disease modelling and therapeutic testing- potential ... · advantages of direct conversion...
TRANSCRIPT
![Page 1: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/1.jpg)
2nd Annual International SLC6A1 Symposium -12/5/2019
Disease modelling and therapeutic testing- Potential
implications for SLC6A1 research
Julieth Andrea Sierra Delgado, MD, MSc.
Meyer lab
Center for Gene Therapy
Nationwide Children’s Hospital
Columbus, Ohio, USA
![Page 2: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/2.jpg)
Reasons for in vitro modelling on neurological
diseases
• Neurological diseases: Difficult to study• Neurological diseases: Difficult to study
• Access to tissue/patients limited: post-mortem
• Neurological diseases: Difficult to study
• Access to tissue/patients limited: post-mortem
• Complex phenotype. Not always genotype-phenotype correlation
In vitro modelling allows us to understand physiological pathways,
cell interactions and get close to the pathophysiology of different
diseases
![Page 3: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/3.jpg)
Non-cell autonomous component of neurological
diseases
http://www.clontech.com/CA/Support/Clontech_Cartoons/2013/September_2013
Neuron disease phenotype and progression is influenced by other cell types
Astrocytes
Microglia
Oligodendrocytes
![Page 4: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/4.jpg)
Why are Astrocytes important?
• Neurosupportive
• Vasomodulation
• Part of Blood-Brain
barrier.
• Regulation of:
Glucose,
neutrotransmiters,
Ions.
![Page 5: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/5.jpg)
Amyotrophic Lateral Sclerosis
• Adult onset neurodegenerative disease
• Approx. 20’000 individuals in USA
• Degeneration of Motor Neurons
• Paralysis and ultimately death
• ~ 90% sporadic, 10% familial cases
• Various disease causing genes identified
Vast Majority of cases: cause unknown
How do we study this disease?
https://alsnewstoday.com/
![Page 6: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/6.jpg)
Cell culture model using human post-mortem cells
Astrocytes from human ALS patients are toxic to motor neurons
![Page 7: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/7.jpg)
Post mortem astrocytes are a limited source
• Post-mortem samples = limited availability + represent only end-stage of
disease
Human skin fibroblasts?
• No gain for the donor
![Page 8: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/8.jpg)
Kim, Curr Opinion in Neurobiol 2012
Current reprogramming methods in use
![Page 9: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/9.jpg)
Advantages of direct conversion compared to
classical reprogramming
• Direct conversion is much faster
• No clonal selection – no clonal variation
• Maintenance much less time consuming
• Able to generate several disease
relevant cell types (Neurons,
Astrocytes, Oligodendrocytes) in
parallel from the same source
Kim, Curr Opinion in Neurobiol 2012
![Page 10: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/10.jpg)
Advantages of direct conversion compared to
classical reprogramming
• Reprogrammed cells can be used on
different assays
• Co-Culture model system
• Metabolic assays
Kim, Curr Opinion in Neurobiol 2012
![Page 11: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/11.jpg)
iAstrocytes are similarly toxic to motor
neurons compared to primary cells
Meyer et al., PNAS 2014
![Page 12: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/12.jpg)
Astrocytes and Oligodendrocytes derived with
this method express cell type specific genes
Ferraiuolo et al., PNAS 2016
Microarray RNA sequencing shows different expression patterns between primary
fibroblast lines and the astrocytes and oligodendrocytes differentiated from them.
![Page 13: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/13.jpg)
Converted fibroblasts are used to model
neurological disease and drug screenings
Modified from L. Ferraiuolo
Goal 2: Can we identify markers that might help sub-classify
patient groups for potential treatments?
Goal 1: Can this system be used to screen potential therapeutics?
NPC colonies
Differentiate NPC into
Astrocytes
iAstrocyte
cultureALS sick
Astrocytes
![Page 14: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/14.jpg)
Patient subpopulations react differently to
treatments
Identification of responders and non-responders to select optimal treatment option
for each patient
Sporadic Familial FamilialHealthy
![Page 15: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/15.jpg)
• Currently more than 50 different cell lines from various neurodegenerative
and neurological disorders with mutations and different disease courses:
o Lysosomal storage diseases (Batten disease)
o Neurodevelopmental diseases (Rett Syndrome)
o Ion channelopathies (SCN2A)
• Disease controls, sibling controls and age matched healthy controls
Expanding the model system to different
diseases
![Page 16: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/16.jpg)
• CLN3: Neurodegenerative disorder:
Dysfunction of lysosomes:
Accumulation of storage material.
• Clinically:
o Seizures
o Progressive deterioration of cognition (dementia)
o Motor function impairment
o Rapid vision loss
o Affects other systems (CV).
Juvenile batten disease (CLN3)
https://bdsra.org/family-profiles/
![Page 17: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/17.jpg)
CLN3 patient Astrocytes are toxic to neurons
0
50
100
150
iAstrocyte Cell line
% o
f M
oto
r N
eu
ron
S
urv
ival
CLN3 3A
CLN3 4A
Healthy
Controls CLN3
0
50
100
150
iAstrocyte Cell line
% o
f M
oto
r N
eu
ron
S
urv
ival
CLN3 3A
CLN3 4A
Healthy
Controls CLN3
0
50
100
150
iAstrocyte Cell line
% o
f M
oto
r N
eu
ron
S
urv
ival
CLN3 3A
CLN3 4A
Healthy
Controls CLN30
50
100
150
% o
f M
oto
r N
eu
ron
S
urv
ival
He
alth
yC
LN
3
![Page 18: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/18.jpg)
Rett Syndrome
• X-linked neurodevelopmental Autism Spectrum Disorder (ASD)
• Clinically:
o Loss of motor function
o Loss of speech
o Stereotypic hand gestures.
o Seizures
Cellular Phenotype: global compaction of neurons with
shortened/fewer neurites
![Page 19: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/19.jpg)
Astrocytes from Rett patients show various severity
towards motor neuron morphology and survival
Healthy Controls Mild Severe
RETT-1
RETT-2
RETT-3
RETT-3 (clone)
CTRL B
CTRL A
RETT-5
RETT-4
![Page 20: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/20.jpg)
SCN2A mutations
• SCN2A: Sodium channel Helps propagate
the electrical signals that neurons use to
communicate
• Primarily expressed in excitatory neurons:
Also in astrocytes.
• Neurodevelopmental disorders:
o Epilepsy
o Nonsyndromic intellectual disability
o Autism spectrum disorders.
https://www.scn2a.org/personal.html
![Page 21: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/21.jpg)
Healthy SCN2A lines
SCN2A Astrocyte toxicity
NOT all SCN2A astrocytes are toxic to motor neurons.
![Page 22: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/22.jpg)
Pharmacological treatment restores mitochondrial activity of SCN2A astrocytes.
SCN2A patient astrocytes show alterations in
mitochondrial function that can be reversed by
compound treatment
AG
155
S3
S3+ S
4S4+
1104
1104
+
TIGER
TIGER+
0
2
4
6
ATP
OC
R (
pm
ol/
min
/to
tal p
rote
in)
*
*
****
Ctr-1
(-)
Ctr-2
(-)
Ctr-3
(-)
Ctr-3
(+)
SCN2A
-1(-)
SCN2A
-1(+
)
SCN2A
-2(-)
SCN2A
-2(+
)
SCN2A
-3(-)
SCN2A
-3(+
)
0
50
100
150
200
% M
oto
r N
eu
ron
Su
rviv
al
**
![Page 23: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/23.jpg)
Glia contributes to the pathophysiology of epilepsy
Devinsky, O et, al, Trends Neurosci., 2013.
![Page 24: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/24.jpg)
GAT1 is expressed by presynaptic neurons and
Astrocytes
Schousboe, Arne, et al. "Astrocytic gaba transporters: pharmacological properties and targets for antiepileptic drugs." Glial Amino Acid Transporters. Springer, Cham, 2017. 283-296..
![Page 25: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/25.jpg)
Take home message
• Direct conversion is a promising tool to generate cell types of
the CNS
• These cells can be used to study disease mechanisms, analyze
commonalities and differences between individual patient
subpopulations in different disease mechanisms.
• Useful tool to test potential new targets on a broad variety of
backgrounds
• Could be a valuable tool to study SLC6A1
![Page 26: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/26.jpg)
Acknowledgements
Kathrin Meyer LaboratoryShibi Likhite Ph.DCassandra Dennys-Rivers Ph.DShrestha Sinha Ray Ph.DYacidzohara Rodriguez Ph.DKrizelle Alcantara M.ScAmy Huffenberger BSAnnalisa Hartlaub, BSRochelle Rodrigo BSXiaoJean Zhang BSSamantha Powers M.ScMaura Schwartz BSRicardo Pineda BSFlorence Roussel M.ScMegan Baird BS, MBALeah Schmelzer Stefanik BSJoseph Caporale BA
Laura Ferraiolo PhD.University of Sheffield
![Page 27: Disease modelling and therapeutic testing- Potential ... · Advantages of direct conversion compared to classical reprogramming • Direct conversion is much faster • No clonal](https://reader035.vdocument.in/reader035/viewer/2022071213/604195f4d424f12dd27d4598/html5/thumbnails/27.jpg)
Support