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

Choi et al. (2014). Mol Brain DOI: 10.1186/1756-6606-7-17 Citations: 196

AAV's ~4.7 kb limit is squeezed by regulatory elements like WPRE and polyA signals. This study shrank WPRE to 247 bp and paired it with a short SV40 polyA signal. The resulting CW3SL cassette matched full expression while freeing 399 bp for larger transgenes.

Parts used

WPREbGH PolyA SignalEGFPtdTomato

Key findings

  • The WPRE3 element, containing minimal gamma and alpha regulatory sequences, reduced WPRE size from 600 bp to 247 bp (41.2% of original) while maintaining 83.4% transgene expression efficiency compared to full-length WPRE in cultured hippocampal neurons. Western blot analysis showed chimeric intron insertion yielded only 16.6% expression efficiency, while complete WPRE deletion reduced expression to 28.6%, confirming WPRE3 as optimal for balancing size reduction with maintained posttranscriptional enhancement.

  • The CW3SL cassette, combining WPRE3 with SV40 late polyadenylation signal plus tandem upstream element, achieved 103.4% expression efficiency relative to the original CWB cassette (WPRE + bGHpA) while reducing element size by 399 bp. Systematic comparison of six polyadenylation configurations showed CW3SL outperformed synthetic polyA signals (49.7-76.4% efficiency) and matched performance of 223 bp bGHpA using only 169 bp total sequence. In vivo hippocampal CA1 injections confirmed 86.2% expression efficiency compared to CWB.

  • The optimized CW3SL cassette enabled packaging of p110γ-EGFP fusion protein (4.03 kb coding sequence, 5.2 kb total construct) within AAV's strict 5.2 kb packaging limit, whereas CWB-p110γ-EGFP exceeded capacity at 5.6 kb total length. Despite comparable viral titers (6.61×10¹² vs 7.01×10¹² GC/ml), CW3SL drove 5-fold higher p110γ-EGFP expression than CWB. The 399 bp reduction expands available coding capacity from 3.6 kb to 4.0 kb, encompassing 488 additional hippocampal transcripts (1.82% of expressed genes).

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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.