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, lon

g 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 AAV2C

UhCLN2 to the brain of rat and African green monkey was carried out

Manufacture and Quality Control of the AAV2C

UhCLN2 Vector

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

Freeze / thaw - Benzonase digestion

Heparin agarose, concentration

Iodixanol step gradient

AAV2CUhCLN2

Plasmid with genome of AAV2CUhCLN2 vector

Cotransfection

Ad / AAV helper plasmid

72 hr

AAV2CUhCLN2 Genome

Lot Release for Toxicology Grade Vector

Human CMV

enhancerHuman

CLN2 cDNA

Chicken -actinpromoter/

splice donor

Rabbit-globin / splice acceptor

Rabbit-globinPoly A

Splice

AAV2 invertedterminal repeat AAV2 inverted

terminal repeat

293 cells from GMP validated

cell bank

TPP-I Distribution After AAV2CUhCLN2 Delivery to Different Regions of Rat

BrainStriatum

Frontal cortex

Parietalcortex

AAV2CUhCLN21010 particle units in 5 l at each site

Evaluate TPP-I expression by

immunohistochemistry

4 wk

TPP-I Expression Following AAV2CUhCLN2 Gene Transfer to the Brain of Non-human Primates

Caudate nucleus

Naïve Cortex

Caudate nucleus

Hippocampus Hippocampus

AAV2CUhCLN2 (3.6x1011 particle units)6 sites / hemisphere

13 wk

African green monkey

Evaluate TPP-I expression

(immunohistochemistry)

Design and Status of Rat Toxicology Study

Evaluate

13 - 78 wk

Male and female Fisher 433 rats

General safety Complete blood count Serum chemistry Histology

AAV2CUhCLN2 (1010 particle units, intrastriatum)

PBS Time (wk)

General safety CBC Chemistry Histology

13

26

52

78

= complete, = pending

Effect of CNS Administration of AAV2CUhCLN2 on Rat Safety Parameters (Selected fro

m n=33)

0

100

200

300

400

500

Wei

ght (

g)

0

1

2

3

4

5

6

WB

C (1

03 /l)

0

30

60

90

120

150

ALT

(U/L

)0.2

0.4

0.6

0.8

1.0

1.2

Bra

in w

eigh

t (%

bod

y w

eigh

t)

6

7

8

9

RB

C (1

06 /l)

0

10

20

30

40

Ure

a ni

trog

en (m

g/dL

)Time post-administration (wk)

13 26

13 26

13 26

13 26 13 26

13 26

Male, PBSMale, AAV2CUhCLN2

Female, PBSFemale, AAV2CUhCLN2

Histochemical Examination of Rat Brain Following Administration of AAV2CUhCLN2

PBS – 13 wk post-administration AAV2CUhCLN2 – 13 wk post-administration

Brains were normal except for gliosis (blue arrow) and hemosiderin (green arrow) in both AAV2CUhCLN2 and PBS groups, and therefore were not vector related.

Injection tract

Injection tract

Design of Primate Toxicology Study Days

pre 0 3 7 14 28 56 90 182 365Vector administration1

3.6x1011 pu AAV2CUhCLN2n=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

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 n=4 n=3

112 injections through 3 burr holes per hemisphere at 2 depths/burr hole

Effect of Intracranial AAV2CUhCLN2 on Primate Safety Parameters (Selected from n=28)

0

4

8

12

16

Hem

oglo

bin

(g/d

l)

0

2

4

6

8

10

Whi

te b

lood

cel

l cou

nt (x

103 /

l)

0

10

20

30

Ure

a ni

trog

en (m

g/dl

)

150

175

200

225

250

Wei

ght (

Oun

ces)

0

150

300

450

600

Plat

elet

( x1

06 /l)

0

50

100

150

Ala

nine

am

inot

rans

fera

se (U

/l)

261384210Pre

Time (wk)261384210Pre

261384210Pre

261384210Pre

261384210Pre

261384210Pre

AAV2CUhCLN2, 3 x1011 pu

AAV2CUhCLN2, 3 x1010 pu Control

Summary of Primate Toxicology Parameters

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

Use ANOVA at each time point to test hypothesis that combined control group (AAV2CUNull, PBS and Sham) differs from AAV2CUhCLN2 groups

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 AAV2CUhCLN2 group at high dose at 7 and 14 days post-vector; the significance of this, if any, is unexplained

Effect of Intracranial AAV2CUhCLN2 on Primate Behavioral Parameters

Group Anxiety(chewing + penile erection + scratching + self-groom + yawn)

Arousal (bipedal lookout + shift + tail flag + threaten outside + climb + vocalization)

Quiet(cage pick +self-groom +drink + eat)

Healthyanxiety +arousal +quiet

Abnormalany observable changes in gait, posture, eating problems, sedation

p value1 0.074 0.36 0.97 0.76 Not applicable

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

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 AAV

2CUhCLN2

AAV2CUNull 1 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

AAV2CUhCLN21 wk post-administration

AAV2CUhCLN213 wk post-administration

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 r

esulted 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