Recruiting endogenous adenosine deaminases using exogenous guide RNAs to edit cellular RNAs is a promising therapeutic strategy, but editing efficiency and durability remains low using current guide RNA designs. We engineered c ircular AD AR recruiting guide RNAs (cadRNAs) to enable more efficient programmable A-to-I RNA editing without requiring co-delivery of any exogenous proteins. Using these cadRNAs we observed robust and durable RNA editing across multiple sites and cell lines, in both untranslated and coding regions of RNAs, and high transcriptome-wide specificity. Additionally, we increased transcript-level specificity for the target adenosine by incorporating interspersed loops in the antisense domains, reducing bystander editing. In vivo delivery of cadRNAs via adeno-associated viruses enabled 53% RNA editing of the mPCSK9 transcript in C57BL/6J mice livers, and 12% UAG-to-UGG RNA correction of the amber nonsense mutation in the IDUA-W392X mouse model of mucopolysaccharidosis type I-Hurler (MPS I-H) syndrome. cadRNAs enable efficient programmable RNA editing in vivo with diverse protein modulation and gene therapeutic applications,

SUMMARY When non-malignant cells were reacted for glucose-6-phosphate dehydrogenase activity, with neotetrazolium chloride as the indicator of the activity, oxygen competed with the neotetrazolium and nullified the reaction. In contrast, about 30% of the activity was retained in malignant cells, in sections and in smears, from cancer of the stomach or colon. This could provide the basis of a qualitative (black-or-white) functional test for distinguishing malignant cells in these conditions.

Little is known concerning the mechanism of myeloid differentiation. A human promyelocytic cell line (HL-60) differentiates to granulocytes or macrophage-like cells when cultured with a variety of agents. How these agents trigger myeloid differentiation is not understood. This study shows that 1.0-10.0 micrograms/ml bromodeoxyuridine (BrdU) induced myeloid differentiation of HL-60 in liquid culture. After 7 days, BrdU (3.0 micrograms/ml) produced only moderate inhibition of HL-60 growth, but induced myeloid maturation with 40% of the cells becoming morphologically more mature; 41% developed the ability to reduce nitroblue tetrazolium (NBT); 19% phagocytized Candida albicans; and 18% developed Fc receptors. The action of BrdU was mimicked by 5-iodo-deoxyuridine. Thymidine (Td) (1- to 10-fold excess) competitively inhibited incorporation of [3H]BrdU into DNA of HL-60 and inhibited the triggering of HL-60 differentiation by BrdU. The BrdU-induced maturation of HL-60 correlated with the incorporation of BrdU into DNA of HL-60. DNA buoyant density studies showed that about 46% of the Td was replaced by BrdU in each DNA strand of HL-60 as the cells differentiated in culture containing 3 micrograms/ml BrdU for 7 days. We established 20 thymidine kinase (TK)-deficient HL-60 clones. The HL-60 TK-deficient cells were unable to phosphorylate Td, to incorporate either [3H]Td or [3H]BrdU or differentiate in the presence of BrdU (1-1000 micrograms/ml). The HL-60 TK-deficient cells retained the ability to differentiate in the presence of other HL-60 inducers. Taken together, the studies suggest myeloid differentiation of HL-60 is triggered because of incorporation of BrdU into DNA of the cells.

Akin to short-hairpin RNAs and antisense oligonucleotides which efficaciously recruit endogenous cellular machinery such as Argonaute and RNase H to enable targeted RNA knockdown, simple long antisense guide RNAs (1) can recruit endogenous adenosine deaminases acting on RNA (ADARs) to enable programmable A-to-I RNA editing, without requiring co-delivery of any exogenous proteins. This approach is highly specific, however the efficiency is typically lower than observed with enzyme overexpression. Conjecturing this was due in part to the short half-life and residence times of guide RNAs, here we engineer highly stable circular ADAR recruiting guide RNAs (cadRNAs), which can be delivered not only by genetically encoding on DNA vectors, but also via transfection of RNA molecules transcribed in vitro. Using these cadRNAs, we observed robust RNA editing across multiple sites and cell lines, in both untranslated and coding regions of RNAs, vastly improved efficiency and durability of RNA editing, and high transcriptome-wide specificity. High transcript-level specificity was achieved by further engineering to reduce bystander editing. Additionally, in vivo delivery of cadRNAs via adeno-associated viruses (AAVs) enabled robust 38% RNA editing of the mPCSK9 transcript in C57BL/6J mice livers, and 12% UAG-to-UGG RNA correction of the amber nonsense mutation in the IDUA-W392X mouse model of mucopolysaccharidosis type I-Hurler (MPS I-H) syndrome. Taken together, cadRNAs enable efficacious programmable RNA editing with application across diverse protein modulation and gene therapeutic settings.

A human acute myelogenous leukemia cell line that forms colonies in soft-gel culture (KG-1) was used to test the effect of various schedules and combinations of chemotherapeutic agents. For comparison, the drug sensitivity of normal human marrow myeloid clonogenic cells was tested. Cytosine arabinoside inhibited both the KG-1 and normal human colony-forming cells (CFC) approximately 25% after a 2-hour exposure, 50% after a 5-hour exposure, and 90% after a 24-hour exposure. Daunorubicin had nearly an equal cytotoxic effect on KG-1 and normal marrow CFC after a 2- to 72-hour exposure to the drug. Daunorubicin at 0.15 micrograms/ml produced nearly complete inhibition of colony-forming cells. Amphotericin B also inhibited colony formation. Amphotericin B and daunorubicin, when combined in culture, produced a synergistic suppression of normal and leukemic CFC. The antileukemic agent 5-azacytidine at a concentration of 0.1 micrograms/ml produced approximately 60% inhibition of colony formation. Cytidine partially rescued CFC when the nucleoside was added in seven-fold excess to cultures containing 5-azacytidine. Leukemic and normal marrow clonogenic cells have nearly the same sensitivity to each chemotherapeutic agent and combination. Human acute myelogenous leukemia lines may provide useful models for the development of new chemotherapeutic schedules and combinations.