Provided are compositions and methods for using the compositions to modify eukaryotic chromosomes. The methods involve iteratively inserting DNA payloads into a chromosomal locus, or into multiple chromosomal loci. The methods utilize positive and negative selection approaches in combination with one or more recombinases to select cells that contain a payload, eliminate cells that do not contain a payload, and sequentially replace contiguous segments of the chromosome with subsequent payload insertions. Modified cells, and modified mammals containing modified cells, are included.

A CRISPR-Cas system targeting a genomic target sequence in a eukaryotic cell and capable of promoting death of the eukaryotic cell is provided. A viral particle and an isolated eukaryotic cell including the CRISPR-Cas system, a method that uses the CRISPR-Cas system, and use of the CRISPR-Cas system as a medicament in the treatment of neoplastic diseases are also provided.

The present disclosure provides nucleic acid constructs for the treatment of cancer, comprising a cancer-specific promoter and one or more therapeutic genes.

The invention relates generally to a method of treatment for a human genetic disease, such as diseases characterized by unbalanced nucleotide pools, e.g., mitochondrial DNA depletion syndromes, and more specifically, thymidine kinase 2 (TK2) deficiency, using gene therapy. The gene therapy may involve administration of one or more constructs, such as a viral vector, containing a nucleic acid encoding a functional protein. The functional protein may correspond to a nuclear gene. For treatment of TK2 deficiency, the gene therapy may involve administration of one or more constructs, such as a viral vector, containing a nucleic acid encoding a functional TK2 enzyme. The treatment may also involve the administration of pharmacological therapy in conjunction with the gene therapy. The treatment protocols of the disclosure, such as those involving gene therapy alone or in combination with pharmacological therapy, can be used to treat, prevent, and/or cure various other disorders of unbalanced nucleoside pools, especially those found in mitochondrial DNA depletion syndrome.

Disclosed are a universal CAR-T cell knocking out one or more of CD3 delta, CD3 gamma, CD3 epsilon and CD3 zeta, and simultaneously introducing the HSV-TK gene. Also disclosed are a method for preparing the above-mentioned CAR-T cell, a preparation comprising the CAR-T cell, and the use of the CAR-T cell.

The present invention relates to a mutant thymidine kinase, specifically a mutant human thymidine kinase 1 (Tkl1). The activity of the mutant thymidine kinase is increased compared to the activity of wildtype thymidine kinase. Provided herein are uses of the mutant thymidine kinase in therapy, such as in cancer therapy. Also methods of treating cancer comprising administering the mutant thymidine kinase are disclosed herein. The present invention relates, inter alia, to a nucleic acid for use hi treating cancer, wherein said nucleic acid comprises a nucleotide sequence, wherein said nucleotide sequence encodes a mutant thymidine kinase.

A recombinant oncolytic virus with improved safety and anticancer effect and a use thereof are disclosed. The recombinant oncolytic virus is obtained by inserting an HSV-TK fragment-encoding gene into a TK gene region to delete TK of Vaccinia virus. The oncolytic virus expresses an HSV-TK fragment to phosphorylate GCV so that cancer cells infected with the oncolytic virus and their neighboring cancer cells can be killed. GCV is also involved in the suppression of viral proliferation and thus can control side effects caused by a virus even upon the administration of a high dose of the virus.

The present disclosure provides a replication-competent, recombinant oncolytic vaccinia virus, compositions comprising the vaccinia virus, and use of the vaccinia virus or composition for inducing oncolysis in an individual having a tumor.

Nucleic acid sequences encoding improved Herpes Simplex Virus Thymidine Kinases are provided, including their use in diagnostic and therapeutic applications. The thymidine kinases may be mutated using conservative mutations, non-conservative mutations, or both. Also provided are gene therapeutic systems, including viral and retroviral particles.

A DNA plasmid useful for diagnostic and therapeutic gene therapy is disclosed. Improvements to gene therapy methods known in the art are provided to ensure cancer-targeting, high efficacy, and long durability of expression. The DNA plasmid is combined with compositions of polymeric nanoparticles for non-viral gene therapy to treat cancer, including hepatocellular carcinoma and prostate cancer.