Supplementary_information_NC_revision_3 - 41467_2020_15818_MOESM1

Supplementary information

Positron emission tomography

imaging

novel

AAV capsid

maps rapid brain

accumulation

J. W.

o et al.

Contents:

Supplementary Methods

Supplementary Tables 1

Supplementary Figures 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′

etramethyl

(1H

benzotriazol

yl)uronium hexafluorophosphate, O

(benzotriazol

yl)

N,N,N′,N

′

tetramethyluronium

hexafluorophosphate

DIPEA

(Sigma

Aldrich, 496219,

N,N

iisopropylethylamine

)

TIPS

(Sigma

Aldrich, 233781,

triisopropylsilane

Fmoc

PEG

(Polypure, 15137

2790), Fmoc

PEG

OH (Chem

Impex International Inc.,07310),

methyl

piperidine (Acros, 127515000, 99%)

TFA

(Acros,

13972, trifluoroacetic acid),

NOTA

bis(t

Bu ester)

(

Macrocyclics, B

620

1,4,7

riazacyclono

nane

1,4

bis

tert

butyl acetate

acetic acid)

, t

etrazine

PEG

NHS ester (Click Chemistry Tools, 1143)

ranscyclooctene

PEG

NHS ester: (Click Chemistry Tools, A137)

SM(PEG)

(ThermoFisher Scientific,

22102

, s

uccinimidyl

[(

maleimidopropionamido)

diethy

leneglycol] ester

)

. Alexa555

S (ThermoFisher

Scientific, A20009), AF555

maleimide (Fluoroprobes, 1168)

Alexa555

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

forward:

CATCGATGGTGTTGCCCGGGCTGCTGTAAGACTTTCgctcctggaaagaagaggcctgtag and

AAV9

reverse:

GAAAGTCTTACAGCAGCCCGGGCAACACCATCGATGcgtcttagccgcttcctcaaccaga).

AAV9

TC and AAV9 packaging

CAG

mNeonGreen

were

produced following protocol outlined in Challis,

et al

Briefly, HEK293T cells (ATCC) were triple transfect

ed using polyethylenimine (PEI); virus was

collected after 120

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

. P

HP.eB packaging

CAG

mNeonGreen

CAG

DIO

GFP

ere

also

prepared

similar to above.

Titration of single

and multichelator

with Cu

63/Cu

64 for

Supplementary Fig. 3

A 20 pmol of single

chelator in double distilled water (2

L) was added to two

fold serial dilutions of

CuCl

solution (3

L) from

mM to 0.3 n

, spiked with 0.33 pmol of

CuCl

L). The titration of 20 pmol of multichelator was

performed with

CuCl

solution (3

L) from

0.1

mM to 0.3 n

, spiked with 14 pmol of

CuCl

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

) in 1xPBS (180

L, pH adjusted to 8 with 0.1 M Na

solution (pH 9.1)), 0.7

L of 1, 5, or 10 mM tetrazine

PEG

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

. To each reaction, 0.7

L of 0.1, 0.5, or 1 mM

(NOTA)

PEG

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

justed to 300

L with 1xPBS

as shown in Supplementary Fig. 4A

The transduction of

the

CAG

GFP

gene by

PHP.eB and PHP.eB were compared in HEK293T cells seeded at a density of 70% in

well plate (3x10

cells/well) with 500

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

for 24

, media was aspirated and replaced

with

fresh

DMEMc

(250

L) at

1x10

/cell

of unmodified control (UMC, n=4) PHP.eB

or modified PHP.eB with

(NOTA)

PEG

TCO in

the following ratios (100, 500, and 1000 (NOTA)

PEG

TCO/AAV ratio, n=4 for

each ratio). Cells were then incubated at 37 °C in a 5% CO

chamber

overnight in virus

loaded media

before aspiration and replacement with 500

L fresh DMEMc.

Cells were cultured

for one additional day

prior to collection

Media was aspirated, and PBS (300

L) was added. Cells were trypsinized and

transferred

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

ith 1xPBS (500

L), centrifuged, and supernatant was removed. This washing cycle was repeated two

times. After cells were resuspended in 1xPBS (500

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

software (TreeStar).

In vitro t

ransduction assay for

(NOTA)

8

AAV9

TC before and after tetracysteine modification

AAV9

CAG

mNeonGreen

samples

(5.

8x10

, 10 pmo

in 1xPBS (100

L) were incubated for 30

minutes after the addition of 5 mM TCEP (5 nmol, 1

L). To each reaction

, 1

L of PBS only, 0.1, 0.5, or 1

mM (NOTA)

PEG

MI (0.1, 0.5, and 1 nmol) in 1xPBS was added. After incubation for 1

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

cells/well)

with 500

L of DMEMc

(Invitrogen). After incubation for 24

media was aspirated

and replaced fresh DMEMc

(

250

) at

1x10

/cells

of unmodified control (n=4) AAV

or treated AAV9

TC with (NOTA)

PEG

MI in the following ratios (

, and

140

multichelator/AAV ratio, n=4 for each

ratio). Cells were then incubate

d at 37 °C in a humidified CO

chamber

overnight in virus

loaded media

before aspiration and replacement with 500

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.

MS/MS analysis of Tz

PHP.eB capsid

For

mass

analysis

of modified capsid protein,

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

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

urther pep

tide extraction was performed by the addition of 60% acetonitrile, 39.9% water, 0.1% formic

acid and incubation for 10

min. The peptide pools were dried in a speed vac. Samples were

reconstituted in 12.5 μl reconstitution buffer (2% acetonitrile with 0.

ormic acid) and 3 μl of each sample

were injected into the instrument.

For

ass spectrometry

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

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

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

. Summary of AAV

labeling condition

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

particle

s/mL

mg/

mL)

Cy3

Cy2

NHS

1.6

2.3

0.1 M

NaHCO

(pH 9.3)

30 min

Dialysis

3,4

AAV2

1x10

particle

Alexa488

TFP

3x10

0.1 M

NaHCO

(pH 9.3)

2 h

alysis

AAV2

5x10

particle

(1 mL)

Cy3

NHS

0.1 M

NaCO

(pH 9.3)

30 min

Dialysis

AAV

Alexa488

NHS

Dialysis

AAV

vg/mL

μ

L used)

SPA

PEG

(20ul total

volume)

1000

(

PEG/lysine)

HEPES

(pH7.4)

3 h

NS: not

specified

Supplementary Table

. 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

and 5

columns.

Frequency of modified lysine

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

(693

702)

VP1

VP2

VP3

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

(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

(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

(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

(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

(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

(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

(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

(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

(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

(688

697)

Supplementary Table

Characterization of the number of

label

per AAV

Amine

directed

reaction

Vg (pmol)

NHS

(nmol)

(NOTA)

A555

TCO

(pmol)

ea (label)/Vg

(particle)

(mean

d.)

PHP.eB

1.2E12 (2)

0.4

0.7

5.4 ± 2.3

AAV9

2.0E12 (3.3)

0.7

3.5 ± 3.0

Thiol

directed

reaction

Vg (pmol)

TCEP (nmol)

A555

MI (pmol)

AAV9

1.7E12 (3)

0.3

0.5 ± 0.3

Supplementary Table

AAV clearance half

lives and 21

hour AUCs following tail

vein administration

mice

Vector

Mouse strain

No. of mice

1/2

(h)

AUC

21h

(h.%

ID/cc)

AAV9

C57BL/6

5.0

0.97

581.9

AAV9

C57BL/6

2.4

0.99

344.3

PHP.eB

C57BL/6

3.1

0.96

391.1

BALB/c

2.4

0.96

276.9

BALB/c

(neuraminidase)

1.00

445.

PHP

C57BL/6

4.1

0.96

8.29

(NOTA)

A555

PHP.eB

C57BL/6

5.3

0.94

8.88

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.

Area un

der curve

represents a

fraction of remaining AAVs in blood

Supplementary

Table

Radioactivity (% ID/cc) in blood pool measured from PET/CT images at 0, 4,

and 21

C57BL/6

BALB/c

Time

(

)

AAV9

(n=3)

AAV9

(n=3)

PHP.eB

(n=3)

PHP.eB

(n=3)

PHP.eB

(n=

, neuraminidase)

mean

s.d.

mean

s.d.

mean

s.d.

mean

s.d.

mean

s.d.

48.4

3.7

51.0

2.5

48.9

4.9

42.8

5.8

33.6

3.1

19.3

2.3

23.3

5.9

15.6

4.0

29.4

15.5

2.0

4.6

1.0

5.7

1.0

42.8

5.8

5.0

Supplementary Table

Radioactivity (% ID/cc) in whole brain measured from ROI analyses of PET/CT

images at 0, 4, and 21

C57BL/6

BALB/c

Time

(

)

AAV9

(n=3)

AAV9

(n=3)

PHP.eB

(n=3)

PHP.eB

(n=3)

PHP.eB

(n=

neuraminidase)

mean

3.94

4.21

10.7

3.1

2.81

3.46

3.03

2.31

19.2

4.0

1.30

1.91

1.40

15.8

3.9

0.78

Supplementary Table

. Blood clearance of PHP.eB and

(NOTA)

A555

PHP.eB assessed by classical

PK analysis with qPCR.

Classical PK analysis

Time (h)

PHP.eB

(NOTA)

A555

PHP.eB

mean

0.57

0.19

0.55

0.15

0.034

0.025

0.12

0.064

Mean values are presented as a fraction of the initial viral vector genome

Supplementary Table

. B

iodistribution of PHP.eB and (NOTA)

A555

PHP.eB with

qPCR

organs

PHP.eB

(NOTA)

A555

PHP.eB

ean

mean

Brain

341.83

287.37

405.15

302.70

Heart

7.27

2.69

13.40

6.94

Liver

18.14

13.07

42.86

34.12

Spleen

208.33

311.67

137.06

114.43

Kidney

24.43

19.55

40.24

49.52

Blood

29443.33

17206.82

26225.00

4714.78

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

Biodistribution of AAVs in organs (% ID/cc) at 21

and brain/blood ratio

C57BL/6

BALB/c

AAV9

(n=3)

AAV9

(n=3)

PHP.eB

(n=3)

PHP.eB

(n=3)

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

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

0.2

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

rain/blood

0.010

0.003

0.172

0.020

8.188

2.442

0.102

0.038

0.204

0.013

Supplementary

Table

Radioactivity

(% ID/cc) in liver measured from PET/CT images at 0, 4, and 21

PHP.eB

Time

(h)

C57BL/6

(n=3)

BALB/c

(n=3)

mean

s.d.

mean

s.d.

26.9

5.3

33.3

4.5

30.5

7.4

45.0

2.7

25.7

6.6

37.0

3.2

Supplementary Table

. 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

Caltech

AAV9

CAG

mNeonGreen

N.A

Fig. 2A

Supplementary

Fig.

Caltech

PHP.eB

CAG

GFP

N.A

Fig. 2A

Supplementary

Fig. 4

Addgene

In vivo

transduction

PHP.eB

CAG

GFP

C57BL/6

Fig. 2D,E

Addgene

PET/CT

AAV9

CAG

mNeonGreen

C57BL/6

Fig. 3B

Caltech

AAV9

CAG

mNeonGreen

C57BL/6

Fig. 3B

Caltech

PHP.eB

CAG

GFP

C57BL/6

Fig. 3B

, Fig.

Addgene

PHP.eB

CAG

mNeonGreen

BALB/c

Fig. 4A

Caltech

PHP.eB

CAG

GFP

BALB/c

(neuraminidase)

Fig. 4G

Addgene

Optical

PHP.eB

CAG

GFP

C57BL/6

Supplementary

Fig. 8,

Movie

Addgene

PHP.eB

CAG

DIO

GFP

C57BL/6

Fig.

Caltech

AAV9

CAG

mNeonGreen

C57BL/6

Supplementary

Fig. 9, 10

Caltech

Classical PK

and

biodistribution

PHP.eB

CAG

GFP

C57BL/6

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

to a multichelator

TCO or

multichelator

maleimide, fo

llowing reaction with tetrazine

conjugated AAVs or with thiol, respectively.

Both reactions afford

labeled AAV

ith similar PK when tested with AAV9.