of 27
Supplementary information
Positron emission tomography
imaging
of
novel
AAV capsid
s
maps rapid brain
accumulation
J. W.
Se
o et al.
Contents:
Supplementary Methods
Supplementary Tables 1
-
11
Supplementary Figures 1
-
1
1
Supplementary References
Supplementary methods
Materials and reagents for multichelator synthesis and AAV labeling.
Rank Amide
-
Chemmatrix®
resin (Biotage®, 7
-
600
-
1310
-
25)
,
HBTU
(
Novabiochem, 851006,
N,N,N′,N′
-
t
etramethyl
-
O
-
(1H
-
benzotriazol
-
1
-
yl)uronium hexafluorophosphate, O
-
(benzotriazol
-
1
-
yl)
-
N,N,N′,N
-
tetramethyluronium
hexafluorophosphate
),
DIPEA
(Sigma
-
Aldrich, 496219,
N,N
-
d
iisopropylethylamine
)
TIPS
(Sigma
-
Aldrich, 233781,
triisopropylsilane
),
Fmoc
-
PEG
27
-
OH
(Polypure, 15137
-
2790), Fmoc
-
PEG
3
-
OH (Chem
-
Impex International Inc.,07310),
4
-
methyl
piperidine (Acros, 127515000, 99%)
TFA
(Acros,
13972, trifluoroacetic acid),
NOTA
-
bis(t
-
Bu ester)
(
Macrocyclics, B
-
620
,
1,4,7
-
t
riazacyclono
nane
-
1,4
-
bis
-
tert
-
butyl acetate
-
7
-
acetic acid)
, t
etrazine
-
PEG
5
-
NHS ester (Click Chemistry Tools, 1143)
,
t
ranscyclooctene
-
PEG
4
-
NHS ester: (Click Chemistry Tools, A137)
,
SM(PEG)
2
(ThermoFisher Scientific,
22102
, s
uccinimidyl
-
[(
N
-
maleimidopropionamido)
-
diethy
leneglycol] ester
)
. Alexa555
-
NH
S (ThermoFisher
Scientific, A20009), AF555
-
maleimide (Fluoroprobes, 1168)
.
Alexa555
-
C2
-
maleimide (ThermoFisher
Scientific,
A20346
)
.
Preparation of AAV9, AAV9
-
TC, and PHP.eB
.
Tetracysteine
motif HRWCCPGCCKTF was cloned into
an AAV9 backbone
(
tTA
-
iCAP
-
AAV9)
using overlap PCR into position 139 of AAV9 capsid region (
AAV9
-
TC
-
forward:
CATCGATGGTGTTGCCCGGGCTGCTGTAAGACTTTCgctcctggaaagaagaggcctgtag and
AAV9
-
TC
-
reverse:
GAAAGTCTTACAGCAGCCCGGGCAACACCATCGATGcgtcttagccgcttcctcaaccaga).
AAV9
-
TC and AAV9 packaging
CAG
-
mNeonGreen
were
produced following protocol outlined in Challis,
et al
1
.
Briefly, HEK293T cells (ATCC) were triple transfect
ed using polyethylenimine (PEI); virus was
collected after 120
h
from both cell lysates and media and purified over iodixanol (Optiprep, Sigma) via
ultracentrifugation.
Maleimide labelling of AAV9
-
TC was confirmed per methods described in Chandran,
el al
2
. P
HP.eB packaging
CAG
-
mNeonGreen
or
CAG
-
DIO
-
GFP
w
ere
also
prepared
similar to above.
Titration of single
-
and multichelator
s
with Cu
-
63/Cu
-
64 for
Supplementary Fig. 3
.
A 20 pmol of single
chelator in double distilled water (2
μ
L) was added to two
-
fold serial dilutions of
CuCl
2
solution (3
μ
L) from
0.
0
1
mM to 0.3 n
M
, spiked with 0.33 pmol of
64
CuCl
2
(1
μ
L). The titration of 20 pmol of multichelator was
performed with
CuCl
2
solution (3
μ
L) from
0.1
mM to 0.3 n
M
, spiked with 14 pmol of
64
CuCl
2
(1
μ
L). After
30 min incubation of reaction mixtures at room temperature, incorporation yields were confirmed by ITLC.
The ratio of Cu/chelator was plotted over incorporation yield as shown in
Supplementary Fig. 3.
In vitro
transduction assay
for
(NOTA)
8
-
PHP
.eB
for
Supplementary Fig. 4,5
.
Evaluation of
PHP.eB
:
CAG
-
GFP
transduction
after multichelator conjugation
was performed based on the following
methods.
To each PHP.eB:
CAG
-
GFP
(4.2x10
1
1
v
g
) in 1xPBS (180
μ
L, pH adjusted to 8 with 0.1 M Na
2
CO
3
solution (pH 9.1)), 0.7
μ
L of 1, 5, or 10 mM tetrazine
-
PEG
5
-
NHS in anhydrous DMSO was added. After
incubation for 1 hour at room temperature, each reaction mixture was dialyzed in a mini
-
dialysis device
(20 kDa molecular weight cut
-
off (MWCO))
at 4 ºC for
6
h
. To each reaction, 0.7
μ
L of 0.1, 0.5, or 1 mM
(NOTA)
8
-
PEG
27
-
TCO in 1xPBS was added, respectively. The reaction mixture was dialyzed overnight at 4
ºC. Purified AAVs were transferred to microcentrifuge tubes, and the volume of each AAV solution was
ad
justed to 300
μ
L with 1xPBS
as shown in Supplementary Fig. 4A
.
The transduction of
the
CAG
-
GFP
gene by
Tz
-
PHP.eB and PHP.eB were compared in HEK293T cells seeded at a density of 70% in
a
24
-
well plate (3x10
4
cells/well) with 500
μ
L of Dulbecco’s Modified Eagle’s medium (DMEM; Invitrogen)
supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% pen
icillin
streptomycin (Invitrogen)
(DMEMc)
.
After incubation at 37 °C and 5% CO
2
for 24
h
, media was aspirated and replaced
with
fresh
DMEMc
(250
μ
L) at
1x10
6
vg
/cell
of unmodified control (UMC, n=4) PHP.eB
or modified PHP.eB with
(NOTA)
8
-
PEG
27
-
TCO in
the following ratios (100, 500, and 1000 (NOTA)
8
-
PEG
27
-
TCO/AAV ratio, n=4 for
each ratio). Cells were then incubated at 37 °C in a 5% CO
2
chamber
overnight in virus
-
loaded media
before aspiration and replacement with 500
μ
L fresh DMEMc.
Cells were cultured
for one additional day
prior to collection
.
Media was aspirated, and PBS (300
μ
L) was added. Cells were trypsinized and
transferred
to
1.5 mL centrifuge tubes
. Cells were centrifuged at 1500 rpm for 3 minutes at room
temperature and then washed with
1xPBS
. This cell washing cycle was repeated two times. 4%
paraformaldehyde (PFA) was added, and the pelleted cells were further kept for 10 min at room
temperature to fix the cells. Fixed cells were centrifuged and PFA solution removed. Cells were washed
w
ith 1xPBS (500
μ
L), centrifuged, and supernatant was removed. This washing cycle was repeated two
times. After cells were resuspended in 1xPBS (500
μ
L) and transferred to 5 mL flow cytometer tubes on
ice, the frequency of cells with green fluorescence was
analyzed using a flow cytometer (BD FACScan
).
All flow cytometry data w
ere
analyzed using FlowJo v10
.1
software (TreeStar).
In vitro t
ransduction assay for
(NOTA)
8
-
AAV9
-
TC before and after tetracysteine modification
.
AAV9
-
TC
:
CAG
-
mNeonGreen
samples
(5.
8x10
12
vg
, 10 pmo
l
in 1xPBS (100
μ
L) were incubated for 30
minutes after the addition of 5 mM TCEP (5 nmol, 1
μ
L). To each reaction
, 1
μ
L of PBS only, 0.1, 0.5, or 1
mM (NOTA)
8
-
PEG
5
-
MI (0.1, 0.5, and 1 nmol) in 1xPBS was added. After incubation for 1
h
at room
temperature, each reaction mixture was filtered through a spin column (Bio
-
rad
, MWCO 6k) and collected
into centrifuge tubes. For the transduction assay, untreated control PHP.
eB was compared to the dialyzed
AAVs as shown
in Supplementary Fig. 5A. HEK293T
cells
were seeded at a density of 70% in 24
-
well
plates
(3x10
4
cells/well)
with 500
μ
L of DMEMc
(Invitrogen). After incubation for 24
h
,
media was aspirated
and replaced fresh DMEMc
(
250
μ
L
) at
1x10
6
vg
/cells
of unmodified control (n=4) AAV
or treated AAV9
-
TC with (NOTA)
8
-
PEG
27
-
MI in the following ratios (
14
,
70
, and
140
multichelator/AAV ratio, n=4 for each
ratio). Cells were then incubate
d at 37 °C in a humidified CO
2
chamber
overnight in virus
-
loaded media
before aspiration and replacement with 500
μ
L fresh DMEM.
Cells were cultured for one additional day
prior to collection
, fixation and analysis of cellular
mNeonGreen
expression
, which
was done using a flow
cytometer (BD FACScan) as above.
LC
-
MS/MS analysis of Tz
-
PHP.eB capsid
.
For
mass
analysis
of modified capsid protein,
Tz
-
PHP.eB capsid were denatured with
SDS
-
sample buffer
(Thermo Fisher Scientific, San Jose, CA) after incubation for 2 min at 85°C. Denatured sample solution
was run on a 12% tris
-
glycine gel (Thermo Fisher Scientific, San Jose, CA) in tris
-
glycine running buffer
(Thermo Fisher Scientific, Sa
n Jose, CA) and resolved by Coomassie staining. Protein gel bands were
excised out into 1.5 ml Eppendorf tubes and further cut into 1x1 mm squares. The excised gel pieces were
then reduced with 5
mM DTT in the presence of 50
mM ammonium bicarbonate at 55°C
for 30
min.
Residual solvent was removed and alkylation was performed using 10
mM acrylamide in 50
mM
ammonium bicarbonate for 30
min at room temperature. The gel pieces were rinsed 2 times with 50%
acetonitrile, 50
mM ammonium bicarbonate and placed in a
speed vacuum for 5
min. Digestion was
performed with Trypsin/LysC (Promega) in the presence of 0.02% protease max (Promega) in a standard
overnight digest at 37°C. Samples were then centrifuged and the solvent including peptides
was
collected
.
F
urther pep
tide extraction was performed by the addition of 60% acetonitrile, 39.9% water, 0.1% formic
acid and incubation for 10
-
15
min. The peptide pools were dried in a speed vac. Samples were
reconstituted in 12.5 μl reconstitution buffer (2% acetonitrile with 0.
1%
f
ormic acid) and 3 μl of each sample
were injected into the instrument.
For
m
ass spectrometry
,
e
xperiments were performed using an Orbitrap Fusion Tribrid mass
spectrometer (Thermo Scientific, San Jose, CA) with liquid chromatography using a Nanoacqui
ty UPLC
(Waters Corporation, Milford, MA). For a typical LCMS experiment, a flow rate of 450 nL/min was used,
where mobile phase A was 0.2% formic acid in water and mobile phase B was 0.2% formic acid in
acetonitrile. Analytical columns were prepared in
-
ho
use with an
inner diameter
of 100 microns pulled to a
nanospray emitter using a P2000 laser puller (Sutter Instrument, Novato, CA). The column was packed
using C18 reprosil Pur 1.8 micron stationary phase (Dr. Maisch) to a length of ~25 cm. Peptides were
d
irectly injected onto the analytical column using a gradient (2
-
45% B, followed by a high
-
B wash) of 80min.
The mass spectrometer was operated in a data dependent fashion using a combination of ETD in Ion trap
and HCD in Orbitrap fragmentation for MS/MS sp
ectra generation.
The
raw
data files were processed using Byonic v 2.14.27 (Protein Metrics, San Carlos, CA) to
identify peptides and subsequently infer proteins using the
Mus musculus
database from
the Universal
Protein Resource
(UniProt,
http://www.uniprot.org
) along with the sequences of capsid proteins.
Proteolysis with trypsin/lys
-
C mix (0.125 ng, mass grade, Promega corporation) was assumed to be
semi
-
specific allowing for N
-
ragged clea
vage with up to two missed cleavage sites. Precursor and
fragment mass accuracies were held within 12 ppm. Proteins were held to a false discovery rate of 1%,
using standard approaches.
Supplementary Tables
Supplementary Table
1
. Summary of AAV
labeling condition
s
from previous literature
Virus
Amount
(volume
used)
NHS
-
ester
Reaction
ratio
(dye:AAV)
Resulting
dye/AAV
ratio
buffer
Reaction
time
Isolation
Ref
AAV
1.33x10
14
particle
s/mL
(1
mg/
mL)
Cy3
-
or
Cy2
-
NHS
NS
1.6
-
2.3
0.1 M
Na
2
CO
2
NaHCO
3
(pH 9.3)
30 min
Dialysis
3,4
AAV2
1x10
10
particle
s
Alexa488
-
TFP
3x10
6
:1
NS
0.1 M
NaHCO
3
(pH 9.3)
2 h
Di
alysis
5
AAV2
5x10
11
particle
s
(1 mL)
Cy3
-
NHS
NS
1
0.1 M
NaCO
3
(pH 9.3)
30 min
Dialysis
6
AAV
2
-
Alexa488
-
NHS
-
2
-
-
Dialysis
7
AAV
10
12
vg/mL
(2
μ
L used)
SPA
-
PEG
(20ul total
volume)
1
-
1000
(
PEG/lysine)
HEPES
(pH7.4)
3 h
-
8
NS: not
-
specified
Supplementary Table
2
. Protein sequence alignment of serotype AAVs compared to
PHP.eB
.
Red
represents the labeled lysine (K) within adjacent amino acids, and
black
is the corresponding lysine (K)
or arginine (R) site located in a similar sequence in serotype AAV1
-
10.
The most frequently modified
lysines in
PHP.eB
were those shown in the 4
th
and 5
th
columns.
Frequency of modified lysine
s
in mass list
of each viral protein
PHP.eB
NGLD
K
GEPVN
(57
-
66)
NPYL
K
YNHAD
(88
-
97)
MASH
K
EGEDR
(524
-
533)
VDAD
K
VMITN
(553
-
562)
EEEI
K
TTNPV
(563
-
572)
PIWA
K
IPHTD
(614
-
623)
ELQ
K
ENS
K
RW
(693
-
702)
VP1
1
1
-
13
8
-
-
VP2
-
-
3
14
10
-
1
VP3
-
-
4
17
11
3
1
Sequence alignment of serotype AAVs with PHP.eB in modified lysine sites
AAV1
NGLD
K
GEPVN
(57
-
66)
NPYL
R
YNHAD
(88
-
97)
MASH
K
DDEDK
(524
-
533)
TALDNVMITD
(553
-
562)
EEEI
K
ATNPV
(563
-
572)
PIWA
K
IPHTD
(614
-
623)
ELQ
K
ENS
K
RW
(686
-
695)
AAV2
NGLD
K
GEPVN
(57
-
66)
NPYL
K
YNHAD
(88
-
97)
MASH
K
DDEEK
(523
-
532)
VDIE
K
VMITD
(552
-
559)
EEEI
R
TTNPV
(562
-
571)
PIWA
K
IPHTD
(616
-
625)
ELQ
K
ENS
K
RW
(685
-
684)
AAV3
NGLD
K
GEPVN
(57
-
66)
NPYL
K
YNHAD
(88
-
97)
MASH
K
DDEEK
(524
-
533)
AELDNVMITD
(553
-
562)
EEEI
R
TTNPV
(563
-
572)
PIWA
K
IPHTD
(617
-
626)
ELQ
K
ENS
K
RW
(686
-
695)
AAV4
NGLD
K
GEPVN
(56
-
65)
NPYL
K
YNHAD
(87
-
96)
MATAGPADSK
(523
-
532)
TVPGTLIFTS
(551
-
560)
EEELAATNAT
(561
-
70)
PIWA
K
IPHTD
(615
-
624)
EIQ
K
ERS
K
RW
(684
-
693)
AAV5
NGLD
R
GEPVN
(56
-
65)
NPYL
K
YNHAD
(87
-
96)
GMTNNLQGSN
(510
-
519)
YLEGNMLITS
(542
-
551)
ESETQPVNRV
(552
-
561)
PIWA
K
IPETG
(606
-
615)
ELK
K
ENS
K
RW
(674
-
683)
AAV6
NGLD
K
GEPVN
(57
-
66)
NPYL
R
YNHAD
(88
-
97)
MASH
K
DDKDK
(524
-
533)
TALDNVMITD
(553
-
562)
EEEI
K
ATNPV
(563
-
572)
PIWA
K
IPHTD
(617
-
626)
ELQ
K
ENS
K
RW
(686
-
695)
AAV7
NGLD
K
GEPVN
(57
-
66)
NPYL
R
YNHAD
(88
-
97)
MATH
K
DDEDR
(526
-
535)
TTLENVLMTN
(554
-
563)
EEEI
R
PTNPV
(564
-
573)
PIWA
K
IPHTD
(618
-
627)
ELQ
K
ENS
K
RW
(687
-
696)
AAV8
NGLD
K
GEPVN
(57
-
66)
NPYL
R
YNHAD
A (88
-
97)
MATH
K
DDEER
(526
-
535)
ADYSDVMLTS
(553
-
562)
EEEI
K
TTNPV
(565
-
574)
PIWA
K
IPHTD
(619
-
628)
ELQ
K
ENS
K
RW
(688
-
697)
AAV9
NGLD
K
GEPVN
(57
-
66)
NPYL
K
YNHAD
(88
-
97)
MASH
K
EGEDR
(524
-
533)
VDAD
K
VMITN
(553
-
562)
EEEI
K
TTNPV
(563
-
572)
PIWA
K
IPHTD
(617
-
626)
ELQ
K
ENS
K
RW
(686
-
695)
AAV10
NGLD
K
GEPVN
(57
-
66)
NPYL
R
YNHAD
(88
-
197)
MATH
K
DDEER
(526
-
535)
VDYSSVMLTS
(555
-
564)
EEEI
K
TTNPV
(565
-
574)
PIWA
K
IPHTD
(619
-
628)
ELQ
K
ENS
K
RW
(688
-
697)
Supplementary Table
3
.
Characterization of the number of
label
s
per AAV
Amine
-
directed
reaction
Vg (pmol)
NHS
-
Tz
(nmol)
(NOTA)
8
-
A555
-
TCO
(pmol)
n
ea (label)/Vg
(particle)
(mean
±
s
.
d.)
PHP.eB
1.2E12 (2)
0.4
-
0.7
20
-
40
6
5.4 ± 2.3
AAV9
2.0E12 (3.3)
0.7
-
1
30
-
50
8
3.5 ± 3.0
Thiol
-
directed
reaction
Vg (pmol)
TCEP (nmol)
A555
-
C2
-
MI (pmol)
n
AAV9
-
TC
1.7E12 (3)
0.3
60
4
0.5 ± 0.3
Supplementary Table
4
.
AAV clearance half
-
lives and 21
-
hour AUCs following tail
vein administration
in
mice
Vector
Mouse strain
No. of mice
t
1/2
a
(h)
R
2
AUC
0
-
21h
b
(h.%
ID/cc)
64
Cu
-
AAV9
C57BL/6
3
5.0
0.97
581.9
64
Cu
-
AAV9
-
TC
C57BL/6
3
2.4
0.99
344.3
64
Cu
-
PHP.eB
C57BL/6
3
3.1
0.96
391.1
BALB/c
3
2.4
0.96
276.9
BALB/c
(neuraminidase)
c
3
5.
7
1.00
445.
2
PHP
.
eB
C57BL/6
4
4.1
0.96
8.29
d
(NOTA)
8
-
A555
-
PHP.eB
C57BL/6
4
5.3
0.94
8.88
d
a. Adeno
-
associated virus clearance half
-
lives from blood, obtained from one
-
phase decay curve fit. b. Area under
curve (AUC) from time radioactivity curve.
c. Neuraminidase was administered to BALB/c mice through nose.
d.
Area un
der curve
represents a
fraction of remaining AAVs in blood
Supplementary
Table
5
.
Radioactivity (% ID/cc) in blood pool measured from PET/CT images at 0, 4,
and 21
h
.
C57BL/6
BALB/c
Time
(
h
)
64
Cu
-
AAV9
(n=3)
64
Cu
-
AAV9
-
TC
(n=3)
64
Cu
-
PHP.eB
(n=3)
64
Cu
-
PHP.eB
(n=3)
64
Cu
-
PHP.eB
(n=
3
, neuraminidase)
mean
s.d.
mean
s.d.
mean
s.d.
mean
s.d.
mean
s.d.
0
48.4
3.7
51.0
2.5
48.9
4.9
42.8
5.8
46
.
9
0.
3
4
33.6
3.1
19.3
2.3
23.3
5.9
15.6
4.0
29.4
0.
9
21
15.5
2.0
4.6
1.0
5.7
1.0
42.8
5.8
5.0
0.
5
Supplementary Table
6
.
Radioactivity (% ID/cc) in whole brain measured from ROI analyses of PET/CT
images at 0, 4, and 21
h
.
C57BL/6
BALB/c
Time
(
h
)
64
Cu
-
AAV9
(n=3)
64
Cu
-
AAV9
-
TC
(n=3)
64
Cu
-
PHP.eB
(n=3)
64
Cu
-
PHP.eB
(n=3)
64
Cu
-
PHP.eB
(n=
3
,
neuraminidase)
mean
s.
d.
mean
s.
d.
mean
s.
d.
mean
s.
d.
mean
s.
d.
0
3.94
0.
39
4.21
0.
27
10.7
3.1
2.81
0.
53
3.46
0.
38
4
3.03
0.
70
2.31
0.
06
19.2
4.0
1.30
0.
37
2.
22
0.
14
21
1.91
0.
31
1.40
0.
12
15.8
3.9
0.78
0.
06
0.
98
0.
10
Supplementary Table
7
. Blood clearance of PHP.eB and
(NOTA)
8
-
A555
-
PHP.eB assessed by classical
PK analysis with qPCR.
Classical PK analysis
Time (h)
PHP.eB
(NOTA)
8
-
A555
-
PHP.eB
mean
s
.
d
.
n
mean
s
.
d
.
n
0
1
0
4
1
0
4
4
0.57
0.19
3
0.55
0.15
4
21
0.034
0.025
4
0.12
0.064
4
Mean values are presented as a fraction of the initial viral vector genome
Supplementary Table
8
. B
iodistribution of PHP.eB and (NOTA)
8
-
A555
-
PHP.eB with
qPCR
.
organs
PHP.eB
(NOTA)
8
-
A555
-
PHP.eB
m
ean
s
.
d
.
n
mean
s
.
d
.
n.
Brain
341.83
287.37
6
405.15
302.70
8
Heart
7.27
2.69
6
13.40
6.94
8
Liver
18.14
13.07
6
42.86
34.12
8
Spleen
208.33
311.67
6
137.06
114.43
8
Kidney
24.43
19.55
6
40.24
49.52
8
Blood
29443.33
17206.82
3
26225.00
4714.78
4
Mean values are
presented as a relative viral gene number (Vg/Gc) with the viral vector gene concentration
(Vg/mL) normalized by the glucagon housekeeping gene concentration (Gc/mL).
Supplementary
Table
9
.
Biodistribution of AAVs in organs (% ID/cc) at 21
h
and brain/blood ratio
C57BL/6
BALB/c
64
Cu
-
AAV9
(n=3)
64
Cu
-
AAV9
-
TC
(n=3)
64
Cu
-
PHP.eB
(n=3)
64
Cu
-
PHP.eB
(n=3)
64
Cu
-
PHP.eB
(n=3, neuraminidase)
mean
s.d.
mean
s.d.
mean
s.d.
mean
s.d.
mean
s.d.
blood
16.9
3.6
3.6
0.3
2.1
0.3
1.7
0.2
2.0
0.2
heart
3.4
0.5
4.7
0.7
3.8
0.9
4.2
0.3
3.9
0.4
lungs
7.1
0.9
7.8
1.7
6.5
0.9
4.1
0.6
5.7
0.5
liver
18.3
1.2
19.3
1.8
35.1
10.4
37.7
1.7
44.4
0.8
spleen
15.8
3.3
6.0
0.5
39.4
9.6
18.3
1.8
18.8
1.4
kidneys
5.6
0.8
8.5
1.0
6.0
1.2
5.3
0.7
7.2
0.7
stomach
4.2
0.2
4.1
0.9
3.5
2.4
1.9
0.9
2.4
0.4
intestine
7.8
0.9
8.0
0.4
7.4
1.5
4.8
0.8
7.1
0.3
muscle
0.8
0.1
0.8
0.1
0.7
0.5
0.5
0.2
0.5
0.0
bone
2.8
0.6
2.2
0.3
2.4
0.7
1.3
0.3
1.6
0.2
brain
0.2
0.0
0.6
0.0
17.0
4.1
0.2
0.1
0.4
0.0
b
rain/blood
0.010
0.003
0.172
0.020
8.188
2.442
0.102
0.038
0.204
0.013
Supplementary
Table
10
.
Radioactivity
(% ID/cc) in liver measured from PET/CT images at 0, 4, and 21
h
.
64
Cu
-
PHP.eB
Time
(h)
C57BL/6
(n=3)
BALB/c
(n=3)
mean
s.d.
mean
s.d.
0
26.9
5.3
33.3
4.5
4
30.5
7.4
45.0
2.7
21
25.7
6.6
37.0
3.2
Supplementary Table
1
1
. AAVs and
reporter
genes
Experiment
Vector
Gene
Mouse strain
No. of
mice
Data
preparation
In vitro
AAV9
CAG
-
mNeonGreen
N.A
N.A.
Fig. 2A
-
C
Caltech
AAV9
-
TC
CAG
-
mNeonGreen
N.A
N.A
Fig. 2A
-
C
,
Supplementary
Fig.
5
Caltech
PHP.eB
CAG
-
GFP
N.A
N.A
Fig. 2A
-
C
Supplementary
Fig. 4
Addgene
In vivo
transduction
PHP.eB
CAG
-
GFP
C57BL/6
6
Fig. 2D,E
Addgene
PET/CT
AAV9
CAG
-
mNeonGreen
C57BL/6
3
Fig. 3B
-
H
Caltech
AAV9
-
TC
CAG
-
mNeonGreen
C57BL/6
3
Fig. 3B
-
H
Caltech
PHP.eB
CAG
-
GFP
C57BL/6
3
Fig. 3B
-
H
, Fig.
4A
-
F
Addgene
PHP.eB
CAG
-
mNeonGreen
BALB/c
3
Fig. 4A
-
G
Caltech
PHP.eB
CAG
-
GFP
BALB/c
(neuraminidase)
4
Fig. 4G
Addgene
Optical
PHP.eB
CAG
-
GFP
C57BL/6
6
Supplementary
Fig. 8,
Movie
4
Addgene
PHP.eB
CAG
-
DIO
-
GFP
C57BL/6
8
Fig.
3I
Caltech
AAV9
CAG
-
mNeonGreen
C57BL/6
Supplementary
Fig. 9, 10
Caltech
Classical PK
and
biodistribution
PHP.eB
CAG
-
GFP
C57BL/6
16
Fig. 3F
Addgene
Supplementary Figures
Supplementary Fig. 1
.
Schematic
illustration
of AAV
radiolabeling via inverse electron demand
Diels
Alder reaction (lEDDA) and maleimide
-
thiol
reaction. Cu
-
64 is incorporated
in
to a multichelator
-
TCO or
multichelator
-
maleimide, fo
llowing reaction with tetrazine
-
conjugated AAVs or with thiol, respectively.
Both reactions afford
a
64
Cu
-
labeled AAV
w
ith similar PK when tested with AAV9.