Five year outcome of lentiviral gene

therapy for human beta-thalassemia,

lessons and prospects

Cyprus TIF 2012 - P. Leboulch

The only curative treatment for the β-thalassemia currently is allogeneic hematopoietic transplantation, although fewer than 25% patients have an HLA-matched related donor, and those who do still face the risks of engraftment failure and Graft-Versus-Host Disease (GVHD)

Ex vivo gene therapy by transfer of a therapeutic β-globin gene into the patient’s own hematopoietic stem cells (HSCs) would alleviate both donor shortage and GVHD.

It is however especially challenging and a paradigm of gene therapy with no intrinsic in vivo advantage for corrected HSCs.

HS2 HS3 HS4

human -globin gene

3’ enhancer

III p

ppt

RRE

cPPT/FLAP 644bp 845bp 1153bp

III

266bp

2 x cHS4Insulator cores

2 x cHS4Insulator cores

Lentiviral vector design with marked β-globin gene

SIN + Insulator(Δ 1 core in ≈ 50%)

SIN + Insulator(Δ 1 core in ≈ 50%) A-T87Q

Why A-T87 marking / mutation?•Provides anti-sickling properties (Leboulch and coll. Science 2001)•In-vivo biomarker for detection therapeutic globin by HPLC

Overview of the clinical protocol

Testingand Release

While Frozen

Maximize% Transduced

HSCs

Vector+

Cytokines

Bone Marrowor PB Harvest

MaximizeMyeloablation

WithoutImmunosuppression

Bone MarrowConditioning

Busulfex

IV InfusionTransduced Cells

(≈ 4x106 CD34+/Kg)(Spontaneous Homing)

CD34+ cells

(2x108 unsorted BM cells/Kgkept for rescue)

Phase I/II clinical trial: Initial focus on severe E/0-thalassemia

In Thailand alone, ~ 3,000 new cases born each year.~ 4% of the 350 million Southern China population carry a E- or a 0- allele.High prevalence among US immigrants.

High worldwide prevalence

Transfusion dependency, iron chelation therapy and often splenectomy.Candidates for allogenic transplant when HLA-matched sibling donor.

Mean spontaneous Hb levels separated by > 2 g/dl.

Severe in ~ 50% cases (similar to 0 Thalassemia major)

Narrow differential Hb levels between severe and well tolerated cases

Transplantation 5 years ago at age 19 on June 7, 2007

Transfusion dependent since age 3 (> 225 ml RBCs /kg/year for Hb > 10 g/dl).

Spontaneous Hb levels as low as 4.5 g/dl.

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

Failure of Hydroxyurea therapy (between ages 5 and 17).

Desferoxamine (5 days/week) since age 8, and oral Exjade since age 18 (although nausea). No liver fibrosis. Moderate iron overload by liver MRI (561 mol/g).

Only child. No related, genoidentical HLA-matched donor. Match strict inclusion and exclusion criteria.

18 year old male with severe E/0-thalassemia (major) and no HPFH or α mutation.

Pre-transplant clinical history of the first gene therapy patient without injection of backup cells

CD15 (neutrophils)CD19 (lymphocytes B)CD45-CD71+ (erythroblasts)CD3+ (lymphocytes T)

Whole blood

CD14 (monocytes)

0.000.020.040.060.080.100.120.140.160.180.20

0 10 20 30 40 50 60Months post-transplantation

Vect

or c

opy

/ cel

lVector copy numbers per circulating cell subsets

(up to 5 years post-transplantation)

LentiglobinTreatment

Published Sept 2010

Transfused Blood

Conversion to transfusion independence for 4.4 years(5.4 years post-transplantation)

Transplantation on June 7, 2007Last RBC transfusion on June 6, 2008

Conversion to transfusion independence (II)

0 10 20 30 40 50 600

2

4

6

8

10

12

HbA

HbAT87Q

HbFHbE

Hem

oglo

bin

(g/d

L)

Months post-transplantation

Last transfusion > 4.4 years ago

≈ 3.5 g/dL vector-encoded AT87Q-globin

Hb totale

Phlebotomies (200 ml each)

Partial correction of dyseythropoiesisand red blood cell maturation/lifespan

Red

blo

od c

ells

(x10

6 /µL)

Ret

icul

ocyt

es(x

105 /µ

L)

Blo

od e

ryth

robl

asts

(x

103 /µ

L)

Months post-transplantation Months post-transplantation

5101520253035

1,01,52,02,53,03,54,0

1,01,52,02,53,03,54,0

10 20 30 40 50 6010 20 30 40 50 60

1000

1500

2000

2500

3000

3500

4 8 12 16 20 24 28 32 36 40 44 48 52 56 60

Ferr

itin

(µg/

L)Phlebotomies

Months post-transplantation

Partial decrease in plasma ferritin levels

Sustained amelioration of the thalassemic phenotype

Complete MCH correction (28.4 pg)(Average MCH for βE/β0-Thal patients = 19 pg)

Calculated expression βAT87Q > 70% endogenous βA-globin on a per gene basis

Calculated increased RBC lifespan > 8.5-fold

Decreased circulating erythroblasts > 3-fold (> 9-fold for vector+ cells)

Significant decrease (> 2-fold) in plasma ferritin levels

Excellent clinical status (full time job)

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 > 80 % cells remain untransduced)

HMGA2

Aberrant splicing of the third intron and polyadenylation within the globin lentiviral vector

ATG51516 51517

TAGGlobin-LV

E1 E2 E3 E4 E5

Let-7 miRNAs

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

Sequencing of the main HMGA2 transcript:Aberrant splicing within the vector insulator + polyadenylationERYTHROID-SPECIFIC EXPRESSION +++++

Cell type distribution of HMGA2 IS (3 months post-transplant)

CFU-GM colonies ~ 7 % positive(~ 67% of vector+)

BFU-E colonies ~ 15 % positive(~ 78% of vector+)

Lymphocytes (thorough search) ~ undetectable(fresh sorted CD3 / CD19 or

lectin expanded)

LTC-IC (7 weeks) ~ 6 % positive(~ 50% of vector+)

First evidence in humans for long-term hematopoietic lineage bias (myleoid vs. lymphoid vs. totipotent)

Further evidence is accumulating in mice …• By limited dilution transplant analyses from C. Eaves lab (e.g., Cell Stem Cell 2012;10:273-83).

• By barcoded transplant analysis from P. Leboulch lab (submitted).

• CD86- marks myeloid restriction from W. Kincade lab (Blood 2012;119:4889-97).

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 countsNo preferred engraftment in NSG mice (coll. C. Eaves)

Asymptotic stabilization of the clone relative dominance

No abnormality detected for MPL. JAK2 and TET2

Natural cases of PNH with HMGA2 dysregulation followed for > 19 years

- Lack of detection in mouse studies- Evidence for initial integration bias ?- Evidence for homeostatic in vivo selection ?- General principle seen with other key genes

HMGA2 IS is frequently retrieved by DNA pyrosequencing in vivo after retroviral and lentiviral human CD34+ gene transfer

HMGA2 in X-SCID trial (γ-RV vector)

> 15 cluster IS in HMGA2 (aggregates of patients data):- several in HMGA2 Intron 3- several with tendency to increase with time and then stabilize- 2 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

Wiskot Aldrich trial (γ-RV vector)

MGMT glioblastoma trial (γ-RV vector)

The percentage of partially dominant HMGA2 site is decreasing over time in whole nucleated blood cells (qPCR – 5 years post-transplant) …

Integration site (IS) analysis by LM-PCR and DNA pyrosequencing4 and 5 years post-transplantation

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

PMS2P1RFX3

ZZEF1HMGA2

SPATS2POLA2

WBC WBC CD45 CD3 CD19 CD15 CD14 Erythro

4 years 5 years post-transplantationIn

sers

ion

site

s (%

of e

ach)

Already the most common after 3 years

38 27 20 10 22 19 23 9Number unique IS

qPCR based kinetics of the four most common ISup to 5 years post-transplantation

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0 10 20 30 40 50 60

Months post-transplantation

Vect

or c

opy

num

ber copies

HMGA2

RFX3

FBXL11

ZZEF1

4 years

ZZEF1 = 1/3 HMGA2

5 years

ZZEF1 = HMGA2

Second E/0-thalassemia (major) gene therapy patient transplanted on November 24, 2011

• 23 year old woman, βE/β0 Thal Major‑

• Transfusion dependent since her 2nd month of life. No HLA matched sibling donor

• The transplantation was uneventful. Engrafted neutrophils by day 20 and had delayed platelet reconstitution (no related complications)

• The patient has returned to full time work

• Peripheral vector copy numbers and therapeutic βAT87Q globin protein in reticulocytes‑ approximately 3-fold greater than in the previous patient at same time-points

• Red blood cell transfusions are now being tapered down to stimulate erythropoiesis and increase βAT87Q globin production‑

PATIENT 1 (specific chain / non- chain)

0

5

10

15

20

0 2 4 6 8

Months post transplant

glob

ins

[%]

87

E

PATIENT 1

0.00.20.40.60.81.01.21.41.6

0 2 4 6 8

Months post transplant

hem

oglo

bins

[g/

dL]

87

E

E

Parallel evolution of therapeutic Hb in the second β-thalassemia patient after lentiviral gene therapy

0

5

10

15

20

0 2 4 6 8

Months post transplant

glob

ins

[%]

87

E

0.00.20.40.60.81.01.21.41.6

0 2 4 6 8

Months post transplant

hem

oglo

bins

[g/d

L]87

E

E

PATIENT 2PATIENT 2 (specific chain / non- chain)

Comparative globin chain analysis by reverse phase HPLC Patient 1 (transfusion independent since 4.5 years ago) and Patient 2

Integration site (IS) analysis by LM-PCR and DNA pyrosequencingin Patient 2 “MHV” 6 months post-transplantation

CD34

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

lowJAK3*WDHD1ACSL4SPOCK3C19ORF47C4ORF45FBXL11(1)MPRIPTNKS2PFASCARM1CEP290DHX8BST2CIP29*C12ORF64

WBC

lowSIRT3RAB5C*EIF4G3GATAD2BSETD2*CHD6,CHD5*AK090973SAFBNCK1*TNFSF12FLJ45340

VWFHORMAD2NBR1*ARID1A*BC031827PLEKHA3

CD45

lowIQGAP1C6ORF106TXNRD3TAFII105DKFZP686B0790C11ORF49B7ARIH1CEP110*FCHSD2TK1*SAPS3CLN3MAGGUSBL2DHDDSTCF12*

CD3

LHX3GUSBL2IMAA

CD19

lowNIP30NAV3SPOPTBLR1RBM5*RPS6KA1*DNAL1C17ORF26MAP1SMALAT1KIAA0827SUV420H1RAB14FBXL11(2)KIF1BFBXL11(3)

DKFZP434N1217

VAPAGTF2IRD2

CD15

lowNR_002821BTBD11IL6ST*GTF3C2SLC19A2

CD14

lowUNQ747MAP3K3VDPC9ORF86PLEKHA3MIER2CUTL1RHOA*NUP88UBA2,SAE2TXNDC11ATP6V1A

DKFZP434 C2019

Erythro

lowCSNK1G1MEX3CSPATS2PMS2P1HMGA2*PB1CRAMP1LPTRFNHN1ZNF280BSF3A3ZNF318*VPS16JUP

Inse

rtion

site

s (%

of

each

)

218 unique sites

Modified vector BB305 (G. Veres & M. Finer – bluebird bio) for continuation French trial and for US trial

VCN ~ 3• Removal insulator and TAT independent

together with novel purification system

• Higher GMP production titers and >2-3-fold better human CD34+ transduction efficiency

• Greater vector copy number (VCN) per cell(important for β0-thal)

• High βAT87Q globin protein expression even in non-‑thalassemic erythroid cells (e.g., βS/ βS)

• Maintained low genotoxic potential in IVIM assays (coll. C. Baum)

• Vector substitution in French trial planned for next patients

• US trial with new vector filed with RAC (unanimous approval) and with FDA in December

Other University Hospitals of Paris (AP-HP): Saint-Louis, Mondor, CHIC, Tenon, Saint Vincent de PaulEliane Gluckman Françoise Bernaudin Gérard Socié

Hopital Necker (AP-HP), ParisMarina Cavazzana-Calvo (current PI clinical trial) Salima Hacein-Bey Abina

Olivier Hermine Felipe Suarez Antoine RibeilBluebird bio, Inc, Cambridge, MA and bluebird bio - FranceOlivier Negre Gabor Veres Maria DenaroBéatrix Gillet-Legrand Yves Beuzard Kathleen Kirby Robert Kutner Dave Davidson Mitchell Finer

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

Institute of Emerging Diseases and Innovative Therapies – INSERM U. 962 – University Paris 11Bluebird bio – France and Harvard Medical School, Brigham and Women’s Hospital, Boston, MAEmmanuel Payen Olivier Negre Leila Maouche-ChrétienAlisa Tubsuwan Soumeya Abed Robert PawliukKaren Westerman Resy Cavallesco Julian DownKathy Hehir Philippe Leboulch

University of Pennsylvania School of Medicine, Philadelphia, PAFrederick Bushman Gary Wang Troy Brady

Mahidol University and Ramathibodi Hospital, Bangkok, ThailandAlisa Tubsuwan Suradej Hongeng Suthat Fucharoen

German Cancer Research Center (DKFZ), Heidelberg, GermanyAnnette Deichmann Manfred Schmidt Kristof Von Kalle

Terry Fox Laboratory, University of British Columbia, Vancouver, CanadaMelanie Kardel Alice Cheung Connie Eaves