Single strand AAV or self-complementary AAV

Adeno-associated viruses (AAVs) can be engineered in two different forms: single-stranded AAV (ssAAV) and self-complementary AAV (scAAV). These forms differ in their genome structure and mechanism of transgene expression, and package capacity.

Single Strand AAV vectors(ssAAV)

In ssAAV vectors, the viral genome is composed of a single-stranded DNA molecule. ssAAV vectors consist of two 145 nucleotide inverted terminal repeats(ITRs) flanking an expression cassette encoding a therapeutic transgene, with deletion of all the viral open reading frames.
When ssAAV infects a target cell, the viral DNA is converted into a double-stranded molecule by cellular machinery before transgene expression can occur. This conversion process is mediated by a second strand synthesis step, which can result in delayed and variable transgene expression.
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. For large coding sequences, the use of dual, overlapping vector strategies may be a good choice for you.



ITR structure

Package Capacity

Gene Expression



Two wt ITRs

~5.0 kb

Low and slow



One wtITR, one mITR

~2.5 kb

High and fast


Self complementary AAV vectors(scAAV)

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[McCarty et al., 2001]. the scAAV vector genome is designed as a single-stranded inverted repeat, which folds back upon itself to form a double-stranded genome when entering into infected cells. A genome <2.5 kilobases can therefore be packaged as a dimer, with the two inverted repeats pairing along their length, closed covalently by a hairpin at the terminal repeat. There is therefore no need for complementary strand synthesis, and this rate-limiting step is bypassed. Dimerization of the genome in this manner can be stabilized by mutation or deletion of one of the two terminal resolution sites (trs; these are Rep-binding sites contained within each inverted terminal repeat, which prevents cleavage by AAV Rep proteins to form monomers. The replication fork initiates at the wild-type trs and proceeds through the genome and through the mutant trs, which is unable to facilitate resolution, causing the replication fork to proceed back across the genome where it terminates at the wild-type trs. The resultant self-complementary molecule is thus flanked by wild-type trs and has a mutant trs in the middle, and dimerizes along its length when packaged into the AAV.
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. Upon transduction of target cells, the scAAV genome exists either as circular genomes or concatemers, the former being much more effective in transgene expression. scAAV vector genomes are more stable and more prone to circularization upon transduction of in vivo tissues than single-stranded AAV (ssAAV) vector.
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). The packaging capacity can be extended to 3.3 kb genomes, but the proportion of single-stranded genomes increases linearly with genome length. Genomes larger than 3.5 kb are packaged almost solely as single-stranded forms[Wu et al., 2007].

Deepak Raj, Andrew M Davidoff, Amit C Nathwani
Self-complementary adeno-associated viral vectors for gene therapy of hemophilia B: progress and challenges. Expert Rev Hematol. 2011 Oct; 4(5): 539–549. doi: 10.1586/ehm.11.48

D M McCarty 1, P E Monahan, R J Samulski. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis
Gene Ther. 2001 Aug;8(16):1248-54.
doi: 10.1038/

Jianqing Wu, Weihong Zhao, Li Zhong, Zongchao Han, Baozheng Li, Wenqin Ma, Kirsten A Weigel-Kelley, Kenneth H Warrington, Arun Srivastava
Self-complementary recombinant adeno-associated viral vectors: packaging capacity and the role of rep proteins in vector purity. Hum Gene Ther. 2007 Feb;18(2):171-82. doi: 10.1089/hum.2006.088.


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