Fig. 5. Targeting IL1RAP via the LNP-Cas9 RNP/MSCM-NF system affects LSC colony-forming ability and reduces long-term leukemic burden.

(A) In vivo imaging system (IVIS) images of in vivo biodistribution. Alexa Fluor 647–labeled free L76-Cas9 or L76-Cas9/MSCM-NF were injected into the right tibia of BALB/c mice (white square). The NP clearance rate was monitored using an IVIS. (B) The ROI (region of interest) (radiant efficiency), as described in (A), showing that free L76-Cas9 NPs were cleared from the bone marrow injection site faster than L76-Cas9/MSCM-NF. (C) CFU, colony-forming unit assay. THP-1 cells were incubated with L76-Cas9 RNP/MSCM-NF for 48 hours before plating in methylcellulose-based medium with recombinant human cytokines. The colony-forming ability was measured after 14 days of culture. n = 2 independent studies. (D) Schematic of mouse xenograft model with ex vivo IL1RAP gene editing treatment. THP-1 cells were treated with L76-Cas9 RNP/MSCM-NF for 2 days, and an equal number of THP-1 cells were transplanted into sublethally irradiated NSG mice. Human leukemic burden in recipient mice was measured by flow cytometry after 8 weeks or when the mice appeared moribund. (E) Percentage of human CD45⁺CD33⁺ population in the bone marrow harvested from recipient mice, as described in (D). (F) Percentage of human IL1RAP expression in CD45⁺CD33⁺ population in bone marrow harvested from recipient mice. (G) Identification of unmodified and modified IL1RAP alleles by amplicon sequencing analysis. THP-1 cells were treated with L76-Cas9 RNP/MSCM-NF as described in (D). DNA was harvested from cells before xenotransplantation for sequencing. (H) Nucleotide distribution near the IL1RAP sgRNA targeting site. The percentage of each base in the amplicon is shown based on the sequencing reads. w/o sgRNA, L76-Cas9/MSCM-NF without IL1RAP sgRNA. w/ sgRNA, L76-Cas9/MSCM-NF with IL1RAP sgRNA. Data are means ± SD. **P < 0.01 and ****P < 0.0001; Student’s t test.