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PRECLINICAL LENTIVIRAL RAG1 GENE THERAPY
Supplemental Data
Supplemental Materials and methods
Vector construction and lentiviral production
Lentiviral gene transfer constructs were derived from pRRL.PPT.PGK.GFPpre(1). The
pRRL.PPT.EFS.RAG1.pre (hereafter: EFS.RAG1), pRRL.PPT.SFFV.RAG1.pre (hereafter:
SFFV.RAG1),
pRRL.PPT.EFS.coRAG1.pre
(hereafter:
EFS.coRAG1),
pRRL.PPT.SFFV.coRAG1.pre (hereafter: SFFV.coRAG1) and pRRL.PPT.UCOE.coRAG1.pre
(hereafter: UCOE.coRAG1) transfer vectors were constructed by replacing the PGK promoter by
a human EFS ( S for short form) promoter, an enhancer–promoter from the spleen focus-forming
virus (SFFV) or a ubiquitously acting chromatin opening element (A2UCOE). The GFP sequence
was replaced by native human RAG1 cDNA or the codon-optimized version of RAG1 (coRAG1).
Codon optimization of the human RAG1 gene was performed by Geneart (Regensburg,
Germany). The WPRE used in these constructs is lacking the X protein ORF and 4 possible
ATGs were mutated(2). Gene transfer constructs were validated by full DNA sequencing of the
transgene. For Supplemental figures 2 and 3 the gene transfer vector was constructed as follows:
The backbone of replication-defective, self-inactivating Trip-ΔU3-EF1α-RAG1 lentiviral vector
has been previously described.(3, 4) The RAG1-containing construct were generated using
BamH1 (New England Biolabs, Ozyme, Saint Quentin Yveline, France) ligation of the coding
sequence of native human RAG1 cDNA. Large-scale helper-plasmid preparations were produced
by PlasmidFactory (Bielefeld, Germany). 293T cells were transiently transfected with transfer
and helper plasmids using the calcium-phosphate method or FuGENE 6 transfection reagent
(Roche, Basel, Switserland). Lentivirus was harvested 24h, 32h and 48h after transfection.
Cleared supernatant was filtered through 0.22 µm pore cellulose acetate filters and stored at –
80°C. Pooled lentiviral supernatant was concentrated by ultracentrifugation for 16 hours at
18,000 x g and 4°C. Pellets were resuspended in StemSpan serum-free expansion medium
(StemSpan-SFEM, Stemcell Technologies Inc, Vancouver, BC, Canada). Because suitable antiRAG1 antibodies were not available, we determined the viral titer using RQ-PCR as described
below.
RQ-PCR
Total RNA from viral supernatant and cells was extracted using the RNeasy Total RNA isolation
kit (Qiagen, Hilden, Germany). RNA was reverse transcribed into cDNA. Genomic DNA was
extracted using the GeneElute Mammalian Genomic DNA miniprep kit (Sigma-Aldrich, Saint
Louis, MO, USA). RQ-PCR reactions were performed on the ABI Prism 7700 Sequence Detector
System (Applied Biosystems, Foster City, CA). The following primers and probes were used.
WPRE:
forward primer, 5’-CCGTTGTCCGTCAACGTG-3’;
reverse primer, 5’AGTTGACAGGTGGTGGCAAT-3’; probe, 5’-FAM-TGCTGACGCAACCCCCACTGGCTAMRA-3’. RAG1: forward primer, 5’- GCCAAACCTAACTCTGAACTGTGTT-3’; reverse
primer, 5’- GCGTCTCGTGGTCAGACTCA-3’; probe, 5’-FAM-CCATTGTGCCTTATGCTTAMRA-3’. coRAG1: forward primer, 5’-CAACTGCAAGCACGTGTTCTG-3’; reverse primer,
5’-GCAGTAGCTGCCCATCACTTT-3’;
probe,
5’-FAMAGAGTGTGCATCCTGCGGTGCCT-TAMRA-3’. For copy number quantification of unknown
samples, standard curves were created by preparing ten-fold serial dilutions of plasmid constructs
of known concentration that contained the WPRE sequence. For samples derived from cellular
RNA, the expression levels were normalized to the expression of the ABL gene: forward primer,
5’-TGGAGATAACACTCTAAGCATAACTAAAGGT-3’;
reverse
primer,
5’GATGTAGTTGCTTGGGACCCA-3’;
probe,
5’-FAMCCATTTTTGGTTTGGGCTTCACACCATT-TAMRA-3’.
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Determining virus titers of non-EGFP vectors
Using a lentiviral RAG2.IRES.EGFP construct, we found that viral titers as determined by RQPCR were approximately 65 times higher (most likely due to detection of intermediates of viral
replication) than transduction-based viral titers (data not shown), allowing us to make a rough
estimation of the multiplicity of infection (MOI) of the non-EGFP vectors.
Flow cytometry studies and cell sorting
Peripheral blood samples were taken periodically from live mice and peripheral blood, spleen,
thymus and bone marrow were obtained from CO2-euthanized mice and cell suspensions were
prepared. Cells were counted and their surface markers stained with the following anti-mouse
antibodies in various combinations: fluorescein isothiocyanate (FITC)-conjugated IgD (1126c.2a) and T-cell receptor β (TCRβ) (H57-597); R-phycoerythrin (PE)-conjugated CD19 (ID3),
IgM (R6-60.2) and TCR γδ (GL3); peridinin chlorophyll protein (PerCP)-conjugated CD8α (536.7); allophycocyanin (APC)-conjugated CD3ε (145-2C11) and CD19 (ID3); PE-Cy7-conjugated
CD4 (L3T4) and CD45R/B220 (RA-6B2); biotin-conjugated CD11b (M1/70), CD43 (S7),
CD45R/B220 (RA-6B2), GR-1 (RB6-8C5), NK1.1 (PK136) and TER-119 (Ly-76) (all antibodies
BD Biosciences, Santa Clara, CA, USA). When necessary, streptavidin-conjugated APC-Cy7
(BD Biosciences) was added as a second step. Data were acquired on a FACSAria or a
FACSCanto II (BD Biosciences). Analyses were performed using FlowJo software (Treestar,
Ashland, OR, USA). CD19+ B cells and CD11b+ cells were sorted from pooled SFFV.RAG1
spleen cells using a FACSDiva (BD Biosciences).
Immunizations and details ELISA
A sandwich enzyme-linked immunosorbent assay (ELISA) was used to determine IgM, IgG1 or
IgG serum levels using unlabeled and peroxidase-labeled anti-mouse IgM, IgG1 and IgG
antibodies (SouthernBiotech, Birmingham, AL, USA) for capture and detection, respectively.
Serially diluted sera were incubated at room temperature for 3 h, and azino-bisethylbenzthiazoline sulfonic acid (ABTS) was used as a substrate. For TNP-specific enzymelinked immunosorbent assay, plates were coated with TNP–KLH and serially diluted pre- and
post-immunization sera were incubated for 2 h. For detection, biotinylated anti-IgG and
streptavidin-coupled peroxidase were used, followed by ABTS as substrate. Antibody
concentrations were calculated by using purified IgM, IgG1 and IgG proteins as standards.
Supplemental Results
Two months after transplantation into Rag1-/- mice, T lymphocytes were found in the peripheral
blood and in the peripheral organs (supplemental figure 2) of 5 out of 30 mice (17%)
(supplemental Table 1). Despite low T-cell counts in all the organs, the cells were functional
(Data not shown; in vitro proliferation assays n=1). Thymic reconstitution was low (4-6%
CD4+CD8+ cells). Of note, we did not observed any reconstitution of the B-cell compartment.
hRAG1 VCNs in central and peripheral lymphoid organs was higher in the reconstituted mice as
compared to the non-reconstituted mice (supplemental figure 3). We observed higher VCNs and
hRAG1 expression in the mice who had the higher T-cell counts (VCN= 8 in the spleen, VCN= 2
in the BM and VCN=16 in LN, VCN=6 in the SCA1 cell before transduction). Altogether, these
data led us to conclude that RAG1 transgene expression was not sufficient to restore the T- and
B-cell development using this particular construct.
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PIKE-OVERZET et al
PRECLINICAL LENTIVIRAL RAG1 GENE THERAPY
Supplemental Table 1.
Overview of B and T cell reconstitution in RAG1-/- mice transplanted with EF-1a-RAG1
transduced Sca-1 cells.
Irradiation
dose
3 Gy
8 Gy
8 Gy
Cells
injected
Sca-1
Sca-1
Sca-1
Sca-1
Sca-1
Sca-1
Sca-1
Sca-1
CD4+/CD3
+%
17
12
7
29
5
20
18
15.7
11.8 ± 5.8
PBL
CD8+/CD3
B220+/IgM
+%
+%
1.6
<1
5.4
<1
3
1.6
20
35
0.3
<1
14
42
6
<1
12.4
47
3.2 ± 2.4
<1
Test PBL
(months)
4
5
5
5
5
Thymus
CD4+/CD8+%
(1*106 cells)
(2*106 cells)
(800 000 cells)
(22*106 cells)
(140 000 cells)
(2.8*106 cells)
Not done
85 (10*106 cells)
4
5
6
76
4
45
Supplemental Table 2.
Overview of B and T cell reconstitution in Rag1-/- mice in figure 2 A-D
Reconstitution
Transplanted cells
WT
Rag1-/EFS.coRAG1
SFFV.coRAG1
# of
mice
7
5
9
7
Success rate
B cells
T cells
B cells
T cells
7/7
0/5
5/9
6/7
7/7
0/5
5/9
6/7
100%
0%
56%
86%
100%
0%
56%
86%
Supplemental Table 3.
Overview of B and T cell reconstitution in Rag1-/- mice in figure 2 E, F and figure 4
Reconstitution
Transplanted cells
WT
Rag1-/UCOE.coRAG1
SFFV.coRAG1
# of
mice
4
2
6
6
Success rate
B cells
T cells
B cells
T cells
4/4
0/2
6/6
6/6
4/4
0/2
4/6
4/6
100%
0%
100%
100%
100%
0%
66.6%
66.6%
3
Reconstitution
3/18
Control mouse
1/3
Control mouse
1/9
Control mouse
5/30 (17%)
PIKE-OVERZET et al
PRECLINICAL LENTIVIRAL RAG1 GENE THERAPY
Supplemental Figure legends
Supplemental figure 1. Partial complementation of the Rag1-/- phenotype by tranduced
lineage negative cells. (a) Flow cytometry analysis of surface IgD/B220 expression in peripheral
blood 6 weeks after transplantation. The numbers in the graphs represent the percentage of
B220+IgD+ cells of MNCs. (b) Average B-cell percentage of lymphocytes 14 weeks after
transplantation, determined by flow cytometery. * P value = 0.0022. Statistical significance was
determined by a two-tailed, unpaired t test. (c) Flow cytometry analysis of surface CD4 and CD8
expression of thymocytes. The numbers in the graphs represent the percentage of total
thymocytes of each quadrant. (d) Genescan analysis of PCR products obtained from
amplifications using Cµ or Cγ-specific primers in combination with a consensus VH primer.
Detection of polyclonal rearrangements on cDNA derived from spleen cells, 14 weeks after
transplantation of transduced lin- cells. (e) 14 weeks after transplantation, IgM and IgG1
concentrations in the serum of mice that received WT, Rag1-/-, EFS.RAG1 or SFFV.RAG1
transduced lin- cells were determined by ELISA.
Supplemental figure 2. Partial complementation of the Rag1-/- phenotype by tranduced
Sca-1+ cells. Two months after transplantation, peripheral blood was analyzed for the presence of
CD4+/CD3+ and CD8+/CD3+ T cells as well as B220+ B cells. (a) T- and B-cell concentrations
in peripheral blood. (b) Absolute numbers of cells of each population in peripheral lymphoid
organs. Absolute numbers were determined based on the total cell count of each organ and the
relative size of the populations as determined by flow cytometry. TG: Gene therapy, ctrl: control
Supplemental figure 3. Vector copy numbers. Two months after transplantation, VCNs were
determined for central and peripheral lymphoid organs in reconstituted and non-reconstituted
mice.
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Supplemental Figures
Supplemental Figure 1
54,08
Supplemental Figure 2
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Supplemental Figure 3
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PIKE-OVERZET et al
PRECLINICAL LENTIVIRAL RAG1 GENE THERAPY
Supplemental References
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Schambach A, Bohne J, Baum C, Hermann FG, Egerer L, von Laer D, et al.
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and promoter sequences enhances retroviral vector titer and expression. Gene Ther. 2006
Apr;13(7):641-5.
3.
Charrier S, Stockholm D, Seye K, Opolon P, Taveau M, Gross DA, et al. A
lentiviral vector encoding the human Wiskott-Aldrich syndrome protein corrects immune
and cytoskeletal defects in WASP knockout mice. Gene Ther. 2005 Apr;12(7):597-606.
4.
Sirven A, Ravet E, Charneau P, Zennou V, Coulombel L, Guetard D, et al.
Enhanced transgene expression in cord blood CD34(+)-derived hematopoietic cells,
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