In vivo delivery of CpG-siRNAs induces RNAi and abrogates target gene expression in TLR9+ leukemia cells. (A) Dose-dependent uptake of IT injected CpG-STAT3 siRNA by TLR9+ MV4-11 cells. Tumor cells were injected SC into NSG mice. After tumors were established, mice were injected IT using 20 or 100 μg of Cy3-labeled CpG-STAT3 siRNA. The percentage of Cy3+ cells was analyzed by flow cytometry in cell suspensions prepared from tumors harvested 3 hours after injection. (B-C) IT injections of CpG(A)-siRNA can effectively silence expression of BCL-XL or STAT3 proteins in xenotransplanted MV4-11 leukemia. NSG mice were injected with MV4-11 AML cells. After tumors were established (day 7), mice were treated 4 times with daily IT injections of CpG(A)-siRNAs targeting BCL-XL (B) or STAT3 (C). Protein levels of both BCL-XL and STAT3 were evaluated using Western blotting with β-actin as a loading control in samples derived from single tumors for both experimental groups. Band intensities were quantified by densitometry using ImageJ Version 1.46 software based on identically exposed images. Shown are results from 1 of 2 independent experiments analyzed for statistical significance. **P = .003; ***P < .0001. (D-E) RNAi-mediated STAT3 gene knockdown in MV4-11 tumors treated using IT injections of CpG(A)-siRNAs as in panel B. (D) STAT3 gene silencing in samples from 4 individual tumors was verified by qPCR and normalized to TBP expression; shown are means ± SE. (E) CpG(A)-STAT3 siRNA-induced cleavage of STAT3 mRNA in MV4-11 tumors in vivo. Tumors injected IT using CpG(A)-siRNAs as indicated were harvested 18 hours later. Total RNA samples were analyzed by the 5′RACE-PCR assay followed by DNA sequencing detecting mRNA cleavage within the targeted region (partial sequences are shown); arrow indicates the major RACE product of the predicted length (300 nt).