Extended Data Fig. 5: Synthetic introns enable mutation-dependent targeting of Sf3b1-mutant hematopoietic cells in vivo.

(a) Schematic of xenograft experiments with MOLM-13 cells expressing doxycycline-inducible SF3B1^WT or SF3B1^K700E. MOLM-13 cells were intravenously injected into sub-lethally irradiated (250 cGy) NOD-scid IL2rg^null (NSG) mice (100,000 cells/mouse). Doxycycline was provided in feed one day before xenograft. (b) Radiance of experiment in (a). Each point represents an individual animal; bars represent means. n = 11 to 15 mice/group. p computed with two-way ANOVA with Tukey’s multiple comparisons test. (c) Schematic of mixed chimeric bone marrow (BM) hematopoietic stem and progenitor experiment. BM cells from CD45.2⁺ Mx1-cre Sf3b1^WT or Sf3b1^K700E/WT mice were mixed 3:1 with BM cells from CD45.1⁺ C57BL/6 wild-type mice. The mixed population was infected with a lentiviral vector encoding GFP-P2A-HSV-TK interrupted by synMTERFD3i1-150. Following two sequential days of infection, cells were treated with GCV in vitro, and GFP⁺ cell percentages were enumerated by FACS. (d) Schematic of in vivo experiment with primary hematopoietic precursors from Mx1-cre Sf3b1^K700E/WT or littermate Sf3b1^WT mice. c-Kit⁺ cells from BM of CD45.2⁺ Mx1-cre Sf3b1^WT or Sf3b1^K700E/WT mice were infected with GFP-P2A-HSV-TK interrupted by synMTERFD3i1-150 and intravenously injected into sub-lethally irradiated CD45.1⁺ wild-type mice. 3 weeks post BM transplantation, CD45.1⁺ recipient mice underwent pIpC and GCV treatment and were then bled for CD45.2⁺, CD45.1⁺, and GFP⁺ content in peripheral blood and BM. n = 5 (WT) and n = 4 (Sf3b1-mutant) mice per cohort. (e) GFP⁺ cell percentages amongst CD45.2⁺ cells in peripheral blood post-pIpC and GCV treatment from cohort in (d). Data represented as mean ± s.d. n = 5 (WT) and 4 (Sf3b1-mutant) mice per cohort. (f) Box plots of GFP⁺ percentages amongst CD45.2⁺ cells in peripheral blood at days 0 and 39 post-pIpC and GCV treatment from cohorts in (d). Points depict individual mice. p computed by an unpaired, two-sided t-test. (g) As (f), but for BM at day 39. Points depict individual mice. p computed by an unpaired, two-sided t-test. For all box plots, the middle line, hinges, notches, and whiskers indicate the median, 25^th/75^th percentiles, and min-max range.

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