GENE THERAPYFOR NEURODEGENERATIVE DISEASES
N CartierINSERM U745 - Génétique et Biothérapies des Maladies Neurodégéneratives Faculté de Pharmacie, Université Paris-Descartes
Ø X-linked adrenoleukodystrophy (ALD)Ø Metachromatic leukodystrophy (MLD)Ø Alzheimer disease (AD)
from genetic leukodystrophies to Alzheimer disease
Ø the brain is a complex organ: interconnections between neuron, glia and other cells
Ø the target areas:- discrete brain region: Parkinson, Huntington- large portion of the brain: lysosomal storage diseases (-> leukodystrophies)
Ø target cells: neurons, sub-population of neurons, glia, endothelial cells
Specific issues
Gene delivery to the Central Nervous System
BBB protects the brain from pathogensHampers the delivery of therapies to the CNS
Modes of gene/protein delivery to the brain
Ø taken up by the cells at the injection site or diffuse away Ø anterograde / retrograde transport to distant sitesØ volume constraints / number of stereotactic injections
Ø astrocytes, macrophages, fibroblasts, neural precursor cells
some cells can migrate away from the injection siteand (neural precursor cells) be incorporated into the cytoarchitecture of CNS
Ø Hematopoietic cell transplantation
GENE THERAPY APPROACHES
STRATEGIES Defective gene
Gene replacement (loss of function)
Symptomatic treatment
Gene correction
Down-regulation of disease genes (siRNA)
Defective gene
The spectrum of vectors used in basic research surpasses the few being used in clinical trials
Adeno-associated virus (AAV)• 20 nm diameter • Linear s/s DNA genome • Maximum insert size - 5 kb • Defective parvovirus dependent on helper virus for replication • Wild type virus integrates into chromosome 19 • Non-pathogenic
Burger et al. (2004) Mol Ther. 2:302-317
TRANSDUCTION EFFICIENCY OF AAV SEROTYPES
AAV: diffusion depends on serotypes
In thespinal cord
AAV-5 vectors: a clear advantage in term of diffusion
AAV5-PGK-ALD
AAV2-PGK-ALD
NeuN + ALDP
AAV vector : neuronal tropism
AAV5-EGFP
AAV2-EGFP
Lenti-EGFP
Gene therapy vectors do not like oligodendrocytes
Needs to develop new vector envelops to target oligo ++(antibody, ligands to specific receptor, chemical modifications..)
Ctx
Cc
Str
Ob
Potential clinical applications
• stroke
• spinal cord injury
• neurodegenerative diseases (Parkinson, Huntington, Alzheimer)
• CNS lysosomal storage diseases
• brain tumors
• pain
Indications addressed by Gene Therapy Clinical Trials
GENE THERAPY CLINICAL TRIALS IN THE CNS
Amyotrophic Lateral Sclerosis
Huntington’s Disease
• Genetically engineered encapsulated cells producing CNTFAebischer et al. (1996) Nat. Med. 2: 696-699
• Genetically engineered encapsulated cells producing CNTFBachoud-Lévi et al. (2000) Hum. Gene Ther. 11: 1723-1729; Bloch et al. (2004) Hum. Gene Ther. 15: 968-975
Alzheimer’s Disease• Autologous fibroblasts infected with an NGF-expressing retroviral
vector (MoMLV) Tuszynski et al. (2005) Nat. Med. 11: 551-555• AAV-NGF (0401-623)
Symptomatic treatments
• Zinc finger DNA binding protein (ZPF-TF, transcription factor), naked DNA, intramuscumlar administration
GENE THERAPY CLINICAL TRIALS IN THE CNS
Parkinson’s Disease
• AAV-hAADC-2 (0307-593) Eberling et al. (2008) Neurology 70: 1980-
1983
• EIAV-TH-AADC-GTPCH Jarraya et al. (2009) Science Transl. Med. 1:
2ra4
• AAV-GAD (104-469) Kaplitt et al. (2007) Lancet 369: 2097-2105
• AAV-Neurturin (0501-689) Marks et al. (2008) Lancet Neurol 7: 400-
408
• AAV2-GDNF
Symptomatic treatments
Canavan (childhood leukodystrophy)
• Cationic liposome containing an ASPA plasmid DNA (9711-222)• AAV-ASPA (0001-381) Janson et al. (2002) Hum. Gene Ther. 13: 1391-1412;
McPhee et al. (2006) J. Gene Med. 8: 577-588
• AAV-CLN2 (tripeptidyl peptidase I) (0312-619) Worgall et al. (2008) Hum. Gene Ther. 19: 463-474
Late infantile neuronal ceroid lipofuscinosis a form of Batten disease(lysosomal storage disease)
GENE THERAPY CLINICAL TRIALS IN THE CNSGene replacement
X-Linked Adrenoleukodystrophy• HIV-ALDCartier et al. (2009) Science 326: 818-823
GENE THERAPYFOR NEURODEGENERATIVE DISEASES
N CartierINSERM U986 / MIRCen CEA
Ø X-linked adrenoleukodystrophy (ALD)Ø Metachromatic leukodystrophy (MLD)Ø Alzheimer disease (AD)
HSC ex vivo gene therapy
Gene Therapy strategies for neurodegeneratives diseases :
Genetic LeukodystrophiesMetachromatic leukodystrophy (MLD)
Adrenoleukodystrophy (ALD)
In situ direct AAV gene transfer to the brain
Adrenoleukodystrophy1/17.000 males, 35 new cases/year in France
• no neurological symptoms
• mild cognitive deficits
• visual deficits• auditive deficits• pyramidal signs• cerebellar signs • seizures
12-18 months
ß-oxidation
VLCFA-CoA
Peroxisomalmembrane
ALDP ALDP
VLCFA-CoA
ATP-binding• ABC peroxysomal transporter
ALD : Accumulation of VLCFA in blood and tissues
The ALD gene and the ALD protein
Hematopoietic stem cell transplantation (HSCT) can arrest cerebral demyelination in ALD
N Engl J Med 1990Lancet 2000
HCT< 18 months
No clinical symptoms
MRI+
Natural evolutionof the disease
The replacement of brain microglia originates from hematopoietic precursors
ü present in the bone marrow after hematological reconstitution following BMT that go into the blood and then penetrate into the brain
ü The replacement of brain microglia is a relatively slow processSome time is needed until a sufficient number of normal microglia cellsin the brain can stop the demyelinating process
Why HSC gene therapy in ALD ?
ü Lack of matched URD or cord blood
üMortality risk of allogeneic BMT in children remains close to 20%
üMortality risk of allogeneic BMT in adults is up to 30-40%
üAny complication of BMT delays the efficacy of BMT in X-ALD ++(GVH, delayed hematopoietic reconstitution)
Objectif : Corriger les propres cellules de la moelle osseuse des enfants
On n’a toujours pas trouvé mieux que….
COMMENT ?
Transférer un gène à l’aide d’un vecteur viral
Which vector ?
• Need for an integrative vector
• Classical retroviral vector
HIV1-derived lentiviral vector
Evaluation ofrecombination risk
MND
Lentiviral ALD gene transferin CD34+ cells from ALD patients
45-60%(ALDP+,CD34+)
SCID/NOD
Human brain microglia(ALDP+, RCA+)
Human monocyte(ALDP+, CD68+)
IL3, SCF, FLT3L, TPO
Peripheral blood Mobilized Cells
CD34+ cells
Ø Development and Production of the clinical grade lenti-ALD vector (Cell Genesys Inc, USA)
Ø Viral safety issues: vector and transduced CD34+ cells
Ø Risk of insertional mutagenesis
Ø Scale-up of the transduction protocol to the clinical conditions
ØAFSSAPS: pre-IND and discussions
Strategic planning towards clinical trial in ALD(2002-2005)
Ø Development and Production of the clinical grade lenti-ALD vector (Cell Genesys Inc, USA)
ØViral safety issues: vector and transduced CD34+ cells
Ø Risk of insertional mutagenesis
AFSSAPS: pre-IND and discussions with experts ++++
Ø Scale-up of the transduction protocol to the clinical conditions
Strategic planning towards clinical trial in ALD(2002-2005)
Ø Final authorization from AFSSAPS : december 2005
Phase I/II studyEx vivo ALD gene transfer to CD34+ cells followed by autologous
transplantation
5 ALD patients (5-15y) with cerebral demyelination, candidate for HSCT, without HLA-matched donor or UCB
• CD34+ cells from ALD patients are purified from PBC, transduced ex vivo, frozen until re-infusion (for RCL assays). Re-infusion of > 4 x 106/kg
--> 3 x 106/kg non-transduced CD34+ cells/kg are preserved (in case of graft failure)
• myeloablating conditioning busulfan ( 4 mg/kg x 4 days: D-10 to D-7)cyclophosphamide (50mg/kg/day x 4 days: D-5 to D-2) followed by re-infusion of ≥ 4 x 106/kg transduced CD34+ cells (D0)
• 3 patients treated
patient 1 : LS score = 2,25gadolinium +7 1/2 y, ALD protein -
patient 2 : LS score = 7gadolinium +7 y, ALD protein -
patient 3: LS score = 2gadolinium +7 y, ALD protein + decreased
HSC gene therapy in X-ALD
Endpoints
Ø Safety
Ø Gene marking and transduction of HSC
Ø Neurological outcome
• Re-infused with: 4.6x106 CD34+/kg (P1), 7.2x106 CD34+/kg (P2), 8x106 CD34+/kg (P3)
• Procedure was well tolerated without complications
• Hematological recovery at day 15, complete for immune functions at 12 months (P1, P2 & P3)
• normal cellularity of bone marrow aspirate P1, P2, P3 at 12 months and P1 & P2 at 24 months
• Safety of lentiviral vector up to 36 months (P1), 30 months( P2) and 16 months (P3)
- all RCL tests negative (gag-pol, HIV1 western-blot, HIV1-ELISA x 2, VSV.G DNA)
- Polyclonal hematopoietic reconstitution without detectable genotoxic effects due to lentiviral vector insertion in or close to genes
HSC gene therapy in X-ALD : Safety
Safety
Any detectable signs of genotoxicity ?
üLentiviral vector integrates into the genome like murine gamma retrovirus
üInsertion of any DNA fragment into, close to or even at distance of gene maymodify gene expression (proto-oncogenes, miR, SNPs acting in cis or transon gene expression) etc..) resulting in the emergence of « dominant clones »
üTracking the frequency of insertion site retrieval allows to assess potentialgenotoxicity effect:
if a dominant clone emerge, the retrieval frequency for lentiviral insertion in such cell clone must be more frequent
Safety
Number of integrated vector copy post-transplantin peripheral monocytes/lymphocytes
day15 M1 M2 M6 M9 M12
0,2
0,4
0,6
0,8
P1
P20,72
0,14
0,20
0,54
Transduced CD34+ cells before injection
M16
P3
M20 M30
Gene therapy
0,63
M36
0,13
months
Preferred Integration in Gene Coding Regions
P1 [#] P1 [%] P2 [#] P2 [%]Unique exactly mappable IS 2217 1380
pre-transplant cells 501 486post-transplant cells 1719 898
IS in Refseq Genes 1612 72.71 1046 75.80IS in Refseq Genes +/- 10 kb 1774 80.02 1144 82.90
0
2
4
6
8
10
10-5 kb 5-0 kb 0-10% 10-20% 20-30% 30-40% 40-50% 50-60% 60-70% 70-80% 80-90% 90-100%
Inte
grat
ion
Site
s [%
]
P1P2TSS
in Gene [%]Upstream [kb]
Characteristic lentiviral integration profile
SafetyClonality of Hematopoietic Repopulation
P1 P2
mar
ker
mar
ker
pre
CD
14 M
6
CD
14 M
12
CD
14 M
21
CD
34 M
24
CD
3 M
24
CD
19 M
24
CD
15 M
24
CFU
-BM
M12
CFU
-BM
M12
-C pre
CD
14 M
6
CD
14 M
9
CD
14 M
20
CD
34 M
20
CD
3 M
20
CD
19 M
20
CD
15 M
20
CFU
-BM
M12
CFU
-BM
M12
-C
IC
IC
Polyclonal hematopoietic repopulation in both patients
Samples: Transduced CD34+ cells prior to re-infusion (pre) Cell sub-populations 6 to 24 months after transplantationBM-derived colony forming units (CFU-GMs)
Objectives
Ø safety
Ø expression of recombinant ALD protein in leucocytes ?
correction of stem cells ?
Ø efficiency on the neurological disease
Expression ?Transduced CD34+ cells before re-infusion
% of ALDP+ % of ALDP+ nb of vector nb of RNAbefore after copy/cell copy/cell
freezing thawing
P1 50,6 51 0,72 5400endogenous: 1320
P2 33 34 0,54 ND
P3 42 44 0,63 ND
Gene marking :ALD protein is expressed
in peripheral blood cells of treated patients
Gene marking: percentage of ALD protein positive monocytes/lymphocytesin peripheral blood after HSC gene therapy
2 4 6 9 12 16
5
10
15
20
P1
P2
20 241
25
30
35
40
0
Months after transplantation
Gene therapy
0
P3
36
Have Hematopoietic Stem Cells been corrected ?
Ø Expression in cells with short half-life ?monocytes et granulocytes (3-4 days)
Ø Expression in bone marrow CD34+ cells ?
Ø Presence of Common insertion sites in lymphoid and myeloid cells ?
M2 M4 M6 M8 M12 M16
5
10
15
20
CD14+
M20 M36
Percentage of monocytes, granulocytes, T and B lymphocytes expressing ALD protein
M1
25
30
35
50
0
CD15+
CD3+
CD19+
P1
CD34+
Gene therapy
M2 M4 M6 M8 M12 M16
5
10
15
20
M30
Percentage of monocytes, granulocytes, T and B lymphocytes expressing ALD protein
M1
25
30
35
40
0
CD14+
CD15+
CD3+
CD19+
P2
CD34+
Gene therapy
Identical insertions of lentiviral vector have been found in at least 4% of both lymphoid and myeloid cells
Next M6 M9 M12 M17 M21 M24
RefSeq Gene 156 374 379 498 218 221
ADRBK2
ARHGEF3
ARID3A
BLID
BRE
BUB1B
C11orf49
C17orf57
C1orf112
C2CD3
CCDC21
CDC25A
CFH
CNTN5
CRADD
ERLIN1
FCHSD2
FRMD8
GLCE
GP6
GTF2A1
HIST1H2BC
HLA-DMB
HOOK1
INPP5A
KIAA0776
KIAA1128
KIAA1267
KIAA1303
KIFC1
LOC93349
LRRK2
LYL1
LYPLAL1
MAPK1
MBOAT5
MBTD1
MECP2
MEIS1
MLZE
M-RIP
MTHFD2L
MYO9A
light blue: IS identified in myeloid cells (CD14 and/or CD15 and/or CFU)
mid blue: IS identified in myeloid and lymphoid cells at the same timepoint
dark blue: IS identified in lymphoid cells (CD3 and/or CD19)
Endpoints
Ø Safety
Ø Gene marking and transduction of HSC
Ø Neurological outcome
Neurologic outcomePatient P1 (M36)• developed mild right hemiparesis and aggravated frontal syndrome at M7• partial regression of hemiparesis at M12 with nearly complete reversal at M16• improvement of frontal syndrome from M12 to M24, stable since• no changes in verbal IQ (108); decrease of performance IQ (99 -> 75)
Patient P2 (M30)• no neurologic signs, excepting bilateral inferior quadranopsia without decreased
visual acuity• no cognitive deficits, excepting moderate decrease of visuo-spatial performances
in respect to other cognitive functions• normal IQ: VIQ= 110, PIQ=114
•Patient P3 (M15)• no neurologic symptoms• « the jury is still out »
MRI lesions progressed and then stablizedas after « classical » successfull allogeneic HCT
<-- Before --> gene therapy
<-- 12 months after -->gene therapy
<-- 16 months after -->gene therapy
<-- 36months after gene therapy
30 months after gene therapy -->
P1 P2
<-- Before --> gene therapy
<--12 months after-->
gene therapy
<--16 months after-->gene therapy
<--36 months after gene therapy
30 months after gene therapy-->
P1
<--12 months after,untreated
<--18 months after,untreated
<--24 months after,untreated
<-- at diagnosis
P2
Untreated ALD patient
HSC gene therapy versus no treatment
Summary of neurologic outcome in HSC gene therapy
• very similar to that observed after allogeneic HCT(100% chimerism, uncomplicated)
• only difference : delayed disappearance of gadolinium enhancement (reflecting neuro-inflammation)
likely due to lower percentage of corrected microglia after HSC gene therapy
No progression of the lesions after M12
HCT or GT12-15 monthsevolution
of the disease
No clinical symptoms
MRI+
• first demonstration that lentiviral vector efficiently transduces HSC in the absence of selective advantage
• no safety concern with respect to HIV infection(mobilization of the vector and recombination)
• no safety concern with respect to insertional mutagenesis risk / genotoxicity
• first successfull gene therapy trial for a severe disease of the CNS
conclusions
ØX-linked adrenoleukodystrophy (ALD)Ø Metachromatic leukodystrophy (MLD)Ø Alzheimer disease (AD)
Gene therapy for genetic leukodystrophies
Metachromatic leukodystrophy (MLD)
Adrenoleukodystrophy (ALD)
Metachromatic Leukodystrophy (MLD)
Lysosomal storage disease due to the deficiency of ArylSulfatase A (ARSA)
Ø 1/40 000
Ø Sulfatides (cerebroside-3-sulfates)
galactosyl-ceramides
Ø CNS and PNS demyelination
Ø Late infantile form (60%)Early Juvenile and Late Juvenile formsAdult form
Golgi
lysosomes
M6Preceptor
SecretionRecapture
lysosomes
Golgi
ARSA
ØARSA can be secreted/recaptured
The rationale of brain gene therapy in MLD
The benefit of allogenic hematopoietic stem cell transplantation is limited to late juvenile (> 6 years) and adult forms of MLD
Enzyme replacement therapy ?lysosomal enzymes do not cross the blood-brain-barrier
Delivering rapidly ARSA enzyme into the brain appears the only potential therapeutic approach to have chance to result in a clinical benefit in rapidly progressive forms of MLD
In situ AAV gene therapy
Intracerebral injections of AAV2/5-ARSAin MLD mice allows strong expression and diffusion of recombinant ARSA
* Injection sites
**
Spinal cord
1500
1000-1500
500-1000
200-500
150-200
S1-S2
S3-S4
S5-S6
Cb
M18
ARSA levels (ng ARSA/mg protein on ELISA)
Human brain: 100-150 Sevin C et al., Hum Mol Genet, 2006Sevin C et al., Gene Ther, 2007
Treated
Untreated
PAS reactivity
Microglial activation
Astrogliosis
Purkinje cell degeneration
MLD MLD controltreated
Intracerebral injection of AAV2-5/ARSA improves brain pathology
18months
Sevin C et al., Hum Mol Genet, 2006Sevin C et al., Gene Ther, 2007
Intracerebral injection of AAV5/ARSA prevents sulfatide storage and motor impairmant
Untreated Treated
Sulfatide storage (Alcian blue)
18 months
Rotarod
Ø DiffusionØTolerance
Preclinical evaluation in large animal
Singe Macacus fascicularis
Efficiency and tolerance in monkeys(AAV5/ARSA-HA)
Expression/diffusion of ARSA-HAin the injected hemisphere
**
Caudate nucleus
Putamen
Pallidum Zona incerta
Cerebral cortex
Diffusion of the AAV5-ARSA vector
Diffusion of the AAV-driven therapeutic protein activity
Injection protocole in primates
GENE THERAPYFOR DEGENERATIVE DISEASES OF THE CNS
Ø X-linked adrenoleukodystrophy (ALD)Ø Metachromatic leukodystrophy (MLD)Ø Alzheimer disease (AD)
from genetic leukodystrophies to … Alzheimer disease
Cholesterol metabolism and Alzheimer Disease
CYP46A1 as a therapeutic target in Alzheimer’s disease
ALZHEIMER DISEASE CHARACTERISTIC LESIONS
Amyloid plaques
Aß peptides
Amyloid plaques
Neurofibrillar tanglesTau
(hyperphosphorylated) NEURON
Three genes identifiedin the familial forms of AD:
- APP (Amyloid Precursor Protein)
- Presenilin 1 et 2 (PS1, PS2)
AST
RO
CYT
E
ABCA1
Cholesterol
Cholesterol
Synaptic vesiclesN
EUR
ON
Blood Brain Barrier
HDL
INTRACEREBRAL CHOLESTEROL METABOLISM
ApoE
AcetylCoA
HMGCR
ABC
HDLApoE
CSF
24-OHC
Cholesterol-24-hydroxylase ( CYP46A1 )LD
LR
excess
INTRACEREBRAL STEREOTACTIC INJECTION OF AAV5-CYP46A1
0 m3 m6 m12
APP23 mouse
- hAPP751 containing the swedish double mutation
- First cognitive deficits as soon as 3 months of age
- First amyloid deposits at 6 months
(Sturchler-Pierrat et al. 1997)
ADENO ASSOCIATED VECTORS
- AAV5-wtCYP46A1 (AAV-CYP)
- AAV5-mtCYP46A1 (AAV-control)
Cyp46 A1 as a potential target for AD
injection of AAV-CYP46 vector results in a significant increaseof the 24-OHC content
Chol
este
rol (
µg/m
g pr
otein
)24
-OHC
(ng/
mg
prot
)
0
100
200
300
400
500
600*** ***
0
50
100
150
200
250
300
350 NSNS
AAV-CYP46 AAV-control
Ctx
Hpc
Thal
Sbcl
CA1/2
CA3
DG
EXPRESSION OF CHOLESTEROL METABOLISM RELATED GENES
Expr
essio
n lev
el (A
.U.)
0
25
50
75
100
125
* *
150
175
AAV-CYP46AAV-control
The injection of AAV-CYP46 vector induces a significant increase of the expression level of Hmgcr and Srebp2
Cholesterol neosynthesis
IN VIVO
AAV-controlAAV-CYP46
Aß42
(ng/
mg
prot
ein)
0
10
20
30
40 **
0
20
40
60
80
100
Aß40
(ng/
mg
prot
ein) *
Signal intensity (A
.U.)
X 12 X 6
Trito
n
X 3 X 1
Tris
*
SDS
Pelle
t* *
*
50
1520
75
MW(KDa)250100
3725
10
Tris
Trito
nSD
SPe
llet
X 12
X 6
X 3
X 1
Tris
Trito
nSD
SPe
llet
Aß OLIGOMERSAß40/42 CONTENT
Aß40/42 peptides and amyloid trimers are decreased in treated mice
The overepression of CYP46 is associated with a significant decrease of AICD production
Non Amyloidogenic
Amyloidogenic
APP
C99
C83
AICD
AICD
AAV-CYP46
0
40
80
120
160
C-te
rmin
al fra
gmen
ts (A
.U.) *
AAV-control
PSEN1
APP
50
150250
PM(KDa)
50
10
15
C83C99
37
10
7.3 AICD
ACTIN
C83
BACE1
APP CLIVAGE PRODUCTS
CYP4
6
Ctx
Hpc
Ctx
Hpc
Cont
rol
CYP46 overexpression prevents amyloïd deposits in APP23 mice
Depo
sits s
urfa
ce(%
)
1,2
1
0,8
0,6
0,4
0,2
0
* * *
0
0,05
0,1
0,15
0,2
0,25* *
020406080
100120140160
Depo
sits n
umbe
r
*
0
2
4
6
8
10
12* *
CORTEX HIPPOCAMPUS
CYP46 overexpression reduces existing amyloïd deposits
in APP23-PS mice
⇒ Decrease of inflammatory cells
MICROGLIOSIS
Iba-
1A
myl
oid
EFFECT ON INFLAMMATION
AAV-controlAAV-CYP46
0
100
200
300
400
500
600
Iba-
1 po
sitiv
e ce
lls/m
m2
NS
**
GFA
P po
sitiv
e ce
lls/m
m2
0
50
100
150
200
250 NS
*
ASTROCYTOSIS
MER
GE
control
ESC
APE
LA
TEN
CY
(SEC
)
8000
6000
4000
2000
012 14 16 18
control
ESCA
PE L
ENGH
T (C
M)
CYP46
600
200
400
012 14 16 18
control
CYP46
Trials
CYP46
SWIM
SPE
ED (C
M/S)
10
15
20
25
5
01 2 3 4 5 6 7 8
Trials
Morris water mazeCOGNITIVE EVALUATION
⇒ AAV-CYP46 injected APP23 mice show an improvment of their cognitive performances
J4
Ø Decreased Aß40/42 productionØ Decreased amyloid plaques and Aß oligomers in APP23 mice (preventive and curative) Ø Decreased inflammationØ Improvement of cognitive deficitsØ Decreased AICD, suggesting that the γ-secretase
cleavage is affectedØ Lower cholesterol content in lipid rafts with a displacement of APP and PSEN1 out of lipid raftsØ no major modification of lipid metabolism
OVEREXPRESSION OF CHOLESTEROL-24-HYDROXYLASE IS ASSOCIATED WITH:
CONCLUSION
CYP AS A THERAPEUTIC TARGET FOR AD :Further steps
Ø Proof of concept
Ø Underlying mechanism of the role of CYP on amyloid pathology
in vivo : microarray APP23/APP23 CYP
in vitro : N2A-APP-CYP subcellular localisation of the different key actorscoll MC Potier, C Duyckaerts
Ø Therapeutic potential
Ø in a more agressive model APP/PS KI : coll B Delatour, C Duyckaerts
ØCYP and Tau pathology : coll Luc Buee
Ø Large animal model : coll N Deglon, P Hantraye
Place des thérapeutiques innovantes pour Alzheimer ?
• Marché actuel : médicaments peu efficaces
• Médicaments en phase II et III prévisionnels connus
• Nouveaux médicaments dans le pipeline connus
Quelle est la place de la thérapie génique aujourd’hui ?
– Nouvelle modalité thérapeutique
– même si paraît plus complexe que les traitements classiques
Challenge : conduire les investisseurs et les pharmas à reconsidérer la TG
2008 et 2009 have been important years for gene therapy….
Parkinson
Déficits immunitaires
Rétinite pigmentaire RP65
Adrénoleucodystrophie
Better perspectives ….
Gene therapy strategies for neurodegenerative diseases
Intracerebral administration, and how in the future ?
INSERM U745, ParisHôpital Saint-Vincent de Paul, ParisDepartment of Pediatric Endocrinologyand NeurologyP. AubourgP.F. Bougnères
S. BenhamidaM. AsheuerJ.-C. Zhao-EmonetI. LaurendeauF. FouquetS. GuidouxB. L’HommeM. Vidaud
C. BellesmeM.C LupterM.C. BlondeauA. De WynterThe nurses
DKFZ, NCTHeidelbergC. von KalleM. SchmidtC. BartholomäHanno GlimmAnne ArensAnnette DeichmannIna KutscheraChristina LulayMichaela Kirchgäßner
Department of BiotherapyHôpital Necker-Enfants Malades, ParisM. Cavazzana-Calvo, S. Hacein-BeyF. Lefrère, L. Dal-Cortico, L. Caccavelli
Department of PediatricImmunology and HematologyHôpital Necker-Enfants Malades, ParisA. FischerN. Malhaoui, S. BlancheC. PicardThe nurses
The ALD teams
Financial supportELA, EEC FP6, AFM,STOP-ALD,Fondation de l’AvenirInserm, AP-HP (PHRC program)
Cell Genesys G. Veres, V. Kiermer, D. Mittelstaedt
Genosafe : M. Audit
INSERM U745, ParisC SevinF PiguetC BouquetP AubourgN Cartier
M ZerahT Roujeau
M. VidaudI. BiècheO AhouansouI. LaurendeauF. FouquetS. Guidoux
INSERM U649, Nantes,F BalterC DarmonP Moullier
UMR INRA 703Ecole Vétérinaire, NantesMA ColleY CherelS Raoul
Cornell Univ, New YorkRonald CrystalDolan SondhiNeil hackettR Zalaznick
INSERM U745 - Génétique et Biothérapies des Maladies Neurodégéneratives
Faculté de Pharmacie, Université Paris-Descartes
INSERM U745, Paris
F DjeltiE GilletC SevinF PiguetP Aubourg
M. VidaudI. BiècheO AhouansouI. LaurendeauF. FouquetS. Guidoux
INSERM U649, Nantes,
F BalterC DarmonP Moullier
UMR INRA 703Ecole Vétérinaire, Nantes
MA ColleY CherelS Raoul
Cornell University, New Yo
Ronald CrystalDolan Sondhi
CEAInstitut d'Imagerie BioMédicale
MIRCen
Nicole DeglonP Hantraye
INSERM UMR975MC PotierJ CossecB DelatourL DauphinotC Duyckaerts
INSERM U649, Nantes,F BalterC DarmonP Moullier
UMR INRA 703Ecole Vétérinaire, NantesMA ColleY CherelS Raoul
Cornell University, New YorkRonald CrystalDolan Sondhi
INSERM U745 - Génétique et Biothérapies des Maladies Neurodégéneratives
Faculté de Pharmacie, Université Paris-Descartes
INSERM U745, ParisHôpital Saint-Vincent de PaulDept of Pediatric Endocrinologyand Neurology
P. AubourgP.F. Bougnères
B L’HommeC BouquetK RuffertF DjeltiF FouquetS GuidouxI BiecheI LaurendeauM VidaudO Ahouansou
C. BellesmeM.C LupterM.C. BlondeauA. De WynterThe nurses
INSERM UMR975, CR-ICMC DuyckaertsM-C PotierJ CossecB Delatour
URA CEA CNRS 2210, MIRCenN DeglonP Hantraye
INSERM U837D BlumL Buee
DKFZ, NCTHeidelbergC. von KalleM. SchmidtC. BartholomäHanno GlimmAnne ArensAnnette DeichmannIna KutscheraChristina LulayMichaela Kirchgäßner
Department of BiotherapyHôpital Necker, ParisM. Cavazzana-Calvo, S. Hacein-BeyF. Lefrère, L. Dal-Cortico, L. Caccavelli
Department of PediatricImmunology and HematologyHôpital Necker, ParisA. FischerN. Malhaoui, S. BlancheC. Picard
INSERM U649, Nantes,F BalterC DarmonP Moullier
UMR INRA 703Ecole Vétérinaire, NantesMA ColleY CherelS Raoul
Cornell University, New YorkRonald CrystalDolan Sondhi
INSERM U745 - Génétique et Biothérapies des Maladies Neurodégéneratives
Faculté de Pharmacie, Université Paris-Descartes
INSERM U745, ParisHôpital Saint-Vincent de PaulDept of Pediatric Endocrinologyand Neurology
P. AubourgP.F. Bougnères
B L’HommeC BouquetK RuffertF DjeltiF FouquetS GuidouxI BiecheI LaurendeauM VidaudO Ahouansou
C. BellesmeM.C LupterM.C. BlondeauA. De WynterThe nurses
INSERM UMR975, CR-ICMC DuyckaertsM-C PotierJ CossecB Delatour
URA CEA CNRS 2210, MIRCenN DeglonP Hantraye
INSERM U837D BlumL Buee
DKFZ, NCTHeidelbergC. von KalleM. SchmidtC. BartholomäHanno GlimmAnne ArensAnnette DeichmannIna KutscheraChristina LulayMichaela Kirchgäßner
Department of BiotherapyHôpital Necker, ParisM. Cavazzana-Calvo, S. Hacein-BeyF. Lefrère, L. Dal-Cortico, L. Caccavelli
Department of PediatricImmunology and HematologyHôpital Necker, ParisA. FischerN. Malhaoui, S. BlancheC. Picard
INTRACELLULAR CHOLESTEROL DISTRIBUTION
N2a-APP17
chol
este
rol c
onte
nt in
eac
h fr
actio
n (%
)
0
10
20
30
40
FLOTILIN-210 9 7 6 5 4 2 1
DRM analysis(Triton X-100 extraction and
purification on a density gradient)
(flotillin-2 positive fractions)
RADEAUX LIPIDIQUES
CHOLESTÉROL
A decrease in the cholesterol content is observed in lipid rafts
N2a-APP17N2a-APP-CYP-A
N2a-APP-CYP-B
% c
hole
stér
ol in
DM
R fr
actio
ns
0
10
20
30
40
FLOTILIN-2
10 9 7 6 5 4 2 1
**
*
INTRACELLULAR CHOLESTEROL DISTRIBUTION
APP AND PSEN1 PROTEINS IN THE DIFFERENT FRACTIONS
APP and PSEN1 are displaced out of the “lipid raft” fractions
N2a-
APP1
7ACTIN
APP
PSEN1
10 9 7 6 5 4 2 1
N2a-
APP-
CYP-
A
ACTIN
APP
PSEN1
ACTIN
APP
PSEN1
N2a-
APP-
CYP-
B % o
f APP
and
PSE
N1
in th
e flo
tilin
-2
posi
tive
frac
tions
0
10
20
30
40
**50
*
APPPSEN1
CYP AS A THERAPEUTIC TARGET FOR AD :Further steps
Ø Proof of concept
Ø Underlying mechanism of the role of CYP on amyloid pathology
in vivo : microarray APP23/APP23 CYP
in vitro : N2A-APP-CYP subcellular localisation of the different key actorscoll MC Potier, C Duyckaerts
Ø Therapeutic potential
Ø in a more agressive model APP/PS KI : coll B Delatour, C Duyckaerts
ØCYP and Tau pathology : coll Luc Buee
Ø Large animal model : coll N Deglon, P Hantraye
2008 et 2009 recent progresses in gene therapy
Parkinson
Déficit en ADA, DICS-X
Rétinite pigmentaire RP65
Adrénoleucodystrophie
L’expression du gène thérapeutique permet la diminution des lésions neuropathologiques et du phénotype clinique
dans le modèle animal
Ce gène est une cible thérapeutique ++++
compléter les données précliniques
pour proposer une thérapie génique
chez des patients Alzheimer
sévèrement et précocément atteints
Murin neuroblastoid N2a cells overexpressing APPsl
ACTIN
N2a-
hAPP
sl-17
Proté
in RN
A
N2a-
hAPP
sl-11
N2a-
hAPP
sl-12
N2a
Actin
APP
APP
Aß40
(ng/
mg
prot
ein)
Aß42
(ng/
mg
prot
ein)
0
510
1520
2530
02
46810
12
******
*
IN VITRO STUDY
stable overexpression of CYP46 is associated with decreased Aß secretionIN VITRO
0
Chol
este
rol
(µg/
mg
prot
ein)
40
80
120
160
N2a-
APP-
CYP-
A
N2a-
APP-
CYP-
B
N2a-
APP1
7
020
4060
80
100120
140 *****
Secr
eted
Aß 4
0/42
Aβ40 Aβ42
N2a-
APP-
CYP-
A
N2a-
APP-
CYP-
B
N2a-
APP1
7
CYP4
6exp
ress
ion
level
(A.U
.)24
-OHC
(ng/
mg
prot
ein)
010203040506070 **
*
10100
1,00010,000
100,000*
**
1
Stable CYP46
Overexpression
inN2A-APP