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