AAV component explorer
Total size of a recombinant AAV genome, and whether it fits the packaging limit.
Total size of a recombinant AAV genome, and whether it fits the packaging limit.
Everything of the virus that is left in a recombinant vector is the pair of inverted terminal repeats. Between them sits a cassette you design: a promoter, the coding sequence, usually a post-transcriptional element, and a polyadenylation signal. The packaging machinery does not care what the sequence says, only how long it is, and it stops at about the length of the wild-type genome.
Worked example. "Try an example" builds the most common research construct: a CAG promoter (1,733 bp) driving EGFP (720 bp), followed by the WPRE (592 bp) and a bGH poly(A) signal (225 bp), with two ITRs at 145 bp each. That totals 3,560 bp, or 76% of the limit, leaving 1,140 bp to spare. Swap EGFP for a 3,500 bp therapeutic gene and the same cassette comes to 6,340 bp, far too large; with an EFS promoter (232 bp), no WPRE and a short synthetic poly(A) (49 bp) the same gene fits at 4,071 bp.
The wild-type AAV2 genome is about 4.7 kb and that is what a capsid is built to hold. Packaging is efficient for genomes of roughly 4.1 to 4.9 kb. Beyond that, capsids fill up to the end and the excess is lost: preparations of oversized vectors contain heterogeneous, 5-prime truncated genomes, so titre and the share of particles able to express the transgene both fall. Since the two ITRs take 290 bp, the working budget for the cassette is about 4.4 kb. At the other end, genomes under about 2 kb package badly as well, and short cassettes are padded with a stuffer.
A self-complementary vector (scAAV) packages the cassette and its own complement as one molecule that folds into a duplex, skipping the second-strand synthesis that limits how fast a standard vector expresses. The price is length: only about half of the 4.7 kb is unique sequence, so the tool compares the total with about 2,350 bp in that mode. One of the three ITRs is mutated at its terminal resolution site, which makes the hairpin form; it is slightly shorter than a full ITR, so the estimate here is on the safe side.
Lengths vary a little between plasmid versions, so treat these as the usual annotated sizes and measure your own plasmid when it matters. Anything not listed, an intron, a Kozak sequence of 6 bp, a tag, a linker, a second cassette or a stuffer, goes into "Other sequence" in the result. An SV40 poly(A) fragment is a case in point: depending on the plasmid it is annotated anywhere from about 120 to 240 bp, so it is worth measuring rather than assuming.
| Element | bp | What it does |
|---|---|---|
| ITR (AAV2) | 145 each | The only viral sequence left in the vector. One at each end, they are the packaging and replication signal and they count toward the limit. |
| CMV promoter | 589 | Strong and ubiquitous, but silenced over weeks in many tissues and rich in CpG. |
| CAG promoter | 1,733 | CMV enhancer, chicken beta-actin promoter and a chimeric intron. Very strong and long lasting, and by far the largest common promoter. |
| CBh promoter | 798 | A shortened hybrid of the CAG parts with much of the CAG strength at less than half the length. |
| EF1a promoter | 1,178 | The human EF1-alpha promoter with its first intron. Resists silencing, ubiquitous. |
| EFS promoter | 232 | The intron-less core of EF1-alpha. The usual choice when a cassette is tight. |
| hSyn1 promoter | 469 | Human synapsin 1 promoter, restricts expression to neurons. |
| gfaABC1D promoter | 681 | A truncated GFAP promoter, restricts expression to astrocytes. |
| WPRE | 592 | Woodchuck hepatitis post-transcriptional element. Raises expression a few fold by stabilising the transcript. |
| WPRE3 | 247 | A shortened WPRE that keeps most of the effect and removes the partial X protein reading frame. |
| bGH poly(A) | 225 | Bovine growth hormone polyadenylation signal, the usual terminator in AAV cassettes. |
| Short synthetic poly(A) | 49 | A minimal synthetic signal, used when every base counts. |
The capsid decides what the vector enters; the genome inside is the same. These are the uses each serotype is best known for, not a guarantee: tropism depends on species, route of delivery, dose and age, and results in mice often fail to carry over to larger animals.
| Serotype | Commonly used for | Notes |
|---|---|---|
| AAV1 | Skeletal muscle, heart, central nervous system | Long used for intramuscular delivery. |
| AAV2 | Neurons, retina, liver | The reference serotype. Binds heparan sulfate, so it spreads less from the injection site. |
| AAV5 | Airway epithelium, central nervous system, retina | Binds sialic acid. Reported to package slightly larger genomes than AAV2. |
| AAV6 | Skeletal muscle, airway epithelium | Close to AAV1 in sequence, better in culture. |
| AAV8 | Liver, skeletal muscle, retina | The usual choice for systemic liver delivery. |
| AAV9 | Liver, heart, skeletal muscle, central nervous system | Crosses the blood-brain barrier after systemic delivery, strongly in neonates. |
| AAVrh10 | Central nervous system, liver, muscle | A rhesus isolate used for brain delivery. |
| AAV-DJ | Broad in cell culture | An engineered hybrid of several serotypes, made for transduction in vitro. |
| AAV-PHP.eB | Central nervous system | An engineered AAV9 variant. Its brain targeting depends on a receptor found in some mouse strains and does not transfer to other species. |
Two things about the sequence itself change how an AAV vector behaves. Unmethylated CpG dinucleotides in the vector genome are read as foreign by innate immune sensing and are associated with loss of expression and inflammation, which is why clinical constructs are often CpG depleted; the CpG depletion tool removes them with silent changes. Codon choice sets how much protein each transcript makes, which matters when the dose is capped by the capsid; the codon optimizer adjusts the coding sequence for the target species. Neither changes the length of the construct, so the budget here stays the same.
About 4.7 kb of single-stranded DNA, the size of the wild-type AAV2 genome, and the two ITRs are part of it. Packaging works well between roughly 4.1 and 4.9 kb; efficiency falls away above that and genomes over 5 kb are packaged as a mixture of truncated fragments, which lowers titre and gives vector that cannot express the full transgene.
Yes. Each AAV2 ITR is 145 bases and both ends are packaged, so 290 bases of the budget are gone before any cassette is designed. That leaves about 4.4 kb for promoter, transgene, regulatory elements and poly(A) signal.
Roughly half. A self-complementary genome carries both strands as one inverted repeat, so the same 4.7 kb of packaged DNA holds only about 2.35 kb of unique sequence, ITRs included. In exchange it skips second-strand synthesis and expresses earlier and more strongly, which is worth it for small transgenes.
In order of how much room they free: swap CAG (1,733 bp) for CBh (798 bp) or EFS (232 bp), drop the WPRE (592 bp) or use WPRE3 (247 bp), use a short synthetic poly(A) instead of bGH, remove introns, tags and linkers, and trim untranslated regions. Beyond that the options are a shortened version of the protein, as micro-dystrophin is for dystrophin, or splitting the transgene across two vectors that recombine in the cell.
Yes, at the other end. Genomes below about 2 kb package poorly and yield particles carrying dimers or partial genomes, so a short cassette is usually padded with a stuffer sequence to bring the total to at least 2.1 to 2.3 kb.