Provided herein are systems, compositions, and methods of introducing protective and/or loss-of-function variants of CCR5 and CCR2. Variants may be introduced using a CRISPR/Cas9-based nucleobase editor or other guide nucleotide sequence-programmable DNA binding protein domain-based fusion protein described herein. Further provided herein are compositions and methods of preventing and treating conditions related to HIV infection and progression as well as to AIDS.

Disclosed is a gene therapy vector in which the occurrence of recombination is minimized. In order to minimize the occurrence of recombination, which is a major problem in the production and infection of a retroviral vector virus that continuously expresses a therapeutic gene during virus replication, a cleaved MCMV promoter was prepared by cutting the MCMV promoter on the basis of a repeat sequence, and the cleaved MCMV promoter was introduced to prepare a vector. It was confirmed that the vector having the cleaved MCMV promoter incorporated therein does not cause recombinations even after being incubated multiple times, and shows a continuous expression of the therapeutic protein, and in cells transfected with the virus containing the vector, cell death effectively occurs when a prodrug is administered thereto. Accordingly, the vector with minimized recombination occurrence of the present invention can be advantageously used for the treatment of cancer.

The present disclosure relates compositions, systems, and methods for treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer or a proliferative disease using a selection gene drive therapy.

The disclosure provides methods and related kits, reagents, and systems for selectively deaminating unmethylated cytosine residues in nucleic acid molecules. In some embodiments, the methods and related kits, reagents, and systems are applied for methods of detecting and/or mapping methylated cytosine residues in nucleic acids. The nucleic can be RNA or DNA. Some embodiments include contacting the polynucleic acid with a bacterial cytosine deaminase, for example DddA or SsdA, or functional fragments or derivatives thereof. Representative DddA and SsdA have sequences set forth in SEQ ID NOS:1 and 2, respectively. The bacterial cytosine deaminases of the disclosure are sensitive to methylation and, thus, deaminate only unmethylated cytosines to provide a cytosine to uracil conversion. The conversion can be detected as a CG-to-TA transitions in subsequent sequencing analysis.

Disclosed are compositions and methods for treating a disease or disorder such as cancer in a subject in need thereof. In some aspects, the method comprises administering to the subject a vector comprising a first nucleic acid sequence encoding a promoter operably linked to each of a second nucleic acid sequence encoding a therapeutic polypeptide, and a third nucleic acid sequence encoding a peptide domain that is stabilized when phosphorylated by kinase activity in a target tissue. The kinase activity can be elevated extracellular regulated kinase (ERK) activity.

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.

Disclosed herein are methodologies and kits for dynamic targeted hypermutation that harness the enzymatic activity of a polynucleic acid-binding protein fused to a nucleobase-editing enzyme to specifically target mutations across a region of interest. These methodologies and kits facilitate the rapid creation of diverse DNA libraries in vivo or in vitro.

The present invention provides novel systems, methods and compositions for making and using recombinantly engineered novel Cas9 enzymes optimized for human cells, for nucleic acid targeting and manipulation. The present invention is based on the discovery of novel Cas9 enzymes from Streptococcus constellatus, Sharpen spp. isolate RUG017, Veillonella parvula, Ezakiella peruensis, Lactobacillus fermentum strain AF15-40LB strain and Peptoniphilus sp. Marseille-P3761 bacteria that were codon-optimized and recombinantly produced for use in human cells. In some embodiments, novel Cas9 enzymes can be used for base editing. In some embodiments, the novel engineered Cas9 enzymes are used to treat human diseases.

A method is disclosed for decreasing or retarding an increase in the size of a localized or metastatic tumor by using a combination of an immune stimulating cytotoxic gene therapy and immune-checkpoint modulating agent, in conjunction with other therapies, including radiation therapy, chemotherapy, surgery, and immunotherapies.