Gene
therapy
            Definition:
delivery
and
expression
of
genetic
             material
leading
to
an
alteration
in...
Key
goals
for
a
gene
therapy
strategy
          Easy
to
administer.

          Long‐term
production
of
a
therapeutic
pro...
Where
to
delivery
it?
                                     During & Tipene-Hook,                                     New E...
Getting
genes
into
cells:
gene
delivery
  Ex
vivo
‐
gene
transfer
performed
   in
cell
culture
before
   transplantation
...
Gene
delivery
‐
viral
vectors
  Exploits
natural
ability
of
viruses
to
infect
   mammalian
cells
  Genetically
engineere...
Life
cycle
of
a
viral
vector
      VECTOR                                  GENE OF INTEREST                               ...
AAV
vector
transgene
cassette
  

          e.g AAV expression cassette                              ITR     rep         ...
  Retrovirus
      7‐11kb
ss
RNA;
Following
target
cell
entry,
RNA
genome
is
reverse
         transcribed
into
ds
DNA
wh...
  Herpes
simplex
virus
(HSV1)
     152kb
linear
ds
DNA
     Broad
host
cell
range
     Main
disadvantage
‐
intrinsic
t...
Achieving
specificity
‐
vector
targeting
  Needed
for:
     Safety
–
minimises
potential
toxicity
of
gene
product
in
hea...
Gene
expression
                                  Recombinant protein  Level
of
transgene
expression
    determined
by
pr...
Regulatory
systems
    Endogenous
promoter‐regulated
transcription
systems
that
are
responsive
to
     physiological
stim...
How
to
test
a
gene
therapy
strategy?
       Test
functionality
of
therapeutic
gene
cassettes

       In
vitro
and
in
viv...
Viral
vectors:
tools
for
gene
function
                                        studies
  Gene
overexpression
     Expres...
Combined
use
of
germline
and
viral
                transgenic
methods
        Can
be
combined
with
transgenic
mouse
model...
Kirik D & Bjorklund A, Trends Neurosci. 26(7)                                       2003, 386-392                         ...
Biochemical
mechanism
of
RNAi
                                         1.  dsRNA
is
introduced
into
the
cell
             ...
siRNA
   21‐23
bp
of
double‐stranded
RNA
with
a
3’
dinucleotide
overhang
   Algorithms
are
used
to
design
RNAi
sequences...
shRNA
                                   siRNA template           Termination sequence      Promoter/enhancer           • ...
MicroRNA
(miRNA)
  Endogenous
RNAi
pathway
in
animal
cells
  Endogenous
~21‐mer
small
RNA
molecules
from
non‐coding
RNA
...
miRNA
reduce
steady
state
protein
levels
    Translational
repression
          Imperfect
duplex


          Reduces
pr...
Potential
Applications
of
RNA
Interference       Testing
hypotheses
of
gene
function

       Functional
screening
and
ta...
Gene
therapy
for
Parkinson’s
disease
                     Parkinson’s
disease
                           Progressive
neu...
What
causes
these
dopamine
cells
to
die?
  Still
largely
unclear.
Possible
contributors:
      Oxidative
stress
      M...
Treatment
strategies
    Goals
       Alleviate
motor
symptoms
       Prevent
ongoing
cell
death
process
in
the
SNpc
 ...
Gene
therapy
strategies
for
PD
    Three
main
strategies:
          Biochemical
augmentation
‐
alleviating
symptoms
    ...
Neuroprotection
                  Introduce
growth
factor
genes
that
promote
cell
survival
and/                    or
reg...
Methods
used
to
assess
whether
gene
therapy
has
                              therapeutic
effect
and
benefit
             ...
  Rate
of
rotation
(e.g
turns/min)
is
directly
proportional
to
                     severity
of
the
lesion
and
dopamine
l...
Before
deep
brain
stimulation
 After
deep‐brain
stimulation
                                 8
  Use
a
genetic
approach
to
silence
STN
neurons
by
introducing
    GAD
(glutamic
acid
decarboxylase)
‐
enzyme
involved
in...
Deep
brain
stimulation
vs
STN
gene
therapy
                          Study
design
                                        ...
Functional
assessment
in
humans
‐
GAD
gene
                                    therapy

        Fluoro‐deoxyglucose
PET
i...
Improvement
in
movement
on
treated
side
            Functional
PET
imaging
                                           12
Neural
network
activity
discriminates
PD
and
                                     controls
Increased
metabolism
in
SMA
aft...
Current
status
of
PD
gene
therapy
    Unravelling
pathways
involved
in
mediating
death
of
      dopaminergic
neurons
will...
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Gene therapy 2010

  1. 1. Gene
therapy
   Definition:
delivery
and
expression
of
genetic
 material
leading
to
an
alteration
in
the
instruction
 set
of
a
cell
for
a
therapeutic
purpose
   1960’s
‐
idea
of
gene
therapy
proposed
   1990
‐
first
human
gene
therapy
clinical
trial

   2010
‐
still
no
gene
therapy
product
commercially
 available

   Somatic
vs
germ‐line
gene
therapy
 Gene
therapy
‐
making
it
work
 What genetic material to deliver?Where to deliver it? How to deliver it?How to control it? How to test it? 1
  2. 2. Key
goals
for
a
gene
therapy
strategy
   Easy
to
administer.

   Long‐term
production
of
a
therapeutic
protein
or
 therapeutic
effect
following
a
single
application.
   Highly
specific,
targeted
to
disease
cells/tissue
only,
 thereby
little
or
no
side‐effects.
 What
genetic
material
to
deliver?
   Genes
and
RNA
interference
sequences
   Correcting
faults
by:
   Replacing
a
non‐functional
gene
with
a
functional
copy
   Silencing
an
abnormally
functioning
gene
   Altering
the
phenotype
of
a
cell
   Need
to
know
something
about
the
disease
process
   Inherited
or
acquired,
acute
or
chronic
   Treatment
outcome
   Cure,
alter
natural
history,
alleviate
symptoms
 2
  3. 3. Where
to
delivery
it?
 During & Tipene-Hook, New Ethicals 1997 May, 57-65 Factors
to
consider
  Many
diseases
affect
multiple
sites
  Effects
on
circuitry
e.g
brain
  Gene
may
need
to
be
delivered
to
widespread
areas
 or
restricted
to
a
specific
organ/group
of
cells
 3
  4. 4. Getting
genes
into
cells:
gene
delivery
  Ex
vivo
‐
gene
transfer
performed
 in
cell
culture
before
 transplantation
  In
vivo
‐
gene
transfer
performed
 in
situ
  Therapeutic
gene
‐
transgene
  Success
of
gene
therapy
 dependent
on
efficient
gene
 transfer

  Gene
transfer
mediated
by
gene
 delivery
vehicles
(=vectors)
 www.biochem.arizona.edu/. ../Lecture25.html Gene
delivery‐
non‐viral
vectors
   Plasmid
DNA
   Liposomes

   DNA
mixed
with
cationic
lipids
and
polymers
 (polylysine,
protamine)
becomes
encased
 within
a
lipid
bubble.

   Cellular
uptake
via
an
endocytic
process.
   No
restriction
on
size
of
gene,
easy
and
 cheap
to
manufacture.
   Main
disadvantages
are
relative
inefficiency
 in
gene
delivery
and
transient
expression
 4
  5. 5. Gene
delivery
‐
viral
vectors
  Exploits
natural
ability
of
viruses
to
infect
 mammalian
cells
  Genetically
engineered
to
carry
a
transgene
 cassette
and
deposit
it
within
a
cell.
  Engineered
for
a
single
round
of
host
cell
infection
 by
removal
of
viral
genes
involved
in
replication.
 VIRUS VIRAL PROTEINS ASSEMBLY SPREAD OF INFECTION VIRAL GENES 5
  6. 6. Life
cycle
of
a
viral
vector
 VECTOR GENE OF INTEREST DISPLACED VIRAL GENES PROTEIN OF INTEREST NO VIRAL GENES NO NEW VIRUS OR VIRAL PROTEINSEngineering
vectors
from
viruses
   Molecular
cloning
techniques
used
to
genetically
 engineer
a
virus
to
carry
a
transgene
cassette
   e.g.
AAV
rep
and
cap
genes
are
removed
and
 replaced
with
a
transgene
gene
cassette
   Therapeutic
genes
are
controlled
by
promoters,
 regulatory
elements
   Cell
lines
are
used
to
package
vector
particles

 6
  7. 7. AAV
vector
transgene
cassette
  

 e.g AAV expression cassette ITR rep cap ITR Wild-type AAV ITR transgene expression cassette ITR Recombinant AAV 145 1100 <1600 600 300 145bp ITR promoter transgene RE pA ITR ITR – AAV inverted terminal repeat RE – regulatory element, e.g. woodchuck postregulatory transcriptional element (WPRE) pA – polyA signal Viral
vectors
‐
types
   Retrovirus
   Herpes
simplex
virus
   Adenovirus
   Adeno‐associated
virus
(AAV)
 7
  8. 8.   Retrovirus
   7‐11kb
ss
RNA;
Following
target
cell
entry,
RNA
genome
is
reverse
 transcribed
into
ds
DNA
which
integrates
into
the
cell
chromatin
   Efficient
integration
of
transgene
into
chromosome
leading
to
stable
 gene
expression
which
persists
in
parent
and
daughter
cells.
   Good
for
ex
vivo
approaches.
   Main
disadvantage
‐
potential
for
random
integration
near
an
 oncogene.
Also
unable
to
infect
non‐dividing
cells
as
it
requires
one
 mitotic
division
for
integration
and
expression
of
the
transgene.

   Lentivirus
(HIV)
‐
subclass
of
retroviruses,
infects
both
dividing
and
 non‐dividing
cells,
stable
and
long‐term
gene
expression.

   Adenovirus
   36kb
dsDNA
   Transduces
non‐dividing
and
dividing
cells
   High
titer
vector
produced
   Main
disadvantage
‐
transient
transgene
expression
 due
to
immune
responses
directed
against
 adenoviral
proteins;
may
be
overcome
by
producing
 “gutless”
adenoviral
vectors
 8
  9. 9.   Herpes
simplex
virus
(HSV1)
   152kb
linear
ds
DNA
   Broad
host
cell
range
   Main
disadvantage
‐
intrinsic
toxicity
and
contamination
of
vector
 stocks
with
wild‐type
(pathogenic)
virus.
Difficulties
in
maintaining
 long‐term
gene
expression
  Adeno‐associated
virus
(AAV)
   4.7kb
ss
DNA
   >40
serotypes
isolated
from
human,
primates
   Non‐pathogenic
   Transduces
non‐dividing
and
dividing
cells

   Long‐term
gene
expression
(>2.5
yrs)
   Main
disadvantage
‐
small
size
 How
do
you
choose
the
right
vector
for
 the
job?

   Dependent
on
cell
targets

   Dividing
vs
non‐dividing
cells,
e.g
stem
cells
and
 post‐mitotic
cells
difficult
to
transduce,
rapidly
 dividing
epithelial
and
cancer
cells
easy
to
transduce

   Transduction
=
vector‐mediated
gene
transfer
   Vector
tropism
=
selectivity
for
a
cell
type
   Dependent
on
the
ligand
present
on
a
vector
and
 whether
the
target
cell
expresses
the
appropriate
 cell‐surface
receptor
   Gene
insert
size
 9
  10. 10. Achieving
specificity
‐
vector
targeting
  Needed
for:
   Safety
–
minimises
potential
toxicity
of
gene
product
in
healthy
cells
   Increases
gene
transfer
efficiency
–
want
to
minimise
amounts
of
 vector
needed
to
produce
gene
product
at
therapeutic
levels
  Achieved
by:
   Mode
of
delivery
e.g.
direct
injection
into
site
of
interest
   Vector
selection
   Exploit
or
modify
specificity
of
viral
vectors
for
certain
cell
types
 e.g
AAV
serotypes
(human,
primate)
   Pseudotype
vectors
   Modify
capsid
shell
that
allows
binding
to
different
receptors
 AAV2 AAV1/2eGFP expression in the hippocampus brain region following AAV2vector or AAV1/2 vector-mediated gene transfer How
to
control
gene
expression?
   Why
is
it
necessary?
 Figure 2. Transduction of the target cell.The vector particle containing the therapeutic gene sequences binds to a cell, generally through a receptor-mediated process and then enters the cell, allowing the genome to enter the nucleus. The vector genome may go through complex processes but ends up as dsDNA that, depending on the vector, can persist as an episome or become integrated into the host genome. Expression of the therapeutic gene follows. Kay et al. 2001. Nat. Med., 7, 33-40. 10
  11. 11. Gene
expression
 Recombinant protein  Level
of
transgene
expression
 determined
by
promoter
choice
 and
inclusion
of
other
cis‐acting
 DNA
elements.
  Aim
is
to
achieve
stable,
long‐ term
transgene
expression.
 Gene therapy Regulating
transgene
expression
 Gene therapy  Essential
in
maintaining
 transgene
protein
expression
at
 steady
therapeutic
levels
  Enables
flexibility
in
adjusting
 dosage
as
disease
evolves
or
for
 therapies
tailor‐made
for
 individual
patients
  Built
in
safety
mechanism
 Gene therapy - regulated Clackson, Gene Therapy, 7, 120-125, 2000. 11
  12. 12. Regulatory
systems
  Endogenous
promoter‐regulated
transcription
systems
that
are
responsive
to
 physiological
stimuli
   e.g.
glucose
responsive
element
‐
control
insulin
release
  Exogenous
drug‐regulated
transcription
system
   e.g.
Rapamycin,
ecdysone,
tet
system
  Ideal
regulatory
system
   Low
basal
expression
   Be
inducible
to
high
levels
over
a
wide
dose
range
to
provide
a
useful
dose
 responsiveness
   Induction
should
be
a
positive
effect
‐
i.e
administer
drug
to
switch
it
on
   Drug
should
be
active
following
oral
administration
and
have
no
pleiotropic
 effects
in
mammalian
cells
   Regulatory
protein
should
have
no
effects
on
endogenous
gene
expression
 and
be
of
human
origin
to
minimise
immunogenicity
 Tet
On/Off
regulatory
system
   Transactivator
‐
regulatory
 element

–
expresses
the
 tetracycline‐controlled
 transactivator
(rtTA)
which
is
 a
chimeric
protein
composed
 of
the
Tet
repressor
fused
to
 the
VP16
activation
domain
 of
HSV.
   Tet‐responsive
element
 (TRE)
is
upstream
of
a
silent
 promoter
driving
the
 transgene
of
interest.
 Adapted from: http://www.bdbiosciences.com/clontech/tet/index.html 12
  13. 13. How
to
test
a
gene
therapy
strategy?
   Test
functionality
of
therapeutic
gene
cassettes

   In
vitro
and
in
vivo
animal
models
of
disease.
   How
adequate
are
those
models
and
do
they
reflect
 disease
processes
in
humans?

   Need
measurable
endpoints
to
assess
whether
 gene
therapy
has
therapeutic
effect
and
benefit
   Assays
for
quantifying
and/or
visualising
 therapeutic
protein
levels
 Current
status
of
gene
therapy
   Toxicity
of
some
viral
vector
systems
   Immune
responses
directed
against
cells
containing
foreign
 proteins
(viral)
leading
to
elimination
of
the
therapy.
Thus
the
 therapy
may
be
short‐lived.
   Enhanced
immune
responses
against
vectors
encountered
 previously
may
mean
problems
in
re‐dosing.
   Potential
for
some
vectors
to
recover
ability
to
cause
disease.
   Vector
targeting
not
optimised
–
transgenes
expressed
in
 healthy
and
diseased
cells
   Safety
mechanisms/control
of
gene
expression
not
sufficiently
 optimised
and
long‐term
effects
unknown
   Multigene
disorders
   Best
candidates
for
gene
therapy
are
those
that
arise
from
 single
gene
mutations.
Many
common
diseases
(e.g
Alzheimer’s,
 heart
disease)
involve
effects
on
a
variety
of
genes.
 13
  14. 14. Viral
vectors:
tools
for
gene
function
 studies
  Gene
overexpression
   Express
poorly
characterised
genes
to
gain
a
better
 understanding
of
their
physiological
effects
   Generating
animal
models
of
disease
e.g.Parkinson’s
 Huntington’s,
Alzheimer’s
diseases
  Gene
knockout
   
RNA
interference
 Advantages
over
transgenic
animals
  Can
be
engineered
to
express
single
or
two
foreign
genes,
different
 regulatory
elements
and
promoters
  Can
be
administered
at
any
developmental
stage
‐
from
in
utero
to
adult
 to
senescent
animals
  Either
short
or
long‐term
CNS
gene
expression
  Expression
can
be
obtained
in
crucial
brain
regions
while
possible
side‐ effects
associated
with
widespread
overexpression
of
the
gene
can
be
 avoided
  Not
host‐specific
‐
rats,
primates,
mice
  Inexpensive
and
rapid
to
generate
compared
with
classical
transgenics
  Higher
levels
of
transgene
expression
obtained
 1
  15. 15. Combined
use
of
germline
and
viral
 transgenic
methods
   Can
be
combined
with
transgenic
mouse
models
for
 studies
of
interactions
of
disease‐causing
proteins
 with
other
cellular
proteins
   Confirm
gene
specificity
with
transgenic
knockouts.
 Use
viral
vector‐mediated
gene
transfer
of
the
 missing
gene
to
rescue
the
phenotype
   Observe
further
potentiation
or
downregulation
of
 gene
effects
   Affect
regulation
of
genes
in
different
tissues
or
 brain
regions
Rat
and
primate
models
of
Parkinson’s
 disease
   AAV
vectors
used
to
overexpress
wild‐type
and
 mutant
forms
of
α‐synuclein
in
the
substantia
nigra
 of
rats
and
primates
   Show
many
of
the
pathological
features
of
PD

 •  protein
inclusions

 •  dystrophic
neurites
 •  progressive
loss
of
TH
cells
in
the
SNpc
 •  drug‐induced
rotation
and
motor
deficits
 2
  16. 16. Kirik D & Bjorklund A, Trends Neurosci. 26(7) 2003, 386-392 RNA
interference
(RNAi)
   A
form
of
post‐transcriptional
gene
silencing
in
which
specific
sequences
 of
double‐stranded
RNA
(dsRNA)
can
be
used
to
knock
down
the
 expression
of
a
gene
target
   Adapted
as
a
tool
to
investigate
gene
function
   History
of
RNAi
http://www.invitrogen.com/content.cfm?pageid=10088 3
  17. 17. Biochemical
mechanism
of
RNAi
 1.  dsRNA
is
introduced
into
the
cell
 2.  DICER
digests
dsRNA
into
~21bp
 dsDNA
(short‐interfering
RNAs;
 siRNAs)
 3.  The
siRNAs
are
integrated
into
 the
RNA
Induced
Silencing
 Complex
(RISC)
 4.  siRNAs
undergo
strand
 separation.
The
antisense
strand
 binds
to
its
complementary/ target
mRNA
 5.  Argonaute
‐
endonuclease
 within
the
RISC
degrades
the
http://www.ambion.com/techlib/tn/101/7.html targeted
mRNA
 Using
RNAi
as
a
tool
for
manipulating
 gene
expression
in
mammalian
cells
   In
mammalian
cells,
introduction
of
long
dsRNA
initiates
 a
cellular
interferon
response
that
ultimately
causes
cell
 shutdown
and
leads
to
apoptosis
   RNAi
can
be
achieved
in
mammalian
cells
by
direct
 delivery
of:
   synthetic
short‐interfering
RNA
(siRNA)   synthetic
short‐hairpin
RNA
(shRNA)
   microRNA
(miRNA)
 4
  18. 18. siRNA
   21‐23
bp
of
double‐stranded
RNA
with
a
3’
dinucleotide
overhang
   Algorithms
are
used
to
design
RNAi
sequences
(guidelines
provided
e.g
 30‐50%
G/C
content).

   3‐5
siRNA
sequences
covering
the
gene
of
interest
are
selected
and
tested
 for
the
degree
of
suppression
of
gene
expression.
 siRNA
‐
synthesis,
delivery,
effect

  Synthesis
in
vitro
   Chemical
synthesis
   In
vitro
transcription
systems
  Delivery
   Cell
culture
‐
transfection,
electroporation
   In
vivo
models
‐
injection
or
direct
 application

  RNAi
effect
‐
transient
(3‐7
days),
partial
to
 full
knockdown
of
gene
expression


 Davidson BL, Paulson HL. Lancet Neurol. 2004 Mar;3(3):145-9. 5
  19. 19. shRNA
 siRNA template Termination sequence Promoter/enhancer • Hairpin siRNA • Pol III: string of 3-5 T’s • Pol III, (U6, H1) • Sense and antisense • Minimal SV40 poly A • Pol II, (CMV) signal strand of siRNA shRNA
  Delivery
   Plasmids
   Viral
vectors
  Effect
   Cell
culture
‐
transient
knockdown
   In
vivo
‐
long‐term
(>1
week)
effects
 using
viral
vectors
 Davidson BL, Paulson HL. Lancet Neurol. 2004 Mar;3(3):145-9. 6
  20. 20. MicroRNA
(miRNA)
  Endogenous
RNAi
pathway
in
animal
cells
  Endogenous
~21‐mer
small
RNA
molecules
from
non‐coding
RNA
(introns,
 independent
miRNA
genes)
  Regulate
gene
transcription
by
binding
to
the
3’‐untranslated
regions
of
 specific
mRNAs
  Key
regulators
of
early
development,
cell
proliferation,
cell
death,
 apoptosis,
cell
differentiation,
brain
development
  miRBASE
‐
http://microrna.sanger.ac.uk/

   3000
miRNA
sequences
from
various
species
   Sequences
of
many
miRNAs
are
homologous
between
species

   800
unique
miRNAs
in
humans
with
400‐500
conserved
in
mice
   Regulate
expression
of
at
least
30%
of
protein‐coding
genes
 miRNA
processing
   Transcription
of
pri‐miRNA
   Processing
into
pre‐miRNA
in
the
 nucleus
by
Drosha
   Export
of
pre‐miRNA
to
the
 cytoplasm
   Dicer
complex
processing
   miRNA
strand
selection
by
RISC
 http://www.ambion.com/techlib/resources/miRNA/mirna_pro.html 7
  21. 21. miRNA
reduce
steady
state
protein
levels
   Translational
repression
   Imperfect
duplex


   Reduces
protein
expression
 without
impacting
on
 corresponding
mRNA
 levels.
(mechanism
still
 unclear)
   mRNA
degradation

   Perfect
duplex
   Transcriptional
regulation
   guiding
chromatin
 methylation
http://www.ambion.com/techlib/resources/miRNA/mirna_fun.html Using
artificial
miRNA
as
a
tool
for
gene
 function
studies
   Similar
to
applications
for
siRNA/shRNA
   Invitrogen
‐
BLOCK‐iT™
system
https://catalog.invitrogen.com /index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=12492 8
  22. 22. Potential
Applications
of
RNA
Interference   Testing
hypotheses
of
gene
function

   Functional
screening
and
target
identification

   Target
validation
for
drug
development

   Potentially
new
therapeutic
approaches
to
treating
 diseases
‐
a
new
approach
to
antisense
and
new
 possibilities
for
gene
therapy
 9
  23. 23. Gene
therapy
for
Parkinson’s
disease
   Parkinson’s
disease
   Progressive
neurodegenerative
disorder
affecting
 ~1%
of
the
population
over
the
age
of
65
   Two
types:
sporadic
and
familial
   Clinical
symptoms:
disorders
in
movement
(resting
 tremor,
rigidity,
akinesia,
bradykinesia).
Non‐motor
 symptoms
include
depression
and
dementia.
 Pathology
  Selective
degeneration
of
dopaminergic
neurons
within
the
substantia
nigra
 pars
compacta
(SNpc)
that
project
axons
to
the
striatum
  Lewy
bodies
‐abnormal
protein
aggregates
in
the
cytoplasm
of
neurons
  Dopamine
depletion
in
the
striatum
causes
disorders
in
movement
  Focal
pathology
but
has
impact
on
overall
basal
ganglia
circuitry
 Adapted from Lindvall & Bjorklund. Nat. Med. 2000. 6, 1207-1208 1
  24. 24. What
causes
these
dopamine
cells
to
die?
  Still
largely
unclear.
Possible
contributors:
   Oxidative
stress
   Mitochondrial
abnormalities
   Excitotoxicity
   Disturbances
in
calcium
homeostasis
   Toxins
   Environmental
e.g
pesticides
   Cellular
e.g.
dopamine,
α‐synuclein
 Animal
models
of
PD
   Models
have
predictive
value
with
regard
to
dopamine
deficiency

   Rodent
models

   6‐hydroxydopamine
(6‐OHDA)
injected
unilaterally
into
the
 striatum
or
SNpc
   MPTP
(systemic
injection)
‐
MPTP
converted
to
toxic
MPP+
which
 is
selectively
taken
up
by
dopamine
neurons,
inhibits
 mitochondrial
respiration

   Rotenone
(chronic
infusion
of
low
doses)
‐
mitochondrial
complex
 I
inhibitor
   transgenic
mice
(e.g
α‐synuclein)
 Non‐human
primate

   MPTP
 2
  25. 25. Treatment
strategies
  Goals
   Alleviate
motor
symptoms
   Prevent
ongoing
cell
death
process
in
the
SNpc
  Current
pharmacological
treatments
   Focus
on
augmenting
striatal
dopamine
levels
   L‐Dopa/carbidopa
‐
crosses
the
blood
brain
barrier
and
is
 converted
to
dopamine
by
aromatic
amino
acid
 decarboxylase
(AADC)
   Dopamine
agonists
and/or
monoamine
oxidase
B
inhibitors
 which
prevent
dopamine
breakdown,
antioxidants,
 glutamate
antagonists
may
provide
some
benefits
   Problems
   Loss
of
efficacy
over
time,
on‐off
effects,
dyskinesias,
 hallucinations
   Do
not
affect
ongoing
cell
death
process
 3
  26. 26. Gene
therapy
strategies
for
PD
   Three
main
strategies:
   Biochemical
augmentation
‐
alleviating
symptoms
   correct
dopamine
deficiency
   Neuroprotection
‐
altering
natural
history
of
the
 disease
   
growth
factors
(e.g.
GDNF)
   Resetting
basal
ganglia
circuitry
‐
alleviating
 symptoms/preventing
further
cell
death?
   silence
overactive
neuronal
circuits
by
expressing
glutamic
 acid
decarboxylase
(GAD)
 Biochemical
augmentation
  Express
genes
involved
in
dopamine
biosynthesis
 
 Tyrosine


 
 
 
Tyrosine
hydroxylase
(TH),
tetrahydrobiopterin
 
 L‐Dopa

 
 

 
AADC 

 
 dopamine
  Vector
systems
used:
HSV,
Ad,
AAV
  Main
disadvantage
is
inability
to
maintain
dopamine
concentrations
 at
appropriate
therapeutic
level

 4
  27. 27. Neuroprotection
   Introduce
growth
factor
genes
that
promote
cell
survival
and/ or
regeneration
of
remaining
neurons
e.g.
glial‐derived
 neurotrophic
factor
(GDNF),
brain‐derived
neurotrophic
factor
 (BDNF),
sonic
hedgehog
   Useful
in
early
stages
of
the
disease
when
there
is
still
a
 significant
dopamine
neuron
population
in
the
SNpc
   GDNF
gene
therapy
in
particular,
looks
promising
‐
 improvements
in
neurochemical
assessments
and
motor
 symptoms
in
rodent
and
primate
models
of
PD
using
AAV
and
 lentiviral
vector
systems
GDNF
promotes
survival
and
regeneration
  GDNF
promotes
axon
regeneration
(sprouting)
and
correction
of
dopamine
 deficiency
in
striatum
  This
effect
only
occurred
when
the
vector
was
injected
directly
into
the
 striatum
 Bjorklund et al., Brain Res., 886 (2000), 82-98 5
  28. 28. Methods
used
to
assess
whether
gene
therapy
has
 therapeutic
effect
and
benefit
   Neurochemistry
‐
measure
dopamine
levels
(tissue
 punches,
microdialysis)
   Assays
for
quantifying
and/or
visualising
therapeutic
 protein
levels
‐
e.g
immunohistochemistry,
RT‐PCR,
 Westerns,
ELISA
   Cell
survival
‐
e.g
TH
as
a
marker
of
dopamine
neurons
   Non‐invasive
imaging
e.g.
PET,
MRI
   Behavioural
‐
changes
in
motor
function
as
a
functional
 endpoint
 Functional
assessment
in
rodents
  Spontaneous
motor
activity
  Drug‐induced
motor
activity
‐
 rotational
behaviour
   Unilateral
lesions
produce
an
 imbalance
in
striatal
dopamine
 levels
between
the
right
and
left
 brain
hemispheres
and
 upregulation
of
postsynaptic
 dopamine
receptors
and/or
signal
 transduction
sensitivity
on
the
 lesioned
side.
 6
  29. 29.   Rate
of
rotation
(e.g
turns/min)
is
directly
proportional
to
 severity
of
the
lesion
and
dopamine
loss.

   Assess
rotation
behaviour
before
and
after
treatment.
 Expectation
is
that
if
a
therapy
works,
will
see
a
reduction
in
 rotation
rate.
 Blue
squares
=
AAVGDNF
in
striatum
 Red
squares
=
AAVGDNF
in
SN
 Green
=
AAVGDNF
in
SN
and
striatum
 Open
squares
=
control
 Bjorklund et al., Brain Res., 886 (2000), 82-98 Resetting
brain
circuitry
  Dopamine
deficiency
in
striatum
has
consequences
on
overall
basal
ganglia
 circuitry
  Overactivity
in
the
subthalamic
nucleus
(STN)
 7
  30. 30. Before
deep
brain
stimulation
 After
deep‐brain
stimulation
 8
  31. 31.   Use
a
genetic
approach
to
silence
STN
neurons
by
introducing
 GAD
(glutamic
acid
decarboxylase)
‐
enzyme
involved
in
GABA
 synthesis
in
these
cells
  Principle
is
similar
to
deep
brain
stimulation,
an
established
 treatment
for
PD
in
humans
  May
also
have
a
neuroprotective
effect
 GAD
gene
therapy
in
humans
  World’s
first
gene
therapy
trial
for
PD
approved
by
the
U.S
FDA
 in
2003
  Open
label,
safety
and
tolerability
trial
of
AAV‐GAD
injected
 unilaterally
into
STN
of
12
patients
  First
patient
treated
August
2003
with
surgery
on
12
patients
 completed
by
July
2005
  1
year
follow‐up
completed
July
2006
  Built‐in
safety
mechanism
‐
ablation
of
the
STN
is
an
 established
treatment
for
PD.
 9
  32. 32. Deep
brain
stimulation
vs
STN
gene
therapy
 Study
design
 10
  33. 33. Functional
assessment
in
humans
‐
GAD
gene
 therapy

   Fluoro‐deoxyglucose
PET
imaging
   MRI
   Motor
assessments
‐
Unified
Parkinson’s
disease
rating
scale
 (UPDRS),
Glosser
QOL
   Neuropsychological
evaluation
   Blood
for
haematology
and
chemistry,
urinalysis,
ECG
   Blood
for
antibodies
to
AAV
and
GAD
 GAD
gene
therapy
led
to
improvement
in
 overall
movement
(UPDRS
score)

 “off “state “on” state 11
  34. 34. Improvement
in
movement
on
treated
side
 Functional
PET
imaging
 12
  35. 35. Neural
network
activity
discriminates
PD
and
 controls
Increased
metabolism
in
SMA
after
GAD
gene
 therapy
 13
  36. 36. Current
status
of
PD
gene
therapy
   Unravelling
pathways
involved
in
mediating
death
of
 dopaminergic
neurons
will
lead
to
identification
of
new
 targets.
   Areas
to
target:
Ubiquitin‐proteosomal
pathway
(e.g
parkin),
 heat‐shock
proteins

 14

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