Adeno-Associated Viral Gene Therapy for Retinal Disorders - Gene Delivery and Therapy for Neurological Disorders - page 212

Fig. 2 Strategies for production of transgenes >5 kb. ( a ) The partially packaged strategy whereby the virus
packages an undefi ned length of DNA from each 3 end of the ITRs, resulting in capsids carrying varying

lengths of genomes. Those containing overlapping segments have the potential to form the full-length trans-
gene; ( b ) the overlapping strategy in which two defi ned fragments of the larger transgene are packaged
between two ITRs, the resulting genomes undergo homologous recombination within the host cell to form the
full-length transgene; ( c ) the trans-splicing approach in which the large transgene is again split into two
defi ned fragments, but this time the generation of the full-length transgene depends on concatamerization
between the two viral genomes following which the unwanted ITR junction is spliced out via the splice donor
(SD) and splice acceptor (SA) sites

′

3. Trans -splicing approach (Fig. 2c )

A variant on the overlapping approach is the trans -splicing
method. This involves splitting the transgene into two 5 kb cas-

settes and packaging them separately, as in the alternative overlap-
ping approach. The difference is the addition of splice donor/
acceptor sites so that the fi rst transgene carries a splice donor site
following on from the split CDS and the second transgene carries
a splice acceptor site prior to the split CDS. Following cotransduc-
tion, the AAV genomes are believed to undergo a recombination
via the ITRs with the splice signals allowing for subsequent removal
of the ITRs, creating a complete transgene exceeding 5 kb; proof
of principle of the trans -splicing approach has been shown in

≤