immune
response has also been reported to enhance liver accumulation in
the BALB/c strain
41
,
42
. Further, the circulation time of PHP.eB
(3.1 h) in the BALB/c mouse (2.4 h) was slightly lower than that
in C57BL/6 mice (Fig.
4
a, Supplementary Table 4). The results
reaf
fi
rm the reduced brain uptake in BALB/c mice, which, unlike
C57BL/6 mice, lack the LY6A receptor that the engineered PHP.
eB binds to
10
; however, other differences also exist in the PK
between strains.
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Previously, pretreatment with neuraminidase (NA) in vitro and
in vivo, was reported to expose terminal N-linked galactose and
enhance AAV9 binding
43
,
44
. In our study, nasal administration of
NA was followed by IV injection of
64
Cu-PHP.eB and
biodistribution at 21 h in BALB/c mice. Lung and brain
accumulation were increased by NA administration 1.4 (
n
=
3,
P
=
0.0229) and 2.0-fold (
n
=
3,
P
=
0.0364), respectively (Fig.
4
f).
The treatment by NA also increased the PHP.eB circulation time
from 2.4 h to 5.6 h (Fig.
4
a). However, the increased accumulation
was relatively small as compared to the differences in brain
accumulation of PHP.eB between strains.
Discussion
We found that radiolabeling AAVs with a unique multichelator
construct allows for a detailed and quantitative study of AAV
biodistribution and pharmacokinetics. Following conjugation of a
dendrimer-like radioactive tag to novel AAVs, the fraction of
a
c
f
g
hi
de
b
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7
radioactivity delivered to the brain was extraordinarily high, thus
demonstrating the potential use of AAVs as carriers. PET, with
appropriate analytic methods, was applied to noninvasively
quantify the endothelial receptor binding with a resolution of
minutes. We found that Logan analysis is particularly important
for characterizing the binding of novel AAV capsids as a rapid
and largely irreversible accumulation occurs on the endothelium
of the target tissue. No alternative technique can quantify binding
of viral capsids in real time; such information is critically
important for capsid engineering and for the identi
fi
cation of the
endothelial receptors responsible for AAV accumulation and
transcytosis.
Recent reports indicate that enhanced brain transduction by
PHP.eB in C57BL/6 mice requires an LY6A receptor-mediated
pathway that is independent of galactose and is absent in BALB/c
mice and non-human primates
10
.
64
Cu-PHP.eB can be exploited
as a non-invasive tool to measure endothelium binding mediated
by the LY6A receptor in various mouse strains and other species.
Furthermore, the binding of serotype AAVs to the AAV receptor
(AAVR), identi
fi
ed as a critical host factor for infection of
naturally-occurring AAVs
45
, can be assessed by a simple blocking
study with novel AAVs.
Optical imaging of the tagged capsid validated the accumula-
tion of the capsids on the brain endothelium within minutes and
Fig. 3 PET and optical imaging-based assessment of AAV pharmacokinetics in C57BL/6 mice. a
Experimental setup for region of interest (ROI) analysis
(0, 4, and 21 h) and biodistribution (21 h) of
64
Cu-PHP.eB, -AAV9, and -AAV9-TC. PET images are acquired at 0, 4 and 21 h after AAV tail vein
administration.
b
Projected PET/CT images at 4 (left) and 21 h (right) (H heart, L liver, S spleen, B brain).
c
Time activity curves (over 21 h) and
d
maximum
brain uptake (at 4 h) of
64
Cu-PHP.eB (magenta triangle),
64
Cu-AAV9 (black circle), and
64
Cu-AAV9-TC (turquoise square) from the ROI analysis of blood
and brain (
n
=
3) after tail vein administration.
e
Representative projected PET/CT image at 4 h of
64
Cu-PHP.eB within the brain (B brain, JV jugular vein).
f
PK (left) and 21-h biodistribution (right) of PHP.eB (
n
=
3, black circle) and (NOTA)
8
-A555-PHP.eB (
n
=
4, black squares) obtained by qPCR.
g
Sliced
PET/CT, autoradiography and GFP images of sagittal section of mouse brain (CB cerebellum, M midbrain, Th thalamus, CC cerebral cortex, S striatum)
acquired at 21 h, 21 h and 3 weeks, respectively, after tail vein injection of
64
Cu-PHP.eB for PET/CT and autoradiography and non-radioactive
63
Cu-PHP.eB
for the GFP image.
h
64
Cu-AAVs brain accumulation (
n
=
3 per group) measured 30 min after tail vein administration (left) and Logan plots (right) of brain
uptake rate after AAV administration.
i
Representative confocal images of (NOTA)
8
-A555-PHP.eB (red) on brain endothelium (green) acquired 4, 24, and
48 h after tail vein injection. White arrows indicate (NOTA)
8
-A555-PHP.eBs (red). Data are shown as mean ± SD. One-way ANOVA with Tukey
’
s multiple
comparison test (
c
,
d
, and
h
(left)) compared means of the three groups. Multiple unpaired
t-
tests with the Holm-Sidak method with alpha
=
0.05
compared the means in
f
. Signi
fi
cance: n.s. (not signi
fi
cant), *
P
≤
0.05, **
P
≤
0.01, and ***
P
≤
0.001.
P
values are shown in the source data. Intensity values
in
b
,
d
,
e
, and (
g
, left) are percent injected dose per cubic centimeter (% ID/cc). Scale bars: 2 mm (
g
), 25
μ
m(
i
).
a
de
f
bc
Fig. 4 Strain and neuraminidase-dependent pharmacokinetics of
64
Cu-PHP.eB. a
Time activity curves of PHP.eB obtained from region of interest (ROI)
analysis of blood (left) and brain (right) from C57BL/6 (black circle), BALB/c (turquoise square), and neuraminidase-treated BALB/c (magenta tria
ngle)
mice over 21 h (
n
=
3 per group). Radioactivity from ROI analysis is presented as % ID/cc.
b
Representative PET/CT projection images (B brain, H heart, L
liver) acquired over 30 min after tail vein injection of
64
Cu-PHP.eB to C57BL/6 and BALB/c mice.
c
Biodistribution (% ID/g) of
64
Cu-PHP.eB in brain (left)
and blood (right) in C57BL/6 (gray bar with black squares) and BALB/c (white bar with black circle) mice at 21 h (
n
=
3).
d
Sliced PET/CT image (H heart,
L liver) at 4 h after tail vein injection of
64
Cu-PHP.eB.
e
Time activity curve of
64
Cu-PHP.eB measured from C57BL/6 (black circle) and BALB/c (turquoise
square) livers (
n
=
3).
f
Biodistribution (21 h) of
64
Cu-PHP.eB in blood (left), brain (middle), and lung (right) from BALB/c with no treatment white bar with
black circle) versus BALB/c mice treated with neuraminidase (gray bar with black square) (
n
=
3). Data are shown as mean ± SD. For statistical analysis, a
one-way ANOVA with Tukey
’
s multiple comparison test in
a
was performed to compare means of three groups (C57BL/6 vs BALB/c: turquoise, C57BL/6
vs BALB/c (neuraminidase): magenta, BALB/c vs BALB/c (neuraminidase): black) at each time point. Unpaired two-tailed Welch
’
s
t
-test was performed in
c
,(
e
, 0, 4, and 21 h) and
f
. Signi
fi
cance is presented as *
P
≤
0.05, **
P
≤
0.01.
P
values are shown in the source data. Maximum and minimum intensity
values of PET/CT images in
b
,
d
are presented as percent injected dose per cubic centimeter (% ID/cc).
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the elimination of the punctate
fl
uorescence on the endothelium
over 24 h. Given the very small size of the AAVs (25 nm),
tracking of their
fl
uorescence within the brain was not feasible.
PET imaging complemented the information in the optical image
by demonstrating that the accumulated radioactivity remains
within the brain over 21 h. Classical pharmacokinetic analysis
based on a PCR further validated the brain accumulation. Given
that the radioactivity was tagged to the capsid surface, the dis-
tribution of radioactivity throughout the brain suggests that the
capsids were transcytosed across the BBB. This observation
supports previous reports of AAV transcytosis across the BBB, as
observed in vitro
46
. Combined PET and optical reporter gene
imaging demonstrated that the pattern of transduction within the
brain was similar to the distribution of the radioactive tag.
The stoichiometry of
fl
uorophore-TCO conjugates on the
surface of AAV9 and PHP.eB was on average four labels per
particle. Labeling of a
fl
uorescence-maleimide conjugate on
AAV9-TC conferred 0.6 label per capsid. CryoEM of AAV9
conjugated with PEG(40 kDa) similarly showed 2
–
3 copies of
extended PEG-string density per capsid. Our AAV surface
modi
fi
cation with tetrazine-NHS ester retained transduction
ef
fi
ciency in HEK cells and in in vivo transduction studies. Fol-
lowing conjugation with the 5 kDa multichelator at a multi-
chelator:AAV ratio of 1:2 PHP.eB, binding to the brain
endothelium and transduction were maintained. Future work will
focus on optimizing the number and size of the conjugated tags.
Here, we minimized the number of tags per AAV in the in vivo
studies of transduction to minimize any effect on transport. Given
the relatively small loading capacity of AAVs, the potential to
conjugate additional cargo to the capsid could be transformative.
Multiple gene editing components, complementary therapeutics
or additional imaging tags can be attached to the surface.
The predominantly-labeled lysines of each viral protein within
PHP.eB were K557 and K567. K61, K92, K528, K618, K696, and
K700 were modi
fi
ed in a smaller fraction of capsids (Supple-
mentary Table 2). We speculate that K557 and K567 are sus-
ceptible to reaction with the NHS-ester and that this is the basis
of their enhanced modi
fi
cations. The variable region VII (aa545-
aa558), including K557, is located within a region of the AAV
capsid associated with liver transduction
47
and delayed blood
clearance
30
,
33
; however, to our best knowledge, there is no report
on the direct involvement of K557 or K567 in host receptor
binding. Recently, unnatural amino acids (UAA) bearing an azide
were site-speci
fi
cally engineered in AAV2 (at aa 587) and AAV-
DJ (a derivative of AAV8, at aa 589) capsids and utilized to
conjugate oligonucleotides
48
. Speci
fi
c protocols can also be
developed for labeling of other capsids. For example, there is a
lysine adjacent to K557 on AAV2 and adjacent to K567 on AAV1,
6, 8, 9, and 10 (Supplementary Table 2). Therefore, addition or
substitution of lysines, cysteines or UAAs within these sequences
or other AAVs can also provide a unique labeling site.
Finally, the PET method developed here to monitor binding
and pharmacokinetics will be paired with PET reporter gene
imaging in future work. A PET reporter gene based on pyruvate
kinase (PKM2) has been shown to have low background in the
brain and can be packaged within AAVs
49
. PKM2 can be used
with the reporter probe [
18
F]DASA-23
49
, which is permeable to
the blood-brain barrier in order to monitor brain transduction
over months or years. In the future, we will couple this tracer with
the PET tag described here.
Methods
Materials and reagents
. All solvents were purchased from Fisher Chemical,
Sigma-Aldrich and Acros. The reagents and materials for multichelator synthesis
were purchased from Novabiochem, Click Chemistry Tools and Biotage. PEG
27
spacers were purchased from Chem-Impex International Inc. and Polypure. PEG
(40 kDa)-amine (Creative PEGWorks) was purchased from Fisher Scienti
fi
c. For
the capsid SDS-PAGE, the gels, buffer, standard ladder and protein staining
reagents were purchased from ThermoFisher Scienti
fi
c. AFDye555-maleimide
(Fluoroprobes), AlexaFluor555-NHS ester (ThermoFisher Scienti
fi
c) and
AlexaFluor555-C2-maleimide (ThermoFisher Scienti
fi
c), each with 555 excitation
max and 580 emission max, are denoted as A555 throughout. The detailed list of
materials and reagents is in the Supplementary Methods.
Cell line and AAVs
. Human embryonic kidney cells (HEK293T) were obtained
from ATCC (CRL-1573). AAV9, AAV9-TC, and PHP.eB packaging including
CAG-mNeonGreen
or
CAG-DIO-GFP
were prepared as described in the Supple-
mentary Methods section entitled
“
Preparation of AAV9, AAV9-TC, and PHP.eB
”
.
All AAVs were used within two months of preparation. AAV-PHP.eB packaging
CAG-GFP
was purchased from Addgene (#37825-PHP.eB). All AAVs used for
in vitro/in vivo transduction, PET/CT, and optical studies are summarized in
Supplementary Table 11.
Synthesis of (NOTA)
8
-NH
2
. Branched (NH
2
)
8
-NH
2
was synthesized on rink
amide resins (0.49 mmol/g, 0.15 g) in a microwave-assisted solid phase synthesizer
(Initiator
+
Alstra, Biotage) as shown in Supplementary Fig. 2a. Sequential cou-
pling reaction was programed to be performed at 75
o
C for 5 min with Fmoc-lys
(Boc)-OH (3 equivalents, 1.47 mmol, 113 mg), 0.2 M Fmoc-PEG
27
-OH (3
equivalents, 1.47 mmol, 372 mg), 0.2 M Fmoc-lys(Fmoc)-OH (3 equiv., 1.47 mmol,
131 mg), 0.2 M Fmoc-lys(Fmoc)-OH (5 equivalents, 2.45 mmol, 219 mg), and 0.2
M Fmoc-lys(Fmoc)-OH (4.9 equivalents, 439 mg) with 0.1 or 0.5 M HBTU (one
equivalent of each amino acids) and 0.2 M DIPEA (two equivalents of each amino
acids). The volume of each coupling reaction was maintained to be 3
–
5 mL DMF.
After drying resins under a vacuum, NOTA-bis(
t
-bu ester) (10 equivalents of
primary amine on resin, 100 mg, 0.24 mmol) was manually further coupled to the
lysine residue (eight amines per mole loading level, 0.25 mmol/g, 100 mg, 0.025
mmol) on resin with HBTU (89 mg, 0.24 mmol) and DIPEA (83 mg, 0.64 mmol) in
DMF (2 mL). NOTA-bis(
t
-bu ester) conjugation was monitored by TNBS assay.
When the TNBS test was positive, NOTA-bis(
t
-bu ester) conjugation was per-
formed one more time. After the cleavage of the (NOTA)
8
-NH
2
mixture from resin
in a cocktail of TFA (95%), water (2.5%), and TIPS (2.5%), (NOTA)
8
-NH
2
(
n
=
2,
13 ± 2 mg, 2.8
μ
mol) was isolated by HPLC (acetonitrile gradient from 5% to 60%
with 0.1%TFA solution for 30 min, retention time: 15.5 min). The mass of
(NOTA)
8
-NH
2
was con
fi
rmed by MALDI-TOF ([M
+
H
+
], exact mass was cal-
culated at 4627.62 and found at 4627.20 Da) (Supplementary Fig. 2a).
Synthesis of (NOTA)
8
-transcyclooctene (TCO)
. To a solution of (NOTA)
8
-NH
2
(1, 3.3 mg, 0.7
μ
mol) in 1x PBS (1 mL, pH 7.8), transcyclooctene-PEG
4
-NHS
(TCO-PEG
4
-NHS, 10 mg, 19
μ
mol,) dissolved in DMSO (80
μ
L) was added. pH
was readjusted to 8, and the reaction mixture was stirred in a vortex mixer at
room temperature for 2
–
3 h. The solution was diluted with double distilled-water
(3
–
4 mL) and concentrated using a 3 kDa MWCO spin
fi
lter unit. Dilution and
concentration steps were repeated. (NOTA)
8
-TCO (2, 1 mg, 0.4
μ
mol) was isolated
by HPLC (acetonitrile gradient from 5% to 60% with 0.1%TFA solution for 30 min,
retention time: 24.4 min). Mass of (NOTA)
8
-TCO was con
fi
rmed by MALDI-TOF
(Supplementary Fig. 2a).
Synthesis of (NOTA)
8
-A555-TCO
. (NOTA)
8
-cys(SH)-NH
2
(4) was similarly
synthesized by adding cysteine and a mono-PEG sequence between PEG
27
and the
lysine from (NOTA)
8
-TCO as shown in Supplementary Fig. 2b. After isolation of
the product with HPLC, the MALDI-TOF spectrum con
fi
rmed the mass of
(NOTA)
8
-cys(SH)-NH
2
. (NOTA)
8
-cys(SH)-NH
2
(2 mg, 0.41
μ
mol) was reacted
with AF555-maleimide (1 mg, 0.79
μ
mol, Fluoroprobe, Az) in PBS, then the iso-
lation with 3 kDa MWCO spin
fi
lter and HPLC afforded (NOTA)
8
-A555-NH
2
(5,
1 mg, 0.17
μ
mol). (NOTA)
8
-A555-NH
2
(1 mg, 0.17
μ
mol) reacted with TCO-PEG
4
-
NHS (3 mg, 5.8
μ
mol) in 1xPBS (1 mL, pH 8) gave (NOTA)
8
-A555-TCO (6, 350
μ
g, 0.06
μ
mol) after HPLC puri
fi
cation (Supplementary Fig. 2b). The AF555, the
fl
uorophore in AF555-MI, is denoted as A555 in the conjugated form.
Synthesis of (NOTA)
8
-maleimide (MI)
. To a solution of (NOTA)
8
-NH
2
(1, 2 mg,
0.7
μ
mol) in 1x PBS (0.5 mL, pH 7.4), 0.1 M EDTA (10
μ
L) and SM(PEG)
2
(NHS-
PEG
2
-MI, 4 mg, 9.4
μ
mol) dissolved in DMSO (30
μ
L) were added. The reaction
mixture was stirred in a vortex mixer at room temperature for 1 h. The solution
was then diluted with 0.05% TFA in water (3
–
4 mL) and concentrated using a 3
kDa MWCO spin
fi
lter unit. Dilution and concentration steps were repeated.
(NOTA)
8
-MI (3, 1 mg, 0.2
μ
mol) was isolated by HPLC (acetonitrile gradient from
5% to 60% with 0.1%TFA solution for 30 min, retention time: 16.6 min). Mass of
(NOTA)
8
-MI) was con
fi
rmed by MALDI-TOF ([M
+
H
+
], exact mass was cal-
culated at 4937.74 and found at 4937.66) (Supplementary Fig. 2a).
Titration and LC-MS/MS
. Detailed methods for the titration of single- and
multichelators with Cu-63/Cu-64 (shown in Supplementary Fig. 3) and for LC-MS/
MS analysis of the Tz-PHP.eB capsid (shown in Supplementary Table 2) are
available in the Supplementary Methods section.
NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15818-4
ARTICLE
NATURE COMMUNICATIONS
| (2020) 11:2102 | https://doi.org/10.1038/s41467-020-15818-4 | www.nature.com/naturecommunications
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