2014 Choi — Optimization of AAV expression cassettes to improve packaging capacity and transgene expression in neurons
WPRE3 (247 bp) achieves 83.4% of full-length WPRE (600 bp) expression in AAV-transduced neurons, enabling ~353 bp payload capacity gain while preserving posttranscriptional boost.
Citation
Choi JH, Yu NK, Baek GC, Bakes J, Seo D, Nam HJ, Baek SH, Lim CS, Lee YS, Kaang BK. “Optimization of AAV expression cassettes to improve packaging capacity and transgene expression in neurons.” Molecular Brain. 2014 Mar 11;7:17. DOI: 10.1186/1756-6606-7-17. PMID: 24618276. PMCID: PMC3975461. Open access.
NOTE: Paper is in Molecular Brain, NOT Mol Ther Methods Clin Dev as misidentified in the task brief. Kaang lab (Seoul National University). Freely accessible via PMC. Addgene plasmids deposited.
TL;DR
Systematically optimized AAV expression cassettes by shrinking or replacing regulatory elements (WPRE, polyA signals, promoters) to maximize transgene size capacity without sacrificing expression in neurons. Key finding: WPRE3 (247 bp, gamma+alpha regions of woodchuck hepatitis PRE) achieves 83.4% of full-length WPRE (600 bp) expression. Best cassette (CW3SL) achieves 103.4% of original expression with 399 bp total size reduction, expanding usable transgene capacity from 3.6 kb to ~4.0 kb.
Numbers that matter
| Parameter | Value | Units | Source (fig/table) | Uncertainty |
|---|---|---|---|---|
| WPRE3 size | 247 | bp | text/Table | exact — gamma+alpha fragments of WHV PRE |
| Full-length WPRE size | 600 | bp | text | exact |
| WPRE3 expression vs full WPRE (CW3B cassette) | 83.4% | % of control | Fig 3 | in neurons (CaMKIIα promoter) |
| CW3SL cassette expression (WPRE3 + SV40 late pA) | 103.4% | % of control CWB | Fig 4 | best-performing compact cassette |
| CW3SL total size reduction vs CWB | ~399 | bp | text | enables ~4 kb transgene vs ~3.6 kb |
| bGHpA size | 223 | bp | text | standard bovine growth hormone polyA |
| SV40 late pA size | 135 | bp | text | smaller alternative to bGHpA |
| CaMKIIα promoter (0.4 kb version) | ~400 | bp | text | neuron-specific; not used in cochlear context |
Method essentials
- Cell type: primary hippocampal neurons (rat), PC12 cells
- Assay: EGFP fluorescence intensity, flow cytometry, confocal imaging
- AAV serotype: AAV1 (for transduction); plasmid transfection for initial screens
- WPRE3 sequence: gamma region (γ) + alpha region (α) of woodchuck hepatitis virus PRE, excluding the potentially oncogenic X protein open reading frame
- Addgene: plasmids deposited for community use (see Addgene listing for this article)
Limitations
- Validated in neurons (CaMKIIα promoter context); not directly validated in cochlear OHCs
- 83.4% expression retention means ~17% expression loss vs full WPRE — acceptable for most applications
- In cochlear context (Omichi 2020 uses WPRE; Iranfar 2026 design uses WPRE3 variant per h03 script comment): direct cochlear OHC validation not in this paper
- WPRE3 retains some CpG content noted in h03 script: “partially depletable — Choi 2014 core has CpG-containing miRNA seed; some positions structurally constrained for RNA folding”
Relevance to STRC
Primary citation for WPRE3 (247 bp) used in the h03 Ultra-Mini construct (ultramini_vector_cpg_audit.py line 88-95). The 247 bp size is confirmed. The h03 script already correctly cites “Choi 2014 Cell 157” — note: the journal is Molecular Brain, not Cell 157 (Cell 157 refers to a different paper from 2014). The PMID 24618276 / DOI 10.1186/1756-6606-7-17 are the correct identifiers. Script journal citation needs correction: “Choi 2014 Mol Brain 7:17” not “Choi 2014 Cell 157.”
WPRE3 blocker is now closed: 247 bp confirmed from primary source.
Connections
[part-of]aav-cochlear-delivery[supports]STRC Ultra-Mini Promoter Shortlist[applies]h03 hub