2014 BDSRA Davidson LINCL
1
Beverly Davidson Lab: CLN2 Gene Transfer for Late Infantile Batten Disease in the dog model Luis Tecedor 1 , Yong Hong Chen 1 , Martin L Katz 2 , Fred A Wininger 2 ; Colleen S Stein 3 , Joan R Coates 2 , Melissa Carpentier 2 , Beverly L Davidson 1 , 1 Center for Cell and Molecular Therapy, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, 2 Washington University, St. Louis, MO and 3 Internal Medicine, University of Iowa, Carver College of Medicine, Iowa City, IA, 52242, contact e-mail: [email protected] CLN2 BDSRA 2014 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 AAV2 as a vehicle to get the CLN2 gene into the brain Cross-correction: 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. pressenter.net adapted from www.dunesciences .com 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. • 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. Volume (mm 3 ) Gene therapy reduces ventricular enlargement 0 4 8 12 • 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. 1 2 3 4 5 6 Norma l Affecte d Treat ed TPP1, secreted by genetically- corrected cell CLN2 gene mRNA TPP1 AAV2-CLN2 genetically- corrected cell cross- corrected cell thalamus hippocampus cerebellum spinal cord occipital cortex rostral ependyma caudate Errors 4 5 6 7 8 9 No treatment Age (months) 0 10 8 6 4 2 12 Gene therapy treatment Learning and memory test
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Transcript of 2014 BDSRA Davidson LINCL
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: [email protected]
CLN2BDSRA 2014
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
AAV2 as a vehicle to get the CLN2 gene into the brain
Cross-correction:
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.pressenter.net
adapted from www.dunesciences.com
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.
• 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.
Vol
ume
(mm
3 )
Gene therapy reduces ventricular enlargement
Normal Affected Treated0
2
4
6
8
10
12
• 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
pm
ol T
PP
1/m
g p
rote
in
In untreated affected dogs, ventricular spaces enlarge to ten times the size of normal dogs. AAV2-CLN2 gene therapy significantly reduces this effect.
INTRODUCTIONThe 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.
1
2
3
4
5 6Normal Affected Treated
TPP1, secreted by genetically-corrected cell
CLN2 gene
mRNA
TPP1
AAV2-CLN2
genetically-corrected cell
cross-corrected cell
thalamus hippocampus
cerebellum
spinal cord
occipital cortexrostral ependyma
caudate
Errors
4 5 6 7 8 9
No treatment
Age (months)
0
10
8
6
4
2
12
Gene therapy treatment
Learning and memory test
