Figure 2.

Alternative approaches for the delivery for large cilial genes. (A) Dual AAV trans-splicing: Two independently delivered viral genomes containing either the 5′ or 3′ coding sequence (CDS) recombined via concatemerization or homologous recombination mediated by the homology recombination domains (HRDs). The inclusion of splice donor (SD) and splice acceptor (SA) sequences permits RNA splicing to produce a mature mRNA encoding for full-length protein. (B) Protein trans-splicing: The CDS is split across two independent viral vectors resulting in the expression of either N- or C-terminal polypeptides of the desired full-length protein. Adjoined to these peptides are either the N- or C-terminal of a split-intein; post-translationally, the split-intein spontaneously recombines and self-excises, resulting in full-length protein. (C) mRNA trans-splicing: dual AAVs result in the transcription of two independent mRNAs encoding each half of the desired sequence, and the inclusion of complementary binding domains (BDs) allows for recombination. Flanking SD and SA sites facilitate RNA splicing and the removal of the BD sequences, resulting in a mature, full-length mRNA. (D) High-capacity adenovirus has a large cargo capacity of up to 36 kb; this facilitates the delivery of large genes in their entirety with a single vector. Promoter (blue arrow); polyadenylation signal (pA) (created with BioRender.com).