Ineffective recombinant adeno-associated virus (AAV) products can not be packed alone. They require certain adenoviral gene products on ground for their packaging. The Adeno-Associated Helper Free system helps to get rid of the need for a adenovirus to help as this make the system safer as well as less difficult. This addgene AAV guide will focus on everything researchers should know about AAV packaging and serotypes.
Common Uses of AAV
Researchers employ AVV expression system in optogenetics experiments. AVV comes above other viruses including lentiviruses. This is because they do not change as primary episomal. On the other hand, lentiviruses intergrate into the genome. This is very crucial because the structure chromatin at genome site integration can alter transgene behavior, including that of the closer genes. The short coding sequences of channelrhodopsins, halorhodopsins, and other optogenetic genes allows AVVs-packagimg.
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. The presence of rep bindin element favors this site, however, there may e further integration at a very much lower frequency. AAV would not gain access to lytic cycle as a being aided as a replication-incompetent virus. Herpes simplex virus or a genotoxic agent such as ultraviolet radiation hydroxyurea are needed to activate the lytic cycle.
When researchers use AAV (rAAV) in research work, there is supply of rep protein on trans, getting rid of the ability of rAAV for the integration into its choice of genomic site integration n human chromosome 19, termed AAVS1. Instead, there is typical of rAAV genome processing into a double-stranded circular episome via double-stranded synthesis.
There can be contactemerizing of these epsiones, resulting in the production of the structure of molecular weight that is maintained with an extra chromosome. When it comes to the integration of non-homologous sites on the genome, rAAV is more likely than the wild type of AAV. However, this will happen at a frequency of approximately 0.1%. Many of the particles of rAAV, regardless, are thought to remain under maintenance either concatemers or episomes.
During the cycle of lytic, there is a profound difference in episomes from viral particles produced. Episomes can develop an organization that looks like chromatin, remaining in non-dividing cells for a longer period, even up to years and will not damage the host cell. On the other hand, there is a quick release of viral particles produced during lytic cycles through cell lysis. If episomes are stable, they allow the expression of transgene for a longer time in non-dividing cells and is a key advantage of rAAV.
Adeno-Associated Virus Serotypes
So far, researchers have been able to identify adgene AVV stereotypes and at the same time, they come with the best characterized that AAV2 uses most. Their stereotypes are not the same in tropisms or the cells they infect as this makes AAV stronger system when it comes to preferential transducing of some kind of cells. However, here is a chat that summarizes the tropism of AAV stereotypes, showing the optimal serotype (s) for a specific organ transduction.
Tissue Optimal Serotype
CNS AAV1, AAV2, AAV4, AAV5, AAV8, AAV9
Heart A AV1, AAV8, AAV9
Kidney 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
Methods of increasing packaging capacity
Increasing the capability of packaging may require a longer transgene splitting between two AAV plasmids, the first with a 3′ splice donor, as well as the second with a 5′ splice acceptor. When there is co-infection of these viruses on a cell, there is concatemers formation spiced together and there can be a full expression of transgene. With this method, transgene can be expressed for a longer time. However, there is less efficiency associated with his expression than with single AAV virus (∼5%).
Another method to increase the capability of packaging is based on the recombination homologous. Using this method, there is division of genes between two transfer plasmids, but with a very large overlapping of sequence. Co-expression induces homologous recombination and expression of the full-length transgene at very low efficiency (<1% of wild type). If researchers can find any of these methods to be efficient, Adeno-Associated virus would be unlimited to small transgenes, allowing more AAV applications to develop.
Ways to package Transgenes That Exceed the Size Limit of Adeno-associated Virus
Many great features make adeno-associated virus a suitable option for viral vector. However, it does not have a better capacity when it comes to packaging it. Its packaging capacity is limited to only -4.7kb or roughly half the packaging limits of lentiviral and adenoviral vectors. On other hand, a lot of transgenes will find this limit suitable, but some prime editing’s PE2 enzymes do not. Researchers often wonder if it is possible for a big gene to into a smaller vector-like AAV. Fortunately, there is a way to make this happen and this is done by breaking the transgene into smaller sizes. The question is? How can this be possible? There are four different ways to package transgenes that exceed the size limit of AAV.
- Sequential homology directed repairs; This approach helps in delivering large donor templates with the use of double sequential homology directed repairs.
- Overlapping: This approach has a region of homology, typically ~400-1400 bp long between part A and B of the transgene to reconstitute the full-length transgene
Trans-splicing: This approach is based on splice donor and acceptor reconstituting the two transgene pieces - Hybrid: An approach that cobins boh overlapping and trans-splicing.
Conclusion
A great drawback of AAV is the limited capability of packaging. However, researchers do not have to worry as they can employ any of the packing methods to make it fits into their needs such as addgene aav purification.
