A non-human primate model of Parkinson’s disease based on...

A non-human primate model of Parkinson’s disease based on viral vector mediated overexpression of alpha-synuclein

James Koprich1, Tom Johnston1, John Seibyl2, Ken Marek2, Yilong Ma3, David Eidelberg3, Chuantao Zuo4, Yi Hui Guan4, Jeffrey Kordower5, Susan Fox6 and Jonathan Brotchie1

1Atuka Inc. Toronto, Canada, 2Molecular Neuroimaging, New Haven CT, 3Feinstein Institute for Medical Research, Manhasset, NY, 4PET Center, Huashan Hospital, Shanghai, PRC,

5Rush University Medical Center, Chicago IL, 6Movement Disorders Clinic, University Health Network, Toronto, Canada.

Introduction and Aim •  Irrespective of etiology and varying upstream

mechanisms, aSyn deposition is a defining pathological feature of PD.

•  Rodent models, transgenic mice and AAV rats, have proven useful in better understanding aSyn function, pathology and biochemistry.

•  As in vivo platforms for testing efficacy, the rodent models are currently being, or have been, used to evaluate several classes of therapies ranging from immunotherapy, autophagy enhancing, aggregate clearing, etc.

•  Currently lacking in the field is a robust, well-characterized non-human primate model based on aSyn overexpression to optimally transition therapeutic development between rodent and human.

•  Our aim in the current study was to develop a primate model of alpha-synucleinopathy that could be used to fill this gap in preclinical drug efficacy testing using a species with a proven track record in preclinical drug screening, the cynomolgus macaque.

Materials & Methods Animals and viral vector delivery. Female cynomolgus macaques (~9 y, ~3 kg) were injected via MRI-guided stereotaxy with 28 µl of 1.7 x 1012 A53T α-syn or a empty vector into the SN over 4 sites within each side of the SN. Behaviour was assessed every month. PET scans (AV-133 and FDG) were conducted every other month. Postmortem measures. Animals were perfused with saline 8 months after surgery and brain portions were dissected fresh or post-fixed. Brains were processed for immuno-labeling and HPLC. Double label immunofluorescence was conducted to reveal tyrosine hydroxylase (TH), human alpha-synuclein or HA to provide detail regarding colocalization. B r i g h t f i e l d m i c r o s c o p y w a s c o n d u c t e d independently on TH and α-synuclein stained sections to reveal axonal morphology and to evaluate protein expression. TH stained sections of the SN will be used for stereological estimation of dopamine neuron numbers

Results

Summary & Conclusions

1.  The model is in a position to assess therapeutics aimed at reducing or preventing aSyn accumulation in the nigrostriatal system in a 4 or 8 month timeframe. Endpoints include: striatal neurochemistry and DAT, aSyn load per DA neuron, striatal aSyn levels, number of TH neurons remaining

2.  The model shows a behavioural phenotype that includes reduced locomotor activity in the absence of overt disability, representing a pre-motor phase of the early PD patient

3.  Robust aSyn expression throughout the nigrostriatal system allows for proof of concept PET studies to screen potential aSyn ligands

aSyn

H AAV-α-syn

p129 aSyn aSyn

TH/ aSyn

TH/ aSyn

TH/ aSyn

AAV1/2 A53T aSyn TH

TH Empty vector control AAV1/2 A53T aSyn

TH

SNpc SNpc

putamen SN SN

SN

putamen

SNpc

Empty vector control

0

20

40

60

spec

ific R

TI-1

21 b

indi

ng (n

Ci/g

tiss

ue)

DATputamen

AAV1/2 Empty Vector

AAV1/2 A53T aSyn

*39% ↓

0

20

40

60

spec

ific R

TI-1

21 b

indi

ng (n

Ci/g

tiss

ue)

DATcaudate

AAV1/2 Empty Vector

AAV1/2 A53T aSyn

*33% ↓

0

50

100

150

dopa

min

e (n

g/m

g pr

otei

n)

dopamineputamen

AAV1/2 Empty Vector

AAV1/2 A53T aSyn

**42% ↓

0

50

100

150

dopa

min

e (n

g/m

g pr

otei

n)

dopaminecaudate

AAV1/2 Empty Vector

AAV1/2 A53T aSyn

0

2

4

6

8

DO

PAC

(ng/

mg

prot

ein)

DOPACputamen

AAV1/2 Empty Vector

AAV1/2 A53T aSyn

*50% ↓

0

2

4

6

8

DO

PAC

(ng/

mg

prot

ein)

DOPACcaudate

AAV1/2 Empty Vector

AAV1/2 A53T aSyn

*34% ↓

DAT putamen

DAT caudate

dopamine putamen

dopamine caudate

DOPAC putamen

DOPAC caudate

0

1

2

3

SUV

r

empty vector controlA53T aSyn

2 4

months postsurgery

6 8

*

baseline

18F AV133 VMAT-2

-1

0

1

2

3

FDG

PrP

sco

re

EVA53T aSyn

2 4

months postsurgery

6 8baseline

FDG PET Parkinson brain

network analysis (PRP)

control aSyn month

-1

+2

+4

+6

+8

18F AV133 VMAT-2

0

1000

2000

3000

4000

activ

ity

empty vector controlA53T aSyn

5 6

months postsurgery

*

7

**

8

*

Absent

Mild

Moderate

Marked

Severe

MPP

rs

Disability(totals, 120 min period)

empty vector controlA53T aSyn

baseline 1 2 3

months postsurgery

4 5 6 7 8

0

10

20

30

time

(s)

empty vector controlA53T aSyn

baseline 1 2 3

months postsurgery

4 5 6 7 8

time to complete mMAP, level D

Distribution of transgene and degenerative changes in the nigrostriatal system produced by exposure to A53T α-synuclein 8 months following injection of AAV1/2

Reduced activity without overt disability

Reductions in striatal DAT and dopamine neurochemistry following exposure to A53T aSyn

AV133 VMAT-2 and FDG PET shows changes over time following exposure to A53T aSyn

33% *

39% *

42% **

50% *

34% *

empty vector control A53T aSyn

empty vector control A53T aSyn