Provided herein are methods for facilitating or inducing stable transgene integration and expression in a proliferating cell, comprising administering to the cell (i) a recombinant AAV (rAAV) vector comprising the transgene flanked by transposon-derived inverted terminal repeat sequences, which sequences are in turn flanked by AAV-derived inverted terminal repeat regions, and (ii) a source of a transposase that recognises said transposon-derived inverted terminal repeat sequences and directs the genomic integration of the transgene into the genome of the proliferating cell. Also provide are methods and transgene delivery systems for the treatment or prevention of diseases affecting, associated with or characterised by proliferating cells.

The present invention provides methods and compositions for stable genetic modification of immune cells. The genetic modifications can be used to produce immune cells for therapeutic or diagnostic purposes.

An in vitro method for integrating an exogenous DNA sequence into the genome of a cell using a transposon system. The transposon system includes a first vector carrying inverted repeat sequences and an exogenous DNA, and a second vector that expresses a transposases. Also provided is a kit including the transposon system for integrating an exogenous DNA into the genome of a cell. A method for treating a subject is described that includes engineering an immune cell to carry an exogenous DNA sequence using the method and/or the kit described above and administering an effective amount of the engineered immune cell to the subject.

Aspects of the disclosure relate to compositions and methods for expressing one or more Ganglioside GM3 synthase (GM3S) isoforms in a cell or subject. In some aspects, the disclosure relates to methods for treating GM3 synthase deficiency in a subject in need thereof.

Genome editing tools for use in systems designed to deliver large genetic elements are disclosed herein. A genome editing system is described, which includes i) an R2 element enzyme or other non-LTR site specific retrotransposon element and ii) a payload RNA, wherein the payload RNA comprises an insertion region and optionally one or more of a 5′ homology region, a 3′ homology region, and a protein binding element, wherein the insertion region comprises a template for a small or large nucleic acid insertion into the genome, and wherein the R2 element enzyme or other non-LTR site specific retrotransposon element comprises a targeting domain, a reverse transcriptase domain, and a nickase domain. Also disclosed are cells edited using such a genome editing system, methods for editing a genome, and compositions comprising cells edited with this genomic editing system.

Two or more conditional, dominant, lethal gene expression systems provide high levels of penetrance in insects. Lethality is induced at an earlier stage of development and the risk of biochemical resistance is reduced, as compared to a single insect conditional, dominant, lethal gene expression system. The invention is useful for the control of insect populations.

The Sleeping Beauty (SB) transposon is an efficient non-viral tool for inserting transgenes into cells. Its broad utilization in gene therapy has been hampered by uncontrolled transposase gene activity and the inability to use transposase protein directly. The present invention concerns the finding that SB transposase spontaneously penetrates mammalian cells and can be delivered with transposon DNA to gene-modify various cell lines, embryonic, hematopoietic and induced pluripotent stem cells. The invention provides methods and compounds to apply the cell penetrating function of transposase in methods of genetically engineering cells as well as using the transposase as a shuttle for delivering cargo into a target cell or even into a target cell organelle. Genomic integration frequency can be titrated using the technology of the invention, which adds an additional layer of safety, opening opportunities for advanced applications in genetic engineering and gene therapy.

Provided are compositions and methods for transposon-CRISPR-based DNA editing. The compositions and methods include a modified TnsA protein that is altered from its original sequence found in Aeromonas salmonicida, and provides improved transposition frequency, and is functional in a heterologous system. The TnsA protein is used in a system with transposon proteins TnsB, TnsC, TniQ, Cas proteins Cas8f, CasSf, Cas7f, Cas6f, a transposable DNA cargo sequence that is flanked by left and right transposon sequences, and at least one guide RNA that contains at least one spacer targeted to a target DNA sequence in the chromosome or the extrachromosomal element.

The present invention relates to a polypeptide comprising a piggyBac transposase or a fragment or a derivative thereof having transposase function comprising at least one amino acid substitution. Further, the present invention relates to a transposable element comprising a piggyBac or piggyBac-like left repeat sequence and left internal repeat sequence, wherein the left internal repeat sequence comprises at least one nucleotide modification. Furthermore, the present invention relates to a kit comprising the above transposase and/or transposable element. In addition, the present invention relates to a targeting system comprising the above transposase and/or transposable element.

Gene therapy compositions and methods are provided that make use of novel transposases and/or chimeric transposases for targeted transposition.