Odile Cohen-Haguenauer, M.D., Ph.D.

New insulated lentivectors mediating
efficient & safe transduction of HSCs
& Directed integration to the
heterochromatin
Odile Cohen-Haguenauer, ENSC
Odile Cohen-Haguenauer
RAC-CliniGene Symposium Dec 2010

Part 1. Design & Evaluation:
Odile Cohen-Haguenauer, ENSC
Insulated gammaretro &
lentiviral vectors

Odile Cohen-Haguenauer, ENSC
Objectives and Rationale
• Objective:
– Preventing insertional mutagenesis related SAEs
• Rationale: GIEs as cis-acting sequences
– Enhancer-blocking activity
– Preventing transgene silencing: Boundary
• Custom design: new GIEs
Small streches fitting RVVs bio
Multimers of core sequences
• Insulator 1: 268 bp
• Insulator 2: 101 bp

100,00%
90,00%
80,00%
70,00%
60,00%
50,00%
40,00%
30,00%
20,00%
10,00%
0,00%
RSV
Efficient, safe & stable Lentivectors
U3 R U5
Follow-up % GFP+: HeLa MOI 5
0 10 20 30 40 50 60 70 80 90
Time (days)
cPPT
Odile Cohen-Haguenauer, ENSC
Pro
Test 1
Ref 1
DCaro4.23
DCaro6.14
DCaro8.22
12weeks: stable GFP+ cells with Insulator 2
eGFP
3
2,5
2
1,5
1
0,5
0
Lentiviral vector 4xIns2 DCaro4.23
R
∆ U3-INSUL
VCN 4.23 VCN Test1 VCN Ref1
2 4 6 11 12
Weeks
VCN: stable with 4xIns2
hPoly A
140%
120%
100%
80%
60%
40%
20%
0%
% GFP+ cells

4xIns2 lentis expression in CD34+cells
Stable over time >12wks:
both phenotype and
genetically
Independent of MOI
Cell-lines
Primary cells
Intensity and specificity
depend on promotor
• According to cell
characteristics
• Different internal promotors
e.g.: CD4 specific: 1200 bp
Odile Cohen-Haguenauer, ENSC
Expression in Cord Blood CD34+ does not vary with MOIs
12 weeks follow-up
in vitro
0,91%
GlyA-APC
GFP
0,41%
36,89%
CD34+
Dapi
1400
1200
1000
800
600
400
200
Follow-up Mean GFP: human CB-HSCs MOI 50
0
0 5 10 15 20
Time (days)
25 30 35 40
1400
1200
1000
800
600
400
200
0
MOI 50 5
Follow-up Mean GFP: human CB-HSC MOI 5
0 5 10 15 20 25 30 35
Time (days)
Test 1
Ref 1
DCaro4.23
DCarohP4
Test 1
Ref 1
DCaro4.23
DCarohP4

100%
10%
1%
70,00%
60,00%
50,00%
40,00%
30,00%
20,00%
10,00%
0,00%
In vivo reconstitution of humanised NOG mice
% GFP+ cells over 24 weeks
Evolution of CD45+/GFP+ cells
Mouse 7 Mouse 8 Mouse 9 Mouse 10 Mouse 11 Mouse 12
0 5 10 15 20 25
Time (weeks)
Evolution of CD45+ cells
Mouse 7 Mouse 8 Mouse 9 Mouse 10 Mouse 11 Mouse 12
0 5 10 15 20 25
Time (weeks)
Odile Cohen-Haguenauer, ENSC
(collaboration with D. Klatzmann)
Dcaro 4xIns2
Control
24 weeks
Follow-up
CD45+
CD45+CD34
+
In vivo genetic stability of insulator
in hu-HSCs : BM samples week 24
100,00%
10,00%
1,00%
Mouse 7 Mouse 8 Mouse 9 Mouse 10 Mouse 11 Mouse 12
0 5 10 15 20 25
Time (weeks)

In vitro test:
- on 293T
- on Jurkat
-MOI: 5
-3 days after
infection
hCD4 specific promoter in hu-CD4+ cells:
293T
Jurkat
Odile Cohen-Haguenauer, ENSC
-
160
140
120
100
80
60
40
20
0
ProT4-eGFP-Ins
ProT4-eGFP
Expression ratio between Jurkat and 293T
cells
Insulator No Insulator
GFP GFP
GFP

Evaluation of potential genotoxicity
• In vitro: high-throughput IS analysis:
collaboration with Christof von Kalle & Manfred Schmidt:
– Evolution of integration patterns ingenuity in
human cells sequentially sampled over 3 months
• no shift in IS distribution
• no common integration site emerging
– Ongoing studies: in hu primary CD34+ cells
• In vivo: cancer-prone mice ongoing studies
collaboration with Eugenio Montini
Odile Cohen-Haguenauer, ENSC

In vitro genotoxicity studies: design
Odile Cohen-Haguenauer, ENSC

Overview of IS data on pSIN-18, pLENP29 and 4xIns2
week
Samples were analyzed by nrLAM- and LAM-PCR (MseI and Tsp509I). IS: Integration Site;
SC: Sequence Count
All 3 vectors show a substantial decrease in the number of IS after 9 weeks:
with DCaro4.23 less than with two others while % transduced cells idem
Odile Cohen-Haguenauer, ENSC
DCaro4.23 Test1 Ref1
IS SC
IS (SC
adjusted
to 1000)
IS SC
IS (SC
adjusted
to 1000)
IS SC
IS (SC
adjusted
to 1000)
1 544 860 633 546 2253 242 997 1592 626
2 512 955 536 626 2406 260 925 1633 566
5 320 580 552 377 2620 144 463 1251 370
6 339 853 397 427 2800 153 301 1321 228
9 92 514 179 121 2001 60 70 1414 50
11 92 1241 74 109 1457 75 39 1701 23
12 74 859 86 54 1249 43 35 1805 19
Total unique
(ex.
mappable)
1736
(1570)
1927
(1734)
2624
(2344)

Number of Unique Integration Sites
Integration Sites [%]
1000
14
12
10
8
6
4
2
0
100
10
1w 2w 5w 6w 9w 11w 12w
Odile Cohen-Haguenauer, ENSC
Number of Unique Integration Sites Over Time
(Sequence Count adjusted to 1000)
Timepoint
Chromosomal Distribution
No shift in chromosomal distribution
DCaro4.23
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
Chromosomes
DCaro4.23
Test1
Ref1
Test1
Ref1

4xIns2: less Identical Integration Sites Over Time
% identical IS
20
15
10
5
0
IdenticalIS found over time
Significantly more identical IS over time were found in Test1 compared
to DCaro4.23 and Ref1, respectively
No identical IS were found between between 1w and any later
timepoint with DCaro4.23; to a lesser extent with Ref1
Significantly more identical IS were found between 1w and any later
timepoint in Test1 compared to DCaro4.23 and Ref1, respectively
Odile Cohen-Haguenauer, ENSC
p = 3.0E-05 p = 3.2E-11
DCaro4.23 Test1 Ref1
Number of identical IS
20
15
10
5
0
IdenticalIS found
between 1w and any later timepoint
p = 2.2E-04 p = 1.3E-04
DCaro4.23 Test1 Ref1
p- value was determined by Chi square test

Integration Sites [%]
10
8
6
4
2
0
10-5 kb
upstream
5-0 kb
upstream
TSS
Upstream [kb]
0-10% in
Gene
10-20% in
Gene
20-30% in
Gene
Odile Cohen-Haguenauer, ENSC
Similar distribution: in gene and upstream
Distribution in Gene and Upstream
30-40% in
Gene
40-50% in
Gene
50-60% in
Gene
% in Gene
60-70% in
Gene
Similar distribution
in gene and
in gene ± 10 kb
70-80% in
Gene
DCaro4.23
Test1
Ref1
80-90% in
Gene
Integration Sites [%]
100
90
80
70
60
50
40
90-100%
in Gene
62,10
Distribution In Gene and +/- 10kb
64,71
62,46
68,47
69,95
In Gene In Gene +/-10 kb
DCaro4.23
Test1
Ref1
67,28

Systemic vector injection into Cdkn2a -/- mice
Vector tested by temporal vein injection in newborn Cdkn2a-/- mice
LV.SF.LTR GFP PRE
LV.SF.LTR
MOI 100
p

-18
Systemic vector injection into Cdkn2a -/- mice
SFFV GFP PRE SFFV
p vs Mock
cPPT
SD SA
-18
SIN.LV.SF.GFP (1*10 8 TU/m)
SIN
cPPT
SD SA
SFFV GFP PRE
p < 0.0001 p = 0.0095
p = ns
Odile Cohen-Haguenauer, ENSC
So far: no difference with mock
SIN
-18
4xIns2.SIN.LV.SF.GFP (2*10 8 TU/m)
4x
Ins2
cPPT
SIN
SD SA
SFFV
GFP
PRE
4x
Ins2
SIN

Full phenotypic correction of FA-cells
With insulated vectors shuttling FANCA-cDNA: 2 different promoters
40
35
30
25
20
15
10
Odile Cohen-Haguenauer, ENSC
5
0
MMC-induced G2 block totally suppressed
in FANCA-vector GM-cells expressing FancA
from U3-promoter; lesser extent from hPGK
WT FA-A FA-A U3-
FancA
- MMC +MMC 100nM
FA-A hP-
FancA
FA-A U3-
GFP
FA-A hP-
GFP
Phenotypic
correction
of FA phenotype
after a 2 weeks
chronic treatment
with 10 mM MMC

Conclusion 1: Design and evaluation
of high titres optimised insulated vectors
• High titers produced with Ins2 contructs (not Ins1)
• Nb of Ins2 repeats greatly impacts on vector titres &
transgene expression: optimal is 4x
• Homogeneous expression profile with Ins2 vectors
– Strong boundary effect with Ins2 (not Ins1)
– Stable transgene expression with Ins2: internal promoter
strength determines expression level; inducibility retained
• High efficiency & stability of 4xIns2-lenti in hu-HSCs
– Both in vitro (12 weeks) and in vivo (over 6 months)
• No genotox evidenced so far
– In vitro: High-throughput IS profile
– In vivo: cancer-prone mice model
Odile Cohen-Haguenauer, ENSC

Odile Cohen-Haguenauer, ENSC
Part 2: Targeting integration
of insulated lentiviral vectors to
the heterochromatin

Chimeric integrases: study design
Two
lentivectors
Test1:
non-insulated
control
DCaro4:
Ins2 insulated
Odile Cohen-Haguenauer, ENSC
ML 3
ML 6
ML 10
ML 21
4p
Control
∂-2x
pack
wild type integrase with control for
flag impact on integrase activity
Histone H3 mediated targeting of
heterochromatin
Methylated CpG islands targeting of
heterochromatin
ML6 negative control : mutation in
targeting domain
Basic 4 plasmids system
with native integrase
Chimeric integrases complemented
with double integrase mutant

100,00%
90,00%
80,00%
70,00%
60,00%
50,00%
40,00%
30,00%
20,00%
10,00%
Chimeric integrases: 12 weeks follow-up HeLa
Evolution of % GFP+ cells : Test 1 (control)
0,00%
0 10 20 30 40 50 60 70 80 90
Odile Cohen-Haguenauer, ENSC
Control 4 plasmides
Control Pack muté
ML 3
ML 6
ML 10
ML 21
100,00%
90,00%
80,00%
70,00%
60,00%
50,00%
40,00%
30,00%
20,00%
10,00%
0,00%
Evolution of % GFP+ cells : DCaro4.23 (Ins2)
0 20 40 60 80 100
Control 4 plasmides
Control Pack muté
ML 3
ML 6
ML 10
ML 21

4p
∂Pack
ML 3
Chimeric integrases wk8: expression with Ins2 vector
Controls Chimeras
Test 1 DCaro4.23 Test 1 DCaro4.23
Odile Cohen-Haguenauer, ENSC
ML 6
ML 10
ML 21
Inactivating mutation

4p
∂Pack
ML 3
Chimeric integrases 10 5 HeLa cells: week 12
Test 1 DCaro4.23 Test 1 DCaro4.2
3
0,55%
34,30%
Odile Cohen-Haguenauer, ENSC
99,34% 93,57%
0,41%
78,79%
ML 6
ML 10
ML 21
4,69%
19,77%
4,53% 36,99%
0,86% 0,31%

• Sustained expression of insulated vector only
• Buffered expression: decreased vs control
• Currently ongoing:
– Integration sites analysis
– huCB-CD34+ cells
• HDACI or proliferation
– e.g. Valproic Acid
– Cytokines
• Is it at the expense of efficacy ?
– In vivo reconstitution in SCIDs mice
– Improved production & scale-up: collaboration with IBET
Odile Cohen-Haguenauer, ENSC
Conclusion 2: Preliminary
data on heterochromatin targeting

ENSC/LBPA Pasteur Institute
Alexandre Artus Marc Lavigne
Caroline Duros Yaïr Botbol
Yaïr Botbol
Emilie Bayart
Aurore Malric UNIL Lausanne
Odile Cohen-Haguenauer Armelle Gaussin
Nic Mermod
DKFZ
Simone Scholtz IBET
Manfred Schmidt Ana-Sofia Corhoadina
Christof von Kalle Pedro Cruz
Paula Alves
APHP-INSERM Manuel Carrondo
Isabelle Ragon
Sofien Dessolin HCSR-TIGET
David Klatzmann Daniela Cesana
Eugenio Montini
EC FP6 LSHB CT-2006-018933
Odile Cohen-Haguenauer, ENSC