Positron emission tomography imaging of novel AAV capsids maps rapid brain accumulation

ARTICLE

Positron emission tomography imaging of novel

AAV capsids maps rapid brain accumulation

Jai Woong Seo

1,2

, Elizabeth S. Ingham

, Lisa Mahakian

, Spencer Tumbale

,BoWu

, Sadaf Aghevlian

Shahin Shams

, Mo Baikoghli

, Poorva Jain

, Xiaozhe Ding

, Nick Goeden

, Tatyana Dobreva

Nicholas C. Flytzanis

, Michael Chavez

, Kratika Singhal

, Ryan Leib

, Michelle L. James

David J. Segal

, R. Holland Cheng

, Eduardo A. Silva

, Viviana Gradinaru

Katherine W. Ferrara

✉

Adeno-associated viruses (AAVs) are typically single-stranded deoxyribonucleic acid

(ssDNA) encapsulated within 25-nm protein capsids. Recently, tissue-speci

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,

1/2

=

12.7 h) is coupled to the viral capsid. We

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

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

Molecular Imaging Program at Stanford (MIPS), Department of Radiology, School of Medicine, Stanford University, Stanford, CA, USA.

Department of

Biomedical Engineering, University of California, Davis, CA, USA.

Department of Molecular and Cellular Biology, University of California, Davis, CA, USA.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.

Department of Bioengineering, Stanford University,

Stanford, CA, USA.

Stanford University Mass Spectrometry, Stanford, CA, USA.

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)

, 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

–

. 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

. 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

and more than 200

clinical trials have been conducted since 1989

. Recently, AAVs

have been shown capable of delivering CRISPR-Cas9 gene

silencing in vivo

, 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)

. 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

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

Many of these methods are invasive, relying on small quantities of

tissue at a single site and/or time point

. 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

. 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

. 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

. 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

–

to PEG

, or adding

peptides

, antibodies

, or small molecules

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

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

. Alternatively, reporter gene imaging

has been used to measure transduction but cannot quantify PK

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

. 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 (

1/2

of 110 and 68 min, respectively); thus,

limiting their utility (Fig.

a). The dose for systemic adminis-

tration of AAVs in mice is low; ~10

–

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

; however, combining

CuCl

(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

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

–

. Based on surface solvent accessibility in the X-ray

structure of the AAV9 capsid

, 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.

b). This includes 7

–

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

−

−

) orthogonal reaction

We modi

fi

ed a small number of the surface lysines with Tz-NHS

ester, followed by conjugation of the

Cu-multichelator-

transcyclooctene (TCO), (NOTA)

-TCO (Fig.

c). Multi-

chelator-maleimide, (NOTA)

-MI, was employed (Fig.

d) to

label AAV9-TC. Notation describing the labeled AAVs (e.g.

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Cu-PHP.eB) is de

fi

ned in Fig.

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

The multichelators, (NOTA)

-TCO and (NOTA)

-MI, were

synthesized through a solid phase reaction. Multistep coupling of

Fmoc-Lys(Fmoc)-OH from polyethylene glycol(PEG)

-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)

-NH

(Supplementary Fig. 2a, 1) from the resin, 1 was

further functionalized to (NOTA)

-TCO (Supplementary Fig. 2a,

2) and (NOTA)

-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)

-A555-TCO with a

cysteine introduced to conjugate A555-maleimide was synthe-

sized as shown in Supplementary Fig. 2b. The mass (M

+

+

)of

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.

Solvent accessible surface of AAV9 capsid

(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.

are the labeling schemes of AAV-PHP.eB, AAV9, and AAV9-TC.

Surface modi

cation with multichelators (MC) on lysine residues in capsids. (NOTA)

-TCO (incorporating a PEG

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

-NHS (Tz-NHS) ester, 1x PBS (pH 8), 4 °C,

overnight dialysis in 20 kDa molecular weight cut-off (MWCO) membrane).

The site-speci

c radiolabeling on cysteine residues in AAV9-TC was

employed with the multichelator-maleimide conjugate ((NOTA)

-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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(NOTA)

-cys(SH)-NH

(4), (NOTA)

-A555-NH

(5) and

(NOTA)

-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.

fi

ciency of Cu-64 incorporation on single and multichelator

To assess the incorporation of copper on the multichelator,

increasing amounts of (NOTA)

-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)

-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)

-TCO that can be incubated with PHP.eB:

CAG-GFP

(and similarly (NOTA)

-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.

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

, 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

(v:v,

8:2), as is routinely exploited in preparation of antibody con-

jugates. Under this reaction condition, PHP.eB (4.2 × 10

vg, 0.7

pmol particles) was incubated with Tz-NHS and followed an

IEDDA reaction with (NOTA)

-TCO (Supplementary Fig. 4a).

SDS-PAGE analysis of PHP.eBs labeled with (NOTA)

-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)

-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

vg, 9.6

pmol) after reduction to HS-AAV9-TC by TCEP was reacted

with (NOTA)

-MIs at 14, 70, and 140 pmol (Supplementary

Fig. 5a). Multiple bands of over-labeled VPs were found when the

incubated (NOTA)

-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

. 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

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

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.

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.

b). Most

importantly, the transduction ef

fi

ciency of

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

vg). The GFP mean

fl

uores-

cence intensity (MFI) within the brain was similar (Fig.

c, d,

=

4, n.s.) following injection of the unmodi

fi

ed (PHP.eB) or the

labeled AAV (

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.

e). The band location of the three VPs (blue bands)

between Tz-PHP.eB and

Cu-PHP.eB, Tz-AAV9 and

Cu-

AAV9 and HS-AAV9-TC and

Cu-AAV9-TC were similar. The

relative radiolabeling of VP3 was greater than VP1 or VP2 for

PHP.eB and AAV9 (Fig.

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

amino acid

. As a result, the gel image from auto-

radiography of

Cu-AAV9-TC showed the VP2 band as the

major radiolabeled VP whereas the protein staining (blue) of

Cu-AAV9-TC was similar to the ratio of each VP (VP1:VP2:

VP3, 1:1:10) (right column image of Fig.

e).

PHP.eB size was unchanged after

Cu-multichelator labeling

The size of

Cu-labeled and unlabeled PHP.eB was 27.9 ± 0.64

and 27.1 ± 0.68 nm (

=

3), respectively, and detected as a single

peak (Supplementary Fig. 6).

Proteomic analysis of modi

fi

ed lysines on the capsid protein

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

, remained unreacted. Based on the

crystal structure of AAV9

, 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.

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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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

ed AAVs (lower row, Tz-PHP.eB, Tz-AAV9,

and HS-AAV9-TC) at 1 × 10

AAV/cell.

Percentage of green

uorescent positive (GF

+

) HEK293T cells 2 days after incubation with unmodi

ed AAVs

(PHP.eB, AAV9, and AAV9-TC, white bar with black circles) and the corresponding modi

ed AAVs (gray bar with black squares), assessed by

cytometry. The frequency of GF

+

cells treated with unmodi

ed and modi

ed AAVs was similar and distinct from non-treated (NT, black triangles) cells

(

=

4 per group).

Representative GFP images of sagittal brain sections from a C57BL/6 mouse at 3 weeks after tail vein administration of

Cu-PHP.eB,

PHP.eB (1.5 × 10

vg) or saline (negative control) and

mean

uorescence intensity (MFI) of sagittal brain sections (

Cu-PHP.eB: gray bar with black

squares, PHP.eB: white bar with black circles, saline: black triangles,

=

4).

SDS-PAGE of modi

ed AAVs (Tz-PHP.eB, Tz-AAV9, and HS-AAV9-TC; lane

1) and radiolabeled AAVs (

Cu-PHP.eB,

Cu-AAV9, and

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.

Illustration of AAV9 capsid with modi

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.

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 (

). Signi

cance is presented as n.s.

(not signi

cant), *

≤

0.05, **

≤

0.01, and ***

≤

0.001. Whole gel and gel autoradiography images and

values are shown in the source data. Scale bars:

100

μ

), 2 mm (

), 50 nm (

, left), 20 nm (

, 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

–

equivalents of (NOTA)

-A555-TCO gave 5.4 ± 2.3 (

=

6) and

3.5 ± 3.0 (

=

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 (

=

4) ea/vg of A555-AAV9-TC

(Supplementary Table 3).

Furthermore, since the 5 kDa size of the (NOTA)

-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.

g).

AAV radiolabeling was achieved with high radiochemical

purity

. The 20

–

35 pmol of (NOTA)

-TCO and (NOTA)

-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

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

Cu-AAV9, -AAV9-

TC, and -PHP.eB capsids (as de

fi

ned in Fig.

) were assessed in

C57BL/6 mice (

=

3/group) with PET/CT as illustrated in

Fig.

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.

b, Supplementary

Movies 1

–

3). Blood circulation of AAV9 (

1/2

=

5.0 h) was longer

than that of AAV9-TC (

1/2

=

2.4 h) and PHP.eB (

1/2

=

3.1 h)

(Fig.

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

can

reduce stability, potentially resulting in a protein corona or

aggregation over time

. 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 (

=

=

0.0096 at 0 h,

=

0.0004 at 4 h,

=

0.0007 at 21 h) and

AAV9-TC (

=

=

0.0116 at 0 h,

=

0.0003 at 4 h,

=

0.0006

at 21 h) (Fig.

c, Supplementary Table 6). Maximum uptake (%

ID/cc) of

Cu-PHP.eB in the entire brain was 35% ID/cc, with

the spatial maximum observed in the midbrain (Fig.

d) and

strong midbrain uptake clearly visualized in the projected PET/

CT brain image (Fig.

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)

A555-PHP.eB cleared at a similar rate from the blood pool

over 21 h (

1/2

=

4.8 h vs 5.3 h, respectively) (Fig.

f, left,

Supplementary Tables 4 and 7) and the blood clearance was

similar to that observed by PET (Fig.

c). The biodistribution of

PHP.eB and (NOTA)

-A555-PHP.eB was similar in the major

organs such as the brain, heart, liver, spleen, kidney and blood

(Fig.

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.

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.

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.

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.

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)

-A555-TCO) conjugated to the

multichelator construct in a manner similar to the (NOTA)

TCO conjugate (Supplementary Fig. 7a). The binding of A555-

PHP.eB, A555-AAV9 (Supplementary Fig. 9), (NOTA)

-A555-

PHP.eB (Fig.

i) or (NOTA)

-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

, the PK, brain uptake and biodistribution of

Cu-PHP.eB were assessed by comparing BALB/c and C57BL/6

mice (Fig.

a, Supplementary Table 9). Dramatically-reduced

brain uptake of

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.

a) (

=

=

0.0048 at 0 h,

=

0.0002 at 4 h,

=

0.004 at

21 h) and PET/CT images at 0 h (Fig.

b). Similar results (% ID/g)

were observed in the brain radioactivity from the biodistribution

at 21 h (Fig.

=

=

0.0193). In addition, the uptake of

PHP.eB was greater in the liver of BALB/c than C57BL/6 mice

(

=

=

0.0627 at 4 h) (Fig.

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

. 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.

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

; 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

. In our study, nasal administration of

NA was followed by IV injection of

Cu-PHP.eB and

biodistribution at 21 h in BALB/c mice. Lung and brain

accumulation were increased by NA administration 1.4 (

=

=

0.0229) and 2.0-fold (

=

=

0.0364), respectively (Fig.

f).

The treatment by NA also increased the PHP.eB circulation time

from 2.4 h to 5.6 h (Fig.

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

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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

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

, 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

Cu-PHP.eB, -AAV9, and -AAV9-TC. PET images are acquired at 0, 4 and 21 h after AAV tail vein

administration.

Projected PET/CT images at 4 (left) and 21 h (right) (H heart, L liver, S spleen, B brain).

Time activity curves (over 21 h) and

maximum

brain uptake (at 4 h) of

Cu-PHP.eB (magenta triangle),

Cu-AAV9 (black circle), and

Cu-AAV9-TC (turquoise square) from the ROI analysis of blood

and brain (

=

3) after tail vein administration.

Representative projected PET/CT image at 4 h of

Cu-PHP.eB within the brain (B brain, JV jugular vein).

PK (left) and 21-h biodistribution (right) of PHP.eB (

=

3, black circle) and (NOTA)

-A555-PHP.eB (

=

4, black squares) obtained by qPCR.

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

Cu-PHP.eB for PET/CT and autoradiography and non-radioactive

Cu-PHP.eB

for the GFP image.

Cu-AAVs brain accumulation (

=

3 per group) measured 30 min after tail vein administration (left) and Logan plots (right) of brain

uptake rate after AAV administration.

Representative confocal images of (NOTA)

-A555-PHP.eB (red) on brain endothelium (green) acquired 4, 24, and

48 h after tail vein injection. White arrows indicate (NOTA)

-A555-PHP.eBs (red). Data are shown as mean ± SD. One-way ANOVA with Tukey

’

s multiple

comparison test (

, and

(left)) compared means of the three groups. Multiple unpaired

tests with the Holm-Sidak method with alpha

=

0.05

compared the means in

. Signi

cance: n.s. (not signi

cant), *

≤

0.05, **

≤

0.01, and ***

≤

0.001.

values are shown in the source data. Intensity values

, and (

, left) are percent injected dose per cubic centimeter (% ID/cc). Scale bars: 2 mm (

), 25

μ

Fig. 4 Strain and neuraminidase-dependent pharmacokinetics of

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 (

=

3 per group). Radioactivity from ROI analysis is presented as % ID/cc.

Representative PET/CT projection images (B brain, H heart, L

liver) acquired over 30 min after tail vein injection of

Cu-PHP.eB to C57BL/6 and BALB/c mice.

Biodistribution (% ID/g) of

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 (

=

3).

Sliced PET/CT image (H heart,

L liver) at 4 h after tail vein injection of

Cu-PHP.eB.

Time activity curve of

Cu-PHP.eB measured from C57BL/6 (black circle) and BALB/c (turquoise

square) livers (

=

3).

Biodistribution (21 h) of

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) (

=

3). Data are shown as mean ± SD. For statistical analysis, a

one-way ANOVA with Tukey

’

s multiple comparison test in

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

’

-test was performed in

, 0, 4, and 21 h) and

. Signi

cance is presented as *

≤

0.05, **

≤

0.01.

values are shown in the source data. Maximum and minimum intensity

values of PET/CT images in

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

. 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

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

and delayed blood

clearance

; 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

. 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

. PKM2 can be used

with the reporter probe [

F]DASA-23

, 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

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

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)

-NH

. Branched (NH

)

-NH

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

C for 5 min with Fmoc-lys

(Boc)-OH (3 equivalents, 1.47 mmol, 113 mg), 0.2 M Fmoc-PEG

-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(

-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(

-bu ester) conjugation was monitored by TNBS assay.

When the TNBS test was positive, NOTA-bis(

-bu ester) conjugation was per-

formed one more time. After the cleavage of the (NOTA)

-NH

mixture from resin

in a cocktail of TFA (95%), water (2.5%), and TIPS (2.5%), (NOTA)

-NH

(

=

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)

-NH

was con

fi

rmed by MALDI-TOF ([M

+

+

], exact mass was cal-

culated at 4627.62 and found at 4627.20 Da) (Supplementary Fig. 2a).

Synthesis of (NOTA)

-transcyclooctene (TCO)

. To a solution of (NOTA)

-NH

(1, 3.3 mg, 0.7

μ

mol) in 1x PBS (1 mL, pH 7.8), transcyclooctene-PEG

-NHS

(TCO-PEG

-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

–

4 mL) and concentrated using a 3 kDa MWCO spin

fi

lter unit. Dilution and

concentration steps were repeated. (NOTA)

-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)

-TCO was con

fi

rmed by MALDI-TOF

(Supplementary Fig. 2a).

Synthesis of (NOTA)

-A555-TCO

. (NOTA)

-cys(SH)-NH

(4) was similarly

synthesized by adding cysteine and a mono-PEG sequence between PEG

and the

lysine from (NOTA)

-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)

-cys(SH)-NH

. (NOTA)

-cys(SH)-NH

(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)

-A555-NH

(5,

1 mg, 0.17

μ

mol). (NOTA)

-A555-NH

(1 mg, 0.17

μ

mol) reacted with TCO-PEG

NHS (3 mg, 5.8

μ

mol) in 1xPBS (1 mL, pH 8) gave (NOTA)

-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)

-maleimide (MI)

. To a solution of (NOTA)

-NH

(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)

(NHS-

PEG

-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)

-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)

-MI) was con

fi

rmed by MALDI-TOF ([M

+

+

], 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.

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