Otsu et al.
Supplemental Materials
Outcomes in Two Japanese Adenosine Deaminase-Deficiency Patients Treated by
Stem Cell Gene Therapy with No Cytoreductive Conditioning
Otsu et al.
Supplemental Methods
Retroviral vector supernatant.
GCsapM-ADA -retroviral vector [1] supernatant was
prepared using the PG13 packaging cell line capable of pseudotyping viral particles
with the Gibbon Ape Leukemia Virus (GALV) envelope. Clinical-grade supernatant
was prepared at the National Human Genome Research Institute, NIH, as described [2].
A defined lot of supernatant, released after a series of rigorous quality analyses, was
transferred to the Hokkaido University Hospital and stored at < -80oC until use.
BM CD34+ cell isolation procedures.
PEG-ADA replacement was withdrawn ~5
weeks before bone marrow (BM) harvest.
BM was harvested from the posterior iliac
crests under general anesthesia with total collection volumes of 17.4 ml/kg and 18.7
ml/kg for pt 1 and pt 2, respectively.
Leukocyte-rich suspensions were prepared by
hydroxy ethyl starch-mediated erythrocyte sedimentation, followed by further lysis
using ammonium-chloride-potassium buffer. These leukocyte-rich suspensions were
processed with the Isolex 300i cell separation system (Baxter, Deerfield, MA) for
enrichment of CD34+ cells, following the manufacturer’s instructions. For pt 1 (1.40 x
109 nucleated cells containing 2.4% CD34+ cells), 1.96 x 107 cells were recovered
(purity 88.9% CD34+). For pt 2 (2.82 x 109 nucleated cells containing 1.8% CD34+
cells), 2.80 x 107 cells were recovered (purity 82.1% CD34+). BM aspirates were
introduced into a sterile collection bag, with all subsequent procedures in sterile bags
with the TSCDR Sterile Tubing Welder device, thus allowing a fully closed system.
Detailed protocol of retroviral-mediated ADA gene transduction.
The CD34+ cells
were transferred into a gas-permeable LifecellR X-foldTM bag (Nexell, Irvine, CA) (day
-5) and pre-stimulated for two days in serum-free X-VIVO15TM medium (Lonza,
Walkersville, MD) supplemented by 1% human serum albumin (HSA) and a cocktail of
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Otsu et al.
cytokines consisting of 50 ng/ml stem cell factor, 50 ng/ml thrombopoietin, 300 ng/ml
Flt3-ligand, 100 ng/ml interleukin (IL)-6, and 500 ng/ml soluble IL-6 receptor (sIL-6R)
(all R&D Systems, Minneapolis, MN). Two days later (day -3), pre-cultured cells
were collected and transferred into an X-fold bag pre-coated with the fibronectin
fragment CH296 (Takara Bio, Otsu, Japan) and pre-loaded with the GCsapM-ADA
supernatant (pre-loading was performed 3 times with 30 ml supernatant used for each
cycle for a 85-cm2 bag).
For the initial phase of transduction, cells were kept in the
same culture medium as described except for the concentration of each cytokine, which
was 2-times higher than the above mentioned final concentration (2X medium). Six
hours later, the culture was fed with fresh GCsapM-ADA supernatant at the equal
volume with the 2X medium, and cultured overnight for the second phase of
transduction on the same day. On day -2, cells were collected, resuspended in a 50:50
mixture of 2X medium and the GCsapM-ADA supernatant, and returned to the original
culture bag. The same transduction procedure was performed on day -1. On day 0,
transduced cells were collected and characterized, with a final cell yield of 2.88 x 107
(98.0% viable) and 5.66 x 107 (99.0% viable) for pt 1 and pt 2, respectively. After
sampling for analysis, the remaining cells were resuspended in ~20 ml of normal saline
containing 1% HSA, and infused intravenously, with 2.74 x 107 cells (70.7% CD34+)
and 5.52 x 107 cells (66.6% CD34+) administered to pt 1 and pt 2, respectively.
Flow-cytometry analysis of intracellular ADA expression. ADA protein expression
was assessed by multi-color flow-cytometry analysis using previously described
procedures [3] with some modifications. PBMNCs were first stained for cell surface
markers.
The antibodies used were Cy-Chrome-anti-CD3 (clone HIT3a, mouse
IgG2a; BD PharMingen, San Diego, CA) and FITC-anti-CD56 (clone MEM188, mouse
IgG2a; Southern Biotechnologies, Birmingham, AL).
After washing, cells were
fixed/permeabilized using Cytofix/Cytoperm reagent (BD PharMingen) according to
manufacturer’s instructions. Cells were split into two tubes, with one used for staining
with a mouse monoclonal antibody (1C5) specific to ADA protein, and another with
isotype mouse IgG1 (BD PharMingen) [3]. To amplify ADA-specific fluorescence
intensity, these stained cells were incubated with biotin-anti-mouse IgG1, followed by
reaction with phycoerythrin (PE)-conjugated streptavidin.
The data were acquired
with a FACSCalibur (BD Biosciences) and analyzed using FlowJo software (Tree Star,
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Otsu et al.
Ashland, OR).
Primer/probe sequences used for vector copy number assessment.
Shown below are
the nucleotide sequences of the primers and probes used in this study.

Primer & probes (5’ -> 3’)



For GCsapM-ADA

Forward:
GGT GGA CCA TCC TCT AGA CCG

Reverse:
GGT TTC AGG CTT GAT GGA TCC

Probe (FAM-TAMRA): CCC GCC TTC GAC AAG CCC AAA
For both vectors (ADA cDNA)

Forward:
GGA ACT GCA TGT CCA CCT AGA CG

Reverse:
TCC ATG CCA ATG ACG TTC AG

Probe (FAM-TAMRA): CCC TCC CAG CTA ACA CAG CAG AGG G
-actin

Forward: ACA CTG TGC CCA TCT ACG AGG

Reverse: GGT GAG GAT CTT CAT GAG GTA GTC A

Probe (FAM-TAMRA): ATC CTG CGT CTG GAC CTG GCT GGC
The -actin primer / probe pair was used to normalize the VCN readout.
Linear amplification mediated (LAM)-polymerase chain reaction (PCR) analysis.
To
investigate clonality of vector integration sites in patient samples, LAM-PCR analysis
was conducted following an original protocol [4] with some modifications. Genomic
DNA samples were prepared using the QIAamp DNA blood Mini Kit (Qiagen, Valencia,
CA).
Linear amplification was performed for 50 cycles using biotinylated primer,
followed by enrichment of amplicons containing the vector-specific sequence by using
streptavidin-coupled magnetic beads.
Following double-strand DNA synthesis,
digestion with the enzyme Tsp509I, and addition of the linker cassette, the fragments
containing a vector-genome junction were specifically amplified by two rounds of PCR
in which nested primers were used for the 2nd amplification. The amplified products
were visualized, using ethidium bromide, after electrophoresis on a 3% agarose gel.
To maximize assay reliability, each reaction was performed in duplicate.
details are available upon request.
3
Further
Otsu et al.
Vector integration site determination.
Precise integration sites and frequencies of each
vector integrant were determined after excision of selected bands from each agarose gel
shown in Fig. 7. Subjected to analysis were in total 9 bands from pt 1 and 4 bands
from pt 2, as shown by white arrowheads in the images. Each band was cloned using a
TA-cloning vector and standard techniques of colony formation with transformed E.
coli.
The cloned plasmids were amplified, and the sequence of each fragment
containing a genome-vector junction was sequenced by a 3730xl DNA Analyzer (Life
Technologies, Carlsbad, CA). Genomic insertion sites were determined by BLAST
analysis using the obtained reads.
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Otsu et al.
Supplemental Table
Summary of vector integration site analysis
Pt 1
ID
Chr.
Location
Orientation
PBMNC
PBMNC
PBG
BM
21 mo
68 mo
68 mo
51 mo
1-A
16
3,708,243

+
+
+
+
1-B
17
5,036,831

+
+
+
+
1-C
3
49,354,365

+
+
+
+
1-D
2
61,621,250

+
+
+
1-E
6
35,123,914

+
+
# of
miscellaneous
IS
-
-
-
0
1
15
5
0
Otsu et al.
Pt 2
PBG
ID
Chr.
Location
PBMNC
Orientation
73 mo
37 mo
52 mo
2-A
11
116,421,276

+
+
NA
2-B
15
40,700,483

+
NA
+
# of
miscellaneous
IS
-
-
-
2
0
1
Integration sites found shared by different lineages are shown with ID.
Chr., chromosome; PBMNCs, peripheral blood mononucle ar
cells; PBG, peripheral blood granulocytes; BM, bone marrow; NA, not applicable.
numbers in a “location” column; cf. UCSC Genome Browser (https://genome.ucsc.edu).
Genomic insertion sites are shown by the
Direction of vector integrants for genomic
sequence is shown by either an arrow to the left (antisense orientation) or an arrow to the right (sense orientation).
is placed where the corresponding vector insertion is identified.
miscellaneous insertion sites (IS) only for their total numbers.
6
Minor reads
The s ymbol “+”
found unshared between lineages are shown as
Otsu et al.
Supplemental References
1.
Onodera M, Nelson DM, Yachie A, Jagadeesh GJ, Bunnell BA, Morgan RA, et al.
Development of improved adenosine deaminase retroviral vectors. J Virol.
1998;72(3):1769-74.
2.
Candotti F, Shaw KL, Muul L, Carbonaro D, Sokolic R, Choi C, et al. Gene
therapy for adenosine deaminase-deficient severe combined immune deficiency:
clinical comparison of retroviral vectors and treatment plans. Blood.
2012;120(18):3635-46.
3.
Otsu M, Hershfield MS, Tuschong LM, Muul LM, Onodera M, Ariga T, et al.
Flow cytometry analysis of adenosine deaminase (ADA) expression: a simple
and reliable tool for the assessment of ADA-deficient patients before and after
gene therapy. Hum Gene Ther. 2002;13:425-32.
4.
Schmidt M, Zickler P, Hoffmann G, Haas S, Wissler M, Muessig A, et al.
Polyclonal long-term repopulating stem cell clones in a primate model. Blood.
2002;100(8):2737-43.
5.
Kato S, Yabe H, Yabe M, Kimura M, Ito M, Tsuchida F, et al. Studies on transfer
of varicella-zoster-virus specific T-cell immunity from bone marrow donor to
recipient. Blood. 1990;75(3):806-9.
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Otsu et al.
Supplemental Figure Legends
Supplemental Fig. 1 Kinetics of systemic detoxification for the first 2 years after
SCGT.
(a) Activities of liver enzymes aspartate transaminase (AST) and alanine
transaminase (ALT) in patient serum before stem cell gene therapy (SCGT) and after
(up to ~26 weeks). The time of the last administration of PEG-ADA is indicated.
(b) Patient erythrocyte %dAXP values before and after SCGT.
To facilitate
comparison, the x-axis scale is the same as that of the x axis in (a).
Supplemental Fig. 2 Kinetics of hematopoietic reconstitution for the first 2-3 years
after SCGT.
(a) Lymphocyte reconstitution.
Absolute lymphocyte counts in patient
peripheral blood; the time of stem cell gene therapy (SCGT) is indicated.
The better
to represent initial development kinetics, the data are shown in a time frame ranging
from before SCGT to the times at which counts reached peak value for each patient.
(b) Neutrophil reconstitution. Absolute neutrophil counts in patient peripheral blood;
cf. (a) for manner of data presentation.
Supplemental Fig. 3
SCGT.
Lymphocyte subset reconstitution for the first 2-3 years after
(a) Absolute CD4+ and CD8+ T cell counts in peripheral blood before and
after stem cell gene therapy (SCGT).
(b) Absolute CD20+ B cell and CD16+/CD56+
NK cell counts in peripheral blood; cf. (a) for manner of data presentation.
Supplemental Fig. 4
Long-term follow-up of ADA-expressing T/NK cells in pt 1.
Shown are flow-cytometry analysis data on frequencies of ADA-expressing cells
among CD3+ T cells and CD56+ NK cells (6.5 years after SCGT).
Open histograms,
isotype control; gray histograms, ADA-specific fluorescence. The frequency (%) of
ADA-positive cells is shown.
Supplemental Fig. 5
Intracellular levels of ADA expression in pt 2’s T/NK cells.
Shown are the results obtained by flow-cytometry analysis to assess intracellular
expression of ADA in peripheral blood CD3+ T cells and CD56+ NK cells.
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Patient 2’s
Otsu et al.
data obtained 6 months after stem cell gene therapy are shown. Dashed histograms
represent isotype control; gray histograms show ADA-specific fluorescence. Control:
Data obtained from a healthy individual. Frequency (%) of ADA-expressing cells is
shown.
Despite signal amplification (see supplemental Methods), relatively low
levels of ADA expression often led to underestimation of %ADA+ cells as exemplified
here in control CD3+ cells that show a value of 35.9% (theoretical readout 100%).
Note that clear two-peak histograms are lacking, unlike those obtained for pt 1’s
samples (Fig. 4), and that virtually all CD3+ T cells are supposed to be LASN+ at this
time, perhaps hindering the detection of GCsapM-ADA-expressing cells.
Supplemental Fig. 6
T cell receptor (TCR) spectratyping analysis.
Clonality of T
cells within each V family is shown as TCR spectratyping results.
PBMNCs
obtained from both patients 2 years after stem cell gene therapy were analyzed with
cord blood mononuclear cells (“Cord Blood”) as control.
Representative data for 4
TCR families show polyclonal distribution of complementarity-determining region 3
(CDR3) lengths in both patients’ T cells.
Supplemental Fig. 7
T lymphocyte functions after stem cell gene therapy.
Proliferative responses of T cells measured ~2.8 years after SCGT are shown. Note
that pt 1 had chickenpox one month before the assay date. PBMNCs were stimulated
with allogeneic PBMNCs (mixed lymphocyte reaction, MLR), or with antigen derived
from varicella-zoster virus (VZV), cytomegalovirus (CMV), or adenovirus (Adeno) as
described [5]. A dotted line shows the cut-off point determined effectively to separate
negative and positive values.
9