Background

1. 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

2. 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

3. 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 Rabbitb-globin / splice acceptor AAV2 inverted terminal repeat Human CMV enhancer Chicken b-actin promoter/ splice donor Rabbit b-globin Poly A Human CLN2 cDNA 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

4. TPP-I Distribution After AAV2CUhCLN2 Delivery to Different Regions of Rat Brain Striatum Frontal cortex Parietal cortex • AAV2CUhCLN2 • 1010 particle units in 5 ml at each site 4 wk Evaluate • TPP-I expression by immunohistochemistry

5. TPP-I Expression Following AAV2CUhCLN2 Gene Transfer to the Brain of Non-human Primates Caudate nucleus Caudate nucleus Hippocampus Hippocampus Naïve Cortex • AAV2CUhCLN2 (3.6x1011 particle units) 6 sites / hemisphere African green monkey 13 wk Evaluate • TPP-I expression (immunohistochemistry)

6. Design and Status of Rat Toxicology Study • AAV2CUhCLN2 (1010 particle units, intrastriatum) • PBS Male and female Fisher 433 rats 13 - 78 wk Evaluate • General safety • Complete blood count • Serum chemistry • Histology  = complete,  = pending

7. Effect of CNS Administration of AAV2CUhCLN2 on Rat Safety Parameters (Selected from n=33) Female, PBS Male, PBS Female, AAV2CUhCLN2 Male, AAV2CUhCLN2 500 150 6 5 400 120 4 Weight (g) WBC (103/ml) 300 90 ALT (U/L) 3 200 60 2 100 30 1 0 0 0 13 26 13 26 13 26 1.2 40 9 1.0 30 8 0.8 Urea nitrogen (mg/dL) Brain weight (% body weight) 20 RBC (106/ml) 0.6 7 10 0.4 0 0.2 6 13 26 13 26 13 26 Time post-administration (wk)

8. 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.

9. Design of Primate Toxicology Study 112 injections through 3 burr holes per hemisphere at 2 depths/burr hole

10. Effect of Intracranial AAV2CUhCLN2 on Primate Safety Parameters (Selected from n=28) AAV2CUhCLN2, 3 x1011 pu AAV2CUhCLN2, 3 x1010 pu Control 250 150 600 225 450 Weight (Ounces) 100 Alanine aminotransferase (U/l) Platelet ( x106/ml) 200 300 50 175 150 0 0 150 Pre 0 1 2 4 8 13 26 Pre 0 1 2 4 8 13 26 Pre 0 1 2 4 8 13 26 10 30 16 8 12 20 6 White blood cell count (x103/ml) Urea nitrogen (mg/dl) Hemoglobin (g/dl) 8 4 10 4 2 0 0 0 Pre 0 1 2 4 8 13 26 Pre 0 1 2 4 8 13 26 Pre 0 1 2 4 8 13 26 Time (wk)

11. 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 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

12. 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 1 Using type III mean squares analysis testing the hypothesis that the vrariable is unaffected by treatment group

13. 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

14. 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

15. 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

16. 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