Terapia Génica mediada por Vectores Lentivirales para la

Terapia Génica mediada por Vectores Lentivirales para la

17 de Mayo de 2016
Terapia Génica mediada por Vectores Lentivirales para la
Deficiencia en Piruvato Quinasa
Dr. José Carlos Segovia
División de Terapias Innovadoras en el Sistema Hematopoyético
Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas / Centro de Investigación
Biomédica en Red de Enfermedades Raras (CIEMAT-CIBERER). Madrid. Spain
Unidad Mixta de Terapias Avanzadas
Fundación Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (FIIS-FJD). Madrid. Spain
jc.segovia@ciemat.es
Pyruvate Kinase Deficiency
PEP
2
2ADP
2ATP
Mg+2
K+
PYRUVATE
2
PYRUVATE KINASE
•
•
•
Autosomal recessive disorder
Chronic non-spherocytic
hemolytic anemia (CNSHA)
Prevalence: 1-9 cases/105 in
Caucasian population
Treatment
Clinical signs
•
•
•
•
RPK activity <25%
Anemia
Reticulocytosis
Splenomegaly
•
•
•
Periodic transfusions
Splenectomy
Allogeneic bone
marrow trasplant
Pyruvate Kinase Deficiency suitable for Gene Therapy
 Monogenic genetic disease
 Can be cured by allogeneic bone marrow transplant
 The gene responsible for the disease is known and has
been cloned
Pyruvate Kinase Deficiency Mouse model
Acb55 (B19) mouse
strain: pklr -/-
Healthy
PKD
Min-Oo, G. et al., Nature Genetics 2003 (4): 357-62
Erythrocyte
parameters
Mouse strain
Healthy mouse
PKD mouse
RBC
10,5 x 1012/L
6,39 x 1012/L
HGB
HCT
13,8 g/dL
9,7 g/dl
46,2%
38,9%
Healthy
PKD
Preclinical gene therapy protocol
SIN-LENTIVIRAL VECTORS (pCCL)
BM harvest
day 0
♂
Transduction
EXPANSION
Transplantation
Day 3
Day 40
2.105 cells/mouse
•
•
•
•
•
•
•
day 1 and day 2
90 %
MOI 10
LinPKD donor
rhIL11
rmSCF
4.75 Gy
+
4.75 Gy
♀
PGK-EGFP-Wpre
PGK-coRPK-Wpre*
Analysis
PKD recipient
Day 100
Day 140
Hematological parameters
Reticulocytes
Erythroid differentiation
Spleen and Liver
coRPK expression in PB
Metabolic parameters
Vector integration analysis
Correction of hematological parameters
Red Blood Cells
Hemoglobin
**
***
18
HGB (g/dL)
RBC (x106/ml)
*
40
100
140
280
Days post-transplantation
Retyculocytes
*
*
***
6
0
40
100
140
Days post-transplantation
***
**
% Retyculocytes
C57 (non transplanted), n=5
PKD (non transplanted), n=6
PKD PGK-EGFP, n= 8
PKD PGK-coRPK, n=16
100
140
Days post-transplantation
280
*
12
*
***
40
*
280
Normalization of PK activity in RBC
Correction of splenomegaly
C57
Garcia-Gomez et al., Molecular Therapy, In press
PKD
EGFP
coRPK
Reduction of erythroid clusters and iron
deposits in the liver
Wild Type
H&E
Fe
PKD
PKD hPGK-eGFP
PKD hPGK-coRPK
No changes in WBC metabolism
WBCs
WBCs
Mass spectrometry
METABOLITE
PROFILING
High-mass-resolution
(Q-ToF)
Untargeted METABOLITE
array
RBC
WBC
Garcia-Gomez et al., Molecular Therapy, In press
Molecular parameters
Vector Copy Number (VCN/cell)
transduction %
donor chimerism %
Groups
WBC
total BM
indiv. CFU
provirus+ CFUs
SRY+ PB cells
PKD PGK-EGFP (n=2)
0.83 ± 0.05
0.42 ± 0.03
0.42 ± 0.00
57.73 ± 12.28
n.d
PKD PGK-EGFP (n=6)
4.56 ± 0.50
1.51 ± 0.28
n.d
91.13±4.49
61.82 ± 3.61
PKD PGK-coRPK (n=3)
4.76 ± 0.28
3.58 ± 0.34
3.07 ± 0.76
91.08 ± 3.67
n.d
PKD PGK-coRPK (n=14)
1.65 ± 0.08
0.99 ± 0.13
n.d
65±0.75
63.66 ± 4.45
2nd coRPK (n=4)
1.44±0.08
n.d.
n.d.
65.13±0.31
62.89±5.61
Viral integration studies
Mice analyzed:
Samples analyzed:
Nº Reads:
Integrations detected:
# integrations/sample:
27
85
5236283
2220
85.1
Garcia-Gomez et al., Molecular Therapy, In press
 No clonal dominance
 No integrations close to genes involved
in cancer, cell cycle, apoptosis
 No common retroviral integration sites
Orphan Drug Designation
EMA: EU/3/14/1130
FDA: DRU-2016-5168
Healthy
RPK gene
Lentiviral vector carrying
the healthy RPK gene
The gene therapy drug
EMA: EU/3/14/1130
FDA: DRU-2016-5168
HEMATOPOIETIC STEM CELLS
CORRECTED BY THE THERAPEUTIC
VECTOR
Healthy
gene
Towards a Gene Therapy Clinical Trial for PKD
EMA:
EU/3/14/1130
FDA:
DRU-2016-5168
HEMATOPOIETIC STEM CELLS
CORRECTED BY THE THERAPEUTIC
VECTOR
Healthy
gene
Registered drug
1 Design and development of the
4 ORPHAN DRUG
THERAPEUTIC VIRUS
6
designation
CIEMAT-CIBER-FJD
Gene therapy development
2
In vitro validation
3
Preclinical studies in mouse models
(EFFICACY and SAFETY)
5
CLINICAL TRIALS
European PKD gene therapy consortium
ENERCA
PATIENTS SIDE
P2:
R van Wijk
UMCU
Utrecht- The
Netherlands
P3:
P Bianchi
IRCCS
Milan - Italy
P4:
JL Vives
HClinB
Barcelona Spain
P5:
L Ribeiro
CHUC
Coimbra Portugal
P6:
S Pissard
INSERM
Paris - France
P7:
I Badell
HPS
Barcelona Spain
P1:
JC Segovia
CIBER
Madrid - Spain
P8:
J Sevilla
SERMAS
Madrid - Spain
Integration site
analysis
DRUG SIDE
P9:
GMP viral
production facility
VIRAL VECTOR PLATFORM
P11:
J Mountford
Univ Glasgow
Glasgow – UK
CRO
EU PKD Patient’s groups / Spanish Association for Rare Diseases
Patients susceptible of being enrolled in the GTCT
Mild-Moderate
Severe
6/65 (9%)
EHA Learning Center. Grace R. Jun 12, 2015; 100615
Patient’s support
www.pyruvatekinasedeficiency.com
Patient’s support
Link to PKD
I'm a
patient
with PKD
4% (37)
I'm a
parent of a
child/adult
with PKD
6% (48)
Other
23% (195)
I'm a family
member of
someone with
PKD
16% (131)
I'm a friend of
someone with
PKD
51% (421)
Summary
• HSC
transduced with LV expressing a codon optimized
version of the RPK cDNA restore normal erythroid function
and compensate hemolytic anemia in a mouse model of
PKD (ODD 3/14/130 ; DRU-2016-5168)
• PKD
progenitors (CD34+) can be transduced with the
therapeutic LV
• Up to 100% of CFCs can be transduced with a pre-GMP
control vector
• Gene Therapy Clinical Trial for PKD is being developed