GENE THERAPY-BASED ORPHAN DRUGS...Gene transfer vector Autologous gene modifed HSC-Autologous...
Transcript of GENE THERAPY-BASED ORPHAN DRUGS...Gene transfer vector Autologous gene modifed HSC-Autologous...
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Alessandro Aiuti
GENE THERAPY-BASED ORPHAN DRUGS
igetTELETHON INSTITUTEFOR GENE THERAPY
UNIVERSITA’ DI ROMA TOR VERGATA
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Ex vivo approaches
RetrovirusAdenovirusLentivirus
In vivo approaches
AdenovirusAAV
DNA/LiposomesLentivirus
Gene therapy strategies
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Ex vivo
SCID-X1ADA-SCID
Wiskott-Aldrich SyndromeBeta-thalassemia
Metachromatic Leukodystrophy
In vivo
Duchenne Muscular Distrophy Alpha-Sarcoglycanopathy
Gamma-Sarcoglycanopathy
Glycogen storage dis. type II (Pompe's disease)
Retinitis pigmentosa Leber’s amaurosis Stargadt’s disease
Epidermolysis bullosa
Gene therapy-based EU designated Orphan Drugs
Muscle
Multiple organs
Eye
Skin
HematopoieticSystem
CNS &PNS
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Primary immunodeficiencies
• Alterations in development and/or functions of adaptive/innate immunity• Higher susceptibility to infections• Failure to thrive• Increase risk of autoimmunity and cancer
ADA-SCID
INNATE IMMUNITY ADAPTIVE IMMUNITY
WISKOTT-ALDRICH SYNDROME
ADA-SCID
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Gene transfervector Autologous gene modifed
HSC
-Autologous procedure (No rejection/ GVHD)
- Reduced toxicity
- Selective advantage for gene corrected cells
- Data on safety and efficacy from preclincial studies and pilot studies
HSC gene therapy for primary immunodeficienciesMedicinal Product
Starting material
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Adenosine Deaminase-deficient SCID
Adenosinedeaminase
dAdo, AdodAXP
Bone Marrow Transplantation Enzyme Replacement Therapy (PEG-ADA) HSC Gene Therapy
TREATMENT OPTIONS
• T, B, NK, lymphopenia• Severe recurrent infections• Autoimmunity
• Bone and growth abnormalities• Organ toxicity (lung, liver)• Neurological and behavioral alterations
Autosomal recessive1:375,000-1,660,000
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RATIONALE FOR GENE THERAPY
Unmet medical need • 90% of children lack an histocompatible donor in the family • High risk of bone marrow transplant from alternative donors (30-65%
survival)
• Treatment with bovine enzyme (PEG-ADA) (80% survival) not a definitive cure, not always effective, very expensive
Scientific rationale • The ADA gene is constitutively and ubiquitously expressed • Gene-corrected lymphocytes have an advantage over ADA-deficient
cells.
• 10% of normal ADA expression may be sufficient
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Day -4: Purification of BM CD34+ cells
Gene transfer protocol into autologous bone marrow CD34+ cells
Day-4: Prestimulation (TPO, FLT3-ligand, SCF, IL-3)
Days -3 to -1: 3 cycles of transduction on retronectin + cytokines
Busulfan 2 mg/Kg/day x 2 (days -3, -2)
Day 0: Infusion
ADA SV Neo
No PEG-ADA
A. Aiuti, MG Roncarolo, C. Bordignon, 2002
MLV LTR
BM Harvest
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CD15+ granulocytes
Years after GT0 1 2 3 4 5 6 7
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0,0
5,0
10,0
15,0
20,0
0 20 40 60 80 100 120 140
4 yrs after infusion
Diversity of integrations in T lymphocytes
Prop
ortion
of d
ifferen
t clone
s
No in vivo skewing
POLYCLONAL VECTOR INTEGRATIONS and REPERTOIRE %
TCR
Vbet
a in
CD3
+ ly
mph
ocyt
e su
bset
TCR Vbeta Repertoire
0246810
12
Pt2
Pt3
Pt5
Pt6
02468
1012
02468
1012
02468
1012
Pt4
02468
1012 Pt7
02468
1012
02468
1012
Pt9
Pt10
0246810
12
Pt1 ND (n=46)
02468
1012
Aiuti et al., JCI 2007 and unpublished results
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Immune reconstitution after GT
Thymus 3 yrs post-GT
Median (n=9)
CD3+CD4+CD8+
1500
+3 years (n = 8) (n = 6)
+1 year +2 years00
500
1000
Cells
/µl
+3 years
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T- cell reconstitution after gene therapy
PRE-GT 1y FU 2y FU >3y FU BMT HC
10
100
1000
10000 *
Cop
ies /
100
ng
of D
NA
Recent thymic emigrants
Recovery of thymic functions
anti-CD3 mAb
cpm
pre-GT 6 months 1 year 2 years 3 years Healthy controlsn=9 n=9 n=9 n=7 n=5 n=114
100
5000
50000100000200000300000
T-cell functions (anti-CD3)
Aiuti et al., NEJM, 2009
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• IVIg discontinued in 6 pts with proven antibody response
(TT, DT, Pertussis, Haemophilus, Pneumo)
2 Pts ongoing IVIg discontinuation
• MMR vaccine in 1 pt led to protective antibodies
• Four patients experienced varicella
without complications
Immune response and protection from infections
05
1015
Rate of severe infections
N. e
vent
s/10
per
son-
mon
ths
Pre GT Post GT20 infections/ 215.4 person-month 4 infections/ 394.5 person-month
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Systemic detoxification and growth improvement
dAXP metabolites
Years after GT0 1 2 3 4 5 6 7
nmol
es/m
l
0
100
200
300
400
500
*
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T cell
B cell
NK cell
Monocyte
Erythrocyte
Granulocyte Platelet
Outcome of ADA-SCID GT
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Clinical translation in ADA-SCID
First attempts Pilot studies Phase I/II
EMEA ODD 2005 1991 2000 2002
Towards registration
Enrolment closed
2008
Nonclinical
FDA ODD 2009
EMEA protocol assistance
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Wiskott-Aldrich Syndrome (WAS)
Autoinhibited WASP
Active WASP
PH-EVH1 GBD
Pro
A C V V
PIP2Plasmamembrane GTP
Cdc42
SH3NckTecGrb2
Actin monomer
Actin polymerization
Arp2/3Cytoplasm
WIP
CELLULAR DEFECTSHSC migrationT cells migration, immune syn.B cells migration, Ig productionPlatelets reduced size / numberDendritic cells migrationMacrophages adhesion, antigen uptakeNK cells cytotoxic activity
Eczema Bleeding Infections Autoimmunity Tumors
X-linked, 1,250.000 newborn male
Life expectancy: 15 years in severe forms (WAS-negative)
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Current Treatment
Filipovich, Blood 2001; Kobayashi, 2006; Ozsahin, Blood 2008
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GA RREcPPT PREwas WASP CMV
LENTIVIRAL VECTORS
• HIV derived, self-inactivating system
• Safer integration profile
• Physiological promoter
• Improved GT efficiency into HSC
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WAS‐/‐ donor (male/CD45.2)
BM harvest
Transplantation i.v. 2.5x105 ‐ 106 lin‐ BM cells
700 rads (sublethal)
WAS‐/‐ recipient (female/CD45.1)
LV transduction
12 hrs
w1.6W_WPREmut
GA RRE cPPT WPRE h1,6WASP hWASP RSV
SAFETY AND EFFICACY OF WAS GT IN WAS KO MICE
Low MOI High MOI
CD45+ CD11b+ B220+ CD8+ CD4+0
25
50
75
100
SpleenBM
* ***
% W
ASP+
c
ells
CD45+ CD11b+ B220+ CD8+ CD4+0
25
50
75
100 * **
SpleenBM
% W
ASP+
c
ells
No long-term toxicity
No vector derived tumors
No increase in tumor incidence
98 mice followed for 4-16 months
Engraftment and selective advantage
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FUNCTIONAL CORRECTION OF T-CELLS AND B CELLS
anti-CD3 2 µg/ml
Pneumo23 vaccine challenge TP23 - P23 immunization
Ab TITER d=7
TP23 WT TP23 UT TP23 GT0
50
100
150
200
250
300
350 TP23 WT
TP23 GTTP23 UT*
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T-cell functions(n
g/m
l)
1
10
100
1000
10000 ** ****IL-2
SI
1
10
100
1000
10000 ** ****PROLIFERATION
wt
Was-/-w1.6W high MOI
w1.6W low MOI
Lin- Was-/-F. Marangoni, A. Villa, M. Bosticardo
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300+250 rads
Rag2‐/‐ γc‐/‐ (neonates)
SUMMARY OF TOXICITY AND SAFETY STUDIES (CD34+ cells)
In vitro growthColony assay (CFC,LTC-IC)Vector shedding
Vector integrations
BiodistributionVector sheddingGermline transmission
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The path to clinical trial in WAS
Vector & Proof of concept
Preclinical studies
Validation
2002 2004 2006 2008
Manufacturing and quality
Phase I/II trial
2010
• Lack of toxicity
• Safety and efficacy in the animal model
• Selective advantage for gene corrected cells
• Efficient gene transfer in human CD34+ stem cells
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Design: non-randomized, open label, single center
Population: 6 patients -Severe WAS mutation or WAS clinical phenotype
-No HLA-identical sibling -No HLA-matched UR BM or UCB donor (Pts
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Very rare population!
“Personalised” therapy
Single curative injection
“Old” therapy approaches Gene/cell therapy for rare diseases
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• Often very rare populations• Long-term safety• Mainly academic-driven, high costs• Limited interest for pharma company’s investment• Manufacturing and standardization • Rapidly evolving scientific field and regulation
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Funding agencies
Regulatory Agencies (National level)
Manufacturing (Biotech or Academic)
Investigators (PRECLINICAL AND CLINICAL)
EC Regulatory Agencies
Manufacturing (Biotech)
Industrial partner
National Health system
European Community
Partners required for clinical development of gene therapy-based ODD
Patients’ organization
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San Raffaele Telethon Institute for Gene Therapy iget
TELETHON INSTITUTEFOR GENE THERAPY
S. Marktel B. Cappelli L. Callegaro M . Casiraghi V. Bergamante F. Ferrua
I Brigida B. Cassani S. Scaramuzza A. Ripamonti L. Biasco A. Sauer
M. Bosticardo A. Villa
L. Naldini M.G. Roncarolo
Pediatric Clinical Research Unit
MolMed S.p.A M. Salomoni M. Dieci
Main European collaborators (WAS) A. Thrasher (London) A. Galy (Paris)
All participating physicians worldwide
HSR BMT Unit F. Ciceri
U. Bicocca MG Valsecchi S. Galimberti
Univ. of Siena A. Tabucchi F. Carlucci
Univ. Wien M. Eibl