Emerging evidence suggested that acute myeloid leukemia stem cells (AML LSC) are enriched in the CD34+CD38-CD123+ subpopulation.Due to low expression of coxsackie-adenovirus recptor (CAR) in both AML LSC and blasts,the utility of type 5 conditionally replicative adenovirus (CRAd) in AML treatment is limited.In this report, we constructed a recombinant sCAR-IL3 fusion protein, which successfully transduced Ad5 into CD34+CD38-CD123+ Kasumi-1 and KG-1 cells.Based on this observation, a novel CRAd, Ad.sp-E1A-sCAR-IL3, was constructed, in which the viral E1A was controlled by a survivin promoter, and a sCAR-1L3 fusion gene expression cassette was inserted into the viral genome.As compared to the control virus Ad.sp-E 1 A, Ad.sp-E 1A-sCAR-IL3 successfully infected KG1 cells and induced cell apoptosis, as detected by a transmission electron microscope.The infection was further determined by the CAR and E1A expression in KG-1 cells.Interestingly, the sCAR-IL3 fusion protein was also detected in HEK293 cells during the viral amplification, suggesting that the viral fiber might have been modified by the sCARIL3 fusion protein during the packaging in HEK293 cells.To improve the therapeutic effect of this recombinant adenovirus, the E1B region of the adenovirus was deleted and an IL-24 expression cassette was inserted.Results showed that the Ad.sp-E1A-sCAR-IL3-△E1B-IL24 adenovirus dramatically inhibited the in vitro proliferation of KG1 cells at a dosage and time dependant manner.Our data suggest that the recombinant adenovirus expressing sCARIL3 fusion proteins could be effective to target leukemia stem cells expressing CD123, and expressing sCAR-ligand through the Ad5 genome may be a novel strategy to transduce oncolytic adenovirus into leukemia cells expressing specific membrane receptors.