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 PEG-ADA replacement was withdrawn ~5 weeks before bone marrow (BM) harvest.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 1 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, 2 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. 4 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. 7 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. 8 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