Investigating the molecular basis of NCL: a genetic research path towards drug development

1. CLN2,
CLN3,
CLN5,
CLN6,
CLN12,
Unknown
NCL
The
MGH
Center
for
Genomic
Medicine
Ba?en
Disease
Research
Program
InvesGgaGng
the
molecular
basis
of
NCL:
a
geneGc
research
path
towards
drug
development
Susan
L.
Cotman,
Ph.D.
(Principal
InvesGgator),
Uma
Chandrachud,
Ph.D.,
Anna-­‐Lena
Hillje,
Ph.D.,
Ursula
Ilo,
M.Sci.,
Abigail
Nowell,
Madeline
C.
Klein
Center
for
Genomic
Medicine,
Department
of
Neurology,
Massachuse?s
General
Hospital,
Harvard
Medical
School
Introduc)on
and
Laboratory
Objec)ves
!
DNA
mutaGons
in
one
of
at
least
13
different
genes
lead
to
the
clinical
symptoms
of
Ba?en
disease,
or
NCL
(for
neuronal
ceroid
lipofuscinosis).
In
some
cases,
idenGfying
the
geneGc
cause
of
disease
remains
a
significant
challenge.
! In
many
forms
of
NCL,
how
the
DNA
mutaGons
lead
to
the
disrupted
cellular
processes
is
not
yet
completely
understood.
It
is
also
sGll
not
well
understood
which
disrupted
processes
lead
to
the
disease
symptoms.
!
Understanding
the
steps
in
the
disease
process,
from
geneGc
trigger
(DNA
mutaGon)
to
clinical
onset
and
progression,
is
important
for
designing
therapies.
!
Our
laboratory
uses
geneGc
model
organisms
as
well
as
human
cell
culture
systems
to
formulate
and
test
hypotheses
regarding
NCL
protein
funcGon
and
the
NCL
disease
process.
! We
also
parGcipate
in
collaboraGve
efforts
to
improve
the
methods
for
idenGfying
the
DNA
mutaGons
and
to
further
improve
the
availability
of
paGent
samples.
Conclusions
! The
increasingly
well
characterized
disease
models
that
now
exist,
which
recapitulate
NCL
DNA
mutaGons,
are
contribuGng
to
important
advances
in
our
understanding
of
the
molecular
basis
of
the
NCLs
! Research
with
these
model
systems
is
leading
to
new
candidate
drug
targets
that
are
currently
being
studied
for
drug
development
! Screening
of
drug
libraries
is
idenGfying
new
informaGon
and
new
candidate
drugs/drug
targets
! Our
understanding
of
the
funcGons
of
the
NCL
proteins
is
increasing,
which
will
lead
to
be?er
targeted
therapies
and
biomarker
tools
for
monitoring
treatment
! New
methods
for
determining
the
underlying
NCL
DNA
mutaGons
are
leading
to
an
increasing
awareness
of
shared
disease
biology
with
other
forms
of
human
disease
and
in
a
greater
appreciaGon
of
how
mutaGons
in
NCL
genes
affect
human
health
more
broadly.
This
knowledge
will
increase
awareness
and
correctly
idenGfy
more
paGents
and
the
underlying
genes
causing
their
disease
! There
is
an
increasing
uGlizaGon
of
paGent
samples
linked
to
geneGc
and
clinical
informaGon
and
a
greater
effort
to
deepen
this
important
resource
Acknowledgements: We thank our numerous scientific and clinical collaborators and supporters, as well as the organizations who’ve provided funding to support our research. We would
also like to expressly thank the families and patients who’ve donated samples and participated in our research studies. Recent funding sources include the Batten Disease Support and
Research Association, the National Institutes of Health: National Institute for Neurological Diseases and Stroke, the MGH Executive Committee on Research, Catherine’s Hope for a Cure,
Beyond Batten Disease Foundation, Beat Batten, and Our Promise to Nicholas.
A
research
tool-­‐kit
for
protein
func)on
and
drug
discovery
Drug screening
to identify
disease
modifiers/drugs
-unbiased drug libraries
-candidate drug testing
Protein detection assay development
for isoform-specific quantification of NCL
proteins in biological samples
Facilita)ng
the
gene)c
research
cycle
for
all
forms
of
NCL
Conceptualiza)on
of
the
NCL
disease
process
Model
systems
we
have
developed
and/or
use
for
NCL
research
GeneGc
Studies
to
IdenGfy
‘Unknowns’
and
GeneGc
Modifiers
• Next
GeneraGon
Sequencing
of
Whole
Exomes/Genomes
• Candidate
Gene
Screening
• Adult
NCL
Gene
Discovery
ConsorGum
• AnalyGc
and
TranslaGonal
GeneGcs
Unit
of
MGH
(Dr.
Mark
Daly,
Dr.
Daniel
MacArthur)
Mouse
models
and
cell
culture
models
• Useful
in
idenGfying
possible
early,
pre-­‐
clinical
symptoms
• Biomarkers
development
• Improved
descripGon
of
the
disease
process
Screening
for
drugs
using
mouse
and
human
neuronal
cells
• Unbiased
screen
of
a
large
drug
library
• CollaboraGng
partners
with
other
academic
labs
and
pharmaceuGcal/biotech
companies
to
test
candidate
treatments
Systems
for
translaGon
of
findings
to
human
paGents
Fibroblasts
Lymphoblasts
**Human
induced
pluripotent
stem
cells
(hiPS
cells)—can
be
differen:ated
into
affected
cell
types,
like
neurons
and
glia
MGH-­‐Ba?en
Disease
Center
(Dr.
Kathryn
Swoboda,
Dr.
Winnie
Xin,)
• MGH
NeurogeneGcs
DNA
Lab
• NCL
Registry
and
Biorepository
• CollaboraGve
efforts
with
Dr.
Jon
Mink
and
others
to
develop
merged,
searchable
clinical
database
linked
to
biorepository
samples
Cln3∆ex7/8 knock-in mice
• Genetic replica of the ~1-kb
deletion mutation most
frequently observed in CLN3
patients
• Cln6nclf mice
CbCln3∆ex7/8 and
CbCln6nclf mouse
neuronal precursor cells
Patient fibroblasts and
reprogrammed human induced
pluripotent stem (hiPS) cells
Can be turned into brain cells and
other relevant cell types
• Phenotyping
(characterizing abnormalities at
the cellular and whole
organism level)
• Disease modifier studies
(cell-based screening and mouse
modifier studies)
• Molecular analysis
(single gene and genomic level)
Potential modifiers:
Mitochondrial pathways
Intracellular Ca2+
Autophagy pathway modifiers
êAutophagy clearance
êendocytosis
êlysosomal protein trafficking
Mitochondrial changes
Subunit c
storage
Sensorimotor
processing affected
Gliosis
Motor function decline
Working
chronology of the
disease process in
NCL genetic
models
cln3 knockout Dictyostelium
discoideum
• Social amoeba, single cell
stage to multicellular stage
developmental life cycle
• Expression of human
CLN3 in the cln3- Dicty
cells rescues
abnormalities
demonstrating conserved
function across evolution
Conception
NCL gene status
= two abnormal copies of an NCL gene
Lifeline of a person with two NCL mutations
Clinical Diagnosis
End-stage
disease
Conception
NCL gene status
= at least one normal copy of NCL gene
End-of-life
Lifeline of an unaffected individual
• Different genetic or environmental modifiers could act at
different stages and affect the progression towards end-stage
disease, which primarily affects the brain and eyes. However,
new research indicates other organ systems may also ultimately
become affected.
• Identifying these modifying factors and then targeting them
through interventions/drugs (blue arrows) could slow or halt
further advancement of disease progression. We also have to
develop better ways of monitoring the effects of treatments,
which are a key component of successful clinical trials and
reaching new drug approval.
CLN3
Drug libraries (e.g. >2000
FDA-approved drugs)
1. Assays are developed that measure a difference between
unaffected and affected cells. In this example, there are
more green dots (a lysosome-related structure called an
autophagosome, labeled by a fluorescent marker) in
affected cells than in unaffected cells.
2. Automated screen performed
3. Hits identified that make the
affected cells look more like the
unaffected cells (e.g. potential drugs,
also tool compounds for research)
4. Follow-up studies and
optimization are performed, which
often leads to new rounds of
modified drug library testing
Unaffected Affected