The 411 on the Adeno Associated Virus

November 12, 2012

The 411 on the Adeno Associated Virus

An adeno associated virus (AAV) is a small virus in the parvovirus family that has a genome of DNA with a single strand. This virus can always place genes into a certain area on chromosome 19 without fail. There are several disadvantages associated with AAV, though. For starters, it can only carry a small DNA amount and it has trouble producing it, as well. Plus, the majority of people who get treated with the adeno associated virus won’t build any immune responses to get rid of the virus or the cells that were effectively treated with it, either. However, this virus is generally used because it isn’t pathogenic.

Various AAV trials are currently being prepared and some are already ongoing with hopes of treating eye diseases and muscle diseases because this is where AAV seems especially helpful. Some clinical trials also involve delivering genes into the brain, though. This works because the adeno associated virus is able to infect non-dividing cells.

Overall, the genome of AAV consists of negative-sensed or positive-sensed ssDNA with single strands, which are around 4.7 kilobase in length. This genome also consists of ITRs with four genes that overlap and encode the rep proteins needed for the AAV’s life cycle, as well as ORFs with overlapping capsid protein sequences of nucleotides that work together to create an icosahedral symmetric capsid.

The ITR sequences each have 145 bases that were named for their symmetry – something that is needed to effectively duplicate the adeno associated virus genome. These sequences can also create hairpins that help with self-priming and allow the synthesis of the other DNA strand. In general, ITRs are also needed to integrate the AAV DNA to the main cell genome and then save it from it, and to efficiently encapsidate the AAV DNA with the generation of complete AAV particles.

When it comes to gene therapy, though, ITRs are the only known sequences aside from therapeutic genes that are needed in cis: packaging and structural genes can be sent in trans. Assuming this, a lot of methods were created to efficiently produce recombinant AAV vectors with therapeutic or reporter genes.

On the left of the genome of the AAV, two promoters exist known as p19 and p5, which can produce two mRNAs of various lengths. Each one of these mRNAs has an intron in them, which could be spliced out. With this possibility, it is therefore possible to synthesize four different mRNAs and four different rep proteins of overlapping sequences. Their names will then depict their overall sizes in terms of kilodaltons.

On the right of the genome of a positive-sensed adeno associated virus, the encoding of three capsid protein sequences happens that start from a single promoter, p40. The three proteins are translated from a single mRNA, which can be spliced out differently: either shorter or longer intron can be excised to create two mRNA pools. Normally, longer intron would be preferable, though, most of all when an adenovirus is present. In this particular form, an AUG codon is cut out, from which the VP1 protein synthesis begins and the VP1 protein synthesis level will then drop in response. This initial AUG codon stays in the main splice afterwards and acts as the first codon for the VP3 protein.

Unfortunately, the ACG sequence can be found just on top of that codon and it encodes threonine, even though it shares the same reading frame as the codon. The best Kozak context surrounds this sequence, though, thus contributing to a low VP2 protein synthesis level – which, in actuality, is simply a VP3 protein that has extra residues of N terminal. More recently, a brand new protein involved in the assembly of viral particles was also identified as AAP, which began its coding in VP2.

Since the larger intron is preferably spliced out and because the ACG codon in the main splice is much weaker in terms of sending out an initiation signal, the overall ratio wherein the structural proteins of the adeno associated virus are synthesized within the vivo stands at around 1:1:10 – similar to the mature particle of the virus. Also, the unique N terminus fragment of the VP1 protein seems to have PLA2 activity, which is needed to release adeno associate virus particles out of late endosomes.

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