Fig. 1: Dual Vectors are inefficient.

A Schematic of recombinant adeno-associated viral (rAAV) vector genome (VG) processing. Single-stranded DNA VGs are converted to double-stranded (ds)DNA and VGs circularize and concatenate by inverted terminal repeat (ITR)-ITR joining, through unclear mechanisms. VGs associate with histones, but when this occurs during these processes is unknown. B The dual vector approach can double payload size by exploiting the natural concatenation process. A large gene is split into separate Left (L) and Right (R) VGs; inclusion of a splice donor (SD)/acceptor (SA) excludes the ITR junction from the final transcript. C Reporter constructs to assay split transgene reconstitution. LacZ is split between L and R VGs in a trans-splicing (TS) pair that requires ITR-ITR concatenation for expression, or a Hybrid (HB) trans-splicing plus Alkaline Phosphatase (AP) partial sequence previously demonstrated to increase split transgene reconstitution efficiency. An unsplit LacZ VG is used as a control. CMV = cytomegalovirus. BSD = Blasticidin resistance. pA = poly A signal. F2A, P2A, T2A = 2A self-cleaving peptides. D LacZ Activated Fluorescence Assay (LAFA) schematic: cultured cells are transduced with equimolar doses of L and R vectors. After 48 h, split transgene reconstitution is assayed by Beta-galactosidase (LacZ) activity cleaving 4-Methylumbelliferyl-beta-D-galactopyranoside (MUG), a fluorogenic beta galactose analog. E U2-OS cells were treated with 1e4 VG/cell of the split pairs or unsplit LacZ diagrammed in (C). LAFA was performed at 48 hours post transduction and quantified by flow cytometry: L + R pairs (magenta, purple), unsplit vector (blue, green), or empty capsid (black) negative control are plotted as a histogram. All capsids and ITRs are AAV2.