AAV Packaging Capacity

One of the main considerations in the rational design of an AAV vector is the packaging size of the expression cassette that will be placed between the two ITRs. 

Packaging capacity of single strand AAV

wtAAV has a single strand DNA genome about 4.7kb. Regular single strand AAV(ssAAV) has a packaging capacity of ~5.0 Kb. Thus, as a starting point, it is generally accepted that anything under 5 kb (including ITRs) is sufficient. Since the two ITRs of AAV are about 0.2-0.3Kb total, the foreign sequence between two ITRs should be smaller than 4.7 Kb. When the length of inserted DNA is close to the maximum, the packaging efficiency decreases significantly. If the insert DNA is over 4.7 Kb, only partial of DNA is packaged into AAV vectors[Dong et al., 2010][Lai et al., 2010][Wu et al., 2010]. For large coding sequences, the use of dual, overlapping vector strategies may be a good choice for you.

Packaging capacity of self-complementary AAV

scAAV is a viral vector engineered from the naturally occurring AAV to be used as a tool for gene therapy. This lab-made progeny of AAV is termed “self-complementary” because the coding region has been designed to form an intra-molecular double-stranded DNA template. A rate-limiting step for the standard AAV genome involves the second-strand synthesis since the typical AAV genome is a single-stranded DNA template. However, this is not the case for scAAV genomes. Upon infection, rather than waiting for cell mediated synthesis of the second strand, the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription. The caveat of this construct is that instead of the full coding capacity found in rAAV (5.0 kb). scAAV can only hold about half of that amount (≈2.5kb)[Wu et al., 2007].

Oversize AAV packaging

Researchers have shown successful expression of 9 kb gene following transduction with AAV vectors in which they had attempted to package oversize transgenes. This success was elucidated to have resulted from the packaging of fragmented transgenes. When a transgene is large, packaging that begins from the 3’ ITRs of both the plus and minus strands and becomes truncated at an undefined point, therefore each capsid carries an incomplete fragment of transgene. This results in a mixed population of AAV vectors carrying different truncated lengths of the transgene plus and minus strands. 

Oversize AAV Packaging

The successful generation of target product despite this heterogeneous vector population was deduced to result from the plus and minus strands carrying overlapping regions of the original therapeutic transgene that could undergo homologous recombination (HR) or annealing at the complementary regions prior to second-synthesis.

Oversize AAV packaging

Dual functional AAV vectors

Some large genes can be separated into two functional fragments, such as FVIII. For those genes, one AAV vector is used to carry the N terminal with an addtional stop codon. Another AAV vector is used exclusively to express the C terminal with additional start codon. The two AAV vectors can be produced separately. The combination of the two vectors can generate a functional full protein .

Dual AAV vectors

Overlapping dual AAV vectors

An advancement on the fragmented dual vector approach is the overlapping approach. In this strategy, there are two defined transgenes that each carry a demarcated fragment of the therapeutic gene CDS that includes a portion of specified sequence overlap in each transgene. The overlapping strategy relies on the same premise that enables the fragmented approach, whereby a region of sequence overlap initiates joining of two separate fragments into a single larger one.

Overlapping dual AAV vectors

Trans-splicing dual AAV vectors

This strategy has no region of sequence overlap and therefore the two transgenes are completely distinct and contain two different fragments of the therapeutic gene. The approach relies on the tendency of ITRs to concatemerize as it has been shown that following transduction and second-strand synthesis, AAV transgenes form stable episomal structures through joining of their ITR structures, a process known as concatemerization. The trans-splicing approach piggy-backs on this process and so with appropriate dual vector design, following joining of the ITRs from the dual vectors, the concatemerized ITR structure that would lie in the middle of the therapeutic gene can be removed by native cellular mechanisms during transcription due to the inclusion of a splice donor site following the 3’ end of the gene contained in the upstream transgene and a splice acceptor site prior to the 5’ end of the gene contained in the downstream vector.

Splicing dual AAV vectors

Trans-splicing and overlapping dual AAV vectors

With the trans-splicing approach, there is a concern that the dual vector transgenes will join in an undesirable way or not concatemerize at all. With the overlapping approach, a concern is that concatemerization would occur at all as there would be no feature to remove an unwanted ITR structure present in the middle of a CDS. The hybrid strategy counters both these concerns by combining the two approaches. This hybrid dual vector strategy incorporates both an overlap region and splice donor/splice acceptor sites in the dual vector transgenes.


The packaging capacity of AAV vectors plays a pivotal role in the success of gene therapy and vector design. Researchers and scientists need to carefully consider the size of the genetic material they intend to deliver using AAV vectors. If the inserted DNA exceeds the packaging capacity, it can lead to inefficient packaging, reduced delivery efficiency, and potentially compromised therapeutic outcomes.

By staying within the packaging limits of AAV vectors, researchers can maximize the chances of successful gene delivery, expression of the desired gene, and achievement of the intended therapeutic effects. This emphasizes the importance of optimizing the size of the expression cassette and considering alternative strategies, such as dual vectors or split constructs, when dealing with larger genetic material. Overall, understanding and respecting the packaging capacity is a fundamental aspect of gene therapy research to ensure safe and effective clinical applications.



Self-complementary recombinant adeno-associated viral vectors: packaging capacity and the role of rep proteins in vector purity. Wu J, Zhao W, Zhong L, Han Z, Li B, Ma W, Weigel-Kelley KA, Warrington KH, Srivastava A.Hum Gene Ther. 2007 Feb;18(2):171-82. doi: 10.1089/hum.2006.088. 

Effect of genome size on AAV vector packaging. Wu Z, Yang H, Colosi P.Mol Ther. 2010 Jan;18(1):80-6. doi: 10.1038/mt.2009.255. Epub 2009 Nov 10.

Evidence for the failure of adeno-associated virus serotype 5 to package a viral genome > or = 8.2 kb. Lai Y, Yue Y, Duan D.Mol Ther. 2010 Jan;18(1):75-9. doi: 10.1038/mt.2009.256. Epub 2009 Nov 10.

Characterization of genome integrity for oversized recombinant AAV vector. Dong B, Nakai H, Xiao W.Mol Ther. 2010 Jan;18(1):87-92. doi: 10.1038/mt.2009.258. Epub 2009 Nov 10.



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