Gene therapy of severe -thalassemiaβ

Yves Beuzard Thalassemia/ENERCA conference, Madrid, November 2010

yvesbeuzard@yahoo.fr

2

Overview of the Clinical ProtocolAutologous transplantation of transduced CD34+ cells

Testingand Release

While Frozen

Maximize% Transduced

HSCs

Vector+

Cytokines

Bone MarrowHarvest

MaximizeMyeloablation

(2x108 unsorted BM cells/Kgkept for rescue)

Bone MarrowConditioning

Busulfex

IV InfusionTransduced Cells

CD34+ cellsMobilized BloodCells

or

Autologous transplantation at age 18, on June 7, 2007 (3.5 years ago)

Fully transfusion dependent since age 3 (225 ml RBCs /kg/year).

Spontaneous Hb levels as low as 4.5 g/dl.

Major hepato-splenomegaly (splenectomy at age 6) and growth retardation.

Failure of Hydroxyurea therapy.

Deferoxamine (5 days/week) since age 8. No liver fibrosis. Iron overload by liver MRI (561 µmol/g). No cardiac toxicity.

No related, genoidentical HLA-matched donor.

Male with severe βE/β0-thalassemia. Diagnosis: 1 year old.

Clinical History of Patient "PL"

HS2 HS3 HS4

human β-globin gene

3’ enhancer

III βp

ppt

RRE

cPPT/FLAP 644bp 845bp 1153bp

III

266bpΨ+

2 x cHS4Insulator cores

2 x cHS4Insulator cores

βA-T87Q

Lentiviral vector design with marked -globin geneβ

SIN + Insulator(Δ 1 core in ≈ 50%)

SIN + Insulator(Δ 1 core in ≈ 50%)

Conversion to transfusion independence (I)

RBC transfusionsMonths post-transplantation

Hem

oglo

bin

(g/d

L)

6

7

8

9

10

11

12

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Last RBC transfusion 2.5 years ago Phlebotomies (2200 ml)

Phlebotomies

appendicitis

Exjade DeferiproneDesferal

HPLC analysis of globin chains in whole blood(3 to 33 months post-transplant)

βE

βA

γG γA

α

Abs

roba

nce

220

nm

3 months

33 months10.0

7.5

5.0

2.5

0.0

-2.5

-5.0

Abs

roba

nce

220

nm

βE

βA-T87Q

α

γG γA

βA-T87Q

Σ (all β + γ)x 100 = 34 - 37 %

10.0

7.5

5.0

2.5

0.0

-2.5

-5.0

Abs

roba

nce

220

nm βA-T87Q

βE

α

γG γA

12 months

βA

10.0

7.5

5.0

2.5

0.0

-2.5

-5.0

Conversion to transfusion independence (II)H

emog

lobi

n (g

/dL)

HbAHbAT87Q

HbFHbE

HbA

HbAT87Q

HbF

HbE

Months post-transplantation

0.2 0.3 0.9 0.7 1.0 1.5 1.8 2.0 1.7 2.2 2.7 2.7 2.7 2.7 3.0

0.6 0.9 1.6 1.3 1.5 2.1 2.3 2.4 2.1 2.8 3.1 2.9 3.0 2.8 3.0

0.4 0.3 0.4 0.8 1.2 1.3 1.3 1.8 2.0 2.4 2.8 2.8 2.9

10.9 6.9 6.7 7.7 5.6 2.7 3.1 1.3 3.7 1.2 0.1

3.0

3.3

3.2

2.8

2.9

3.2

3.1

3.2

3.7

2.9

2.8

3.6

3.0

3.1

3.5

0

2

4

6

8

10

12

-3 3 4 5 6 9 11 12 13 15 16 18 19 20 21 22 24 25 27 28 29 30 31 32 33 33.5

35 35.5

36.5

2.7

3.1

3.2

2.6

3.3

3.3

2.5

3.0

3.0

2.9

3.5

3.4

2.9

3.3

3.7

3.0

3.0

3.2

2.7

3.0

3.2

2.8

3.0

3.3

2.9

3.2

3.4

Decreased dyserythropoiesis and increased RBC survival

Near normal endogenous level of βA-T87Q -globin expression in circulating RBCs on a per gene basis

MCH correction:Patient's MCH 27-33 months post-transplant = 28.7 pg (within normal range 27-32 pg)Average MCH for βE/β0-Thal patients = 19 pg

MCHN

MCHP x HbA-T87Q (%) 2x= 0.69

βA-T87Q-globin

βA-globin (normal) =

Gene expression output on a per gene basis = 69 – 100 %

Decreased circulating erythroblasts

1.01.52.02.53.03.54.0

0 5 10 15 20 25 30 35

RB

C (x

106 /

µL)

1.01.52.02.53.03.54.0

Ret

ic (x

105 /µ

L)

05101520253035

0 5 10 15 20 25 30 35

Eryt

hrob

last

s

(x10

3 /µL

)

Increased RBC lifespan

Months post-transplantation Months post-transplantation

Integration site (IS) analysis by LM-PCR and DNA pyrosequencing(whole nucleated blood cells and purified sub-populations)

Low total number of different IS (< 300)In actively transcribed regions, similar to generic HIV vector

24 IS both myeloid and lymphoid

Months post-transplantation (whole nucleated blood cells)

020406080

100%

3 5 9 13 16 18 19 20 24

HMGA2FBXL11TBC1D5PILRBMKLN1IRAK1ZZEF1RFX3NUP98ATXN10EPB41L2EIF1PHF16SAE1GNA12POLA2

Relative dominance of IS at the HMGA2 locus(dominance relative to other IS, but 90% blood cells remain untransduced)

HMGA2

ATG51516 51517

TAGGlobin-LV

E1 E2 E3 E4 E5

Let-7 miRNAs

Vector integration in the third intron of the HMGA2 gene

Intron 3 (~ 113 kb)Deletion of 1 copy insulator core

Physiological regulation of HMGA2 expression

Expression largely restricted to embryonic and stem cells

Normal degradation of RNA by Let-7 miRNAs (multiple targets in E5)

Decreased expression in HSCs correlated with aging

Quantification by qPCR of the number of copies of vector provirus in circulating blood cells (overall and HMGA2 IS)

(qPCR for whole nucleated blood cells and purified sub-populations)

Whole nucleated blood cells

Total vector copy number (black) and copy number at specific HMGA2 site (blue)

10-12%

2-4%

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0 5 10 15 20 25 30 35 40Months post-BMT

Vect

or c

opy

num

ber

Proposed target of Globin LV-HMGA2 IS clone formation

LT-HSC

Lymphoid-restrictedLT-HSC (?)

Myeloid-restrictedLT-HSC (?)

" cells"α

Common Myeloid Progenitor

BFU-E CFU-GM

BM Erythroblasts

Granulo-Mono

HMGA2 IS 7 %≈HMGA2 RNA = 0Δ

HMGA2 IS 15 %≈HMGA2 RNA > 10,000xΔ

Erythroblasts

"Stemness"acquisition

LTC-ICHMGA2 IS 5 %≈

HMGA2 IS undetectablein lymphocytes

• High expression of therapeutic globin gene– 60-100% normal endogenous βA-globin on a per gene basis– No evidence of secondary repression (PEV)– Correction of MCH (19 pg 29 pg/GR)– Therapeutic gene contributes # 3.5 g / dL Hb in blood

• Conversion to transfusion independence for the last 27 months– Major increase in survival of corrected RBCs (life span x 8,7)– Hb 9-10 g/dL– Reduction in reticulocytosis and erythroblastosis

• Much improved quality of life– Excellent tolerance: feeling cured, full time cook.– Oral iron chelation + phlebotomies:

major decrease in blood ferritin and liver iron levels.

CONCLUSIONS (I)

• HMGA2 Clone in myeloid cells, not present in lymphocytes- Transcriptional activation by LCR, in erythroblasts only.- Failure of single core cHS4 insulator in the erythroid precursors, but efficiency in granulocytes/monocytes. - truncation of HMGA2 mRNA and loss of Let 7 micro-RNA repression

• Presumptive homeostasis: stable HMG2A2 clone, normal caryo-cytology, to be monitored for 15 years

• Hypothesis: Amplification of myeloid-biased LT-HSC with lineage priming

• Other cases of IS in HMGA2: SCID-X1 (RV), ALD (LV)

• Benign expression of truncated HMGA2 gene (Lipomas and other benign tumors, PNH)

• Importance of HSC dose: increased risk vs. increased appearance of polyclonality

CONCLUSIONS (II)

Other University Hospitals of Paris (AP-HP): Saint-Louis, Mondor, CHIC, Tenon, Saint Vincent de PaulEliane Gluckman (PI) Françoise Bernaudin Gérard SociéRobert Girot Jean Soulier Nabil Kabbara

Hopital Necker (AP-HP), ParisSalima Hacein-Bey Abina Laure Cacavelli Marina Cavazzana Calvo (PI)

Bluebird Bio, Cambridge, MAKathy Hehir Maria Denaro Julian Down

Indiana Vector Production Facility, Indianapolis, INKen Cornetta Scott Cross Chris Ballas

Harvard Medical School, Brigham and Women’s Hospital, Boston, MARobert Pawliuk Karen Westerman Resy cavallesoPhilippe Leboulch

Institute of Emerging Diseases and Innovative Therapies CEA – INSERM U. 962 – Universities Paris 7 and 11and Bluebirdbio - FranceEmmanuel Payen Olivier Negre Béatrix Gillet-LegrandFloriane Fusil Céline Courne Yves BeuzardPhilippe Leboulch

University of Pennsylvania School of Medicine, Philadelphia, PAGary Wang Troy Brady Frederick Bushman

Percentages of vector bearing cells in blood and bone marrow(qPCR – 3 years post-transplant)

BLOOD (35 months)All nucleated cells 8.9 %

Granulo-Monocytes (CD15+) 18.6 %

B Lymphocytes (CD19+) 9.4 %

T Lymphocytes (CD3+) 2.3 %

Erythroblasts (CD45- CD71+) 1.1 %

BONE MARROW (36 months)All nucleated cells 21.0 %

CD34+ 25.0 %

CD45+ 19.4 %

Erythroblasts (glycoA+ CD71+) 34.6 %

Memory T cellsnot exposed

Correctiondyserythropoiesis

≈ 20%

Viral titer cGMP lot = 1.3 x 108 TU / ml during CD34+ cell transduction

Pre-transplant transduction efficiency = 0.6 vector copy / CD34+ cell (qPCR)

Pre-translant conditioning: BUSULFEX alone (at myeloablative dose)

Dose of CD34+ cells injected = 3.9 x 106 CD34+ cells / kg

Transduction / transplantation parameters

Fresh marrow for transduction and mobilized CD34+ cells for rescue (back up)

Expression of βA-T87Q - globin in whole blood(HPLC analysis)

Months post-transplantation

βAT8

7Q T

hera

peut

ic H

b (%

)

0

5

10

15

20

25

30

35

40

Hb

βAT8

7Q (

g/dL

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0 10 20 30 40

p < 0.0001r2 = 0.9872

Months post-transplantation

Rat

ios b

lood

glo

bins

/ βE

(nor

mal

ized

HPL

C A

reas

)

βE

β87

γA+γG

Kinetics of -globin expressionγ

0,0

0,5

1,0

1,5

2,0

2,5

3,0

0 10 20 30 40

Hematopoieticreconstitution with change in blood

Granulo-Mono (GM) / Erytho (EB) ratio

Kinetics of bloodvector-bearing cells

Vect

or c

opy

num

ber

0.00

0.020.040.060.08

0.100.12

0 5 10 15 20 25 30 35Months post-transplantation

Reconstitution

Change in GM/ EB ratio

0

5000

10000

15000

20000

25000

0 5 10 15 20 25 30 350

150000

300000

450000

600000

750000

Neutro

PLT

EB

GM

Neu

tro,

GM

, EB

/ µL

Plat

elet

s / µ

LMonths post-transplantation

30000900000

Integration site (IS) analysis by DNA pyrosequencing(Blood cell subsets)

T lymphocytes(CD3+)

B lymphocytes(CD19+)

Dominance relative of the HMGA2 IS in both granulocytes-monocytes and erythroblasts, but not lymphocytes (negative qPCR after lymphocyte expansion)

Granulocytes – Monocytes (CD15+)

Erythroblasts(CD45-/CD71+)

Blood CD15+cells (GM)

Blood CD45-/CD71+cells (E)

16-20%

8-9%

2-3%

1-2%

0.000.040.080.120.160.20

0 5 10 15 20 25 30 35 40

0.0000.0050.0100.0150.0200.0250.0300.035

5 10 15 20 25 30 35 40

Months post-BMT

0Months post-BMT

Evidence of truncation of themain HMGA2 transcript (E1 – E3)by staggered RT-qPCR

Sequencing of the main HMGA2 transcript:Aberrant splicing within the vector insulator + polyadenylation

Is partial clonal dominance linked to HMGA2 activation ?

RT-qPCR

Only expressed in erythroid cells (not granulo-mono) in spite of similar percentage vector at HMGA2 IS (qPCR HMGA2/LV junction)

LCR-mediated effect vs. loss of Let-7 miRNA targets?

"run-on"

Increased transcription = 371 fold (likely LCR-driven with insulator failure)Increased RNA stability = 36 fold (likely loss of Let-7 miRNA targets)

36

26

20

Detection of the HMGA2-derived protein – Western blot

Detection of the protein in erythroid cells derived from BFUes in vitro

PLB

β-th

al in

divi

dual

Tru

ncat

ed

Full-

leng

th

Ponceau staining

WB: anti-HMGA2

Rec HMGA2

36

26

20

PLB

β-th

al in

divi

dual

Stabilization of HMGA2 mRNA expression in whole blood (erythroblasts) – RT-qPCR analysis

Red dots: RNA was extracted from whole blood before (0) and after transplantation.

Absence of major position effect variegation (PEV) regardless of integration site (IS) dominance

60

0

10

20

30

40

50

HbA

-T87

Q (%

)

Globin HPLC analysis of individualvector-bearing (PCR+) BFU-Es

12 – 20 months post-transplantation(during HMGA2 IS dominance)

0 – 6 months post-transplantation(before HMGA2 IS dominance)

Homogenous -globin expression γin single BFU-E colonies regardless of vector integration sites (HMGA2 or not)

Proposed target of Globin LV-HMGA2 IS clone formation

LT-HSC

Lymphoid-restrictedLT-HSC (?)

Myeloid-restrictedLT-HSC (?)

" cells"α

Common Myeloid Progenitor

BFU-E CFU-GM

BM Erythroblasts

Granulo-Mono

HMGA2 IS 7 %≈HMGA2 RNA = 0Δ

HMGA2 IS 15 %≈HMGA2 RNA > 10,000xΔ

Erythroblasts

"Stemness"acquisition

LTC-ICHMGA2 IS 5 %≈

HMGA2 IS undetectablein lymphocytes

Hematopoietic homeostasis is maintained

Normal blood and bone marrow cytology – Normal cytoflurometry analysis

Normal karyotype and high resolution CGH-array chromosomal analysis –No Trisomy 8 with specific probe

Lack of cytokine-independence in CFU-C assays

Normal LTC-IC counts

Asymptotic stabilization of the clone relative dominance(in agreement with mathematical modeling and the natural cases of PNH with HMGA2 dysregulation followed for > 17 years)

Importance of the HSC dose:

- In deterministic mathematical models,increasing transduced HSC dose results in increased riskwith paradoxical increase in appearance of polyclonality

- In certain stochastic mathematical models,increasing transduced HSC dose results in balanced co-suppression

Recent evidence of HMGA2 IS in other gene therapy trials("hotspot" or evidence of homeostatic in vivo advantage?)

HMGA2 in X-SCID trial (γ-RV vector)> 15 cluster IS in HMGA2 (aggregates of patients data):

- 12 in HMGA2 Intron 3- 11 in same orientation - Increase abundance with time and then stabilize- 2 (at least) with truncated RNA by aberrant splicing Intron 3 into vector

HMGA2 in ALD trial (LV vector)1 IS in HMGA2 Intron 3 in patient P1:

- only in B lymphocytes and 1 time-point (9 months)

Interpretation of polyclonal patterns of IS (ALD, ADA trials)

True lack of genetic dysregulation resulting in growth advantage

"Peaceful co-existence" of cells with minor genetic dysregulation