AAV Vector & Packaging
AAV vector construction and packaging involve several plasmids whose maps are labeled with common elements: ITR, Rep, Cap, Promoter, GOI, and PolyA.The ITRs support genome replication and packaging, Rep proteins drive AAV genome processing, Cap proteins determine capsid assembly and tropism, the promoter controls transgene expression, and the GOI provides the therapeutic or experimental payload, so each element must be designed and verified as part of one connected vector-production system.
Review the ElementsDiscuss Your ConstructElement Reference
A quick reference for the common labels on AAV plasmid maps.
| Label | Full name | Main function | Typical location |
|---|---|---|---|
| ITR | Inverted Terminal Repeat | Cis elements for genome replication, processing, and packaging; define the genome boundary. | Transfer plasmid, at both ends. |
| Rep | Replication proteins | Rep proteins involved in genome replication and packaging. | Rep/Cap plasmid. |
| Cap | Capsid | Encodes VP1, VP2, and VP3; determines the capsid serotype. | Rep/Cap plasmid. |
| Promoter | Promoter | Regulates transcription of the gene of interest. | Transfer plasmid, upstream of the GOI. |
| GOI | Gene of Interest | The transgene to be delivered and expressed. | Between the two ITRs. |
| PolyA | Polyadenylation signal | Directs 3′ processing, polyadenylation, and transcription termination. | Transfer plasmid, downstream of the GOI. |
ITR & Cassette
The ITRs are cis-acting elements, not promoters or coding sequences, and they form the boundaries of the genome that gets packaged.
The ITRs participate in AAV genome replication, processing, and packaging. In a typical rAAV vector, the expression cassette sits between the two ITRs, which constitute the genome boundary. Because ITRs have complex secondary structure, their stability and integrity during cloning, bacterial amplification, and sequencing require careful attention.
The transfer plasmid carries the packaged genome arranged as ITR–Promoter–GOI–PolyA–ITR. This is the sequence that ultimately becomes the rAAV vector genome.
ITR Structure
The ITR is more than a simple repeat; its structure and sequence motifs drive replication and packaging.
The canonical AAV ITR is about 145 nucleotides with palindromic A, B, and C regions that fold into a T-shaped hairpin, plus a D sequence that serves as the packaging signal.
Rep proteins bind the ITR and perform terminal resolution, nicking the DNA to generate the primers needed for replication and to resolve concatemers into unit-length genomes.
The palindromic hairpin makes the ITR prone to deletion, mutation, and rearrangement during plasmid construction and bacterial amplification, which is why ITR integrity must be verified before packaging.
Rep & Cap
The Rep/Cap module supplies the replication machinery and the capsid that defines serotype.
Rep proteins arise from different transcripts and splicing events. The large Rep proteins (Rep78 and Rep68) drive genome replication and regulation, while the small Rep proteins (Rep52 and Rep40) are involved in packaging. Rep is normally co-located with Cap on the Rep/Cap plasmid.
The Cap gene encodes the three capsid proteins that form the viral shell. Different serotypes have different Cap sequences, so Cap is the core determinant of capsid type. AAV2, AAV5, AAV6, AAV8, AAV9, and AAVrh10 differ in their Cap sequences.
Cap determines the capsid, but tissue tropism is also shaped by route, dose, promoter, species, and target tissue. Capsid selection should therefore consider the whole experimental system.
Promoter Reference
A quick reference for commonly used AAV promoters and the tissues or cell types they are often paired with.
| Promoter | Typical use | Notes |
|---|---|---|
| CMV | Broad exogenous expression | Strong in many cell types; may be silenced in some contexts. |
| CAG / CBh | Strong, broad expression | Larger promoters widely used for robust expression. |
| EF1α | Sustained expression | Commonly used in mammalian cells. |
| hSyn | Neurons | Cell-type-preferring for neuronal expression. |
| GFAP | Astrocytes | Cell-type-preferring for glial expression. |
| TBG | Liver | Tissue-specific hepatic expression. |
| MCK | Muscle | Tissue-specific for skeletal and cardiac muscle. |
Promoter & GOI
These three elements work together to control what is expressed and how the transcript is processed.
| Element | Common examples | Key point |
|---|---|---|
| Promoter | CMV, CAG, EF1α, hSyn, GFAP, TBG, MCK | Determines expression level and cell range; choose for the target tissue and cell type, not "strongest is best." |
| GOI | Coding sequence, or other functional genetic element | The transgene to be delivered; must fit within the packaging capacity with all other elements. |
| PolyA | bGH, SV40, rabbit β-globin | Directs 3′ processing and termination; the DNA signal is distinct from the RNA poly(A) tail. |
Regulatory Elements
Beyond the core elements, additional sequences modulate transcript processing and expression.
| Element | Function | Common examples |
|---|---|---|
| poly(A) signal | Directs 3′ processing and transcription termination. | bGH, SV40, rabbit β-globin |
| WPRE | Enhances mRNA stability and expression. | Woodchuck hepatitis post-transcriptional regulatory element |
| Intron | May improve transcript processing and expression. | Chimeric introns |
| Enhancer | Modulates promoter activity. | Tissue- or context-specific enhancers |
These elements add length to the cassette and must be counted toward the packaging capacity.
Why It Matters
Knowing the elements prevents the common mistake of conflating vector and packaging plasmids.
Confirm the ITR–Promoter–GOI–PolyA–ITR arrangement matches the intended design before moving to packaging.
Match the Cap sequence to the intended serotype and the target tissue, species, and route.
Understand that Transfer, Rep/Cap, and Helper plasmids play distinct roles to avoid ordering or designing errors.
Verify ITR and GOI integrity with appropriate methods, since complex structures cannot be judged from a map alone.
Project Support
Creative Biolabs provides AAV vector construction and production capabilities that can be scoped around your transfer cassette, capsid, and packaging strategy.
Build the transfer and packaging components of an AAV production system.
Configure the promoter, GOI, and polyA cassette for your expression goal.
Supply engineered Cap to generate capsids with the desired tropism.
Select the right promoter for tissue- or cell-type-specific expression.
Produce rAAV under a defined process from your transfer and packaging constructs.
Confirm the packaged genome sequence and integrity.
References
FAQ
Share your transgene, target serotype, and expression goal. Creative Biolabs can help configure the transfer cassette and packaging strategy, and verify key sequences before production.
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