Gene Therapy For Lysosomal Storage Diseases

N Cartier Pediatric Neurology Bictre Hospital, Paris INSERM U745 - Facult de Pharmacie, Universit Paris-Descartes

Lysosomal Storage Diseases

More than 40 different inherited metabolic diseases; 1/7000 births Lysosmal proteins are ubiquitously expressed : Multiple organs Most involve the CNS Subclasses : Transporters Majority : deficit in soluble lysosomal hydrolases Progressive course : implication for therapy

Integral membrane proteins (NPC and ceroid lipufuscinosis

Lysosomal enzymes can be secreted and recaptured allowing trans-correction

Sands M and Davidson B, Mol Ther 2006

Lysosomal Storage Diseases

Lysosomal Storage Diseases

Therapeutic options for LSD

Enzyme Replacement Therapy Enzyme Enhancement Therapy Substrate Reduction Therapy HSC Transplantation Gene Therapy
in vivo : systemic (liver, lung) gene therapy CNS targeting ex vivo : HSCT

Treatment of LSD is based on cross-correction

Enzyme is released by normal cells and taken up by deficient cells (mannose 6-phosphate receptor) Low levels of enzyme are presumed to be therapeutic Therapeutic implications:

enzyme replacement strategies engineered tissue sources of enzyme/cell or gene replacement strategies

Therapeutic approaches for LSD

Giancarlo Parenti
EMBO Mol Med 1, 268279

Therapeutic approaches for LSD : ERT (Enzyme Replacement Therapy)

Standard care for Gaucher type 1 Fabry : reduces cardiomyopathie et cerebraovascular complications, and slows progressiion to renal failure MPS I,II I : positive effect on joint, lung, organomegaly but no effect on the CNS involvment Pompe : partially ameliorates muscle strength and heart function

Limitations : access to organs : CNS +++ Immunological tolerance in CRIM patients Combining therapies ? : Enhanced Enzymatic Delivery synthetic oligos containing M6P chaperones (imino sugars)

Therapeutic approaches for LSD : ERT (Enzyme Replacement Therapy)

Giancarlo Parenti EMBO Mol Med 1, 268279

Gene therapy for LSD

LSD are good candidates for GT monogenic diseases no complex regulation mechanisms 15-20% activity are sufficiant for clinical efficacy good animal models (murine and larg animals)

Allow constant delivery of the therapeutic protein to target organs (particularly difficult to rich) : bone, brain +++

In vivo GT (adeno, AAV, lenti) Systemic ++,

Gene therapy for LSD

Secretion of lysosomal enzyme from organs ( enzyme factories ) to blood circulation Liver (MPS1 models) , lung (AAV and Fabry) pb + : brain access Brain ++++ (lipofuscinosis, MPS III, MLD) Ex vivo GT Stem cells , encapsulated fibroblasts HSC transplantation (MPS, MLD, Pompe)

Gene therapy for LSD : limitations access to organs

Specificity of expression toxicity of the vectors immunological response :
How to Minimize immune response ? : induction of tolerance liver restricted expression : liver specific promoter

neonatal delivery low AAV particules administration combined with ERT leads to enhanced response

Koeberl et al Current gene therapy 2009

Gene therapy for LSD

Sands M and Davidson B, Mol Ther 2006

Gene therapy for LSD

Sands M and Davidson B, Mol Ther 2006

Gene therapy for LSD

Metachromatic Leukodystrophy (MLD) paradigm

HSC ex vivo gene therapy

in vivo AAV gene transfer to the brain

Metachromatic Leukodystrophy (MLD)

Severe Demyelination of the CNS and the PNS
Accumulation of sulfatides in glial cells and neurons
SULFATIDE

Frequency 1:40.000 Clinical variants:

Late infantile (< 2 years) : >60%
Early Juvenile (2-6 years) Late Juvenile (6-12 years) Adult (12 years)

Arylsulfatase A ARSA

GALACTO CEREBROSIDE

Prognosis:
fatal within few years from onset

Rationale for Gene Therapy:

HCT poorly efficacious ERT not efficacious

Gene therapy to modulate enzyme release in HCT

7000 HSC Monocytes
RRE

GA SD

cPPT-CTS PGK

L. enzyme WPRE

SA

Lysosomal E activity (nmol/mg/h)

6000

5000

4000

3000

2000

1000

0
normal donor pt pt + LV

Kindly provided by A Biffi

ARSA over-expression in HSC and their progeny allowed

HSC gene therapy for MLD: achievements

Prevention and correction of functional and histopathological disease manifestations upon pre-symptomatic and symptomatic treatment Gene corrected HSC are more effective than WT HSC Dose-dependent benefit
Biffi et al., JCI 2004; Biffi et al., JCI 2006

Safety & toxicology addressed in: MLD mice, ARSA tg mice, tumor prone models and human HSC

Capotondo et al, HST 2007; Montini et al., Nature Biotech. 2006 and JCI 2009

HSC gene therapy for MLD: clinical testing

A Phase I/II clinical trial of hematopoietic stem cell gene therapy for the treatment of Metachromatic Leukodystrophy
Autologous HSC 3rd generation ARSA encoding LV Busulfan-based conditioning non-randomized, open label, prospective, comparative, single centre study Open to recruitment: A. Biffi, M. Sessa

In vivo Brain Gene Therapy for MLD

In situ AAV gene transfer

9 Rapid delivery of ARSA enzyme into the brain to stop progression of the disease in rapidly progressive forms of MLD

Intracerebral injections of AAV2/5-ARSA in MLD mice allows strong expression and diffusion of recombinant ARSA

*
Untreated

Treated

ARSA levels (ng ARSA/mg protein)

1500
S1-S2

1000-1500
S3-S4

500-1000
S5-S6

200-500 150-200

* Injection sites sites

Spinal cord

Cb

Human brain: 100-150

M6

M18 Sevin C et al., Hum Mol Genet, 2006

Sevin C et al., Gene Ther, 2007

Intracerebral injection of AAV2-5/ARSA improves brain pathology

18months

PAS reactivity

Microglial activation

Astrogliosis

Purkinje cell degeneration

Sevin C et al., Hum Mol Genet, 2006 Sevin C et al., Gene Ther, 2007

MLD

MLD treated

control

Intracerebral injection of AAV5/ARSA prevents sulfatide storage and motor impairment

Sulfatide storage (Alcian blue)
Untreated
Cortex

Rotarod

Treated

White Matter

18 months

Preclinical evaluation in large animal

Macacus fascicularis monkey

Diffusion Tolerance

Efficiency and tolerance in monkeys (AAV5/ARSA)

Sparse isolated lymphocytic perivascular cuffs are observed at injection sites

No neuronal loss or demyelination ++ (2 10 12 particules = X 5)

Expression of ARSA in the injected hemisphere

Cerebral cortex

* *

Caudate nucleus Putamen

Zona incerta Pallidum

Diffusion of the AAV-driven therapeutic protein activity

Hemisphere % recombinant ARSA Volume (cm3) of ARSA overexpression of ARSA overexpression activity

Prim1 Prim2 Prim3 Prim4 Prim5 Prim6

1.38 0.01 (0,013)

1.21 0.05 (0,027) 1.24 0.06 (0,029) 1.14 0.04 (0,042) 1.12 0.03 (0,027) 1.16 0.05 (0,049)

18.1 16.3 19 21.1 18.1 21.1

55.7 % 50.2 % 58.5 % 64.9 % 57.7 % 64.9 %

Increasing efficacy with AAVrh10-hARSA ?

(Coll R. Crystal, D. Sondhi, N Hackett)
AAV2/5-ARSA AAV2/rh.10-ARSA

AAVrh10-GFP in the striatum

AAVrh.10-ARSA in MLD mouse

Transduction of CNS cells with AAVrh10 and AAV5

AAVrh.10-ARSA vs AAV5-ARSA in MLD mouse

AAVrh.10-ARSA allows efficient biochemical correction in oligodendrocytes

WT UT 1XAAV5/ARSA 1XAAVrh.10/ARSA

-30%

-63%

Oligodendrocytes express recombinant ARSA

Short chain species: Neurons & astrocytes

Long chain species: Oligodendrocytes

Correction of sulfatides species specific for oligodendrocytes

Efficacy study in 4 non-human primates

9 1.1 10e11 PP / hemisphere = 1x dose for patient 9 3 sites per hemisphere; 2 deposits per site: same device as planned in patients 9No immunosuppression 9Brain imaging (short and long term tolerance) 9Neurological evaluation 9Euthanasia 3 months post injection

AAVrh10 diffusion in unilateral NHP

NHP1 NHP2

Injected hemisphere

Injected hemisphere

Mean: 3.1 +/- 1.0 VGC Max: 147 VGC

73% VG+

Mean: 5.6 +/- 1.3 VGC Max: 124 VGC

62% VG+

Non-injected hemisphere

Non-injected hemisphere

11% VG+

1% VG+

Increased ARSA activity in unilateral NHP

injected

Activity ratio

Noninjected

*
400

C
200

IncreaseinARSAactivityoverthetherapeuticallevel
In comparison with AAV2-5/ARSA injected NHP:
9 Higher levels of ARSA activity in the whole injected hemisphere 9 with ten-fold less vector

Optimizing the neurosurgical protocol

Determination of injection sites using MRI

Anterior site

Median site

Posterior site

Brain MRI after injection

JULES MRIatday3 MRIatday90

T2FLAIRaxial

T2axial

T2axial

T2TSEaxial

MRI at day 3

CHOPIN

T2TSEsagittal

Last steps before clinical application

9 Toxicological study in rat non-human primate with a GLP grade AAVrh10-ARSA vector batch (done) 9 Production of the GMP batch (done) 9 Pre-IND to AFSSAPS (done) 9 Final Submission to AFSSAPS in September 2011

Proposed study (Phase I)

Intracranial Administration of a replication deficient Adeno-Associated Virus serotype rh.10 vector expressing the human ARSA cDNA to children with severe early forms of MLD

9 5 MLD patients (males or females) with onset of symptoms between 1-5 years (CRIM ARSA +) 9 Total dose of injected AAVrh10-ARSA vector: 4. 1012 vg (3.3 1011 vg / deposit) 9 Targeted regions : anterior medial and posterior part of centrum ovale in each hemisphere 9 3 tracks/hemisphere; 2 deposits/track (12 sites injected simultaneously) 9 No immunosuppression 9 2-years follow-up . Safety . Efficiency (motor and cognitive functions, MRI, electrophysiology)

The MLD teams

INSERM U745, Paris Hpital Bictre Dept of Pediatric Neurology

Cornell University, New York

Ronald Crystal Dolan Sondhi Neil Hackett UMR INRA 703 Ecole Vtrinaire, Nantes MA Colle Y Cherel S Raoul INSERM U649, Nantes, F Balter C Darmon P Moullier

TIGET San rafaelle A Biffi M Sessa MG Roncarolo L Lorioli T Plati A Capotondo F Fumagalli E Montini L Naldini

C. Sevin N. Cartier F. Piguet M. Zerah T. Roujeau C. Bouquet F Fouquet I Bieche M Vidaud P Aubourg