Recent Advances in

Severe Combined Immune Deficiency

Luigi D. Notarangelo

Division of Immunology

Children’s Hospital Boston

luigi.notarangelo@childrens.harvard.edu

Belgian Hematological Society

Liege, January 27-28, 2012

SEVERE COMBINED IMMUNE DEFICIENCY (SCID):

A MEDICAL EMERGENCY

• 1/40,000 – 1/100,000 live borns

• early onset (first months of life)

• severe infections

- interstitital pneumonia

- chronic diarrhea

- candidiasis

- opportunistic pathogens often involved

• failure to thrive

• lymphopenia

• presence of maternal T cells is common and may cause GvHD

• lethal within 2 years of age

but……

can be cured with hematopoietic stem cell transplantation

Immunological heterogeneity of SCID

1) T- B+ NK- SCID

2) T- B+ NK+ SCID

3) T- B- NK- SCID

4) T- B- NK+ SCID

Each of these may be due to various gene defects

HSC CLP

Bp B

T/NKp

NKp

NK

DN DP

CD4 CD4

CD8 CD8

myeloid

progenitor

THYMUS

ADA, PNP

AK2

cell survival

gc, IL7R, JAK3

cytokine-mediated proliferation

RAG1/2, Artemis, DNA-PKcs

Lig4, Cernunnos

CD3d,e,z

CD45

expression of pre-TCR

FOXN1

thymus organogenesis

The phenotypic spectrum

of severe congenital T-cell defects

SCID

• early onset

• opportunistic infections

• respiratory tract infections

• diarrhea

• failure to thrive

• extreme T cell lymphopenia

• variable B cell numbers

• rapidly fatal, unless treated

by HCT

• early onset

• severe infections

• erythrodermia

• lymphadenopathy

• autologous, oligoclonal,activated

tissue-infiltratingT cells

• most often, low B cell numbers

• hypogamma, but high IgE

• autoimmunity

• rapidly fatal, unless treated

by HCT

Omenn syndrome

leaky

SCID

null mutations

in RAG1/RAG2

hypomorphic mutations

in RAG1/RAG2

RAG1

RAG2

HMG1

coding ends

signal ends

I

V(D)J recombination

V J

RSS 7-12-9

RSS 9-23-7

lymphoid-specific

V

J

ubiquitous NHEJ DNA repair machinery

signal joint

TdT

NNN V J

coding joint

Nuclease

III

DNA

PKcs

II

Ku70/80 DNA ligIV

XRCC4

Artemis (hairpin opening)

Cernunnos

V

V

J

J

V

J

The extended spectrum of phenotypes

associated with RAG mutations

Phenotype Age at onset Clinical phenotype T- B- NK+ first months severe infections, FTT

Omenn syndrome first months skin rash, infections, hepatosplenomegaly,

lymphadenopathy, activated/oligoclonal T cells SCID with gd T cells < 1 year infections (CMV), autoimmunity, lymphoma,

expansion of gd T cells

Leaky SCID <2 years infections, autoimmunity, lymphoma,

activated/oligoclonal T cells

CID with granuloma 2 - >10 years granuloma, infections,

autoimmunity, lymphoma

Idiopathic CD4+ ≥5 years recurrent episodes of fever and viral pneumonia

T cell lymphopenia CD4 lymphopenia, polyclonal T cell repertoire,

low TRECs, low KRECs

Can we make a sense of the extended

phenotypic variability associated

with RAG mutations?

• genotype-phenotype correlation • immune dysregulation in patients with hypomorphic mutations

L454Q

ZFA

RIN

G

NB

R

ZFB

HB

R

P86fs32X*

R142X*

T174fs26X*

Q248X

R314W

C328Y R373H

K383FS6X

R396C/H/L*

T403P

M435V

M458fs33X

R474C

R404Q

R507W

W522C*

L541fs30X

R559S R561H*

S626X

L732P

R778Q*

P786L

R841W*

Y912C

R975Q

R394W/Q R778W*

R975W*

Q981P

R699W R699Q

100 a.a Rag1 catalytic core

R410W/Q*

K992E

R764P

N476fs16X C730F*

G516A

S601P

W959X

S401P

A444V

RING: Zinc Finger RING type

ZFA: Zinc Finger A

NBR: Nonamer Binding Region

HBR: Heptamer Binding Region

ZFB: Zinc Finger B

CID-Granuloma

Leaky SCID

SCID with gd T cells

Omenn syndrome

T- B- NK+ SCID

hRAG1

47 mutations

LTR I-hCD4 LTR

pBMN (empty) hRAG1 (wt) mRAG1 (wt)

GFP

LTR LTR hRAG1 I-hCD2

+ mRag2 + NHEJ + STI-571

Rag1-/- tg.bcl2 Abelson

virus

Rag1-/- tg.bcl2 pro-B cell line

GFP

I-hCD4 LTR LTR

hCD4

Rag1-/- tg.bcl2 tg. pro-B cell line

GFP

100

101

102

103

104

0

300

600

900

1200

# C

ells

Granuloma

GFP

AS/LS gd T hRAG1 (S626X) hRAG1 (L454Q) hRAG1 (R474C) hRAG1 (R699W) hRAG1 (R975W)

Omenn T- B- SCID

WT

S626X

L454Q

R474C

R699W

R975W

pBMN (empty) hRAG1 (wt) mRAG1 (wt)

GFP

Genotype-phenotype correlation

in human RAG1 deficiency

p<0.01 p<0.01 p<0.01

p<0.001 p<0.005

p<0.0005

Lymphostromal cross-talk, maturation

of thymic epithelium and T cell fate

K5+K8+

K5+K8 Cld4 UEA-1+

cl IIlo aire

K5 K8+ -

K5+K8 cl IIhi

Cld4+ UEA-1+ aire+

(modified from Takahama, 2006)

K5+ K8 Cld4+ UEA-1

negative selection

†

nTreg

-

-

-

- -

-

receptor

editing

bone marrow periphery

BAFF-

mediated

survival

early immature

B cells

immature

B cells

transitional

B cells

naïve mature

B cells

1st

checkpoint

2nd

checkpoint

V(D)J, kl

B cell dysregulation associated with

leaky defects of V(D)J recombination

Printed array 76 autoantigens

and 6 control proteins

1. Probe array with serum

2. Cy3 α-IgG and Cy5 α -IgM 2nd antibodies

Scan

3. Read fluorescence signal

Hybridized array mfi on 635nm (IgG)

mfi on 570 nm (IgM)

Heatmap Summary of all sera

vs autoantigens

4. Optimize data

Zhen Q.L. JCI 2005

HCT for SCID gives optimal survival

if performed early in life

(Buckley, 2008)

THYMUS BLOOD BM

HSC Tpro

CD4+

CD8+

TREC

= signal joint TREC sjTREC

generated at

DP stage

dilute with

subsequent

divisions in

periphery

incidence (at least 1:100,000)

fatal without treatment

early treatment improves outcome

robust feasible test

reasonable “false positive” rate

Secretary’s Advisory Committee on Heritable Diseases in Newborns and Children, May 2011 report

Screening for SCID using dried blood spots

at birth in United States

Date

started

Births/

year

# screened # SCID Incidence

Wisconsin Jan 2008 69,322 243,707 4 ~1:61,000

Massachusetts* Feb 2009 77,022 194,056 4 ~1:48,000

California* Aug 2010 510,000 500,000 7 ~1:71,000

New York* Sept 2010 236,656 239,454 4 ~1:60,000

Louisiana Oct 2010 65,268 31,464 0 -

Total (incl PR

and Navajo)*

1,005,798 1,208,681 19 ~1:58,000

Summary of newborn screening for SCID to date in US (publicly available data through April 2011)

Secretary’s Advisory Committee on Heritable Diseases in Newborns and Children

•up to date to September 2011, courtesy of Anne Comeau and MA SCID Newborn Screening workgroup;

Fred Lorey, Jennifer Puck, Mort Cowan in CA; Michelle Caggana in NY

Summary of treatment and outcome to date

19 patients identified:

3 PEG-ADA/GT

1 awaiting transplant

15 transplanted:

follow-up 1 month-15 months

14/15 alive, 1 died of VOD

donors: 1 sib, 1 haplo/homozygous sib, 3 mother, 7 URD, 1 UCB

conditioning: 2 none

2 ATG alone

10 myeloablation (bu/cy or bu/flu +/- ATG)

(personal communication SY Pai, M Cowan, TN Small, C Seroogy, J Routes, D Kohn, M Porteus)

SCID Transplant Outcomes

• Mortality - 20-40%

• Inadequate antibody production - 30-60%

• Decline in T cell function?

• GvHD

• Autoimmunity

• Growth and development problems

• Cognitive problems

Gene Therapy for SCID

Gene Therapy for SCID: Rationale

• correct the disease at its roots by inserting one normal copy

of the gene into the patient’s hematopoietic stem cells

• no risks of Graft-versus-Host Disease

• selective advantage expected for gene-corrected cells in

T cell development

• no or little chemotherapy needed

• an alternative to MMRD-HCT for patients who lack

HLA-identical donors

• stem cells readily available (patient’s own cells!)

Insertional mutagenesis and

clonal proliferation (5 out 20) Gene therapy for X-SCID:

Experience in London and Paris

- 20 patients (10 at each site)

- 18 alive

- 17 showing persistent immune

reconstitution as the result of GT

gd

gc

gc

periphery

immature B pre B pro B

bone

marrow

NK

CD4

CD8

pro-NK

pro-T

gd

ab

switched B

mature B

thymus

day 0 day 90 day 120 day 180

1

10

100

1000

10000

Days post gene transfer

MoLV U5 U5 Y++

R SD SA

R P

IL2RG

LTR-driven gammaretroviral vector: MFG gC

MoLV

U5 U5 Y

R R Q

Δ SD

PRE* EFS IL2RG

New gammaretroviral SIN vectors: SRS11

No gag, pol or env residues

Δ

Safer vector designed to reduce insertional mutagenesis

Key modifications compared to Paris/London MFG vector:

• removal of viral LTRs to reduce transactivation of neighboring genes

• removal of all gammaretroviral coding regions

• cellular EF1a promoter to drive transgene expression

• modification of PRE (posttranslational regulatory element)

to enhance expression

• other modifications to improve titer

Knock-in of SIN EFS vector is much less

capable of activating LMO2

100

0 0

20

40

60

80

140

Rel

ativ

e LM

O2

mR

NA

(K

56

2)

LMO2

Actin

#1 #31 #16 #5.2 K562 Jurkat #19 #21 #160 #40 #54 #92 #38 #43 #43

GFP Y

71

61

143

R656 gc Y

6.0 12.4

17.7 18.2

MFG gc gc Y

EF1a

1.9 0.4 0.2

EFS gc cHS4 GFP Y

RRE EF1a

cHS4

0.8 0.7 0.3

R707

Vector

transduction

Primary

recipients

4 mos

analysis

Secondary

recipients

12 mos

analysis

PB and BM, deep sequencing

NO DONOR-DERIVED TUMORS (~100 MICE)

Chris Baum (Hannover MS) Chad Harris (CHB)

Martijn Brugman (Hannover MS)

Clonal dominance assay

Insertion in Evi1

MFG: 8 out of 3621 insertions

SRS: 0 out of 2690 insertions

Gene transfer for SCID-X1 using a self-

inactivating (SIN) gammaretroviral vector

A multi-institutional phase I/II trial evaluating the treatment of

SCID-X1 patients with retrovirus-mediated gene transfer

Sites:

Great Ormond Street Hospital, UK

Hôpital Necker Enfants Malades, France

Children’s Hospital Boston, US

Cincinnati Children’s Hospital Medical Center, US

Mattel Children’s Hospital, Los Angeles, US

eligible if no sib donor,

sick or no matched URD

autologous

BM harvest

infuse

SCIDX1 gene transfer protocol

CD34+ selection

SCF

IL3

TPO

Flt3L

3 rounds of transduction in

retronectin coated bags

Upfront therapy

No conditioning

Observe for safety, reconstitution and clinical outcome

d-4 d-2 d-1 d0

24h 24h 6h

Patient P00001

4 mo old. Diagnosed at birth because of Family History

PMH: thrush, therapy-resistant oral ulcers

ALC: 2450 cells/mL

CD3: 5 cells/mL

CD19: 1866 cells/mL

CD16: 86 cells/mL

IL2RG: Y98C

No HLA-matched related

or unrelated donor

diss. BCG

10 0 10 1 10 2 10 3 10 4

0

20

40

60

80

100

10 0

10 1

10 2

10 3

10 4

0

20

40

60

80

100

Patient Control

gc

Gene therapy at 5.5 months

-60 -30 0 30 60 90 120 150 180 210 240 270 300 330 3600

500

1000

1500

2000

2500

3000

3500

4000

CD3+

CD4+

CD8+

CD16/56+

Gene Therapy

days post GT

cells

/ml

Immune reconstitution after gene therapy

for X-SCID in GT 00001

London & Paris old vector

Boston 00001 new vector

day 0 day 90 day 120 day 180

1

10

100

1000

10000

Days post gene transfer

London and Paris data, courtesy of A. Thrasher, S. Hacein-Bey-Abina

Reconstitution kinetics is comparable to

previous trial

CD

3

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

pre- GT day + 9 0 day + 1 3 5 day + 1 8 7

PHA stimulation index

Polyclonal profile of integration sites

Conclusions

• Mutations on SCID-associated genes may result in a diverse

spectrum of clinical and immunological phenotypes. Besides

interfering with lymphoid development, SCID-associated

mutations may also impinge on immune tolerance.

• Hematopoietic cell transplantation is the mainstay of

treatment for SCID. Optimal results are obtained if

the transplant is done early in life. This goal can now be

achieved through newborn screening.

• Novel and safer vectors for gene therapy are being developed

to correct SCID, with promising results. However, long-term

follow-up studies are needed.

Javier Chinen

Silvia Giliani

Mort Cowan

Clinicians around the world

Jack Bleesing

Catharina Schuetz

Srdjan Pasic

Andy Gennery

Waleed Al-Herz

Gehad ElGhazali

Roshini Abraham

Fred Alt

IDI, Boston

Necil Kutukculer

Ghassan Dbaibo

Chaim Roifman

Taco W. Kuijpers

Steve Holland

Jennifer Puck

Sung-Yun Pai

David A. Williams

Children’s Hospital, Boston

Frederic Bushman

UPenn, Philadelphia

Chris Baum

Hannover Medical School

Adrian Thrasher

Bobby Gaspar

Institute of Child’s Health, London

Don Kohn

Lisa Filipovich

Alain Fischer

Marina Cavazzana-Calvo

Anne Comeau

MA Newborn Screening