Addgene: Adeno-Associated Virus (AAV) Guide (2023)

  • scientific guides
  • AAV Guide
  • Components
  • common uses
  • viral integration
  • serotypes
  • pseudotipagem
  • AAV variants
  • References

AAV Components

The small AAV ssDNA genome (4.8 kb) consists of two open reading frames, Rep and Cap, flanked by two 145 base inverted terminal repeats (ITRs). These ITR base pairs allow the synthesis of the complementary DNA strand. Rep and Cap are translated to produce several different proteins (Rep78, Rep68, Rep52, Rep40, required for the AAV life cycle; VP1, VP2, VP3, capsid proteins). When constructing an AAV transfer plasmid, the transgene is placed between the two ITRs and Rep and Cap are trans.

In addition to Rep and Cap, AAV requires a helper plasmid containing adenovirus genes. These genes (E4, E2a and VA) mediate AAV replication. The transfer plasmid, Rep/Cap, and helper plasmid are transfected into HEK293 cells, containing the adenovirus E1+ gene, to produce infectious AAV particles. Rep/Cap and adenovirus helper genes can also be combined on a single plasmid; the separation of Rep and Cap shown in the figure on the right facilitates viral pseudotyping discussed below.

Addgene: Adeno-Associated Virus (AAV) Guide (1)
(Video) AAV Transfer Plasmids - Viral Vectors 101

Common Uses of AAV

AAV is commonly used in optogenetics experiments. These viruses are preferable to lentiviruses because they remain primarily episomal, whereas lentiviruses integrate into the genome. This is important because the local chromatin structure at the genome integration site can affect the expression of the transgene, as well as the expression of neighboring genes. The short coding sequences for channelrhodopsins, halorhodopsins, and other optogenetic genes allow them to be packaged into AAVs.Click here for more information on the optogenetic plasmids available from Addgene.

The popular CRISPR/Cas9 genome editing system has been modified for use with AAV; This system represents a great step forward forlivegenome editing.To learn more about AAV-CRISPR, check out this blog post.Browse Ran's Articleand another, 2015 by Zhang Lab forfind plasmids optimized for use in AAV or containing Staphylococcus aureus (SaCas9).

Addgene: Adeno-Associated Virus (AAV) Guide (2)

AAV is also a promising method for gene therapy. Of the commonly used viruses, AAV produces the lowest immune response, is non-pathogenic even in the wild, and is therefore considered the most suitable virus for therapeutic applications. Clinical trials using AAV for various gene delivery applications are ongoing.

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viral integration

As part of its lysogenic cycle, wild-type AAV integrates into the host genome at a specific site, AAVS1 on human chromosome 19. This site is favored by the presence of a representative linker; however, random integrations can occur at a much lower frequency. As a replication-incompetent virus, AAV cannot enter the lytic cycle without assistance. Activation of the lytic cycle requires another virus, such as adenovirus or herpes simplex virus, or a genotoxic agent, such as UV radiation or hydroxyurea.

(Video) AAV Titration by qPCR

When recombinant AAV (rAAV) is used for research purposes, the Rep protein is providedme trans, eliminating the ability of rAAV to integrate at its preferred genomic integration site on human chromosome 19, called AAVS1. Instead, the rAAV genome is generally processed into a circular double-stranded episome via double-stranded synthesis. These episomes can concatemerize, producing high molecular weight structures that are maintained extrachromosomally. rAAV is more likely than wild-type AAV to integrate into non-homologous sites in the genome and does so with a frequency of about 0.1%. However, it is believed that most rAAV particles are maintained in episomes or concatemers.

Episomes differ profoundly from viral particles produced during a lytic cycle. rAAV episomes can develop a chromatin-like organization and persist in non-dividing cells for a period of years without harming the host cell. In contrast, viral particles produced during a lytic cycle are rapidly released through cell lysis. Episomal stability allows for long-term transgene expression in non-dividing cells and is a major advantage of rAAV.

AAV serotypes

To date, eleven AAV serotypes have been identified, with AAV2 being the best characterized and the most widely used. These serotypes differ in their tropism or in the cell types they infect, making AAV a very useful system for preferential transduction of specific cell types. The following table presents a summary of the tropism of AAV serotypes, indicating the ideal serotype(s) for transduction of a given organ.

Tissue ideal serotype
Heart AAV1, AAV8, AAV9
Should AAV2
Liver AAV7, AAV8, AAV9
Lung AAV4, AAV5, AAV6, AAV9
pancreas AAV8
photoreceptor cells AAV2, AAV5, AAV8
RPE (retinal pigment epithelium) AAV1, AAV2, AAV4, AAV5, AAV8
Skeletal muscle AAV1, AAV6, AAV7, AAV8, AAV9

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Pseudotipagema AAV

The researchers further refined AAV tropism by pseudotyping or shuffling a capsid and genome from different viral serotypes. These serotypes are indicated by a slash, thus AAV2/5 indicates a virus containing the serotype 2 genome packaged in the serotype 5 capsid. The use of these pseudotyped viruses may improve transduction efficiency as well as alter the tropism. For example, AAV2/5 targets neurons that are not efficiently transduced by AAV2/2 and is more widely distributed in the brain, indicating higher transduction efficiency. Many of these hybrid viruses have been well characterized and may be preferred to standard viruses forliveforms

Scientists have also experimented with hybrid capsids derived from several different serotypes, which also alter viral tropism. A common example is AAV-DJ, which contains a hybrid capsid derived from eight serotypes. AAV-DJ exhibits higher transduction efficiencyin vitrothan any wild type serotype;live, exhibits very high infectivity in a wide range of cell types. The AAV-DJ8 mutant exhibits the properties of AAV-DJ but with enhanced brain uptake.

(Video) Optogenetics - Viral Vectors 101

AAV variants

As mentioned above, AAV improvements have included the production of synthetic capsids and the mixing of capsids/ITRs from different AAV serotypes to create hybrid viruses with new properties. Other system variants include:

AAV autocomplementario (scAAV):A disadvantage of AAV is its single-stranded DNA genome. Because the virus depends on the cell's DNA replication machinery to synthesize the antisense strand, expression of the transgene can be delayed. To overcome this rate-limiting step, scAAV contains complementary sequences that can spontaneously hybridize after infection, eliminating the need for host cell DNA synthesis. Unfortunately, this technique further limits the packing capacity of the AAV to 2.4 kb.

Methods to increase packing capacity:To increase AAV packaging ability, a longer transgene can be split between two AAV transfer plasmids, the first with a 3' splice donor and the second with a 5' splice acceptor. When these viruses co-infect a cell, they form concatemers, come together, and the complete transgene can be expressed. This method allows longer expression of the transgene, but expression is much less efficient than with a single AAV virus (∼5%). Another technique to increase packaging capacity is based on homologous recombination. In this method, a gene is split between two transfer plasmids, but with substantial sequence overlap. Co-expression induces homologous recombination and full-length transgene expression with very low efficiency (<1% wild type). If any of these methods can be made more efficient, the use of AAV viruses will no longer be limited to small transgenes, allowing the development of other AAV applications.

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additional resources

web references


Viral serotypes and pseudotyping/gene therapy:
  • Modified gene delivery vectors: molecular engineering and evolution of adeno-associated viral vectors to improve gene transfer. Kwon I, Schaffer DV.Farm Nothing2008 March;25(3):489-99.PubMed.

  • Adeno-associated virus serotypes: vector toolkit for human gene therapy. Wu Z, Asokan A, Samulski RJ.Suggest. Day.2006 September; 14(3):316-27. Epub 2006 Jul 7.PubMed.

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  • Pseudotyped recombinant AAV viral vectors with serotypes 1, 2, and 5 viral capsids exhibit differential efficiency and cell tropism when delivered to different regions of the central nervous system. Burger C, Gorbatyuk OS, Velardo MJ, Peden CS, Williams P, Zolotukhin S, Reier PJ, Mandel RJ, Muzyczka N.Suggest. Day.2004;10(2):302-17.PubMed.

  • From virus evolution to vector revolution: use of naturally occurring adeno-associated virus (AAV) serotypes as novel vectors for human gene therapy. Grimm D, Kay MA.Curr Gene Ther.2003;3(4):281-304.PubMed.

Integration of the vector genome:
  • Integration of adeno-associated virus vectors. Deyle DR, Russell DW.Curr Opin Mol Ther.2009; 11(4):442-447.PubMed.

AVA variants:
  • Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independent of DNA synthesis. McCarty DM, Monahan PE, Samulski RJ.Gene Ther.2001 August; 8 (16): 1248-54 .PubMed.

  • Expansion of AAV packaging capacity with overlapping or trans-splicing vectors: a quantitative comparison. Duan D, Yue Y, Engelhardt JF.Mol Ther.2001; 4(4):383-91.PubMed.

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1. How Open Sharing Speeds Science with Angela Abitua, PhD (Webinar - 06-29-2020)
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