Baboon envelope pseudotyped lentiviral vectors: a highly efficient new tool to genetically manipulate T-cell acute lymphoblastic leukaemia-initiating cells.

Caroline Costa1+, Guillaume Hypolite2+, Ornellie Bernadin1+, Camille Lévy1, Francois-Loïc Cosset1, Vahid Asnafi2, Elizabeth Macintyre2, Els Verhoeyen1,3 and Melania Tesio2

SUPPLEMENTAL DATA

Supplementary figure 1

Supplementary figure 2

Supplementary figure 3

Supplementary figure 4

Supplementary figure 5

Supplementary figure 6

Supplementary table 1

Supplementary table 2

Supplementary table 3

Supplementary table 4

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A. LDL-R, CD150, CD46, ASCT1 and ASCT2 mRNA levels in primary T-ALL cells. Results are expressed as fold change compared to CD7 levels, set as 1 (dotted line).

B-C. Representative FACS plots showing LDLR, CD150 and CD46 expression on CD45+CD7+ blasts (A) and on LIC-enriched CD34+ blasts (B) from a patient-derived sample (UPN763).

D. ASCT1 and ASCT2 expression in LIC-enriched CD34+ blasts in basal conditions (not stimulated) or following 3 days of culture on OP9-DL1 cells in the presence of cytokines and insulin. Data are expressed as fold change to ASCT1 and ASCT2 mRNA levels detected in unstimulated healthy T-cells, set here to 1. * p0.05 (one-tailed Student t test)

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ASCT1 and ASCT2 expression in patients-derived T-ALL cells according to the following different oncogenic abnormalities: NOTCH1/FBXW7 activating mutations (N/F mut) (A-D), PTEN deletions/mutations (PTEN altered) (B-E), CALM-AF10 fusion transcript (CA), SIL-TAL1 fusion transcript (S-T) and TLX1/3 overexpression (TLX1/3) (C-F). Data are expressed as fold change to ASCT1 and ASCT2 mRNA levels detected in unstimulated healthy T-cells, set here to 1.

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GFP expression analysed 4 days post-transduction on leukemic CD45+CD7+ blasts (UPN149, UPN105, UPN489, UPN735, UPN525) transduced with VSVG, HF or BaEV-pseudotyped lentiviral vectors using a range of MOIs (1, 5, 10 and 20). * p0.05, *** p0.001 (one-tailed Student t test).

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A-B. Percentage of GFP expression (A) and GFP mean fluorescent intensity (B) evaluated two and six days post-transduction (UPN633, UPN613).

C-D. Percentage GFP expression (C) and vector copy numbers (D) in blasts (UPN633, UPN613) transduced with increasing MOIs.

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Competitive xenografts. Experimental procedure (A) and vector copy numbers observed in bone marrow cells issued from secondary transplants (B).

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Experimental procedure followed during non-competitive xenografts (A) and during limiting dilutions assays (B).

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Supplementary Table 1

Oncogenic abnormalities and maturation arrest stage of primary T-ALL blasts analysed during in vitro experiments

Patient Origina Maturationarrest stage

Oncogenicabnormalities

UPN525 Xenograft IMG RAS mutationUPN534 Xenograft Pre- PTEN deletionUPN584 Xenograft TCR FBXW7 mutation

SIL-TAL1 fusion transcript

UPN727 Xenograft TCR NOTCH1 mutationFBXW7 mutation

UPN149 Xenograft TCR TLX3 overexpressionNOTCH1/FBXW7

mutation

UPN656 Xenograft TCR NOTCH1 mutation

TLX3 overexpressionPTEN mutation

UPN633 Xenograft TCR PTEN mutationUPN613 Xenograft TCR NOTCH1 mutation

UPN727 Patient-derived

Pre-FBXW7 mutation

PTEN deletionTLX1 overexpression

UPN763 Patient-derived

IMD RAS mutation

a Xenograft means patient-derived T-ALL cells amplified in NSG mice (1 to 2 passages) and patient-derived means T-ALL cells transduced upon direct isolation from patient’s samples.

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Supplementary table 2. Competitive xenografts into NSG mice.

Engraftment of transduced and non-transduced clones into two serial cohorts of NGS mice.

PRIMARY

T-ALL

MATURATIONARREST STAGE &

ONCOGENIC ABNORMALITIES

GFP EXPRESSION IN CD45+CD7+ BLASTS

INJECTED BLASTS

XENOGRAFTED MICE

UPN525IMG

RAS mutation82.5 %

Primary Secondary

Blood: 73.6 ± 6.3 (n=2)

87.2 ± 0.3 (n=5)

Bone marrow: 76.9 ± 2.65 (n=2)

79.4 ± 1.9 (n=5)

Spleen: 73.5 ± 2.75 (n=2)

83.6 ± 2.4 (n=5)

UPN534

Pre-

PTEN deletion49.3 %

Blood: 41.8 (n=1) 44.7 ± 4.2 (n=3)

Bone marrow: 42.9 (n=1) 45.6 ± 4.3 (n=3)

Spleen: 43.2 (n=1) 46.3 ± 1.9 (n=3)

UPN584

TCR

FBXW7 mutationSIL-TAL1 fusion

transcript

59.4%

Blood: 63.0 ± 2.4 (n=2)

61.6 ± 5.7 (n=4)

Bone marrow: 60.7 ± 6.1 (n=2)

55.5 ± 2.7 (n=4)

Spleen: 55.4 ± 1.26 (n=2)

39 ± 9.6 (n=4)10

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Supplementary table 3. Non competitive xenografts

Summary of non competitive xenograft experiments showing the engraftment of sorted GFP negative and GFP positive blasts as well as the percentage of GFP expression in two serial cohorts of NSG mice.

T-ALLMATURATION

ARREST STAGE &

ONCOGENIC ABNORMALITI

ES

PRIMARYXENOGRAFTS

SECONDARYXENOGRAFTS

UPN525

IMG

RAS mutationBlood:

Bone marrow:

Spleen:

Engraftment GFP-

blasts

51 ± 1.3 (n=3)

80.9 ± 2.8 (n=3)

41.4 ± 2.7 (n=3)

Engraftment

GFP+ blasts

45.9 ± 2.3 (n=3)

69.2 ± 7.4 (n=3)

58.9 ± 0.2 (n=3)

% GFPexpressio

n

99.5 ± 0.15

(n=3)

98.8 ± 0.26

(n=3)

99.5 ± 0.05

(n=3)

Engraftment GFP-

blasts

54.3 ± 3.4 (n=3)

82.9 ± 4.8 (n=3)

88.4± 0.6 (n=3)

Engraftment

GFP+ blasts

60.4 ± 3.9 (n=3)

91.9 ± 6.6 (n=3)

90.6 ± 0.7 (n=3)

% GFPexpressio

n

99 ± 0.5 (n=3)

97 ± 0.9 (n=3)

96.5 ± 0.35

(n=3)

Blood: 31.2 ± 5.7 51 ± 17.5 99 ± 1.8 41.5 ± 3.4 50.5 ± 3.0 98.4 ± 0.2

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UPN534

Pre-

PTEN deletionBone

marrow:

Spleen:

(n=2)

77.5 ± 0.15

(n=2)

81.7 ± 5.7 (n=2)

(n=2)

82.3 ± 2.75

(n=2)

85.8 ± 1.7 (n=2)

(n=2)

89.9 ± 0.32

(n=2)

98.7 ± 0.08

(n=2)

(n=3)

89.1 ± 3.3 (n=3)

87.1 ± 1.9 (n=3)

(n=4)

94.2 ± 2.1 (n=4)

86.3± 1.2 (n=4)

(n=4)

96.9 ± 0.2 (n=4)

97.9 ± 0.1 (n=4)

UPN584

TCR

FBXW7 mutation,

SIL-TAL1 fusion transcript

Blood:

Bone marrow:

Spleen:

41.9 ± 1.4 (n=2)

87.8 ± 0.9 (n=2)

63.7 ± 3.3 (n=2)

45.4 ± 2.3 (n=2)

85.5 ± 1.9 (n=2)

59.8 ± 1.4 (n=2)

95 ± 1.3(n=2)

96.9 ± 0.95

(n=2)

97.8 ± 0.9 (n=2)

83.7 ± 1.6 (n=2)

96.5 ± 2.1 (n=2)

86.5 ± 1.3 (n=2)

81 ± 1.5 (n=2)

93 ± 1.7(n=2)

88.8 ± 1.1 (n=2)

98 ± 0.8 (n=2)

99 ± 2.1 (n=2)

98 ± 0.5 (n=2)

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Supplementary table 4. Limiting dilution assay

Cell dose Blasts Number of

injected mice

Number of

engrafted mice

Time to death

(days)

5 x10^5 GFP- 3 3/3 33, 34, 37

GFP+ 3 3/3 35, 37, 38

5 x10^4 GFP- 6 6/6 37,39,44,46,50,62

GFP+ 6 6/6 38, 39, 39, 43, 46,68

1 x10^4 GFP- 8 7/8 40, 43, 47, 52, 53, 61, 68

GFP+ 6 5/6 40, 42, 58, 61, 78

1 x10^3 GFP- 5 2/5 70, 82

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GFP+ 5 3/5 58, 60,110

1 x10^2 GFP- 5 0/5 n/a

GFP+ 5 0/5 n/a

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