Recent Advances in Severe Combined Immune Deficiency · SEVERE COMBINED IMMUNE DEFICIENCY (SCID): A...
Transcript of Recent Advances in Severe Combined Immune Deficiency · SEVERE COMBINED IMMUNE DEFICIENCY (SCID): A...
Recent Advances in
Severe Combined Immune Deficiency
Luigi D. Notarangelo
Division of Immunology
Children’s Hospital Boston
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