ssAAV needs to complete complementary strand synthesis in the cell before forming a transcription template; scAAV folds to form a double-stranded structure, which usually works faster and is more stable, but the available expression cassette capacity is approximately halved. Conventional ssAAV design should calculate the ITR at both ends within a total load of about 4.7 kb; the non-ITR expression cassette of scAAV is usually controlled at about 2.2-2.4 kb. As the upper limit is approached, not only may yields decrease, but the proportion of truncated or heterogeneous genomes may increase. [5-1 to 5-4]
Processing sequence of ultra-long expression cassettes
|
Sequence |
Processing actions |
Judgment Criteria |
|
1 |
Recalculate the complete load |
All sequences between ITRs are counted; confirm promoter, intron, WPRE, tag and polyA |
|
2 |
Compression control element |
Prioritize to use a validated short promoter, short polyA or delete unnecessary tags |
|
3 |
Replacement of compact functional components |
Use shorter coding sequences, split editors, or more compact control systems |
|
4 |
Adopt dual AAV |
Clear the recombination mechanism, linker design, double-vector ratio and co-transduction bottleneck |
|
5 |
Change delivery platform |
When dual carriers cannot meet efficiency or consistency, overload packaging is no longer the default solution |
"Ability to detect packaging" does not equate to "resulting in a product that is uniform, complete and has the intended efficacy." Overloading vectors should be assessed for genomic integrity using long-read sequencing or orthogonal methods covering the entire expression cassette. [5-3]
References for this chapter
[5-1] McCarty DM. Self-complementary AAV vectors: advances and applications. Mol Ther. 2008;16:1648-1656. DOI:10.1038/mt.2008.171. [F]
[5-2] Bennett A, et al. Understanding capsid assembly and genome packaging for adeno-associated viruses. Future Virol. 2017;12:283-297. DOI:10.2217/fvl-2017-0011. [H]
[5-3] Tran NT, et al. AAV-genome population sequencing of vectors packaging CRISPR components reveals design-influenced heterogeneity. Mol Ther Methods Clin Dev. 2020;18:639-651. DOI:10.1016/j.omtm.2020.07.007. PMID:32775498. [H]
[5-4] Davis JR, et al. Efficient prime editing in mouse brain, liver and heart with dual AAVs. Nat Biotechnol. 2024;42:253-264. DOI:10.1038/s41587-023-01758-z. PMID:37142705. [N]