Methods and compositions for modifying the coding sequence of endogenous genes using rare-cutting endonucleases and transposases. The methods and compositions described herein can be used to modify the coding sequence of endogenous genes.

An adenovirus comprising a sequence of formula (I) 5′ITR-B.sub.1-B.sub.A-B.sub.2-B.sub.X-B.sub.B-B.sub.Y-B.sub.3-3′ITR wherein B.sub.Y comprises a transgene cassette containing four transgenes, said genes encoding a FAP-Bispecific T cell activator, CXL10, CXL9, and IFN. The disclosure also extends to a pharmaceutical composition comprising the virus, and use of the virus or formulation in treatment.

A new promoter comprising: (i) an hCMV enhancer sequence; (ii) an hCMV promoter sequence; (iii) a splice donor region; (iv) a cell-derived enhancer sequence; and (v) a splice acceptor region.

Disclosed are methods and constructs for engineering circular RNA. Disclosed is a vector for making circular RNA, said vector comprising the following elements operably connected to each other and arranged in the following sequence:

a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) optionally, a 5′ spacer sequence, d.) a protein coding or noncoding region, e.) optionally, a 3′ spacer sequence, f) a 5′ Group I intron fragment containing a 5′ splice site dinucleotide, and g.) a 3′ homology arm, said vector allowing production of a circular RNA that is translatable or biologically active inside eukaryotic cells. In another embodiment, the vector can comprise the 5′ spacer sequence, but not the 3′ spacer sequence. In yet another embodiment, the vector can comprise the 3′ spacer sequence, but not the 5′ spacer sequence. Also disclosed is a method for purifying the circular RNA produced by the vector and the use of nucleoside modifications in circular RNA produced by the vector.

A circular RNA molecule generally includes at least one coding region and an internal ribosome entry site (IRES) operably linked to the coding region. The RNA may be more resistant to digestion by an RNA endonuclease that a linear form of the circular RNA. In another aspect, a polynucleotide generally includes a transcription unit and a promoter operably linked to the transcription unit. The transcription unit includes a circularizing element, at least one coding region and an internal ribosome entry site (IRES) operably linked to the coding region. When transcribed by a cell, the transcribed RNA forms a circular RNA molecule.

Methods of creating mutations in genomic exons by inserting introns into the genomic exons via homologous recombination. Also, methods are provided for introducing modifications into genomic exons by inserting introns into the genomic exons via homologous recombination such that a mature mRNA transcript produced from a genomic region of the genome comprising the genomic exon does not contain the modification are provided. The methods provide for a rapid method for introducing mutations and/or modifications of any type into a mammalian cell genome.

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.

Adeno-associated virus (AAV) Clade F vectors or AAV vector variants (relative to AAV9) for precise editing of the genome of a cell and methods and kits thereof are provided. Targeted genome editing using the AAV Clade F vectors or AAV vector variants provided herein occurred at frequencies that were shown to be 1,000 to 100,000 fold more efficient than has previously been reported. Also provided are methods of treating a disease or disorder in a subject by editing the genome of a cell of the subject via transducing the cell with an AAV Clade F vector or AAV vector variant as described herein and further transplanting the transduced cell into the subject to treat the disease or disorder of the subject. Also provided herein are methods of treating a disease or disorder in a subject by in vivo genome editing by directly administering the AAV Clade F vector or AAV vector variant as described herein to the subject.

The invention provides a splice control module for sex-specific splicing and expression of a gene of interest. In certain embodiments, a dsx-based splice control module is used to express a lethal gene in an insect that is spliced in a sex-specific manner to impart lethality to female insects but not male insects.

Provided herein are compositions that include at least two different nucleic acid vectors, where each of the at least two different vectors includes a coding sequence that encodes a different portion of an otoferlin protein, and the use of these compositions to treat hearing loss in a subject.