Recombinant adeno-associated virus (AAV) is often the preferred method for delivering genes to target cells due to its high titer, mild immune response, ability to infect a broad range of cells, and overall safety. Native AAV can infect humans and primates; however it has not been reported to cause disease and poses minimal risk to humans. Compared with other viral vectors such as adenovirus, AAV elicits a mild immune response in animal models, making AAV an ideal virus for researchers delivering genes in vivo or who are concerned about safety. AAV infects a broad range of cell types and infection is not dependent upon active cell division. One concern when using other viruses, such as retrovirus or lentivirus, is the random integration events that can disrupt gene function. Because AAV does not integrate into the host cell genome, the risk of insertional mutagenesis is low. AAV can exist long term as concatomers in non-dividing cells, however it will be lost in replicating cells.
Native AAV is replication deficient and requires co-infection with a helper virus for productive AAV infection. The AAV Helper Free Expression System supplies these necessary helper genes on separate plasmids, providing the helper function necessary for infection, while eliminating the need for a helper virus. Recombinant AAV can be generated by co-transfecting 293 cells, which supply the necessary adenoviral E1 genes, with an AAV expression plasmid, a rep-cap plasmid, and a pHelper plasmid. The expression plasmid contains the viral ITR sequences and can be used to clone and express a gene of interest. There are also options available for expressing various reporters to aid in the analysis. The Rep-Cap plasmid contains the rep and cap genes and specifies the serotype. Providing the rep and cap genes on a separate plasmid have helped to increase the cloning capacity of AAV, which is limited due to its small genome size. The pHelper plasmid contains the adenoviral genes VA, E2A, and E4, which are typically found in the helper virus and are required for successful infection. Cells are harvested 48-72 hours after transfection, subjected to a series of freeze/thaw cycles to release the virus from the cells, and collected by centrifugation. The crude lysate can be stored, tittered, or purified.
AAV is able to infect many different cell types, although the infection efficiency varies based upon serotype, which is determined by the sequence of the capsid protein. Several native AAV serotypes have been identified, with serotypes 1-9 being the most commonly used for recombinant AAV. AAV-2 is the most well-studied and published serotype. The AAV-DJ system, developed by Dr. Mark Kay, includes novel serotypes AAV-DJ and AAV-DJ/8. These serotypes were created through DNA shuffling of multiple AAV serotypes to produce AAV with hybrid capsids that have improved transduction efficiencies in vitro (AAV-DJ) and in vivo (AAV-DJ/8) in a variety of cells and tissues.
The AAV genome is single stranded, but must undergo second strand synthesis to produce the double stranded DNA that is required for gene expression. Second strand synthesis is the rate limiting step of AAV infection and can result in low transduction efficiencies. To overcome this limitation, the AAV genome can be packaged as double stranded DNA, rather than single stranded, as self-complementary AAV (scAAV). With scAAV, a region of the ITR sequence is deleted, preventing cleavage of the genome into the monomers that normally get packaged. The double stranded scAAV genome is able to bypass the second strand synthesis step. These vectors result in a greater yield, but cloning capacity is reduced to accommodate for the complementary strand.
Tittering the packaged virus is an important step to ensure that enough virus is delivered to the target cells and for maintaining consistency between experiments. It is recommended to titer the virus after packaging and again after any purification or concentration procedures. A titer kit can be used to quantify the viral nucleic acid of unpurified AAV-2 or AAV-DJ and any purified AAV serotype.
Purification of the virus is essential if using for in vivo studies or if the crude viral lysate could be toxic to the target cells. AAV-2 and AAV-DJ can be purified with a purification kit, which can recover 60% or more of the virus present in the supernatant, or ultracentrifugation can be used to purify any AAV serotype.
Once the virus is packaged and tittered, it is ready to be applied to target cells. The appropriate multiplicity of infection (MOI) should be determined for each target cell line using a GFP or luciferase control virus. The MOI is the ratio of virus (Infectious units) to cells and is calculated based on the viral titer and the number of cells plated. Optimal MOI can vary greatly by target cell. Ideally, the lowest MOI that provides 100% infection should be used. An MOI that is too high will result in cell death and an MOI that is too low will have reduced infection efficiency. An additive such as ViraDuctinTM AAV Transduction Reagent can be used when infecting non-permissive cells or to improve transduction efficiency with any AAV serotype. Gene expression can be analyzed 2-7 days post infection, depending on the gene and the amount of virus used for transduction.