Suppl. Material and Methods
Lentiviral vector
Vector lots were produced using a large scale validated process based on transient
quadritransfection in 293T cells (Merten et al., Hum Gene Ther, 2010; 22:343-356) followed by
purification through endonuclease treatment, anion exchange chromatography with gradient elution,
gel filtration, resuspension in serum free media, 0.22 m filtration and aseptic filling. Each lot was
characterized in terms of potency, purity and safety aspects (reported in Tab. S6 and M. Radrizzani
et al., in preparation). Infectious titer was calculated by qPCR for copy number (see below in this
section) on human T cell line transduced using serial dilution of tested LVV, and dilution with a
VCN approximately near to 0.2 was chosen for the calculation. This value was selected to be sure
that transduced cells presented only one integration per cell. Final titer calculated as Transfer Unit
(TU)/ml, is obtained multiply (LVV VCN) X (vector dilution considered as positive) X transduced
cell number.
CFU-C and LTC-IC assay
Colony forming unit-cells (CFU-C) assay was performed, immediately after transduction, according
to the manufacturer’s procedure in Methocult medium (H4434) (Stem Cell Technologies,
Vancouver, Canada). At day 14, colonies were scored to determine number and type of colonies,
singly picked and analysed by qPCR to evaluate the percentage of transduction. For the long-term
culture-initiating cells (LTC-IC) assay, 6 x 104 CD34+ cells were plated on an irradiated fetal BM
feeder cell line in Myelocult medium (Stem Cell Technologies, Vancouver, Canada) changing half
of the medium weekly. After 5 weeks of culture, cells were harvested and plated in CFU-C assay.
Differentiation of CD34+ cells in megakaryocytes
Megakaryocytes (MKs) were differentiated as previously described42. Briefly, CD34+ cells were
stimulated with TPO (10 ng/ml), FLT3-l (20 ng/ml) and SCF (50 ng/ml) for 11 days. Differentiated
cells were stained with anti-CD41 PE (BD Biosciences, San Jose, CA, USA), anti-WASP antibody
503 (kindly provided by H. Ochs and L. Notarangelo) revealed with Alexa 488 goat anti-rabbit
antibody (Invitrogen, Carlsbad, CA, USA) and 4’-6-diamidino-2-phenylindole dihydrochloride
(DAPI, Merck KGaA, Darmstadt, Germany) for nuclear staining. Analysis was performed by laser
scanner microscopy equipped with appropriate filter combinations (Leica TSC SP2).
Real-time PCR analysis
In order to evaluate the number of lentiviral vector copies integrated per genome a qPCR was
performed using specific primer and probes for human Telomerase, lentiviral vector and murine βactin and reference standard obtained from human T cell line carrying 1 copy of integrated LVV.
Results of integrated vector copies were normalized for the number of evaluated genomes. As
negative control untransduced cells were used. All the reactions were performed according to the
manufacturer’s instructions and analyzed with an ABI PRISM 7900 sequence detection system
(Applied Biosystem, Foster City, CA). Primers and probes sequences were reported in Tab. S7
B2-SINE PCR to specifically detect the presence of integrated lentiviral vector in murine cells
In order to detect the presence of the integrated vector in secondary targets upon transplantation of
transduced human cells in murine recipients, we modified the previously developed B2-SINE PCR
protocol (Follenzi et al, 2002) (Suppl. Fig. 1). We used the forward primers previously designed on
the 3' end of the vector for B2-SINE PCR while we designed 2 new primers in reverse orientation
on the 5' end and 3' end of MBTCON1 B2 elements (Mayorov et al, 2000). In order to enrich for
fragments carrying mouse B2 elements, we added a biotin modification to the 5' end of the B2
antisense 1 primer allowing binding to streptavidine magnetic beads (Dynabeads, Invitrogen). The
sequences of the primers used for the B2-SINE PCR are the following:
Vector primers:
WPRE SENSE: 5'- CTTTCCATGGCTGCTCGC -3'
DNEF SENSE: 5'- CGAGCTCGGTACCTTTAAGACC -3'
LTR8 ANTISENSE: 5'- TCCCAGGCTCAGATCTGGTCTAAC -3'
B2 element primers:
B2 ANTISENSE 1 BIOTINILATED: 5'-ATATGTGAGTACACTGTAGC -3'
B2 ANTISENSE 2: 5'- ACTGAGCCATCTCTCCAGCC -3'
The method is composed by a three-steps PCR. The first amplification was performed with a
forward primer on the wPRE element of the vector and the first biotinilated reverse primer on B2
element under the following conditions:
1st PCR mix
Reagents
Bf 10x
Wpre sense (25uM)
B2 antisense 1 biot (25uM)
dNTPs (25 mM)
Taq
100ng DNA + H20
ul
5
0.5
0.5
0.4
0.5
43
V tot
49.9
1st PCR conditions
temperature
time
95°C
10'
denaturing, annealing, extension
94°C
1'
54°C
1'
72°C
1'
repeat for 30 cycles
72°C
7'
4°C
for ever
The PCR products were then incubated overnight with streptavidine magnetic beads washed twice
for purification and detached from beads with 5ul of NaOH 0.1 M. 1ul of this purified product is
then added to 49ul of mix containing the same wPRE forward primer but the second B2 element
reverse primer. The 2nd PCR was performed under these conditions:
2nd PCR mix
Reagents
Bf 10x
Wpre sense (25uM)
B2 antisense 2 (25uM)
dNTPs (25 mM)
Taq
H20
1st PCR product
ul
5
0.5
0.5
0.4
0.5
42
1
V tot
49.9
2nd PCR conditions
temperature
time
95°C
10'
denaturing, annealing, extension
94°C
1'
54°C
1'
72°C
1'
repeat for 30 cycles
72°C
7'
4°C
for ever
After this step 1ul of the 2nd PCR product was added to 49ul of a third PCR mix with Dnef sense
and LTR8 antisense primers both built on the 3'end of the vector. The 3rd PCR was performed with
these conditions:
3rd PCR mix
Reagents
Bf 10x
Dnef sense (25uM)
LTR8 antisense (25uM)
dNTPs (25 mM)
Taq
H20
2nd PCR product
ul
5
0.5
0.5
0.4
0.5
42
1
V tot
49.9
3rd PCR conditions
temperature
time
95°C
10'
denaturing, annealing, extension
94°C
1'
58°C
1'
72°C
1'
repeat for 20 cycles
72°C
7'
4°C
for ever
The final PCR product was 166bp in lenght.
In order to measure the sensitivity of the technique we built a standard reference with serial
dilutions of transduced DNA into untransduced one from mouse (lin- cells) and human (BM
CD34+cells) samples for a total amount of 100ng per reaction. We diluted transduced mouse DNA
into untransduced mouse DNA (7 dilutions ranging from 1:1 to 0.06:100) to reproduce the presence
of given amounts of secondary transduced targets in recipient murine tissues fully purified from
human cells. We also tested 3 dilutions of transduced human DNA into untransduced mouse DNA
(from 1:1 to 1:100) to virtually recapitulate the presence of human cell contaminants in murine
recipient tissues. For further specificity controls we added 100% pure transduced DNA from mouse
and from human cells with an average vector copy number of 1.8 and 1.5 respectively (Suppl. Fig.
4). Given that the size of a murine genome is 3,420,842,930 bp and that 1pg correspond to 978Mbp
(Dolezel et al, 2003), the weight of a diploid mouse genome should be equivalent to 3,49 x 2 =
6,99pg total. Thus, we calculated that we were able to detect reproducibly around 71 transduced
mouse genomes diluted into 14,000 untransduced ones. In some cases we reached an even higher
detection limit up to 250-125 pg of vector-carrying DNA corresponding to 35-17 transduced mouse
genomes. The variability of this lower limit is probably due to the stochastically variable presence
of vector integrants close enough to a B2 element to be amplified by the technique.