Figure 1.
Schematic illustration of the overlapping and trans-splicing mechanisms to express genes that exceed the packaging capacity of a single AAV vector. (A) Reconstitution of oversized transgene expression cassettes from two vectors with partially homologous genome sequences. In this mechanism, the first vector contains the promoter and the 5′ half of the coding sequence and the second vector contains the 3′ half of the coding sequence and the polyadenylation signal. The 3′ region of the coding sequence of the first vector and the 5′ region of the second vector are homologous. A full-length cDNA expression cassette that includes all the regulatory elements is then reconstituted through homologous recombination of the overlapping regions of the two vectors. (B) Expression of full-length, oversized transgene cassettes through so-called trans-splicing. In this approach, the first vector contains the promoter and the first exon followed by a splice donor sequence (SD). The second vector contains a 5′ splice acceptor sequence (SA) followed by the second exon and a polyadenylation signal. After uncoating and second-strand synthesis, AAV genomes form linear or circular, mostly head-to-tail, concatamers. This concatamerization of a right-hand ITR (shown in red) from the first vector with a left-hand ITR (shown in blue) from the second vector reconstitutes a full-length transcription cassette. Transcription then generates a pre-mRNA that contains the entire cDNA interrupted by an ITR-intron (dashed magenta box) followed by a poly A tail. Splicing leads to the removal of the ITR-intron and the formation of the full-length, mature mRNA. Color image available online at www.liebertpub.com/hgtb