0. Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL)
Rare, autosomal recessive, fatal lysosomal storage disease with extensive CNS neurodegeneration
Caused by mutations in the CLN2 gene, coding for a tripeptidyl peptidase (TPP-I) that normally functions to remove waste membrane proteins
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)1
AAV2 mediated gene transfer to the CNS corrects the storage defect in CLN2 knockout mice2
1 Sondhi et al, ASGT Abstract #660 2 Passini et al. ASGT Abstract #427

1. Clinical Assessment of LINCL
Challenge
To develop a non-invasive strategy to assess the efficacy of direct CNS administration of AAV2CUhCLN2 in clinical studies
Strategy
Assess magnetic resonance spectroscopy as a method to evaluate the status of the CNS in children with LINCL over time

2. Demographic Characteristics of Study Population
Subject
Sex
Genotype
Age at 1st MRS1 scan (yr)
Interval between 1st and 2nd MRS (days)
LINCL rating2 1 M
G3556C / T3016A
8.4 74
3 (severe) 2
G3556C / G3085A
9.2 92 3
6.6
127 4 F
C3670T / T4396G
13.6x
nd1 5
G3556C / C3670T
6.2
336
5 (moderate) 6
G3556C / G3556C
7.9 46 7 nd 8
6.0
4 (severe) 9
G3556C / T4383C
6.8 10
C3670T / ?
5.0 11
5.4 12
C3670T / C3670T
4.4 39
1 MRS = magnetic resonance spectroscopy; nd = not done
2 LINCL rating scale based on Steinfeld et al, Am J Med Gen (2002); 112:

3. Magnetic Resonance Scan of Severe LINCL1 2
Slice 1
Slice 2
Slice 3
Slice 4 3 4

4. Metabolite Levels in Voxels from Parenchyma and Ventricles Determined by Magnetic Resonance Spectroscopy of Subject with Severe LINCL
LAC
CHO CR
NAA
CHO = choline
CR = creatine
NAA = N-acetylaspartate
LAC = lactic acid

5. Definition of Area in Cortex for Assessment by Magnetic Resonance Spectroscopy
Metabolite concentrations assessed in voxels indicated in slice 2

6. Examples of Metabolites in the CortexCR CR
CHO
NAA
LAC
CHO
NAA
LAC
BD001 Age 8
BD009, Age 5
BD005, Age 13
BD002, Age 9

7. Metabolite Levels in the Cortex N-acetyl aspartate / creatine
Subject
Age
(Yr)
Lactate
N-acetyl aspartate
Creatine
Choline
N-acetyl aspartate / creatine 1
8.4
2.94
4.66
3.44
4.99
1.36 2
9.2
2.28
6.33
3.87
6.20
1.65 3
6.6
2.07
8.10
4.59
7.99
1.77 4
13.6x
17.90x
2.51
2.84
0.61 5
6.2
2.41
7.23
4.48
6.96
1.74 6
7.9
8.53
4.76
3.93
7.21
1.22 7
2.34
6.89
5.25
7.81
1.32 8
6.0
nd1 nd 9
6.8
2.63
7.40
6.45
8.99
1.16 10
5.0
1.31
4.72
3.01
5.01
1.59 11
5.4
3.21
10.74x
7.55
9.78
1.43 12
4.4
2.48
11.30x
8.38
11.11x
1. nd – not determined

8. Level of N-acetylaspartate
Spatial Variation of N-acetylaspartate Levels in Subject with Severe LINCL 8 6
Level of N-acetylaspartate 4 2
Caudal /
rostral
Lateral
Due to substantial spatial variation, comparison of two MRS scans for the same subject requires careful alignment of the images

9. Age-dependence of Creatine and Lactate Levels in Cortex
Subject 1 5 10 2 6 11 3 7 12 4 9 10 20 8
y = x
r2 = 0.48 15
y = x
r2 = 0.49 6
Creatine
Lactate 10 4 5 2 2 4 6 8 10 12 14 2 4 6 8 10 12 14
Age (yr)

10. Age-dependence of Choline and N-acetylaspartate Levels in Cortex
Subject 1 5 10 2 6 11 3 7 12 4 9 14 14 12 12
y = x
r2 = 0.56
y = x
r2 = 0.62 10 10 8 8
Choline
N-acetylaspartate 6 6 4 4 2 2 2 4 6 8 10 12 14 2 4 6 8 10 12 14
Age (yr)

11. Reproducibility of N-acetylaspartate Level Between two MRS Scans of the Same Subject
Local increase
Subject #1
Subject #3
Local decrease
6.0
6.0
3.0
3.0
Change in NAA level
(1st scan – 2nd scan)
0.0
0.0
Change in NAA level
(1st scan – 2nd scan)
-3.0
Left Right
-3.0
Left Right
-6.0
-6.0
Caudal Rostral
Caudal Rostral
Changes in N-acetylaspartate level are small and spatially uniform

12. Summary
Magnetic resonance spectroscopy scans of children with LINCL show age-dependent decreases in the levels of choline, creatine and N-acetylaspartate, and an age dependent increase in levels of lactate
N-acetylaspartate provides the most reliable parameter of age-dependent changes
Duplicate MRS on the same subject can be registered, demonstrating similar local metabolite levels
Assessment of local N-acetylaspartate levels in serial MRS scans following gene transfer have the potential to show areas of the brain that are spared from progression of the disease