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. 2014 Mar 11:7:17.
doi: 10.1186/1756-6606-7-17.

Optimization of AAV expression cassettes to improve packaging capacity and transgene expression in neurons

Jun-Hyeok Choi, Nam-Kyung Yu, Gi-Chul Baek, Joseph Bakes, Daekwan Seo, Hye Jin Nam, Sung Hee Baek, Chae-Seok Lim, Yong-Seok Lee, Bong-Kiun Kaang  1
Affiliations

Optimization of AAV expression cassettes to improve packaging capacity and transgene expression in neurons

Jun-Hyeok Choi et al. Mol Brain. .

Abstract

Adeno-associated virus (AAV) vectors can deliver transgenes to diverse cell types and are therefore useful for basic research and gene therapy. Although AAV has many advantages over other viral vectors, its relatively small packaging capacity limits its use for delivering large genes. The available transgene size is further limited by the existence of additional elements in the expression cassette without which the gene expression level becomes much lower. By using alternative combinations of shorter elements, we generated a series of AAV expression cassettes and systematically evaluated their expression efficiency in neurons to maximize the transgene size available within the AAV packaging capacity while not compromising the transgene expression. We found that the newly developed smaller expression cassette shows comparable expression efficiency with an efficient vector generally used for strong gene expression. This new expression cassette will allow us to package larger transgenes without compromising expression efficiency.

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Figures

Figure 1
Figure 1
Comparison of expression cassettes with modification or deletion of WPRE. A. Schematic drawings of expression cassettes tested. Each EGFP expression cassette was transduced into hippocampal neuron cultures together with tdTomato expression cassette, which contains complete WPRE and bGHpA. (ITR, Inverted Terminal Repeat; CaMKIIp, CaMKIIα promoter; WPRE, Woodchuck hepatitis virus posttranscriptional regulatory element, bGHpA: bovine growth hormone polyadenylation signal, INT: chimeric intron) B-C. Representative images of cell fluorescence (B), and western blot analysis of EGFP and tdTomato expression (C). Scale bar, 200 μm. D. Summary graph of western blot analysis shows that the expression level of EGFP driven by CW3B having WPRE3 (247 bp) is comparable (83.4%) to CWB having WPRE (600 bp). In contrast, EGFP expression level was significantly reduced in CIB (16.6%, P < 0.001) and CB (28.6%, P < 0.001) compared to CWB. (***P < 0.001, ANOVA Bonferroni’s post-hoc comparison).
Figure 2
Figure 2
Comparison of transgene expression with modification of polyadenylation signal. A. Schematic drawings of EGFP expression cassettes having modifications in polyadenylation signal. (L: SV40 late polyadenylation signal; SL: SV40 late polyadenylation signal upstream element + SV40 late polyadenylation signal; A: synthetic polyadenylation signal, SA: SV40 late polyadenylation signal upstream element + synthetic polyadenylation signal; SSA: double copy of SV40 late polyadenylation signal upstream element + synthetic polyadenylation signal). B-C. Representative images of fluorescence (B) and western blot analysis (C) of EGFP and tdTomato expression. Scale bar, 200 μm. D. Summary graph shows that CW3SL containing WPRE3, an additional SV40 late polyadenylation signal upstream element followed by SV40 late polyadenylation signal, drives highest EGFP expression level. CW3B and CW3L drive significantly higher expression than CW3A (P < 0.01), while CW3SL drives significantly higher expression than CW3A (P < 0.001), CW3SA (P < 0.01), and CW3SSA (P < 0.05) (*P < 0.05, **P < 0.01, ***P < 0.001, ANOVA Bonferroni’s post-hoc comparison).
Figure 3
Figure 3
The plot of optimized elements’ size against transgene expression efficiency of each expression cassette. A. Common scheme of the expression cassette generation in this study. The target element sites varied for optimization are indicated. B. The normalized EGFP expression level by each cassette plotted against the size of each expression cassette with modification in WPRE and bGHpA sites. Note that size reduction of about 399 bp in these two elements in CW3SL still gives similar level of transgene expression as that of CWB.
Figure 4
Figure 4
Transgene expression in the mouse brains with CW3SL expression cassette. Either CWB-EGFP (A) or CW3SL-EGFP (B) was injected together with CWB-tdTomato into the hippocampal CA1 region. The expression levels of EGFP and tdTomato were detected by confocal fluorescence imaging. Scale bar, 200 μm.
Figure 5
Figure 5
Utilization of CW3SL expression cassette for large transgene expression. A. Western blot analysis of p110γ-EGFP fusion protein expression by CWB-p110γ-EGFP and CW3SL-p110γ-EGFP. B. Quantification of the western blot analysis. p110γ-EGFP expression level was normalized to that of tubulin, which was then normalized to the average expression level by CWB-p110γ-EGFP. C. The percentage of transcripts in mouse hippocampus with coding sequence length within every 0.4 kb. Transcripts expressed in mouse hippocampus were selected based on RNA sequencing data (analyzed based on Ensembl Genes version 67, NCBIM37). The transcripts in the range of 2.4-4 kb (striped bars) and those in the range of 3.6-4 kb (darker and striped bar) that exceed the packaging capacity of conventional AAV expression cassettes and CWB cassette, respectively, become available using CW3SL cassette.
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