Investigating the molecular basis of NCL: a path to improved diagnosis and drug development

1. CLN2,
CLN3,
CLN6,
Adult,
Unknown
NCL
Inves7ga7ng
the
molecular
basis
of
NCL:
a
path
to
improved
diagnosis
and
drug
development
Susan
L.
Cotman,
Ph.D.
(Principal
Inves7gator),
Uma
Chandrachud,
Ph.D.,
Anna-­‐Lena
Hillje,
Ph.D.,
Ursula
Ilo,
M.Sci.,
Abigail
Nowell,
Hyejin
Oh,
Ph.D.,
Center
for
Human
Gene7c
Research,
Department
of
Neurology,
MassachuseQs
General
Hospital,
Harvard
Medical
School
Introduc)on
and
Laboratory
Objec)ves
!
DNA
muta7ons
in
one
of
at
least
13
different
genes
lead
to
the
clinical
symptoms
of
BaQen
disease,
or
NCL
(for
neuronal
ceroid
lipofuscinosis).
In
some
cases,
iden7fying
the
gene7c
cause
of
disease
remains
a
significant
challenge.
! In
many
forms
of
NCL,
how
the
DNA
muta7ons
lead
to
the
disrupted
cellular
processes
is
not
yet
completely
understood.
It
is
also
s7ll
not
well
understood
which
disrupted
processes
lead
to
the
disease
symptoms.
!
Understanding
the
steps
in
the
disease
process,
from
gene7c
trigger
(DNA
muta7on)
to
clinical
onset
and
progression,
is
important
for
designing
therapies.
!
Our
laboratory
uses
gene7c
model
organisms
as
well
as
human
cell
culture
systems
to
formulate
and
test
hypotheses
regarding
the
NCL
disease
process.
! We
also
par7cipate
in
collabora7ve
efforts
to
improve
the
methods
for
iden7fying
the
DNA
muta7ons
and
to
further
improve
the
availability
of
pa7ent
samples.
Conclusions
! The
increasingly
well
characterized
disease
models
that
now
exist,
which
recapitulate
NCL
DNA
muta7ons,
are
contribu7ng
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
iden7fying
new
informa7on
and
new
candidate
drugs/drug
targets
! Our
understanding
of
the
func7ons
of
the
NCL
proteins
is
increasing,
which
will
lead
to
beQer
targeted
therapies
and
biomarker
tools
for
monitoring
treatment
! New
methods
for
determining
the
underlying
NCL
DNA
muta7ons
are
leading
to
an
increasing
awareness
of
shared
disease
biology
with
other
forms
of
human
disease
and
in
a
greater
apprecia7on
of
how
muta7ons
in
NCL
genes
affect
human
health
more
broadly.
This
knowledge
will
increase
awareness
and
correctly
iden7fy
more
pa7ents
and
the
underlying
genes
causing
their
disease
! There
is
an
increasing
u7liza7on
of
pa7ent
samples
linked
to
gene7c
and
clinical
informa7on
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 and Beat Batten.
Drug
screening
in
a
CLN3
model
iden)fies
a
candidate
target
pathway
for
therapy
development
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
Gene7c
Studies
to
Iden7fy
‘Unknowns’
and
Gene7c
Modifiers
• Next
Genera7on
Sequencing
of
Whole
Exomes/Genomes
• Candidate
Gene
Screening
• Adult
NCL
Gene
Discovery
Consor7um
• Analy7c
and
Transla7onal
Gene7cs
Unit
of
MGH
(Dr.
Mark
Daly,
Dr.
Daniel
MacArthur)
Mouse
models
and
cell
culture
models
• Useful
in
iden7fying
possible
early,
pre-­‐
clinical
symptoms
• Biomarkers
development
• Improved
descrip7on
of
the
disease
process
Screening
for
drugs
using
mouse
and
human
neuronal
cells
• Unbiased
screen
of
a
large
drug
library
• Collabora7ng
partners
with
other
academic
labs
and
pharmaceu7cal/biotech
companies
to
test
candidate
treatments
Systems
for
transla7on
of
findings
to
human
pa7ents
Fibroblasts
Lymphoblasts
**Human
induced
pluripotent
stem
cells
(hiPS
cells)—can
be
differen:ated
into
affected
cell
types,
like
neurons
and
glia
MGH-­‐BaQen
Disease
Center
(Dr.
Kathryn
Swoboda,
Dr.
Winnie
Xin,)
• MGH
Neurogene7cs
DNA
Lab
• NCL
Registry
and
Biorepository
• Collabora7ve
efforts
with
Dr.
Jon
Mink
to
develop
merged,
searchable
clinical
database
linked
to
biorepository
samples
Drug libraries (e.g. >2000
FDA-approved drugs)
Phenotypic brain
cell-based assays
are developed
Automated screen performed
Hits identified (e.g. potential
CLN3 drugs)
Follow-up studies to validate
and optimize leads
~2000 drugs screened
Candidate drugs that improve an abnormality
One class of drugs identified as hits targeted certain Ca2+ channels, which
prompted follow up studies on how CLN3 neurons handle Ca2+
Disease-modifying drugs; understanding these effects
can lead to new information about target pathways
Elevated
lysosomal
Ca2+
in
cultured
CLN3
brain
cells
Drugs
that
lower
the
elevated
lysosomal
Ca2+
in
cultured
CLN3
brain
cells
to
normal
levels
are
in
further
tes7ng
as
candidate
treatments
(collabora7on
with
other
groups
including
Dr.
Emyr
Lloyd-­‐Evans)
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
Lifeline of a person with two NCL mutations
Clinical Diagnosis
End-stage
disease
Conception
NCL gene status
End-of-life
Lifeline of a person with at least one normal NCL gene
• Different genetic or environmental modifiers could act at
different stages and affect the progression towards end-
stage disease.
• Identifying these factors and then targeting them through
interventions/drugs (blue arrows) could slow or halt
further advancement of disease progression.
CLN3