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

Synthesized

Pol 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 U5R U5

d.gag d.pol d.envFV 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

En

graf

tmen

t

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-G

FP+

FV-mU6 or H1 vectors reduce marker gene expression in vitro

293T

HT1

080

HeL

a

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

120

Relative GFP expression (MFI)

FV.mU6-RNAi: Stable target downregulation in vivo

Donors: GFP+

d0 BMT

Hosts: 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

AG

LO 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 H5

VCN=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.β

VCN 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

Η1Anti – 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