Center for Ehlers-Danlos Syndrome Research at Baylor College of

Center for Ehlers‐Danlos Syndrome Research
at Baylor College of Medicine
Brendan Lee, MD, PhD
Department of Molecular and Human Genetics
Where are we in EDS research and care?
• Heritable Disorders of Connective Tissue (1956)
– Victor McKusick MD
• Marfan
• Osteogenesis Imperfecta
• Ehlers Danlos syndrome Molecular Mechanisms
• Majority of patients have alterations in one of the two type V collagen genes, COL5A1 and COL5A2
• Most common mechanism is 50% decrease of the amount of Collagen type V
Collagen Fiber
Type V Collagen
• Found in skin, joints and bones
• Controls the thickness of type I collagen fibrils
• Mouse Collagen type V is 94% similar to human
Unanswered Questions
• Why does loss of collagen type V affect skin and joints? • What are the biochemical alterations in tendon, ligaments and skins in EDS • Can we correct EDS by expressing collagen type V or by identifying and then targeting downstream consequences? Classical EDS Mouse Model
• Complete loss of Col5a1 results in lethality and heart failure. • 50% loss of Col5a1:
– Skin
Hyper‐extensible skin
Reduced strength of normal and wounded skin
– Tendon
– Vascular Decreased aortic stiffness and breaking strength (esp. in the descending aorta)
Develop the quantitative tools to assess a mouse model of EDS and to effects of potential treatments
Classical EDS Mouse Model
WT
EDS
Wenstrup R J et al. J. Biol. Chem. 2006
Skin Phenotype of EDS Mice
Histological Assessment
Hair
Epidermis
Collagen
Fibroblast
Blood
vessel
Dermis
Subcutaneous
Fat
Skin Phenotype of EDS Mice
Histological Assessment
WT
EDS
Epidermis
Dermis
Fat Layer
Muscle
Skin Phenotype of EDS Mice
Mechanical Testing
Skin Phenotype of EDS Mice
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Breaking point
CTRL
Breaking point
EDS
0.0
1.5
3.0
4.5
6.0
7.5
9.0
10.5
12.0
13.5
15.0
16.5
18.0
19.5
21.0
22.5
24.0
25.5
27.0
28.5
30.0
31.5
33.0
34.5
Load (N)
Maximum Load to Tear for Skin
Time (sec)
WT EDS
Skin Phenotype of EDS Mice
Wound Healing Model
Visual
Assessment
Splint
Histology
Punch
Wound
Gene
Expression
Skin Phenotype of EDS Mice
Gene Transfer in Wound Healing Model
Control
(no virus)
Treatment
(Virus with normal
Col5a1)
Skin Phenotype of EDS Mice
Helper‐dependent Adenoviral Vectors
• Adenovirus: Can cause common cold
• Helper‐dependent Adenovirus: Adenovirus without viral gene but with the gene we want to express
• Can be generated in our lab using a cell factory HDV-EF1-Col5 Generation
ITR
ITR
Human Stuffer
HDV-EF1-Col5a1
EF1
Col5
HDV-EF1-Col5a2
Skin Phenotype of EDS Mice
Expected Effect of Gene Therapy on Wound Healing
Evaluation of the strength of EDS tendons
Classical EDS Mouse Model ‐
Tendons • Collagen fibrils are smaller in diameter and have irregular cross section
• Flexor digitorum longus tendon in EDS mice has lower stiffness (consistent with increased elasticity) Wenstrup R J et al. J. Biol. Chem. 2011
Tendon Phenotype of EDS Mice
Histological Assessment
Tendon Phenotype of EDS Mice
Maximum Load to Tear for Achilles Tendon
12
Breaking point
CTRL
Breaking point
8
6
4
EDS
2
0
0.00
0.18
0.36
0.54
0.72
0.90
1.08
1.26
1.44
1.62
1.80
1.98
2.16
2.34
2.52
2.70
2.88
3.06
3.24
3.42
3.60
3.78
3.96
Load (N)
10
Time (sec)
WT EDS
Genetic Tools to Study Tendon Development
Pryce B A et al. Development 2009;136:1351-1361
What are the molecular differences in EDS tissues? Gene Expression (RNASeq) Study
DNA
RNA
Control Tendon/Skin
Protein
EDS Tendon/Skin
Evaluation of EDS Bone Phenotype
Classical EDS Bone Observations
• Higher fracture rates
• Lower bone mineral density Bone Phenotype of EDS Mice
Mechanical Testing via 3‐Point Bending
Bone Phenotype of EDS Mice
Stiffness of Femur
Stiffness (GPa)
120
104
100
80
61
60
40
20
0
CTRL
WT
1
HET
EDS
Test results –
•CTRLs are significantly stiffer
•EDS may be more brittle (high absorption of elastic energy with low plasticity)
What can we learn from related or “opposite” syndromes? Bruck Syndrome
Patients have contractures, the opposite of EDS
Mouse Model of Bruck Syndrome
What can we learn from less common EDS types? Whole Exome Sequencing (WES)
• WES identifies changes in genes that may cause disease
• WES facilitate disease gene discovery
/
http://www.ucmp.berkeley.edu
Why Focus on Gene Discovery?
• Allows us to gain greater understanding of the basic science underlying connective tissue biology
• Allows for more precise genetic diagnosis
• May lead to new therapeutic strategies for disorders
Osteogenesis Imperfecta:
A Success Story
Patients with “brittle bone disease” were enrolled Exome studies were performed
New “brittle bone disease” genes identified
(eg WNT1, SERPINF1, FKBP10, CRTAP,)
Greater understanding of unifying disease mechanisms
Targeted therapies underway EDS Type III Clinical Exome Project
• Baylor Human Genome Sequencing Center
• One of 3 large‐scale sequencing centers in U.S.
• Partners with WGL which performs WES on a clinical basis Clinical Studies
• Collaboration with Acer Therapeutics to make Celiprolol available in the US
Celiprolol
• Cardioselective β blocker with β2 agonist
properties
• Brings about dilatation of blood vessels
• Decrease in heart rate is less as compared to
other β blockers
• Effective in treatment of mild-to-moderate
hypertension
• Also used in prevention of angina
• Use associated with improvements in lipid profile
and no adverse effects on glucose metabolism
Celiprolol is effective in EDS type IV % with vascular
free events
100
Celiprolol
75
Control
50
25
0
% with visceral free events
100
Celiprolol
75
50
Control
25
0
0 1 2 3 4 5 Time in yrs
Ong et al Lancet 2010
Acknowledgements
• Ott Family
– David and Pam Ott
– Rogers and Elizabeth Herndon
• Clinical team
– Shweta Dhar
– John Belmont
– Sandesh Nagamani
• The joint and tendon team
–
–
–
–
–
–
Merry Ruan
Matthew Grol
Keren Machol
Ronit Marom
Caressa Lietman
Lindsay Burrage

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