Control of excess α -chains in β- thalassemia
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Control of excess α-chains in β-thalassemia G. Vassilopoulos MD PhD Associate Professor, Hematology and Internal Medicineci U. of Thessalia Medical School Principal Investigator, BRFAA Division of Genetics & Gene Therapy
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Control of excess α -chains in β- thalassemia. G. Vassilopoulos MD PhD Associate Professor, Hematology and Internal Medicineci U. of Thessalia Medical School Principal Investigator, BRFAA Division of Genetics & Gene Therapy. - PowerPoint PPT Presentation
Transcript of Control of excess α -chains in β- thalassemia
Control of excess α-chains in β-thalassemia
G. Vassilopoulos MD PhDAssociate Professor, Hematology and Internal Medicineci
U. of Thessalia Medical SchoolPrincipal Investigator, BRFAA
Division of Genetics & Gene Therapy
Globin Chain Imbalance is the Major Determinat of Disease Severity in β-thalassemia
Disease Morbidity
β/α = 1 β/α < 0.2
β/α = 0.4-0.6Carrier state
normal β-thalassemia
Excess α chains• Inclusion bodies (can trigger immune destruction)• Membrane damage (Mechanical removal)• Phosphatidyl exposure (Hypercoagulability)• Apoptosis (Ineffective erythropoiesis)
β globin mutations
ChronicHemolytic
Anemia
Ineffective Erythropoiesis
Loss of erythrocyte precursors in the
Bone Marrow
60-75% of total erythropoiesis
HYPOTHESISIf alpha - globin excess is ameliorated, a significant portion of ineffective erythropoiesis would be eliminated.
Tool : RNAi
nucleus
B.
Drosha DGCR8
pre-miRNA
pri-miRNA
5 319-23 nt siRNAs
DICER
Exportin-5
SynthesizedPol II miRNA
Pol III shRNA
ChemicalsiRNA
RISC
Ago-25 3
mRNA Target
ΑΑΑΑ
5’- CAP
cytoplasm
Nobel in Medicine 2006: RNA interference
TasPol
IPEnv R U5U3R U5U3 Gag
bel 2&3
Tas
PR RT INenzymes
SP SU TMenvelope
M - C - NC
Foamy Virus wt and derived Vectors
delU3
CMV R U5 R U5d.gag d.pol d.env
FV vector Deleted Foamy (ΔΦ)del U3-LTR (SIN)Minimal cis-acting sequences No Transactivator
Foamy Virus Vectors can transduce HSC
AP-expressing vector
GFP+ CFU
100 102 104 100 102 104 100 102 1040
2000
Coun
ts
WBC RBC PLT
1000
39% 51% 59%
GFP
GFP-expressing vector
0
20
40
60
80
100
Engr
aftm
ent
BMCD45
BMCD19
BMCD33
BMCD34
PBCD45
SP CD45
CFU
(p
reB
MT
)
GFP+
%
43
65 68 71 68
55
39
75
FV vector mediated transduction of huCD34+ cells
Josephson, et al HuGeneTher, 2004
FV.MscvGFP/ΔNGFR
CMV R U5 R U5Mscv
Design of FV-RNAi vectors
ΔNGFRor GFP
FV.mU6/H1.shRNA.MF/NmU6.shRNA
CMV R U5 R U5
H1.shRNA
Mscv ΔNGFRor GFP
shRNA Promoters mU6 or H1 (5’ pol)
Reporter genes GFP or ΔNGFR
0
20
40
60
80
100
Η1 mU6
Unt
x-GF
P+
FV-mU6 or H1 vectors reduce marker gene expression in vitro
293T
HT10
80
HeLa
Η5
shN
Ssh
bcr.a
bl
22%
4.5%
Η7
23%
2.5%
20
40
60
% A
nnex
in
Apoptosis d7
NS bcr.abl
0
20
40
60
80
100
120
3 5 7 15 20 30ΗΜΕΡΕΣ
% G
FP o
ver D
3
shNS
shbcr.abl
FV.H1-RNAi: Targeting bcr.abl induces apoptosis in Κ562 cells
Target: GFP+ K562 cells
Busilvex IP
d-4 d-1
♀ ♀ ♀
0
20
40
60
80
100
120Relative GFP expression (MFI)
FV.mU6-RNAi: Stable target downregulation in vivo
Donors: GFP+
d0 BMTHosts: wt females
FV: mU6.shGFP/NS.ΔNGFR
PB 6w
PB BM SPsc
ram
bled
13 w
Ανθρώπινη
100 200 300 400 500
Μυϊκή
100 200 300 400 500
Mouse cDNA
Human cDNA
TSS
AIMMild (30-50%) reduction in a-globin expression
Β9/C7/D3/E1 FV.mU6.shA.MF
Η1/2/4/5 FV.H1.shA.MF
Assay vector efficiency in eryhtroid lines(MEL, K562)
Pick best performers for assaying in primary cells
% a
glob
insh
AGL
O v
s CM
EL
50
100
B9 C7 D3 E1
Cont
rol
27
53
79
12
FV.mU6.shA.MF: reduction in mouse α-globin in ΜEL cellssc
ram
bled
H1SCR H2 H4 H5
α globin
b-actin
FV.Η1.shA: human α-globin reduction in Κ562
1-4.5 1-4 1-5 1.5-4.5
0
20
40
60
80
100
120
SCR H1 H4 H5
mRN
Ash
A/co
ntro
l
Cont
rol
FV.mU6.shA.MF: α-globin mRNA reduction in murine BFUe
% m
RNA
shA
/ co
ntro
l
0
20
40
60
80
100
B9 C7 D3 E1
Cont
rol
16 28
15 21
Lin-wt cells
Tx o/nwith vector
Grow BFUe
PickGFP+ BFUe
6.8%
Thal3/+ FLC
12.3%
Thal3/+ FLC-GFP+
mU6.shA.MF vectors can improve ineffective erythropoiesis in thal3 mice (in vitro)
TER119
CD71
Nishina et al, BBRC, 2009
10
20
30
Thal
3/+
B9 C7
25
106
# BFUe / 10E4 thal3 Lin-
0
20
40
60
80
100
UnTx H1 H2 H4 H5VCN=10-15 , n= 3-5
Cont
rol
mRN
Ash
A/co
ntro
l
FV.Η1.shA: human α-globin reduction in CD34+ cells
CD34+ transduction with
H1.shA.GFP
Assay RNA RealTimePCRα-globin/GAPDH
Human β-globin
MFI7.44
9.0%
MFI13.9
34.9%
MFI12.8
32.8%
β-globin expression in CD34+ from thal patients transduced with two different
therapeutic FV vectors
Control HS40.β HS2.HS3.β
HS40.β HS2.HS3.βVCN 0.75 1.35
Combination FV vector expressing α-globin shRNA and β-globin
pΔΦ.Η4/HS40.β
a-HS40
β-promoter
123
CMV/LTR
3’UTR
Η1 Anti – αGLO shRNA
β-globin
Amelioration of β/α ratio in CD34+ cells from a thalassemic patient
β/α globin mRNA ratio
Comfortzone
Conclusions
• FV vectors expressing shRNA can provide sustained gene silencing in vitro and in vivo
• Efficient gene silencing of the mouse and human a-globin transcripts
• Amelioration of ineffective hematopoiesis
• Therapeutic effect with the combination vector
• In β-thal, strategies aiming at reducing α-excess are rationale and worth further exploration
There are Howevers…..
• RNAi is often unpredicted
• There is need for tight regulation of expression
• Vector integration is unpredicted and could lead to overactive transgenes with non-desired consequences
• Vector-transduced cells are cells and not medicine; once in, tough to take them out
Future Perspectives
• RNAi is not water and is here to stay
• Since RNAi functions in the cytoplasm, transient effect could be more desirable
• Expect smart delivery methods since vectors and genes and stem cells are costly, need sophisticated infrastrucrure and are not accessible to every patient.
ACKNOWLEDGMENTSMagda PAPADAKI – RNAiJohn MORIANOS - Glo vectorsElena SIAPATI – mom of the labCollaboratorsK. STAVROPOULOU - BRFAAA. KATAMIS - Ag.Sofia HospitalDW RUSSELL - U.WashingtonDW EMERY - U.WashingtonG. STAMATOYANNOPOULOSD. LOUKOPOULOSSupportCONSERT, FP6, EUPENED, GSRT, GRBRFAA, Intramural
