1. Ubiquitous high-level gene expression in hematopoietic lineages provides effective lentiviral gene therapy of murine Wiskott-Aldrich syndrome
by Alexander Astrakhan, Blythe D. Sather, Byoung Y. Ryu, Socheath Khim, Swati Singh, Stephanie Humblet-Baron, Hans D. Ochs, Carol H. Miao, and David J. Rawlings
Blood
Volume 119(19):
May 10, 2012
©2012 by American Society of Hematology

2. WS1.6 promoter results in limited selection of WASp+ cells in vivo.
WS1.6 promoter results in limited selection of WASp+ cells in vivo. (A) Experimental design for in vivo promoter comparison studies. WASp−/− congenically marked HSC were transduced with either MND or WS1.6-containing LV and cotransplanted into lethally irradiated WASp−/− CD45.1+CD45.2+ recipients. (B) WASp expression in peripheral blood mononuclear subsets was examined by intracellular staining at different time points; monocytes (CD11b+GR1low); B cells (B220+CD3−); and T cells (B220−CD3+). (C) Viral copy numbers in sorted CD45.1+ or CD45.2+ BM CD11b+ monocytes were analyzed by qPCR. (D) Representative WASp staining in splenic T-and B-cell subsets, with the summary WASp expression data shown for T cells (E) and for B cells (F). To quantify WASp expression in different subsets, we analyzed MFI of WASp+ cells within the T- (G) and B-cell (H) lineages. Data represent 2 unique experiments with error bars showing standard deviation (SD), n = 8 for MND and 7 for WS1.6 (*P < .05; **P < .01; ***P < .001).
Alexander Astrakhan et al. Blood 2012;119:
©2012 by American Society of Hematology

3. The MND-WASp LV mediates selection of WASp+ T cells and restoration of T-cell functionality.
The MND-WASp LV mediates selection of WASp+ T cells and restoration of T-cell functionality. (A) WASp expression in the splenic CD11b+ monocyte and thymic single-positive T-cell compartment. The dotted line represents WASp expression within splenic monocyte compartment, whereas stars identify significant deviations from monocyte marking. (B) Cumulative data showing the percentage WASp+ cells in distinct CD4+ splenic T-cell subsets. (C) Representative flow cytometry data for CD4+ splenic subsets. The proportion of WASp+ cells is listed in the top right corner, whereas MFI of WASp+ cells is shown in the top left corner. (D) WASp expression in the BM and splenic CD3+NK1.1+ NKT compartment. (E) MFI values were obtained from WASp+ cells. The aggregate MFI values for splenic monocytes and CD4+ T cells are shown. (F) Total splenocytes were stimulated with αCD3 or PMA/Ionomycin and proliferation was quantified using thymidine incorporation. Stimulation index (SI) is the ratio of stimulated compared with unstimulated samples. (G) Total splenocytes were stimulated with αCD3 or PMA/Ionomycin for 6 hours and percent of IL2+CD4+ cells quantified by flow cytometry. With the exception of the MFI data, these findings represent 4 unique experiments with error bars indicative of SD (n = 7 for WTM, 6 for KOM, and 19 for MND). The MFI analysis is derived from 2 unique experiments, n = 4 for WTM mice and 8 for MND animals. Unless specifically indicated, stars indicate significant differences in WASp expression compared with splenic CD11b+ monocytes (*P < .05; **P < .01; ***P < .001).
Alexander Astrakhan et al. Blood 2012;119:
©2012 by American Society of Hematology

4. The MND-huWASp LV restores B-cell development and selection.
The MND-huWASp LV restores B-cell development and selection. (A) Numbers of BM CD11b+GR1hi neutrophils, CD11b+GR1low monocytes, CD11b−B220+ B cells, and CD11b−CD3+ T cells at 40 weeks after transplant. (B) WASp expression in splenic B-cell subsets. As in Figure 2, the dotted line represents WASp expression within splenic monocytes, whereas stars identify significant deviations from monocyte marking. (C) Absolute numbers in splenic B-cell compartments. These data are from 4 independent experiments with error bars indicative of SD (n = 7 for WTM, 6 for KOM, and 19 for MND). (D) Proportion of λ+ cells within the splenic B-cell subsets. Error bars represent SD based on 3 unique experiments; n = 6 for WTM, 4 for KOM, and 13 for MND. Unless specifically indicated, stars indicate significant differences in WASp expression compared with splenic CD11b+ monocytes (*P < .05; **P < .01; ***P < .001).
Alexander Astrakhan et al. Blood 2012;119:
©2012 by American Society of Hematology

5. The MND-huWASp LV transduces long-lived hematopoietic stem cells.
The MND-huWASp LV transduces long-lived hematopoietic stem cells. (A) Representative flow cytometric WASp expression in PB leukocytes from primary or secondary recipients. The primary recipient in top panel was used to reconstitute secondary recipient in the bottom panel. (B-D) Summary of WASp expression in primary versus secondary recipients for splenic total hematopoietic populations (B); T-cell (C) and B-cell (D) subsets. The dotted line represents proportion of WASp+ monocytes in secondary recipients. (E) Number of viral integrations within total spleen or BM was calculated using real-time PCR. (F) Kaplan-Meier curves were generated on the basis of overall animal survival during the nearly 2-year follow-up period including both primary and secondary recipients. Error bars represent SD based on 19 primary and 34 secondary recipients (*P < .05; **P < .01; ***P < .001).
Alexander Astrakhan et al. Blood 2012;119:
©2012 by American Society of Hematology

6. Development of myeloid clonal expansion in a single MND-huWASp LV recipient.
Development of myeloid clonal expansion in a single MND-huWASp LV recipient. (A) Total white blood cell numbers (top) and number of peripheral blood linneg cells (bottom) at various times after transplant. (B) Absolute numbers of hematopoietic subsets in the spleen and the BM 28 weeks after transplant. (C) WASp staining within distinct splenic hematopoietic subsets. (D) LAM-PCR analysis of total splenocytes and total BM from primary and secondary recipients. (E) Atf7ip mRNA expression in BM and spleen isolated from primary recipients. Error bars represent SD based on 3 separate PCR experiments.
Alexander Astrakhan et al. Blood 2012;119:
©2012 by American Society of Hematology

7. The WS1.6-huWASp LV partially restores WASp expression and functionality in T cells.
The WS1.6-huWASp LV partially restores WASp expression and functionality in T cells. WASp expression was analyzed 20 weeks after transplant in MND- versus WS 1.6-huWASp LV recipients. (A) Representative flow cytometry plots showing WASp expression within different PB subsets. Percent WASp+ cells in each subset is shown in the top right corner and the MFI of WASp+ cells is shown in the top left corner. Pooled PB data are shown in panel B. (C) Representative flow cytometry analysis showing WASp expression in CD4+ splenic T-cell subsets. For T-cell functional assays, total splenocytes were stimulated with αCD3 or PMA/ionomycin and proliferation (D) and IL2 production (E) were analyzed as described in Figure 2. As the proliferation and IL2 production were similar for mock treated and nontransplanted controls, these 2 groups were combined for panels D and E to improve statistical power. Except for panel B, the data represent 2 unique experiments, n = 5 for WTM, 5 for KOM, and 8 each for MND and WS1.6. Data shown in panel B represents 5 unique experiments, n = 11 for WTM, 11 for KOM, 20 for MND, and 21 for WS1.6. Error bars represent SD (*P < .05; **P < .01; ***P < .001).
Alexander Astrakhan et al. Blood 2012;119:
©2012 by American Society of Hematology

8. The WS1.6-huWASp LV results in minimal B-cell correction.
The WS1.6-huWASp LV results in minimal B-cell correction. Analysis of WASp expression, B-cell development and function in B cells from MND versus WS1.6 recipients. (A) Pooled flow cytometry data showing WASp expression for different splenic B-cell subsets. (B) Absolute numbers of splenic B-cell subsets in different recipients. (C) BrdU uptake in splenic MZ B cells from different recipients. (D) Analysis of λ LC usage within the splenic FM and MZ B-cell subsets. (E) Absolute numbers of spontaneous GC B cells (FAS+PNA+) and autoimmune prone B cells (CD11c+FAS+) within the spleen. (F) The titers of anti–double-stranded (ds) DNA antibodies were analyzed ∼ 22 weeks after transplant at a 1:200 dilution. (G) For in vitro isotype switching, B cells were isolated and stimulated with LPS for 5 days. The numbers shown reflect percent of IgG2b+ B cells after dead cell exclusion. (H) Number of viral integrations within total spleen or BM was calculated using real-time PCR. Data shown in panels A, B, D, and E are based on 5 unique experiments, (n = 11 for WTM, 11 for KOM, 20 for MND and 21 for WS1.6). Anti-DNA ELISA data in panel F are based on 8 unique experiments (n = 17 for WTM, 16 for KOM, 40 for MND, and 21 for WS1.6). Data in panels C and G are based on 3 experiments, n = 6 for WTM, 6 for KOM, 12 for MND, and 13 for WS1.6. Error bars represent SD (*P < .05; **P < .01; ***P < .001).
Alexander Astrakhan et al. Blood 2012;119:
©2012 by American Society of Hematology