Provided herein are methods of delivering “split” Cas9 protein or nucleobase editors into a cell, e.g., via a recombinant adeno-associated vims (rAAV), to form a complete and functional Cas9 protein or nucleobase editor. The Cas9 protein or the nucleobase editor is split into two sections, each fused with one part of an intein system (e.g., intein-N and intein-C encoded by the dnaE-n and dnaE-c genes, respectively). Upon co-expression, the two sections of the Cas9 protein or nucleobase editor are ligated together via intein-mediated protein splicing. Nucleic acid molecules encoding the N-terminal portion of a Cas9 protein or a nucleobase editor fused to an intein, and nucleic acid molecules encoding the C-terminal portion of a Cas9 protein or nucleobase editor, are provided. Recombinant AAV vectors (e.g, vectors comprising one or more of these nucleic acid molecules each comprising an intein) and particles for the delivery of the split Cas9 protein or nucleobase editor, compositions comprising such AAV vectors and particles, and methods of using such rAAV vectors and particles are also provided. Methods of administering such compositions and AAV particles to a subject are further provided. Cells and compositions comprising these nucleic acid molecules rAAV vectors, and rAAV particles are also provided.

Means and methods for preparing combinatorial libraries of DNA constructs, in particular expression cassettes, including nucleic acid constructs, expression vectors, host cells, methods for preparing host cells, and methods for producing polypeptides of interest, whereby the expression comprises an first intron and a second intron on either side of the polynucleotide to be expressed and a promoter and a terminator. Also claimed is a method of constructing eukaryotic host cells in which the cells are contacted with three polynucleotides and in which the first and second and the second and third are pairwise capable of homologous recombination and of subsequent formation of introns.

The present invention relates to nucleic acid expression cassettes and vectors containing liver-specific regulatory elements and codon-optimized factor IX or factor VIII transgenes, methods employing these expression cassettes and vectors and uses thereof. The present invention is particularly useful for applications using liver-directed gene therapy, in particular for the treatment of hemophilia A and B.

Disclosed herein are nucleic acids comprising multifunctional double-stranded break reporter constructs for stable or transient transfection into a cell, as well as detecting a type of double-stranded break repair mechanism in a cell. Also provided are methods for detecting types of double-stranded break repair mechanism in a cell, as well as vectors and cells comprising nucleic acids comprising multifunctional double-stranded break reporter constructs.

The present disclosure provides systems and methods for modulating the occurrence of chromosomal rearrangements, e.g., translocations, in a cell during genome editing. Embodiments are provided for reducing the occurrence of unwanted chromosomal rearrangements, and for increasing the occurrence of desired chromosomal rearrangements.

A method of preparing a chimeric virus comprising bocavirus capsid protein (VP) and a recombinant adeno-associated (AAV) viral genome, and isolated mutant bocavirus genomes, are provided.

Recombinant constructs, cells and means for improved production of Adeno-Associated Viruses (AAVs) are described. Also described are methods of using the constructs and cells to produce recombinant AAVs.

The invention belongs to the field of biomedicine. Specifically, the present invention relates to improved therapeutic T cells and methods for their preparation. Specifically, the present invention relates to preparing improved therapeutic T cells by co-expression of an exogenous antigen-specific receptor protein and a dominant negative TGF- type II receptor in T cells through lentiviral vector transduction.

Herein is reported that for transient transfections the use of the human elongation factor 1 alpha promoter (with Intron A) provides for an enhanced productivity (in LC-HC-SM organization), the use of the bovine growth hormone polyA signal sequence provides for an enhanced productivity compared to use of the SV40 polyA signal sequence, the addition of the HUT to the bGH PolyA signal sequence results in an increased productivity in vectors containing the hCMV promoter and the vector organization LC(3-5)-HC-SM results in improved expression. For stable pools it is reported that pools generated with vectors containing the hEF1 promoter show an enhanced productivity in batch analysis, clones generated with vectors containing the hEF1 promoter show a reduced number of low producing clones, and clones generated with vectors containing the hEF1 promoter show a higher stability of IgG expression. For single clones it is reported that the vector organization with downstream position of selection marker (LC-HC-SM) has a positive effect on productivity of single clones and that clones generated with vectors containing the bGH polyA signal sequence and the hGT have higher productivities.

The present disclosure is related to an engineered nucleic acid encoding a post-poly A signal RNA 3 to a terminator for expression of protein, and/or non-coding RNA. Also provided herein are methods for reducing epigenetic silencing, genetic modification, transcriptional regulation of the engineered nucleic acid described herein.