The CRISPR system uses Cas9 complexed with a user-defined guide RNA (gRNA) to recognize and cut complementary sequences. The DSBs enhance homologous recombination to insert transgenes at specific sequences but off-target DNA cleavages at unknown remote sites cause inadvertent mutations that require complex genotoxicity screens for detection. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas9) and transcription activator-like effector (TALE) nuclease (TALEN) systems induce double-stranded DNA breaks (DSBs). Nucleases are also being evaluated for use in non-viral human gene therapy. Non-viral vectors have been designed by modifying the surface of a non-viral vector for targeted therapy. Compared to viral vectors, non-viral vectors are generally safer to prepare, and the risk of pathogenic and immunologic complications is diminished. Non-viral vectors (i.e., lipid-based, polymer-based, lipid-polymer based, and poly-lysine) are synthetic tools for encapsulating transgenic DNA or RNA until it reaches the cellular target. In fact, re-administration of viral-based vectors can promote immune responses that can result in life threatening systemic effects and limit gene-transfer efficacy. Accordingly, limitations of viral vectors such as pathogenicity, expensive production, and systemic instability have proved to be major obstacles to the use of viral-based systems. liver growth or other organ growth), limiting therapeutic efficacy. Also, because the AAV genome mainly persists in an episomal form in the nucleus of the infected cells, it can be lost in conditions of cell proliferation (such as, e.g. Viral vector-host interaction can include immunogenicity, and integration of a viral vector DNA in a host genome may have genotoxic effects. Viral systems are also limited in cargo size, restricting the size and number of transgenes and their regulatory elements. Thus, adenoviruses and adeno-associated viruses (AAV) have been shown to evoke host human responses that limit administration or re-administration, while retroviruses/lentiviruses preferentially integrate transgenes into euchromatin thereby increasing the risk of insertional mutagenesis or oncogenesis. Although it is possible to achieve stable genomic integration with high-efficiency using viral vectors, multiple studies have shown serious disadvantages and safety concerns. The ability to safely and efficiently integrate genes into a host genome is essential for successful gene therapy in humans.Ĭurrently, the most commonly used vectors for permanent or transient transfer of genes in gene therapy trials are virus-based. Gene therapy involves replacing or complementing a mutated gene (which causes a disease) with a healthy copy of the gene, inactivating or silencing a mutated gene that is functioning improperly (or any other gene), or introducing a new gene into chromosomes. Human gene therapy is a promising approach that delivers genes for treating and mitigating various diseases and conditions, including inherited and acquired diseases. The Sequence Listing is incorporated herein by reference in its entirety. The ASCII copy, created on May 3, 2021, is named SAL-003PC_ST25.txt and is 182,990 bytes in size. This application contains a Sequence Listing in ASCII format submitted electronically herewith via EFS-Web. DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY 15, 2021, the entirety of each which is incorporated by reference herein. The present application claims priority to and benefit from U.S. The present invention relates, in part, to a dual system using enzymes capable of transposition (e.g., engineered transposases and/or chimeric transposases) and transposons for targeting human genomic safe harbor sites (GSHS).
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