Lentiviral Vectors and Transductions of Haematopoietic Stem Cells

Lentiviral Vectors and Transductions of Haematopoietic Stem Cells

1

Supplementary information

METHODS

Lentiviral vectors and transductions of haematopoietic stem cells

The third generation lentiviral vectors used in these studies have the self-inactivating deletion, which removes the LTR enhancer sequences that were implicated in LMO2 activation and leukaemogenesis in the oncoretroviral vector IL2RG-treated X-SCID patients, Dull et al. J. Virol. 72, 8463-8471. They were generated by substituting the marker gene GFP with either the murine IL2RG, or murine LMO2 cDNAs. Transgene expression was driven from the high-level, ubiquitously expressing CMV promoter hybrid containing the chicken actin enhancer (CAG). For vector production reagents contact Invitrogen, Carlsbad, CA. Murine IL2RG and LMO2 cDNAs were generated from RNA extracts from murine bone marrow and testis respectively, cloning details provided upon request. Lentiviral vector supernatants were prepared as previously described Miyoshi et al., J. Virol. 72, 8150-8157. Efficient transduction of lineage depleted haematopoietic stem cells and progenitors was achieved using a novel transduction technique that involved 2 days of prestimulation in serum-free expansion medium with 50 ng/ml of each stem cell factor, thrombopoietin, and flt-3 ligand (Stem Cell Technologies, Vancouver, BC), followed by a high multiplicity of infection transduction of blood cells by pelleting and removal of excess medium and resuspending in 30 L of concentrated virus with greater than 109 TU/mL for 1 hour, followed by addition of 150 L of medium and cytokines overnight. A second hit was then performed using an additional 30 L of high titer virus directly to the cells in medium and incubated overnight.

Mouse transplantations

Murine haematopoietic progenitor donor cells were harvested from IL2RG-/- knockout mice (originally described by Cao et al. Immunity 2, 223-238) or IL2RG wild-type CD45.1 (both lines back-crossed to C57Bl6J mice and available through Jackson Laboratories, Bar Harbor, Maine). Lineage negative separation of progenitor cells was performed using the StemSep system, Stem Cell Technologies. Transduced cells were injected into lethally irradiated IL2RG-/- mice (900-1000 rads; Cobalt-60 source) via tail vein injection (100,000 starting cells/mouse, cell expansion during 4 day transduction was approximately 1.5-3 fold). In total 76 mice were transplanted for this study, which includes additional control mice such as mock transduced and LV-LMO2 transduced mice whose donor cells were derived from IL2RG-/- mice; as expected these control mice did not develop thymic lymphomas (data not shown). Vector transduction efficiency in mice peripheral blood as measured by flow assisted cell sorting (FACS) for the LV-GFP vector and found to be 5739%, n=15. Since no LV-GFP transduced mice have developed lymphomas in this study, we conclude that constitutive overexpression of IL2RG in haematopoietic cells is perhaps the principle oncogenic determinant in the generation of T-cell lymphomas in this study. For the LV-IL2RG and LV-LMO2 vectors transduction efficiency was measured by semi-quantitative PCR of bone marrow cells and found to be from 30-100% (primers sequences and conditions available upon request). Multiple vector copy integration was detected in thymic tissues (1-5 copies per genome) with lower copy numbers seen in bone marrow and spleen demonstrating an expansion of LV-IL2RG transduced leukaemic clones in the primary lymphomic organ, the thymus. Only one mouse in this study developed a thymic lymphoma where the IL2RG transgene could not be detected by PCR. Transgene expression exceeded endogenous IL2RG levels of wild-type mice by several fold in bone marrow and spleen samples as determined by Western Blot (mouse IL2RG antibody purchased from Santa Cruz Biotechnology, California). However, the levels seen in the thymuses of lymphomic mice were similar to those levels seen in the developing thymus of a 5-week-old mouse. Vector integration site analysis was performed by linear amplification mediated polymerase chain reaction as previously described (Schmidt et al. Ann. N. Y. Acad. Sci. 996, 112-21.)

Characterization of T-cell lymphomas

Determination of T-cell lymphoma was based on the presence of a large thymic mass. FACS analysis of these masses from LV–IL2RG-transduced mice revealed a prevalent, atypical T-cell with both a CD3+ and B220+ cell-surface phenotype. In addition, a less prevalent but definitive second population of larger atypical blast-like cells was also present, which were predominantly CD4/CD8 double-positive. Histological examination of these mice showed neoplastic cells infiltrating the spleen, liver, kidneys, lung and heart. The statistical analysis program GB-Stat was used to generate survival plots and perform the Wilcoxon rank test between vector groups.