1. Beverly Davidson Lab:
CLN2 Gene Transfer for Late Infantile Batten Disease in the dog model
Luis Tecedor1, Yong Hong Chen1, Martin L Katz2, Fred A Wininger2; Colleen S Stein3
, Joan R Coates2, Melissa Carpentier2, Beverly L
Davidson1, 1Center for Cell and Molecular Therapy, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, 2Washington University, St. Louis,
MO and 3Internal Medicine, University of Iowa, Carver College of Medicine, Iowa City, IA, 52242, contact e-mail: davidsonbl@email.chop.edu
Learning and memory test
No treatment
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WHAT THIS MEANS FOR
THERAPY
Acknowledgements
• We thank the Gene Transfer Vector Core
and the Central Microscopy Research
Facility at the University of Iowa, and the
Research Vector Core at the Children’s
Hospital of Philadelphia.
• These studies are funded by the BDSRA,
Blake’s Purpose, Trottin for Taylor,
Partnership for Cures, the NIH
The LINCL dog model
Cross-correction:
genetically-corrected cell
CLN2 gene
mRNA
TPP1
AAV2-CLN2
AAV2 as a vehicle to get the
CLN2 gene into the brain
AAV2-CLN2
AAV2 particles are microscopic
virus-like particles that are very
efficient at delivering genes
into cells. For gene therapy,
the CLN2 gene is packaged
into AAV2 particles. This
preparation is referred to as
AAV2-CLN2.
AAV2-CLN2 injection into
the ventricular space
• In the LINCL dog, AAV2-CLN2 gene transfer results in
TPP1 protein replenishment to many areas of the
brain.
• Our results indicate that AAV2-CLN2 gene transfer is
providing significant therapeutic effects, reducing or
delaying symptoms and improving the quality of life
for the LINCL dogs.
• This gene therapy strategy holds promise for
translation to LINCL patients.
adapted from
http://community.pressen
ter.net
adapted from
www.dunesciences.com
Volume (mm3)
Gene therapy reduces
ventricular enlargement
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Normal Affected Treated
TPP1 spread in LINCL dog brain after AAV2-CLN2 gene transfer
Gene therapy reduces
neurological signs
Miniature dachshund model for LINCL
• The CLN2 gene mutation in the LINCL dog
renders the TPP1 protein non-functional,
and TPP1 protein is undetectable.
• With disease progression, brain tissues
shrink, leading to enlarged ventricular
spaces in the brain.
• Neurological symptoms include decline in
balance and motor functions, loss of vision,
tremors.
STRATEGY
1. Correct copies of the CLN2 gene are packaged into AAV2.
2. AAV2-CLN2 is injected into the brain ventricle (a fluid-filled
space), and the CLN2 gene is delivered into the cells that line
the ventricles.
3. The gene-corrected cells serve as factories to continuously
make and secrete TPP1 protein which can flow within the fluid
and diffuse into the tissue to replenish functional TPP1 to cells
throughout the brain.
occipital cortex
• After AAV2-CLN2 gene transfer to brains of affected dogs, enzyme activity assay indicates above normal TPP1
levels in all but one of the tested brain regions.
• In addition, the TPP1 protein can be detected (using a brown staining method) in tissues slices taken from different
brain regions.
• In untreated LINCL dogs, TPP1 is not detectable by the enzyme activity assay or the staining method.
• Dogs are assessed for LINCL neurological symptoms using many different
tests, covering motor and mental functions. These include measures of
coordination, vision, eye movements, involuntary body movements, and
reflexes.
• Without treatment, affected dogs show signs of disease in all tests by ~30
weeks of age. They reach end-stage disease and must be euthanized
between 45 and 48 weeks of age.
• In dogs that received AAV2-CLN2 gene therapy, the onset and
progression of these clinical changes were delayed and their
cognitive function dramatically improved. Dogs with no treatment
made many errors (exemplified by red line). Dogs with treatment
made very few errors (exemplified by blue and green line)
Normal
AAV2-CLN2 Treated
TPP1 enzyme activity
pmol TPP1/mg protein
In untreated affected dogs, ventricular spaces enlarge to ten
times the size of normal dogs. AAV2-CLN2 gene therapy
significantly reduces this effect.
INTRODUCTION
The CLN2 gene codes for a protein called TPP1. TPP1 is an
enzyme that works inside cells to help degrade unwanted
material. In LINCL (late infantile neuronal ceroid lipofuscinosis)
the CLN2 gene is mutated such that little or no functional TPP1
protein is made. Without TPP1, storage material builds up in
cells, leading to malfunction or death of brain cells and impaired
brain function.
GENE THERAPY: One avenue for treating LINCL is to deliver
good (non-mutated) copies of the CLN2 gene to LINCL brain
cells. These “genetically-corrected” cells would then make fully
functional TPP1.
CROSS-CORRECTION: While much of the TPP1 made within a
cell stays in that cell, a portion is secreted and taken up by
neighboring cells. This property of secretion and uptake is
called cross-correction (Figure 1). Cross-correction is valuable
in the context of gene therapy. If we can transfer the CLN2
gene to strategically situated cells in the LINCL brain, then this
can allow for cross-correction of many cells.
We are testing AAV2-CLN2 gene
therapy in the LINCL dog model
AAV2-CLN2 GENE THERAPY
Affected LINCL pups are given gene
therapy at 3 months of age. For gene
therapy, AAV2-CLN2 is injected at a single
site (lateral ventricle) or at two sites
(lateral ventricle plus cisterna magna) in
the brain.
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Normal Affected Treated
TPP1, secreted by
genetically-corrected cell
cross-corrected cell
thalamus hippocampus
cerebellum
spinal cord
rostral ependyma
caudate
Errors
4 5 6 7 8 9
Age (months)
0
Gene therapy treatment