We reported that T cells with anti-CD38-chimeric antigen receptors (CAR) eliminated B-cell lymphoma cells expressing CD38. To employ anti-CD38-CAR against acute myeloid leukemia (AML) blasts not expressing CD38, it is necessary to induce or increase the intensity of CD38 expression. A lactate dehydrogenase (LDH)-releasing assay and flow cytometry showed that anti-CD38-CAR T cells were cytotoxic against AML lines (THP-1 and CMK) expressing high CD38 levels (>99%), in time- and number of effector-dependent manners. In other AML lines (KG1, U937 and HL60) partially expressing CD38, CD38+ AML cells were killed by CD38-specific T cells, but CD38− AML cells remained survived. Intriguingly, 10 nM all-trans retinoic acid (ATRA) augmented CD38 expression in KG1, U937 and HL60 cells and primary leukemic cells from AML patients. Moreover, the withdrawal of ATRA from the medium decreased CD38 expression in AML cells. Killing effects of anti-CD38-CAR T cells against AML lines and AML cells were limited without ATRA, whereas CD38-specific T cells enhanced cytotoxicity on AML cells by ATRA in association with enhanced CD38 expression. These results indicate that anti-CD38-CAR T cells eliminate AML cells through CD38 expression induced by ATRA.
The present study describes improved properties of 27-nt dsRNAs over 21-nt siRNAs, and accents on the possibility to use their modifications and conjugates for direct long-term gene silencing in viable cells and animals, avoiding conventional transfectants. Using a Renilla Luciferase gene-silencing system and cultured cell lines, we established that 27-nt dsRNAs possessed about three to five times higher "long-term" RNAi activity than 21-nt siRNAs and 21-nt dsRNAs. Moreover, if RNA duplexes were preincubated with cell-cultured medium for several hours before their transfection in cells, 21-mer completely lost its RNAi effect, while 27-mer, its amino modifications, thiol modifications, and cholesterol conjugates manifested a strong gene silencing. In attempts to clarify the reason(s) for the higher RNAi activity of 27-nt dsRNAs, we found that they were approximately 100 times more stable than 21-nt siRNA and 21-nt dsRNA in cell-cultured medium supplemented with 10% inactivated serum, approximately 50 times more stable in 90% inactivated serum, and approximately six times more stable in active serum. The 5' sense modification was selected as the most stable, accessible to Dicer, and with highest RNAi potential. The RNAi activity of 5' sense modifications was higher even than the activity of nonmodified 27-nt dsRNA. The 5' sense amino modification also did not influence the activity of 21-nt siRNA, right overhang 25/27-nt (R25D/27), and 25D/27-nt RNAs. The stability of 5' sense modified R25D/27-nt and 25D/27-nt RNAs in serum was lower than that of blunt 27-nt dsRNA. However, these asymmetric RNAs were more active than modified and nonmodified blunt 27-nt dsRNAs, which demonstrates the superiority of the asymmetric design. The 5' sense modifications were considered as most appropriate for conjugation with small signal molecules to facilitate the intracellular delivery of RNA duplex, to preserve its RNAi capacity, and to ensure a possibility for rapid long-term gene silencing in viable cells and animals. The 5' sense conjugation with cholesterol approved this assumption.
Novel anti-HIV lectin family which shows a strict binding specificity for high mannose glycans has been found in lower organisms. The bacterial orthologue has been identified in the genome of Pseudomonas fluorescens Pf0-1 and the gene coding a putative lectin was cloned, expressed in Escherichia coli and purified by one step gel filtration. Glycan array screening of the recombinant lectin, termed PFL, has revealed that PFL preferentially recognizes high mannose glycans with α1-3 Man that was highly exposed at the D2 position. In contrast, masking of this α1-3 Man with α1-2 Man dramatically impaired lectin-carbohydrate interactions. Reducing terminal disaccharide, GlcNAc-GlcNAc of high mannose glycans was also essential for PFL-binding. PFL showed a potent anti-influenza virus activity by inhibiting the virus entry into cells at doses of low nanomolar concentration. At micromolar concentration or higher, PFL showed a cytotoxicity accompanying loss of the cell adhesion against human gastric cancer MKN28 cells. The cell surface molecule to which PFL bound was co-precipitated with biotin-labeled PFL and identified as integrin α2 by peptide mass fingerprinting using MALDI-TOF mass spectrometry. Intriguingly, upon treatment with exogenous PFL, integrin α2 on the cell surface underwent rapid internalization to the cytoplasm and accumulated to perinuclear region, together with the bound PFL. The resulting loss of cell adherence would trigger a signaling pathway that induced anoikis-like cell death. These events were effectively inhibited by pretreatment of PFL with mannnan, indicating the involvement of high mannose glycans on PFL-induced cell death that was triggered by PFL-integrin α2 interactions.
The development of Dicer-substrate small interfering RNAs (DsiRNAs) has been pursued in recent years because these molecules exhibit a much more potent gene-silencing effect than 21-nucleotide (nt) siRNAs. In the present study, we designed eight different types of amino-modified DsiRNAs and a palmitic acid-conjugated DsiRNA expected to result in improved biological properties of siRNAs, including their stability against nuclease degradation, membrane permeability, and RNAi efficacy. The DsiRNAs were modified with an amine at the 5'- and/or 3'-end of the sense and/or antisense strand. Dicer enzyme cleaved most of the amino-modified DsiRNAs to lead to the release of 21-nt siRNA; some of them, however, were not or partly cleaved. All amino-modified DsiRNAs exhibited strong resistance against nuclease degradations. Among the amino-modified DsiRNAs, the DsiRNA modified with an amine restricted at the 3'-end of the sense strand showed the most enhanced gene-silencing effect and maintained its potent gene suppression after one week of cell transfection against Renilla luciferase activity. For further improvement, palmitic acid was conjugated to DsiRNA at the 3'-end of the sense strand (C16-DsiRNA) to facilitate the membrane permeability and potent gene-silencing activity. The C16-DsiRNA showed enhanced membrane permeability to HeLa cells. The C16-DsiRNA exhibited extremely high inhibition of Renilla luciferase activity.
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