Adeno-associated virus (AAV) has become a popular viral tool for research and clinical applications. AAV can transiently express target genes in a wide range of cells. It was first described about 50 years ago as a contaminant of adenoviral agents, hence its name. AAV is a single-stranded DNA virus belonging to the family of fine viruses. It has a "simple" genome packaged in an icosahedral capsid protein. It does not have a lipid shell, also known as a lipid envelope, and therefore cannot support the addition of glycoproteins, such as VSV-G, to its surface. In research applications, the genome is often modified to provide valuable carrying space for gene delivery and security. By providing genes encoding replicase and coat proteins, you can easily produce virus particles in a tissue culture environment. This allows researchers to produce more viruses in a controlled environment. Even though AAV is isolated from a wide range of organisms, it is not associated with disease and is therefore considered a biosafety level 1 (BSL1) viral agent.

Considerations for Perfect Acquisition of AAV (rAAV) Virus

AAV can be used for a variety of applications including transient gene expression in specific cell types, CRISPR genome editing, optogenetics and chemical genetics experiments. If you are just starting out with rAAV as a gene delivery tool, there are a few things to consider before you begin:

• Carrying capacity

Even the modified version of rAAV has a carrying capacity of ~ 4.7 kb, which is one of the key limitations of the virus for gene delivery. However, if your gene of interest is small enough, you can design single rAAV vectors that contain both genes and use elements such as IRES or 2A to co-express them using a single promoter. Co-infection of different rAAVs is also high if the titer is high, so if you can't put two genes in a single vector, you can do co-infection as well (although this may not always work in vivo).

• Specificity

There are several different rAAV components that can drive gene expression in a given cell/tissue; these include promoters, Flp- or Cre-dependent gene switches, and serotypes (serotypes are discussed further below). Promoters with broad activity, such as CAG (chicken β-actin promoter containing the CMV early enhancer), are a good choice if broad expression is the goal. If you have a specific target cell type, you may want to try a different promoter, such as Camk IIa as a neuron-specific promoter. On the other hand, rAAV vectors with Cre- or Flip-dependent gene expression can be injected into animals that express Cre or Flp in specific cell types, so that the gene is expressed only in those cells.

• Serotype

Capsid proteins are very important rAAV carrier components and drive the biological functions of these carriers. Although several studies have shown that different serotypes differ in their ability to infect different cell types, a recent study has shown that most (or all) serotypes use the same receptor (AAVR). The observed directionality may be due to other factors such as attachment of viral particles to the cell surface, or possibly post-entry steps such as decapsulation.

• Genome

Wild-type AAV is a single-stranded DNA virus. after DNA decapsidation, the virus relies on the host cell replication machinery to synthesize complementary strands of DNA. This step is thought to be a limiting factor in rAAV transduction efficiency. To overcome this problem, McCarty et al. constructed a dimeric or self-complementary AAV (named scAAV) by mutating one of the ITRs. scAAV vectors have the ability to rapidly invade and express transgenes at a higher level than ssAAV. However, its packaging capacity (< 2.5 Kb) is half that of ssAAV (< 4.8 Kb), limiting the number of successfully packaged genes and regulatory elements.

Creative Biogene offers a comprehensive range of recombinant AAV reporter particles featuring over 12 serotypes and engineered capsid variants. These AAVs achieve efficient delivery of fluorescent and bioluminescent reporter genes to diverse cell types in vitro and in vivo.

View more of our reporter AAV particles products!

How to Produce rAAV?

Briefly, we first transfected HEK293T cells with the rAAV vector carrying the target gene, an adenovirus helper plasmid, and a plasmid containing Rep and Cap (often referred to as "triple plasmid transfection"). 2 - 3 days later, the supernatant was collected (or, in some cases, some of the rAAV serotypes were released into the culture medium as a cellular extract while others were not) and the viral particles were precipitated by PEG. PEG to precipitate virus particles. The virus particles were then further purified by density gradient centrifugation using a high-speed ultracentrifuge. The virus particles will form a band due to the high density and this band can be collected from the gradient. The density gradient is then removed by dialysis/buffer exchange and the virus particles can be further concentrated if desired. Using primers and/or protein gels targeting the viral genome, titers of viral particle preparations can be determined by qPCR. These titers are for physical particles, many of which are not infectious. The ratio of physical particles to infectious particles can vary widely, from 1 to > 100.

Fig. 1 Recombinat AAV (rAAV) transfer vectors typically carry a transgene driven by a promoter of choice between ITRs.

In Vivo rAAV Delivery

There are multiple factors that go into the delivery of AAV in the body, depending on your biological system. These factors greatly influence the success of your infection. Regardless of what your purpose may be, listed below are a few parameters that you may want to consider before experimenting before continuing:

• Injection titer: The number of viral particles you can deliver to the tissue will be determined by the titer of your virus and the maximum delivery volume that can be delivered, which is very important. Unfortunately, as mentioned above, the titer of a physical particle does not directly translate to the virus you observe in the body, it is just a "starting point". You can review the literature for recommendations for specific tissues, but it is usually necessary to try a series of dilutions to determine the optimal dose.
• Age of the animal: First, it is important to remember that ultimately differentiated cells (i.e., post-mitotic cells) will have long-term AAV expression; however, expression will be lost in dividing cells. The age of the injected animal will determine what type of cells can be successfully infected (i.e., early in development, unborn cells, even if mitotic cells are present, cannot be infected with AAV).
• Infection visualization: Before starting injections, you should also consider how to visualize your rAAV infection. Is there a reporter gene included in the rAAV vector, such as GFP or mCherry? This will be easily visualized by fluorescence measurements to directly observe where expression occurs. If not, you might consider injecting an AAV carrying a reporter gene under the same promoter to drive the expression of your target gene and will measure the expression of the reporter gene as a representative of your target gene that is not readily detectable. If co-expression or co-infection is not well achieved, then detection of your protein by immunohistochemistry or in situ hybridization may be a better option.
• Time after infection: sufficient time needs to elapse between injection and tissue action to detect AAV-mediated gene expression. The time will be highly dependent on the capsid type and the infected tissue. For many tissues, it may be necessary to wait ~ 2 weeks initially. AAV-mediated gene expression has been reported to be fairly stable, persisting for several years in human clinical trials and in dogs.