The present disclosure relates to nucleobase editors and methods of use thereof. Disclosed herein are fusion proteins, systems, and compositions for editing disease-associated mutations and methods of use thereof. In some aspects, disclosed herein is a fusion protein comprising a Cas9 nickase and a nucleotide deaminase, wherein the Cas9 nickase comprises a first amino acid substitution at a position selected from the group consisting of 262, 324, 409, 480, 543, 694, and 1219 when compared to SEQ ID NO: 11, and wherein the Cas9 nickase comprises a second amino acid substitution at a position selected from the group consisting of 1111, 1135, 1218, 1219, 1322, 1335, and 1337 when compared to SEQ ID NO: 11.

The present invention provides a genetically modified NK cell line prepared by transducing host NK cell line with a gene construction encoding a cancer antigen-specific chimeric antigen receptor (CAR) comprising a FLT3-specific monoclonal antibody or a functional fragment thereof, a transmembrane domain, and a CD3ζ domain of a T-cell receptor for more efficient immunotherapy of acute myeloid leukemia, and use thereof, for more efficient immunotherapy of acute myeloid leukemia.

Provided herein are systems, compositions, and methods for the incorporation of unnatural amino acids into proteins via nonsense suppression or rare codon suppression. Nonsense codons and rare codons may be introduced into the coding sequence of a protein of interest using a CRISPR/Cas9-based nucleobase editor described herein. The nucleobase editors are able to be programmed by guide nucleotide sequences to edit the target codons in the coding sequence of the protein of interest. Also provided are application enabled by the technology described herein.

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.

The disclosure relates to fusion proteins, methods of making fusion proteins, and methods of using fusion proteins, wherein the fusion proteins comprise a functional single-domain antibody (sdAb) or a functional variant thereof and a cytosine deaminase (CD) protein or a functional variant thereof, optionally connected via a peptide linker. The fusion proteins of the disclosure also have CD activity. The disclosure also relates to pharmaceutical compositions or formulations comprising such fusion proteins and pharmaceutically acceptable excipients, as well as medical uses of these fusion proteins.

The present invention pertains to a pharmaceutical composition comprising a recombinant measles virus encoding a suicide gene for use in the treatment of malignant cells with primary or secondary resistances against an oncolytic measles virus without suicide gene activity. Further, the present invention pertains to a recombinant measles virus based on measles vaccine strain Schwarz encoding a suicide gene, which comprises a fusion of a cytosine deaminase, particularly yeast cytosine deaminase, and a uracil phosphoribosyltransferase, particularly yeast uracil phosphoribosyltransferase, to a method and a kit for preparing the recombinant measles virus as claimed herein.

The invention relates generally to methods of generating induced pluripotent stem cells (iPSCs) that do not contain the reprogramming vector. In some embodiments, the invention relates to inducing pluripotency in somatic cells by introducing an episomal vector(s) comprising at least one expression cassette containing reprogramming factors and/or synthetic transcription factors and a suicide gene. In some embodiments, the invention relates to inducing pluripotency in somatic cells by introducing episomal vector(s) comprising expression cassettes containing reprogramming factors and/or synthetic transcription factors and a transcriptionally regulated EBNA-1 gene. In some embodiments, the invention relates to inducing pluripotency in somatic cells by introducing episomal vector(s) comprising expression cassettes containing reprogramming factors and/or synthetic transcription factors and both a suicide gene and a transcriptionally regulated EBNA-1 gene.

Described herein are methods for transfecting mesenchymal stem cells (MSCs) with a nucleic acid construct using a cationic polymer, a first reagent capable of redirecting endocytosed nucleic acids from intracellular acidic compartments, and a second agent capable of stabilizing a microtubular network of the MSCs. The methods are free of virus-based transfection vehicle materials and the transfected MSCs have substantially unchanged multipotent phenotype. In certain embodiments, the transfected MSCs express functional genes comprising suicide gene, such as cytosine deaminase or uracil phosphoribosyltransferase. Also described are methods for the treatment of diseases, such as cancer, using such transfected cells in combination with 5FC, 5FU, GCV, as well as kits and composition relating thereof.

An object of the invention is to provide a novel pharmaceutical composition. The pharmaceutical composition of the disclosure contains a DNA encoding a suicide gene having at least one intron sequence. The intron sequence has a donor sequence or an acceptor sequence to be used in a tumor cell with abnormal splicing not in a normal cell. In a transcript of the DNA, the suicide gene is expressed when the intron is abnormally spliced and the suicide gene is not expressed when the intron is not abnormally spliced.

Engineered base editor variants with reduced RNA editing activity, and methods of using the same.