Gene Transfer and Insertional Mutagenesis
Molecular and Gene Therapy ProgramDivision of Experimental HematologyChildren‘s Hospital Research FoundationCincinnati
Medizinische Universitätsklinik und Poliklinik, Abt. Innere Medizin I
INSTITUT FÜR MOLEKULARE MEDIZIN UND ZELLFORSCHUNG
ALBERT-LUDWIGS-UNIVERSITÄT FREIBURG
Risk/Benefit Assessment of Clinical ApplicationsFor Insertional Vector Systems
As Pharmaceutical Agents
Copies/Cell 0.01 0.1 1 > 10
ModifiedStem Cells 1 100 1000 > 106
TherapeuticEfficacy
Therapeutic WindowPotential
Side Effects
1995 1998 2000 2002
Insertion Site Analysis: Dose Finding in Gene Therapy
• Stem Cell Clonality
• Insertion Site Distribution Analysis
• Cell Type Dependence of Insertion
• Genomic Side Effects of Insertion
• Active Modification of Insertion Behavior
Integration Site Analysis by LAM-PCR
1 18 84
5M 4 H2OH2O5
HeLa Clones:10ngtransd.+1µg00
LC1LTR2
GenomicDNA Proviral DNA GenomicDNA5` 3`
n-Beads
BB
B
LTR1Avidi
RB
BLC
DenaturationSchmidt et al., Hum Gen Ther 2001Schmidt et al., Blood 2002Ailles et al., Hum Gen Ther 2002Woods et al., Blood 2003United States Patent No. 06514706 B1
SCID-X1 Gene Therapy (A. Fischer et al., A. Thrasher et al.)
In vivo Clonality Analysis
Gaspar et al., Lancet in press, Schmidt M et al., Blood, in press
LymphoidProgenitor
MyeloidProgenitor
Hematopoietic Stem Cell
Monocyte/ Macrophage
Platelet
Plasma Cell
T-Lymphocyte
B-Lymphocyte
Erythrocyte
Megakaryocyte
Granulocyte
PluripotentStem Cell
Line
age
Spe
cific
Pro
geni
tors
Non-hematopoieticTissues
√
?? ?
√?
ADA Gene TherapyIn Vivo Clonality Analysis
Patient 2
+80+48 +72+64
+63+53+48 *
+80 +94
+9+1 +32
+49+48 *
+43+28 +48 *
+88+32 +49
Patient 1
Pat .1 + C
Pat.1+ C
Pat.1
C1 C1
C1
PBMC CD 3+ CD 13/14 PBMCSchmidt M et al., Nat Med 2003
The unmodified retroviral VectorLNGFRFused into Evi-1Exon 1,Inducing Constitutive Expression of theProleukemicTranscription Factor
Li et. al, Science 2002
Development of Monoclonality in Human HematopoiesisA
10ng WT
50c. IS10ng WT
500c. IS
CD3 Tγδ PBL
3‘LTR
P4
IS
PBL
6 3113 2417 34 -C 37 M
10ng WT
50c. IS10ng WT
500c. IS
CD3 PBL
3‘LTR
3113 34 M-C 37
IS
PBL
P5
B
******* * *
198 pbs169 pbs
C1 2 3 4 5 6MFG γc
44,218 46,229 54,614 66,467 66,867 71,646 76,883
P5 P4
MFG γc
41,253
Hacein-Bey, Kalle, Schmidt et al., Science 2003
Co-Transcription by RNA FISH
LAM High Throughput Data Pipeline
1
91
Time
Clo
ne 24w16w12w8w4w1w0w-4w
B) RQ2297
1
94
A) RQ2314
-4w 0w 1w 2w 4w 6w 8w 12w 16w 20w 32w
#1212
#1247
#1304
#1318
#1337#1315#1340
Busulfan Conditioning ImpactOn Stem Cell Clone Numbers
RQ2314 RQ2297
Neo
Β -actin
-4w
0 w
1 w
2 w
4
w8
w
10 w
12
w
16 w
20 w
H2O
(-)
200
%20
%2
%0.
2 %
0.02
%
H2O
(-) 20
0 %
20 %
2 %
0.2
%0.
02 %
-4w 0 w
1 w
4 w
8 w
12
w
16 w
G-CSF: Kuramoto et al., Blood 2004;Bu: Kuramoto et al., Blood 2004
Analysis of Retroviral Integration Sites (IS)
23%58IS within +/- 5 kb around transcription start site
41%104IS more than 10 kb away from RefSeq genes
59%148
IS in RefSeq genes including the region 10 kb upstream and downstream of the gene
39%99IS in RefSeq genes
344Total number of integration sites (IS)
Percentage Absolute
Post-Transplantation
Exactly mappable IS 100%252
LAM Characterization of Post-Transplantation Hematopoiesis
M 1 2 3 4 5 C MP4
IC
37m post T.
30ng CD3+ T cells6m post T.
10ng CD3+ T cells
41m post T.
10ng PBL24m post T.
10ng CD3+ T cells
1 2 3 4 5 6 7 8 9 101112
13 14 1516 1718 19 20 21 22 X Y
Chromosomal distribution of posttransplantation RISs
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
chromosome
freq
uenc
y
X Y
Insertions around Transcription Start Sites
0%
1%
2%
3%
4%
5%
6%
7%
8%
-10 to -9
-9 to -8
-8 to -7
-7 to -6
-6 to -5
-5 to -4
-4 to -3
-3 to -2
-2 to -1
-1 to 0
0 to 1
1 to 2
2 to 3
3 to 4
4 to 5
5 to 6
6 to 7
7 to 8
8 to 9
9 to 10
Inte
grat
ions
in %
TSS
Distance from Transcription Start Site TSS (kb)
Gene Ontology Analysis (NCBI)
Level Category List Hits Fisher Exact
2 Kinase activity 14 0.000534
2 Transferase activity 19 0.00173
3 Protein kinase activity 10 0.00403
4Phosphotransferase Activity 12 0.00134
4Protein-tyrosine kinase Activity 5 0.00632
Molecular Function
Total number of RefSeq genes in the list: 136
Insertional Activation – Relevant Parameters to Look for In Vivo
• Insertion Correlation to Expression Pattern
• Regulatory Insertions Should Prevail over Disruptive Insertions
• Increase of Contribution over Time
Vector-induced LMO2 activation
• Our data suggest: - Enhancer effect by the 3’LTR.- Promoter competition for the 5′LTR enhancer
• Position within the LMO2 locus may affect activation.
• Negative regulatory elements upstream of the LMO2 promoter need to be overcome. (Precedence: Differential interactions betweenpromoters of adult and fetal b-globin locus)Townes and Behringer (1990), Crossley and Orkin (1993)
• Hypothesis: A “competent” transcriptional complex at the 5’LTR may be stabilized by components of the RNA processing machinery.
LMO2 Promoter Activation Test ConstructsPatient 5
luciferase-3190
gC
3’ LTR5’ LTR
+494
Patient 4
-3190 +494
luciferaseHd Hd
+5300
Ex2
gC
5’ LTR3’ LTR
IVS1
3’LTR Activates the LMO2 Promoter
100 200 300
Activation in Jurkat cell assay
LUC-3190LMO
LUC3’LTR
gC-3190LMO
-3190LMO3’LTRgC LUC-
+
+
Relative Activity
LTR
gC
5’LTR5’LTR 3’
Activation of the LMO2 promoter by the 5’LTR appears to be modulated by vector elements
LUC-3190LMO
5’LTR
gC-3190LMO
LUC
Activation in Jurkat cell assay
5’LTR -3190LMOLUC
5’LTR
100Relative Activity
200 300
+
-3190LMOLUC
-
5’LTR-
LUCgC
-3190LMO
-
+
SIN Vector Design Prevents LMO2 Promoter Activation
Ex2
-3190luc
+5300
γC+1572
-3190luc
+5300
Ex2γC
+1572
Ex2
-3190luc
+5300
γC+3277
-3190 +424luc
+5300
Ex2IVS
0 100 200 300 400
Ex2
-3190luc
+5300+1572
Ex2γC
-3190luc
+5300+1572
γC
Relative Luciferase Activity
Conclusions
• Preferred integration of MLV-based vectors within or close to genes:
59% within or close to genes,39% within the gene itself, ~50% of these within in the first 20% of the gene
• Insertion peaks around the start of transcription in a narrow segment (+/- 4 kb)
• Clustering in Common Insertion Sites
• Differences before and after engraftment
• Differences between trials
Insertion Site AnalysisAdvancing to the Next Level
• Stem Cell Clonality – Novel Data Quality
• Insertion Site Distribution Analysis- Cell Type Dependence of Insertion- Gene Expression Dependence of Insertion- Genomic Side Effects of Insertion
• Active Modification of Insertion Behavior
Freiburg UniversityManfred SchmidtHanno GlimmManuela WisslerClaudia PrinzArne MüßigNina LemkeCarsten SpeckmannSebastian HaasPhilipp ZicklerGesa HoffmannBirgitta VonderstrassSilke Klingenberg
NHLBI, NIH, BethesdaCynthia E. DunbarHyeoung Joon KimTong Wu HyeounStephanie Sellers
Great Ormond Hospital, LondonAdrian Thrasher
Sidney, AustraliaIan Alexander
NIDDK, NIH, BethesdaJohn Tisdale
Children's Hospital, L.A.Donald B. KohnDenise Carbonaro
FHCRC, SeattleHans-Peter KiemTobias NeffPeter HornRobert G. Andrews
COLLABORATORSHôpital Necker, ParisAlain FischerMarina Cavazzana-CalvoSalima Hacein-Bey
Cincinnati Children‘s HospitalDavid A. WilliamsChristopher BaumPatrick KellyDao PanKathleen AndersonGerlinde Layh-SchmittCharles KlankeChristopher LutzKim BohnDavid Kuhel