Transcript スライド 1 - Weill Cornell Medical College

Background

Late Infantile Neuronal Ceroid Lipofuscinosis
(LINCL) is a rare, autosomal recessive, fatal
lysosomal storage disease with extensive CNS
neurodegeneration

LINCL is caused by mutations in the CLN2
gene resulting in a deficiency of the lysosomal
protease TPP-I (tripeptidyl peptidase I)

Deficiency of TPP-I leads to accumulation of
undigested proteins in lysosomes and neuronal
death
Background (2)

Prior studies have demonstrated high level,
long term TPP-I expression in the brain
following intracranial gene transfer using an
AAV2-based vector expressing the human
CLN2 cDNA (AAV2CUhCLN2)

In order to move AAV2CUhCLN2 to the clinic, a
toxicology study of the administration of
AAV2CUhCLN2 to the brain of rat and African
green monkey was carried out
Manufacture and Quality Control of the
AAV2CUhCLN2 Vector
Ad / AAV helper
plasmid
Plasmid with genome of
AAV2CUhCLN2 vector
AAV2CUhCLN2 Genome
Cotransfection
AAV2 inverted
terminal repeat
Human
CMV
enhancer
Rabbitb-globin /
splice acceptor
Chicken b-actin
promoter/
splice donor
AAV2 inverted
terminal repeat
Human
CLN2 cDNA
Rabbit
b-globin
Poly A
Splice
Lot Release for Toxicology Grade Vector
293 cells from
GMP validated
cell bank
72 hr
Freeze / thaw - Benzonase digestion
Iodixanol step gradient
Heparin agarose, concentration
AAV2CUhCLN2
Parameter
Lot Release
Sterility
Sterile
Endotoxin
< 20 IU / ml
Transgene function
>200 FU / min in transduction assay
Capsid titer
2.0 x 1012 / ml
Genomic titer
0.45 x 1012 genome copies / ml
Mycoplasma
None
TPP-I Distribution After AAV2CUhCLN2
Delivery to Different Regions of Rat
Brain

AAV2CUhCLN2
1010 particle units
in 5 ml at each site
Striatum
Frontal
cortex
4 wk
Parietal
cortex
Evaluate

TPP-I expression by
immunohistochemistry
TPP-I Expression Following AAV2CUhCLN2 Gene
Transfer to the Brain of Non-human Primates

AAV2CUhCLN2 (3.6x1011 particle units)
6 sites / hemisphere
Caudate nucleus
Caudate nucleus
African green
monkey
Hippocampus
Hippocampus
13 wk
Naïve Cortex
Evaluate

TPP-I expression
(immunohistochemistry)
Design and Status of Rat
Toxicology Study


AAV2CUhCLN2 (1010 particle units,
intrastriatum)
PBS
Time
(wk)
General
safety
CBC
13




26




52




78




Chemistry Histology
Male and female Fisher 433 rats
13 - 78 wk
Evaluate




General safety
Complete blood count
Serum chemistry
Histology
 = complete,  = pending
Effect of CNS Administration of
AAV2CUhCLN2 on Rat Safety Parameters
(Selected from n=33)
Female, PBS
Female, AAV2CUhCLN2
400
300
200
150
5
120
4
3
2
13
0
26
1.2
13
26
9
1.0
0.8
0.6
0.4
0.2
13
26
8
7
6
60
13
0
Urea nitrogen (mg/dL)
0
90
30
1
RBC (106/ml)
Brain weight (% body weight)
100
6
ALT (U/L)
WBC (103/ml)
Weight (g)
500
Male, PBS
Male, AAV2CUhCLN2
26
Time post-administration (wk)
13
26
40
30
20
10
0
13
26
Histochemical Examination of Rat Brain
Following Administration of AAV2CUhCLN2
PBS – 13 wk post-administration
AAV2CUhCLN2 – 13 wk post-administration
Injection tract
Injection tract

Brains were normal except for gliosis (blue arrow) and hemosiderin (green arrow) in
both AAV2CUhCLN2 and PBS groups, and therefore were not vector related.
Design of Primate Toxicology Study
Days
pre
0
Vector administration1
3.6x1011 pu AAV2CUhCLN2
n=12
3.6x1010 pu AAV2CUhCLN2
n=12
3.6x1011 pu AAV2CUNull
n=6
PBS or Sham
n=7
General assessment
(pulse, temp, resp, weight)
Blood studies
Behavioral testing
3
7
14
28
56
90
182
365




























Sacrifice for histology
3.6x1011 pu AAV2CUhCLN2
n=3
n=3
n=3
n=3
3.6x1010 pu AAV2CUhCLN2
n=3
n=3
n=3
n=3
3.6x1011 pu AAV2CUNull
n=1
n=1
n=1
n=3
PBS or Sham
112
injections through 3 burr holes per hemisphere at 2 depths/burr hole
n=4
n=3
Effect of Intracranial AAV2CUhCLN2 on Primate
Safety Parameters (Selected from n=28)
Platelet ( x106/ml)
600
225
450
200
300
175
150
Pre 0
1
2
4
8
13
26
16
12
8
4
0
Pre 0
1
2
4
8
13
26
0
Pre 0
1
2
4
8
13
26
10
Urea nitrogen (mg/dl)
150
Hemoglobin (g/dl)
Control
250
White blood cell count (x103/ml)
Weight (Ounces)
AAV2CUhCLN2, 3 x1010 pu
Alanine aminotransferase (U/l)
AAV2CUhCLN2, 3 x1011 pu
8
6
4
2
150
100
50
0
Pre 0
1
2
4
8
13
26
Pre 0
1
2
4
8
13
26
30
20
10
0
0
Pre 0
1
2
4
Time (wk)
8
13
26
Summary of Primate Toxicology Parameters
Use ANOVA at each time point to test hypothesis that combined control group
(AAV2CUNull, PBS and Sham) differs from AAV2CUhCLN2 groups
Parameter
p value1 Parameter
p value1
Parameter
p value1
Parameter
p value1
Body weight
0.23
Hemoglobin
0.06
Glucose
0.31
Calcium
0.11
Pulse
0.05
Hematocrit
0.08
Urea nitrogen
0.06
Phosphoprus
0.19
Respiratory rate
0.02
White count
0.22
Creatinine
0.12
Sodium
0.12
Temperature
0.07
Red count
0.12
Bilirubin
0.14
Potassium
0.03
Platelet count
0.09
Alk Phos
0.07
Chloride
0.03
% neutrophil
0.19
ALT
0.03
Total protein
0.26
% lymphocyte
0.16
AST
0.12
Albumin
0.02
Cholesterol2
0.003
Globulin
0.03
CPK
0.18
1 Most
significant p value for all time points comparing control to high dose AAV2CUhCLN2; Due to
multiple variables, a cut off of p<0.01 was accepted as significant
2 Cholesterol levels were significantly lower in AAV2 hCLN2 group at high dose at 7 and 14 days
CU
post-vector; the significance of this, if any, is unexplained
Effect of Intracranial AAV2CUhCLN2
on Primate Behavioral Parameters

Monkeys were videotaped both at rest and while a series of standard stimuli were
applied

Defined behaviors were quantitated on each videotape by two independent observers
Group
Anxiety
(chewing +
penile erection +
scratching + selfgroom + yawn)
Arousal
(bipedal lookout + shift +
tail flag + threaten
outside + climb +
vocalization)
Quiet
(cage pick +
self-groom +
drink + eat)
Healthy
anxiety +
arousal +
quiet
Abnormal
any observable
changes in gait,
posture, eating
problems, sedation
p value1
0.074
0.36
0.97
0.76
Not applicable
1
Using type III mean squares analysis testing the hypothesis that the vrariable is
unaffected by treatment group
Histochemical Examination of Primate
Brain Following Administration of
AAV2CUhCLN2
AAV2CUNull
1 wk post-administration

AAV2CUhCLN2
1 wk post-administration
AAV2CUhCLN2
13 wk post-administration
Brains were normal except for gliosis observed at injection sites (blue oval)
in PBS, AAV2CUNull and AAV2CUhCLN2 animals, and thus gliosis was
related to injection of vehicle and not vector
Summary – Rat Toxicology

Intracranial injection of AAV2CUhCLN2 into rat
brain resulted in no changes in general
assessment or complete blood count in
AAV2CUhCLN2 group compared to PBS group
up to 26 wk

The mild histopathological changes in rat
brain observed in the AAV2CUhCLN2 group
were observed in both PBS and
AAV2CUhCLN2 groups and resulted from
injection trauma
Summary – Primate Toxicology

Intracranial injection of AAV2CUhCLN2 into primate brain
resulted in no changes in general assessment and
complete blood count correlated with vector

The were no vector related changes in serum chemistry
except for a transient decrease in serum cholesterol at 7
and 15 days post vector in AAV2CUhCLN2 group. Due to
transient nature of change, and absence of any other
serum chemistry changes, this was deemed medically
unimportant

Histopathology of primate brain showed acute gliosis at
the site of injection, not attributable to vector

Behavioral assessment of injected primates showed no
abnormal behaviors in the AAV2CUhCLN2 injected group
Conclusion

In the context that LINCL is a fatal,
untreatable disease, it is safe to preceed
with a clinical trial using AAV2CUhCLN2 to
treat the CNS manifestations of this
disorder