Improving the hybrid adeno-associated viral vectors with the exonic splicing enhancer

Abstract

Adeno-associated virus (AAV) is a rising champion for gene therapy due to its powerful and safe gene delivery properties. Yet the small packaging capacity of AAV limits its application for large therapeutic genes. The hybrid dual vectors show promise in overcoming this limitation (Ghosh 2008). In this approach, the gene of interest is split into 5' and 3' parts. Each part is packaged in a separate AAV virion. The 5' vector carries a splice donor, and the 3' vector a splice acceptor. Both also share an overlapping sequence. Recombination occurs simultaneously under two pathways, either through homologous recombination mediated by the overlapping sequence or head-to-tail ITR-mediated recombination. The recombined genome is spliced to generate mature mRNA. In an effort to further improve the hybrid vectors, we replaced the alkaline phosphatase (AP) overlapping sequence in a previously published LacZ hybrid vector set with an exonic splicing enhancer (ESE) sequence. The ESE sequences are known to increase splicing efficiency (Skordis, 2003). We hypothesized that the ESE sequence should increase splicing efficiency in the hybrid vectors, hence, lead to greater gene expression. To test this hypothesis, we transfected 293 cells with a series of different LacZ constructs. We then measured LacZ protein expression by cytochemical staining and enzymatic assay. Green fluorescent protein (GFP) containing plasmid was co-transfected as an internal control for transfection efficiency. The ESE-intron LacZ, unmodified intact LacZ, intron LacZ and AP-intron LacZ yielded β-galactosidase (LacZ) activity of 475 ± 36, 361 ± 18, 283 ± 9 and 225 ± 35 units/µg protein, respectively. Next, we split the ESE-intron LacZ gene into two separate constructs, which can be used to make recombinant AAV. Encouragingly, co-transfection of the split LacZ ESE-intron constructs resulted in 5.61 ± 0.76 units/µg protein β-galactosidase activity while the previously described split LacZ AP-intron and split LacZ intron constructs resulted in 3.93 ± 0.36 and 3.41 ± 0.36 units/µg protein β-galactosidase activity, respectively. Our results suggest that the ESE-intron vector is operating at par with the intact gene and may provide better gene delivery than the current generation of hybrid vectors. Future study with the corresponding recombinant AAV viruses may further reveal the utility of ESE containing hybrid vectors.