Adeno-associated virus: a Trojan Horse for Transgene Delivery

Adeno-associated virus is a single-strand of DNA that codes for its own replication and protective protein shell. Adeno-associated virus was first identified as a contaminant of adenovirus cultures (it was “associated” with adenovirus). The AAV genome contains a ‘rep’, ‘cap’, and ‘aap’ gene. ‘Rep’ codes for DNA replication, ‘aap’ for protein assembly, and ‘cap’ for protein production and packaging of the DNA fragment into the protein shell. The genome is flanked by inverted terminal repeats (ITRs) which play an important role in replication, packaging, and post-infection stability for transcription. Wild-type AAV will integrate into chromosome 19 at a site called AAVS1 and lay dormant in the absence of a helper virus (i.e. adenovirus) which is required for AAV’s replication.

Recombinant adeno-associated virus (rAAV) is the product of AAV and a transgene that researchers want expressed in a certain tissue. The transgene will be flanked by inverted terminal repeats, allowing it to be packaged into the protein shell that AAV’s genome supplies. When creating a recombinant adeno-associated virus, researchers will insert AAV, their transgene, and a helper virus into a cell line called a ‘packaging cell line;’ it gets its name for the machinery that a cell naturally provides (ribosomes, amino acids, etc.) that are necessary for the protein shell’s creation. The result is a single strand of the ITR-flanked transgene encapsulated by the AAV protein shell. Half of the rAAV will be composed of the 3’-5’ (“plus”) DNA, half the 5’-3’ (“minus”) DNA. Consider the protein shell a cellular trojan horse; the shell contains binding factors that are recognized by a cell’s membrane receptors. The cell receptors recognize the protein shell and bring it into the cell, where it can then transmit the transgene into the cell’s nucleus for transcription.

Just as there are different breeds of horses, there are different ‘serotypes’ of adeno-associated viruses. Each serotype has an easier time gaining access to different types of cell tissue. So far eleven serotypes have been discovered. AAV2 and AAV5 are commonly used for central nervous system tissue. Here is a more detailed list of serotype affinity.

A transgene requires a promoter sequence to “promote” transcription and a termination sequence to end it. The cytomegalovirus (CMV) promoter is commonly used when creating rAAV because it is constitutively active and lends a higher rate of expression. A researcher may use an inducible promoter that is only expressed in the presence of another molecule if they want to tightly regulate gene expression. A common method of inducible transgene expression includes the use of tetracylcine and a tetracycline-controlled transactivator (tTa) or reverse tetracycline-controlled transactivator (rtTa) (Das, Tenenbaum, & Berkhout, 2016).

The rAAV cassette can only contain about 4.7kb (4700 bases) of genetic information, or nucleotides. Three nucleotides code for one amino acid, and proteins are composed of hundreds of amino acids. This severely limits the amount of genetic material that researchers can include in their rAAV transgene. Current research is attempting to expand the amount of genetic information that can be included in a rAAV. One study found that the MeCP2 promoter (229bp long), which is about 570bp shorter than the CMV promoter, drove specific neuronal expression in mice and could potentially be used in place of CMV to allow more space for a larger transgene (Gray et al., 2011). 

Other attempts at delivering larger transgenes includes the dual vector approach which involves using two rAAVs to construct one large transgene. There are currently four methods of dual vector delivery: fragmented, overlapping, trans-splicing and hybrid. Let’s briefly go over each one:

Fragmented AAV dual vectors

The fragmented AAV dual vector approach is done by attempting to package a transgene that is too large to be packaged. The transgene will be cut or “fragmented” at different lengths while it is being replicated and packaged. Some of these fragments will contain overlapping sequences and anneal to each other (image left). Once they are annealed they undergo second-strand synthesis and the transgene is fully constructed. The transgenes may also undergo second-strand synthesis first and then recombination with the other fragment at the spot of overlap (image right).

Overlapping AAV Dual Vectors

Overlapping AAV dual vectors are two distinct transgenes that each carry an overlapping coding sequence. The success of this approach relies mainly on homologous recombination, which exchanges genetic information between two strands of genetically similar DNA. After homologous recombination, both halves of the transgene would be joined and expressed correctly.

Trans-splicing AAV Dual Vectors

Trans-splicing vectors contain splice donor and acceptor sites around the inverted terminal repeats. The splice donor site is located at the end of the upstream transgene and the splice acceptor site at the beginning of the downstream transgene. When the two transgenes concatemerize (when their ITR’s join), whatever is between the two splice sites is excised, leaving the full transgene. However, this approach relies on the two transgenes concatemerizing and doing so in the correct orientation.

Hybrid AAV Dual Vectors

The hybrid AAV dual vector contains an overlapping coding sequence and splice sites to encourage correct orientation of the transgene and prevent any inverted terminal repeats from staying in the coding sequence. Of all dual vector approaches, the hybrid approach seems to be the most effective (McClements & MacLaren, 2017).

The dual vector method is still in the early stages of development and has displayed variable results, but is a step in the right direction in delivering larger transgenes. A refined technique for delivering large transgenes will prove extremely useful in gene therapy and can open up room for greater manipulation in neurons to better understand cellular pathways and neuronal circuits.

In summary, AAV is a tool for packaging and delivering a transgene to a cell’s nucleus and has become a go-to method for researchers studying the central nervous system. By delivering a transgene via AAV, researchers can manipulate neuronal function to better understand how neurons and their circuits work.

works cited

Das, A. T., Tenenbaum, L., & Berkhout, B. (2016). Tet-On Systems For Doxycycline-inducible Gene Expression. Current Gene Therapy, 16(3),

156–167.

Gray, S. J., Foti, S. B., Schwartz, J. W., Bachaboina, L., Taylor-Blake, B., Coleman, J., … Samulski, R. J. (2011). Optimizing Promoters for

Recombinant Adeno-Associated Virus-Mediated Gene Expression in the Peripheral and Central Nervous System Using Self

Complementary Vectors. Human Gene Therapy, 22(9), 1143–1153.

McClements, M. E., & MacLaren, R. E. (2017). Adeno-associated Virus (AAV) Dual Vector Strategies for Gene Therapy Encoding Large

Transgenes. The Yale Journal of Biology and Medicine, 90(4), 611–623.