This study evaluated different methods of gene therapy to treat infantile Batten disease (INCL) in a mouse model. Intrathecal injection targeting the spinal cord alone extended lifespan by 3 months and showed motor deficits at 7 months while decreasing disease markers in the spinal cord but not brain. Intracranial injection targeting the brain alone extended lifespan by 5.3 months and showed motor deficits at 9 months while decreasing markers in the brain but not spinal cord. Combination intrathecal and intracranial injection provided the most effective treatment, extending lifespan by 11.5 months and delaying motor deficits until 15 months by decreasing disease markers in both the brain and spinal cord. Targeting both areas simultaneously provided dramatic improvement over targeting

1. Spinal Cord Disease in Brain-directed Gene Therapy
Figure 1. AAV2/9 is a far superior vector to AAV2/5 but does not correct the spinal cord. A) Lifespan curve
comparing AAV2/9 (13.7 months) and AAV2/5 (10.6 months). B) Axonal loss observed in the spinal cord. Fluorescent
markers (Thy1-YFP) delineating axonal tracts showed a significant decrease in relative fluorescence in the PPT1-/-
spinal cord. C) Examination of the AAV2/9 brain and spinal cord. Decreased AFSM in the brain with intracranial
injection and resolution of neuroinflammation. No decrease in the spinal cord for AFSM and neuroinflammation
A New and Effective Target for Infantile Batten Disease
Charles Shyng1, Hemanth Nelvagal2, Joshua T Dearborn1, Jonathan D Cooper2, Mark S Sands1,3
Departments of Internal Medicine1, Genetics3; Washington University School of Medicine
Pediatric Storage Disorders Laboratory (PSDL), Department of Basic and Clinical Neuroscience2, King's College London
Background
INCL
• The infantile form of Batten disease is the most rapidly progressing form
• INCL is caused by a deficiency in palmitoyl-protein thioeseterase-1 (PPT1), a soluble lysosomal hydrolase
• PPT1 deficiency leads to accumulation of autofluorescent storage material (AFSM), neurodegeneration,
and glial activation
• There is no treatment or cure for INCL
Therapy
• Various gene and cell therapies have been attempted in the INCL mouse model (Stem cell transplant,
Enzyme replacement, Small Molecule Drugs, Gene Transfer)
• Brain-directed gene transfer has shown to be the most effective in improving biochemical, histological,
and clinical features of INCL disease
• However, greater efficacy was expected based on the level of PPT1 expression from brain-directed gene
therapy
• A retrospective analysis of INCL animals treated with brain-directed gene therapy revealed widespread
spinal cord disease that was not effectively treated.
• We show here that targeting both the brain and spinal cord disease dramatically increased therapeutic
efficacy.
PPT1 activity
Figure 2. PPT1 activity in the brain and spinal cord of treated mice. A) Analysis of PPT1 activity
at 1 month in the Brain and Spinal cord. There is supraphysiological levels of PPT1 activity in the
spinal cord following intrathecal injection but little in the brain. The reciprocal distribution is
seen in the intracranial injected animals. B) PPT1 activity in the treated animals show that there
is near WT levels of PPT1 activity in the intracranial alone and combination-treated animals.
There is nearly undetectable brain activity following intrathecal injection (approximately 5% of
WT levels)
Histological Markers
Figure 3. Histological markers of disease are decreased with IC/IT injection. A) AFSM is
reduced in the brain but not the spinal cord following intracranial alone (orange asterick). AFSM
is reduced in the spinal cord and not in the brain following intrathecal alone (red asterick). In
the combination, AFSM is decreased in both the spinal cord and brain (purple asterick). B) CD68
staining for neuroinfllammation. For the regions targeted by gene therapy, there is little to no
CD68 staining. However, in the regions not targeted, there is CD68 staining at levels near PPT1-
/-.
Clinical
Figure 4. Functional and behavioral phenotype is improved with combination treatment. A)
Rotarod test for motor function. The PPT1 -/- mice were unable to stay on the rotarod past 7 mo
(blue). The intrathecal mice showed motor deficits at 7 month and could not stay on past 11 mo
(red). The intracranial mice showed deficits at 9 mo and could not stay on past 13 mo (green). The
combination showed deficits at 15 mo and could not stay on past 19 mo (purple). B) Lifespan. The
median lifespan for PPT1-/- mice was 8.4 mo (blue). Intrathecal injection extended the lifespan to
11.3 mo (+3 mo, red). Intracranial injection extended the lifespan to 13.7 mo (+5.3 mo, green).
The combination intracranial and intrathecal injection extended the lifespan to 19.5 mo (+11.5
mo, purple).
Conclusions
• AAV2/9 is a superior vector compared to AAV2/5
• Simultaneously targeting both the brain (intracranial) and spinal cord
(intrathecal) dramatically increases the therapeutic efficacy of AAV-mediated
gene therapy for INCL
• Intrathecal (IT, spinal cord) AAV2/9-hPPT1
• Extended lifespan: 3 months
• Show motor deficits in motor function: 7 months.
• Decreased AFSM and CD68 in spinal cord but not brain
• Intracranial (IC, brain) AAV2/9-hPPT1
• Extended lifespan: 5.3 months
• Show motor deficits: 9 months.
• Decreased AFSM and CD68 in brain but not spinal cord
• Intrathecal + Intracranial AAV2/9
• significant improvement over IT or IC injection alone
• Extended lifespan: 19.5 months
• Show motor deficits: 15 months
• Decreased AFSM and CD68 in both brain and spinal cord
Brain
Spinal Cord
Intracranial IntrathecalWildtype IC/ITPpt1-/-
IC/IT
Brain
Spinal Cord
Intracranial IntrathecalWildtype Ppt1-/-
Brain
IntracranialWildtype Ppt1-/-
Spinal Cord
IntracranialWildtype Ppt1-/-
AFSM
CD68
Decreased
Disease
No
Decrease
A B
A
B
B
A
BA
C
Axonal Loss
*
*
*
*
*
*
*
*
*
*
*
*
INCL