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Supplementary information Phenotype of leukemic mice Recipient type leukemias

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Supplementary information
Phenotype of leukemic mice
Recipient type leukemias

In observation time of 11 months we noted two cases of precursor T-cell leukemias, one in the SRS.SF.IL2RG.pre transplanted mice in exp.I and one in the MFG.γC group of the exp.II. In both cases no retroviral vector genomes could be detected in the leukemic cells by qPCR and Southern blot (1). We conclude that both of these leukemias were not vector related but irradiation induced, as also observed by others (2). No symptoms of vector-associated leukemia were found in primary recipients, as diagnosed by histopathology, distribution of the leukocyte lineages in bone marrow and blood analysis by FACS and differential cell counts.

Lymphoblastic leukemia (M32-1 and M32-2 and M35-1 and M35-2)

Four mice developed leukocytosis, enlarged lymph nodes (cervical, inguinal, popliteal, mesenterial), enlarged spleen, infiltrations in liver, kidneys and lung (exp.II/M32-1 and exp.III/M32-2, exp.III/M35-1 and exp.III/M35-2). The leukemic cells were negative for most lineage markers tested (CD11b, Gr1, CD19, CD3, CD4, CD8, C25, Ter119, CD41), but positive for B220, Sca-1, ckit, CD44 and CD24. The cyto-morphology of the leukemic cells was lymhoblastic (Figure 3a, b). Importantly, molecular analysis of these four leukemias by ligation-mediated PCR (LMPCR) and Southern blot revealed the same leukemic clone (as characterized by the insertion site) in all four mice, indicating that the founder cell had undergone a self-renewal division already during the short culture phase prior to the first BMT. This lymphoblastic leukemic clone contained its single insertion 5,100 bp upstream of the fourth exon of Evi1 that harbours the translational start site.

Myeloid leukemia with maturation (M34-2)

A fifth leukemia case had a myeloid phenotype by FACS (Gr1+ and CD11b+) and cytology (exp.III/M34-2, Figure 3a). There was no strong involvement of the lymph nodes, but the spleen was enlarged and the liver infiltrated by leukemic cells. Southern blot analysis identified four retroviral vector copies in the leukemic clone and therefore this leukemia was distinctly different to the B220 positive leukemia detected in the four other mice (Figure 3c). This leukemic clone contained a vector insertion -69,696 bp upstream of the second exon of Evi1. The three further insertions of this clone were close by the genes Lcp2, Snx10 and Mrpl39 (suppl. Table 1).

MPD-like myeloid leukaemia (M31-1 and M31-2)

Two further mice (exp.III/M31-1 and M31-2) transplanted from the same primary donor were diagnosed leukemic during necropsy despite not showing any obvious symptoms while being alive. Spleen and bone marrow were infiltrated by CD11b/Gr1 positive myeloid cells that were well differentiated to granulocytes. The leukemia was therefore phenotyped as a MPD-like myeloid leukemia. Southern blot analysis detected the same single vector insertion in both mice (Figure 3c) that was located upstream of the second exon of Evi1 (-109 689 bp),

Myeloid leukemias induced by LTR vectors

M3-1 (exp. I): This mouse presented with massively enlarged spleen and liver and no involvement of the thymus. The BM was pale. There was ten times increase of leukocyte counts. The leukemic cells were positive for the surface marker Gr1 and CD11b as shown in the suppl. figure 3.

M(48-50)-1 and M(48-50)-3 and M(48-50)-2 and M(48-50)-4 (exp. III): All mice has enlarged spleens and liver (see table2) and leukocytosis. Leukemic cells were positive for Gr1, CD11b and ckit (see suppl. figure 3).

Supplementary Materials and Methods
Virus production

Cell–free supernatants were generated by calcium phosphate transfection of 293T packaging cells or Phoenix-gp cells (kindly provided by G. Nolan, Stanford, USA) by co-transfection of a gag/pol construct (MLV or HIV gag/pol) and the ecotropic envelope construct (kindly provided by T. Kitamura, Tokyo, Japan). In addition, for lentiviral vectors, a plasmid encoding Rev was co-transfected. Virus titrations were performed on SC1 fibroblasts. Viral titres were in the range of 1-10x106 t.u./ml.

BM cell purification and transduction

Briefly, Lin- cells were isolated from complete BM by magnetic sorting using lineage- specific antibodies (Gr1, CD11b, CD45R/B220, CD3e, TER-119; Miltenyi Biotech, Bergisch Gladbach, Germany). Prior to retroviral transduction lin. neg. BM cells were prestimulated for 2 days in StemSpan HS2000 medium (CellSystems, St. Katharinen, Germany), containing 50 ng/ml mSCF, 100 ng/ml hFlt-3 ligand, 100 ng/ml hIL-11, 10 ng/ml mIL-3, 1% penicillin/streptomycin, 2 mM glutamine. On day 3, cells were infected on virus preloaded Retronectin (TaKaRa, Otsu, Japan) plates. For preparation of plates, the bottom surface of the wells were coated with 10 µg/cm2 Retronectin and virus loaded by centrifugation of viral supernatants by 900-1000g for 30 minutes at 4C. Virus preloading was repeated up to three times to enrich low titre supernatants (3). Lin- cells were infected on two following days (day 3 and 4). Transduction efficiency was measured before BMT (day 5) by FACS, and after BMT by PCR in genomic DNA of blood leukocytes.

Analysis of leukemic mice

Mice were sacrificed when symptomatic and macroscopically examined for pathological abnormalities during dissection. Enlarged organs were weighted. A panel of tissues was collected, fixed in 4% formalin, and paraffin-embedded for histological examinations. Cells from BM, spleen, thymus (if enlarged) and blood were subjected to flow cytometry using a FACS calibur. After red blood cell lysis, cells were stained with lineage-specific antibodies against Gr1, CD11b (myeloid cells), CD19, B220 (B-cells), Ter119 (erythroid cells), CD3, CD4, CD8, CD25 (T-cells), CD41 (megakaryocytes), Sca1 and c-Kit (Pharmingen, Hamburg, Germany). Antibodies were usually directly linked to FITC, PE or APC fluorochromes. Dead cells were excluded by propidium iodide staining. Leukocyte morphology was also evaluated in Giemsa stained blood smears and cytospins of BM and spleen cells. Blood cell counts were collected by microscopic counting or by using an automatic analyzer (SCIL Vet abcTM blood counter). Mice that did not develop leukemia were analyzed according this protocol after an observation period of 6-11 months.

To amplify the 5’LTR junction of transduced BM cells, DNA was digested with 2.5 U of restriction enzyme Tsp5091 (New England BioLabs, UK) per microgram of DNA for 2 h at 65° C. For primer extension 0.25 pmol of biotinylated retroviral primer LTR1 (5’bioTGCGGTGACCATCTGTTCTTGGCCCCG3’) was used. The first PCR (95°C for 5 min; 95°C for 1 min, 55°C for 30 sec, 68°C for 2 min for 30 cycles; 68°C for 10 min) was performed by using Extensor Hi-Fidelity PCR Master Mix (AB gene), retroviral primer LTR2 (5’GACCTTGATCTGAACTTCTC3’) and linker specific primer OCI (4). The nested PCR was performed under identical conditions, but using retroviral primer LTR3 (5’TCCATGCCTTGCAAAATGGCG3’) and linker specific primer OCII.

For the detection of gammaretroviral SIN vector insertions a wPre specific primer was used for the linear extension (SINPRE: 5’bioGCACTGATAATTCCG TGGTGTTGTC). For the exponential PCRs LTR primers were used as follows: SIN LTR2: 5’AGCGATATCGAATTCACAACC3’, SIN LTR3: 5’CCCAATAAAGCCTCTTGCTGT3’. In the case of amplification of gammaretroviral SIN vector insertion sites in the leukemic clone of mouse M34-2 the SIN LTR2 primer was used as biotinylated primer for the linear extension. The exponential PCR was performed with and additional primer SIN LTR2b: 5’GTCCTCCGATTGACTGCGTCG3’ and SIN LTR3.

For the detection of lentiviral insertion sites the primer were used as published in (4). lvLTR1: 5’bioGAACCCACTGCTTAAGCCTCA3’, lvLTR2 5’AGCTTGCCTTGAGTGCTTCA3’. lvLTR3 5’AGTAGTGTGTGCCCGTCTGT3’.

As reverse primers for the exponential PCR in the gammaretroviral SIN LMPCR and lentiviral LMPCR the linker specific primer OCI and OCII were used.

PCR products were isolated after gel electrophoresis using QIA quick Gel Extraction Kit (QIAGEN, Hilden Germany), subcloned into the pCR2.1 TA vector (Invitrogen, Karlsruhe, Germany) and sequenced using M13 primers.

Quantitative PCR

Quantitative PCR for the PRE and IL2RG was performed on a 7300 Real Time PCR System (Applied Biosystems) using QuantiTect SYBR Green (Qiagen). For quantification of vector copy numbers a qPCR was used detecting a 94 bp wPRE specific sequence (wPRE specific primers forward 5’-GAGGAGTTGTGGCCCGTTGT-3’ and reverse 5’-TGACAGGTGGTGGCAATGCC-3’). The PRE specific signal was normalized by the signal of a housekeeping gene (flk-1 intron enhancer (AF061804, bases 352-459), forward 5'-gtgaattgcagagctgtgtgttg-3' and reverse 5'-attcattgtataaaggtgggattg-3'). The primers have equal efficiencies. Results were quantified using the comparative CT method using a murine hematopoietic cell clones with 9 retroviral genome copies as a standard. The copy number of IL2RG integrated genomes was quantified with IL2RG specific primers (IL2RG forward: 5’-TGCTAAAACTGCAGAATCTGGT-3’; IL2RG reverse 5’-AGCTGGGATTCACTCAGTTTG-3’).

For the specific quantification of the Mds/Evi1 fusion and Evi1 transcript primers were designed as follows:





Chromosome preparation and spectral karyotyping

Metaphase chromosomes were prepared by treating cells with colcemid (0.035µg/ml, 6 hours) followed by incubation in 0.075M KCl for 20min at 37°C and fixation in a freshly prepared mixture of methanol:acetic acid (3:1) at room temperature. Cell suspension was dropped onto glass slides in a climate chamber (Polymer, Kassel, Germany) at 22°C and 48% humidity.

Spectral karyotyping (SKY) was performed as described previously (5) and according to the manufacturer´s instructions (ASI; Applied Spectral Imaging, Ltd., Migdal HaEmek, Israel). Spectral images were acquired using an epifluorescence microscope equipped with an interferometer (SpectraCubeTM ASI), a custom-designed optical filter and the SkyViewTM software (ASI).

1. Will E, Bailey J, Schuesler T, Modlich U, Balcik B, Burzynski B, Witte D, Layh-Schmitt G, Rudolph C, Schlegelberger B, von Kalle C, Baum C, Sorrentino BP, Wagner LM, Kelly P, Reeves L, Williams DA. Importance of murine study design for testing toxicity of retroviral vectors in support of phase I trials. Mol Ther. 2007 Apr;15(4):782-791.

2. Siapati EK, Bigger BW, Kashofer K, Themis M, Thrasher AJ, Bonnet D. Murine leukemia following irradiation conditioning for transplantation of lentivirally-modified hematopoietic stem cells. Eur J Haematol. 2007 Apr;78(4):303-313.

3. Kustikova OS, Wahlers A, Kuhlcke K, Stahle B, Zander AR, Baum C, Fehse B. Dose finding with retroviral vectors: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population. Blood. 2003 Dec 1;102(12):3934-3937.

4. Schmidt M, Hoffmann G, Wissler M, Lemke N, Mussig A, Glimm H, Williams DA, Ragg S, Hesemann CU, von Kalle C. Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples. Hum Gene Ther. 2001 May 1;12(7):743-749.

5. Frank O, Rudolph C, Heberlein C, von Neuhoff N, Schrock E, Schambach A, Schlegelberger B, Fehse B, Ostertag W, Stocking C, Baum C. Tumor cells escape suicide gene therapy by genetic and epigenetic instability. Blood. 2004 Dec 1;104(12):3543-3549.

Figure legend
Suppl. figure 1

DsRED2 transgene expression in mice of exp. III in primary and secondary recipients. SIN vectors and LTR vectors expressing DsRED showed similar long-term expression with the exception of two cases: In the SIN vector group, one mouse died due to bone marrow failure and one other mouse had no transgene positive cells in the bone marrow at necropsy despite an initial high gene marking in the blood at 9 weeks post BMT. The initial lower transduction rate of the SIN.SF transduced cells is apparent also in vivo (blue lines) compared to the LTR transduced cells (red lines).

Suppl. figure 2

(a) The sensitivity of the LM PCR that was developed to detect insertions sites of gammaretroviral SIN vectors was tested in defined DNA samples: DNA of a single clone, containing a single integration, was mixed into a mass culture at different percentages. Starting with 20% input clone DNA the single clone became already apparent.

(b) The absolute sensitivity of the LM PCR for gammaretroviral SIN vectors was tested by titration of the amount of input DNA. DNA of one single clone with multiple vector copies was used as test DNA.

(c) LM PCR amplified in DNA of BM, spleen and peripheral blood cells of one healthy mouse. The pattern was in all samples almost identical, indicating that indeed dominant hematopoietic clones were identified by LM PCR.

Suppl. figure 3

FACS analysis of the three leukemic clones that developed after gene transfer with the SF91 LTR vector. All three leukemias had a similar myeloid phenotype.

Suppl. Figure 4

LMPCR analysis of mice that received BM after prolonged culture time showed stronger selection for single clones (M14-M16) compared to mice that received BM cells directly after the transduction (M28-33). Right hand side picture shows the selection of dominant clones after secondary transplantation including the leukemic mice M31-1 and M31-2.

Suppl. figure 5:

Spectral karyotyping (SKY) analysis of mouse M3-1. No chromosomal alterations were detected.

Suppl. figure 6:

The leukemic clone of M34-2 contained four insertion sites. To verify that the insertions were contained in the same cell, leukemic cells were plated in semisolid methylcellulose medium. After a week clones derived from single cells were picked and expanded in suspension culture for further three weeks. DNA was extracted and LMPCR performed. The detection of the indentical insertion pattern in all four clones derived from the leukemic mouse M34-2 clearly shows that the insertions were within the same cell. (The LMPCR was performed using a biotinylated first primer that spanned the deletion in the U3 region for primer extension. Therefore, an internal band was amplified, as indicated.)

Suppl. figure 7:

Enlarged view of figure 5c: The number of genes with forward orientation was divided by the number of genes with reverse orientation and the resulting ratio was plotted. Here, the data is plotted to a window size of 10 kb giving a better resolution of the distribution around the TSS. Red line: gammaretroviral SIN vectors, blue line: gammaretroviral LTR vectors.

Suppl. figure 8:

LMPCR analysis of mice transplanted with lentiviral transduced bone marrow cells. LMPCR was performed on DNA extracted from peripheral blood of primary and secondary mice taken on the day of sacrifice. Mouse M23-2 was transplanted with BM cell that were expanded in vitro for 5 days prior to transplantation.

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