In alternative embodiments, provided are methods for treating and ameliorating a cancer such as a leukemia such as acute myeloid leukemia (AML) comprising administration to an individual in need thereof a pharmaceutical composition comprising imetelstat, or imetelstat and second drug such as dasatinib, or ruxolitinib, fedratinib, 8-aza-adenosine, raltegravir and/or dolutegravir or any combination thereof. In alternative embodiments, provided are methods for the in vivo inhibition of myeloproliferative neoplasm (MPN) or AML stem cell propagation comprising administration to an individual in need thereof a pharmaceutical composition comprising imetelstat, or imetelstat and second drug. In alternative embodiments, provided are methods for the in vivo inhibition pre-leukemia stem cell (pre-LSC) transformation into leukemia stem cells (LSCs) comprising administration to an individual in need thereof a pharmaceutical composition comprising imetelstat, or imetelstat and second drug such as dastinib, or ruxolitinib, fedratinib, 8-aza-adenosine, raltegravir and/or dolutegravir or any combination thereof.

Provided are fusion proteins that include an apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBEC3A) and a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein, optionally further with uracil glycosylase inhibitor (UGI). Such a fusion protein is able to conduct base editing in DNA by deaminating cytosine to uracil, even when the cytosine is in a GpC context or is methylated.

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing enzymes or domains, are provided.

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing enzymes or domains, are provided.

In alternative embodiment, provided are methods and compositions for treating, ameliorating or preventing diseases and conditions, such as cancer, including cancers associated with stem cells such as, without limitation, myelodysplastic syndrome (MDS) and a myeloproliferative neoplasm like chronic myeloid leukemia (CML) or acute myeloid leukemia (AML), and ablating or killing cancer stem cells. In alternative embodiment, provided are a new set of biomarkers to detect leukemia stem cell reprogramming and CML progression. In alternative embodiment, provided are therapeutic targets for treating myelodysplastic syndrome (MDS) and chronic myeloid leukemia (CML) by targeting edited let-7 transcripts.

An object of the present invention is to provide an antisense guide RNA for editing a polyadenylation signal sequence of a target RNA and a nucleic acid that encodes the guide RNA, which are expected to control expression of the target RNA with high efficiency. The present inventors have intensively examined technologies for controlling expression of a target RNA with high efficiency, thereby completing the present invention by discovering a guide RNA for ADAR-dependent editing of the target RNA, including an antisense region complementary to a portion of the target RNA including a polyadenylation signal sequence.

In some aspects, the present invention provides methods and compositions for modifying target sites within nucleic acid molecules. In some embodiments, the methods comprise using adenosine deaminases that act on RNA (ADARs), and variants thereof, to modify target sites within DNA-RNA hybrid molecules. In other aspects, ADAR2 variant polypeptides as well as fusion proteins comprising an ADAR catalytic domain and a hybrid nucleic acid binding domain are provided, as are methods for use thereof. Methods for preventing and treating genetic disorders are also provided herein.

In alternative embodiment, provided are methods and compositions for treating, ameliorating or preventing diseases and conditions, such as cancer, including cancers associated with stem cells such as, without limitation, myelodysplastic syndrome (MDS) and a myeloproliferative neoplasm like chronic myeloid leukemia (CML) or acute myeloid leukemia (AML), and ablating or killing cancer stem cells. In alternative embodiment, provided are a new set of biomarkers to detect leukemia stem cell reprogramming and CML progression. In alternative embodiment, provided are therapeutic targets for treating myelodysplastic syndrome (MDS) and chronic myeloid leukemia (CML) by targeting edited let-7 transcripts.

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity of RNA-programmable endonucleases, such as Cas9, or for controlling the activity of proteins comprising a Cas9 variant fused to a functional effector domain, such as a nuclease, nickase, recombinase, deaminase, transcriptional activator, transcriptional repressor, or epigenetic modifying domain. For example, the inventive proteins provided comprise a ligand-dependent intein, the presence of which inhibits one or more activities of the protein (e.g., gRNA binding, enzymatic activity, target DNA binding). The binding of a ligand to the intein results in self-excision of the intein, restoring the activity of the protein.

The invention relates to methods and substances for the targeted alteration of genetic information on an RNA level. The substances are artificially produced guide RNAs, which are capable of recruiting endogenous editing enzymes, such as hADAR enzymes, in particular hADAR2 and hADAR1, in order to introduce targeted point mutations in selected mRNAs. The guide RNA consists of multiple segments and is constructed in such a way that individual nucleotides from different segments pair to form a double helix, and the nucleotides of a determined segment form a hairpin structure within the guide RNA. The invention also relates to the method for directed RNA editing, wherein the guide RNA is transfected into the cells in which the RNA editing is to be carried out. The substances and the method can be used for repairing individual, e.g. disease-relevant point mutations, such as those leading to premature stop signals. An advantage of the invention is that endogenous editing enzymes are also used in order to introduce targeted point mutations into the RNA. Only the short guide RNA, used for recruiting endogenous editing enzymes, must be artificially produced for each specific problem and ectopically expressed.