GENE THERAPY FOR NEURODEGENERATIVE DISEASEScoursl3bichat2012-2013.weebly.com/uploads/9/6/0/7/9607940/diapos... · BBB protects the brain ... GENE THERAPY FOR NEURODEGENERATIVE DISEASES

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

http://www.pdfcomplete.com/cms/hppl/tabid/108/Default.aspx?r=q8b3uige22

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




leucodystrophies


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 ?


ALD

Capside

Enveloppe


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


I - Safety


• 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



<-- 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


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





Cornell University, New YorkRonald CrystalDolan Sondhi



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


INSERM UMR975, CR-ICMC DuyckaertsM-C PotierJ CossecB Delatour

URA CEA CNRS 2210, MIRCenN DeglonP Hantraye

INSERM U837D BlumL Buee


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




Cornell University, New YorkRonald CrystalDolan Sondhi



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


INSERM UMR975, CR-ICMC DuyckaertsM-C PotierJ CossecB Delatour

URA CEA CNRS 2210, MIRCenN DeglonP Hantraye

INSERM U837D BlumL Buee


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


Documents

GENE THERAPY FOR NEURODEGENERATIVE DISEASEScoursl3bichat2012-2013.weebly.com/uploads/9/6/0/7/9607940/diapos... · BBB protects the brain ... GENE THERAPY FOR NEURODEGENERATIVE DISEASES