腺相关病毒(AAV)FAQs

What is adeno-associated virus (AAV)
The adeno-associated virus (AAV) is a small, icosahedral and nonenveloped virus that belongs to Parvoviridae family. It was discovered in 1965 as a contaminant of adenovirus (Ad) preparations and helper virus such as Adenovirus or Herpes virus is usually required for a productive infection to occur. The wild AAV has a linear single-stranded DNA genome of about 4.7 kb of either plus or minus polarity. There are two ITRs flanking the two viral genes rep (replication) and cap (capsid). The Rep gene encode four regulatory proteins:Rep78, Rep68, Rep52 and Rep40 on basis of molecular weight. The Cap gene encode three structural proteins (VP1, VP2 and VP3) which assemble at a ratio of approximately 1:1:10, to form a mature AAV particle which is approximately 22nm. The AAV2 DNA termini consists of a 145 nucleotide-long inverted terminal repeat (ITR) that forms a characteristic T-shaped hairpin structure, due to the multipalindromic nature of its terminal 125 bases, which allows its fold on itself via complementary base pairing , forming a secondary structure that provides a free 3' hydroxyl group for the initiation of viral DNA replication via a self-priming strand-displacement mechanism. AAV does not encode its own polymerase so its replication process relies on host cell polymerase activities.

What is recombinant AAV (rAAV)?
Recombinant AAV is the artificial AAV which does not contain any AAV rep and cap genes which encode viral replication and structural proteins, respectively. rep and cap are replaced with a gene or construct of interest which is flanked by the ITRs which contain all the cis-acting elements necessary for replication and packaging.  Though AAV possesses a 4.7 kb genome, efficient packaging of rAAV can be performed with constructs ranging from 4.1 kb to 4.9 kb in size.

What is helper free rAAV?
Because AAV is a replication defective virus, its replication depend on other virus such as adenovirus and herps virus. these virus also called helper virus. Studies identified AAV only need some of Helper virus products (E1A and E1B,, E2A, E4 and VA) which are provided in trans by another vector or by stably expression cell line.

Is the recombinant AAV safe?
The recombinant AAV (rAAV) composed by several plasmids (cis plasmid, Helper plasmid, rep/Cap plasmid). Cis plasmid and Helper do not share any regions of homology with the rep/cap-gene containing plasmid , preventing the production of wild-type AAV-2 through recombination system. To our knowledge AAV is not linked to any human disease.

What are reps and caps?
They are replication proteins and capsid proteins encoded by rep and cap genes respectively. Reps consist of rep 78, 68, 52 and 40. Reps involve the replication of AAV. Caps have three proteins, VP1 (virion protein 1), VP2 and VP3, with molecular weight of 87, 72 and 62 kDa respectively. These capsid proteins assemble into a near-spherical protein shell of 60 subunits. Caps determine the serotype of AAV.

How to produce different serotype of recombinant AAV?
the most used method to produce different serotype of recombinant AAV is the hybrid trans-complementing method in which the reps and ITR are derived from AAV2; E2A, E4 ,VA from Adenovirus proteins provided by Helper plasmid, E1A and E1B are stably expressed in 293 cells. Caps which give rise to the serotypes of AAV are from different plasmids respectively.

What is the maximum insert size for recombinant AAV?
The maximum insert size for rAAV is about 3.0 kb. If you would like to express other tags such as GFP or RFP, the allowed size is even shorter, about 1.5 kb.

Which serotype should I use in my experiment?
Below are some preliminary guidelines based on literatures.
AAV1: CNS, Eye, Heart, Lung, Skeletal muscle
AAV2: CNS, Eye
AAV5: CNS, Eye, Lung
AAV6: Adipose, Heart, Liver, Lung, Skeletal muscle
AAV8: Adipose, CNS, Eye, Liver, Skeletal muscle;
AAV9: Adipose, CNS, Eye, Heart, Liver, Lung, Skeletal muscle

SignaGen provides AAV serotype test kit for pilot serotype selection studies. Keep in mind there is inconsistence of serotype selection studies be performed in vitro and in vivo.

What is the typical titer of a vector preparation?
For custom DNA and reporter gene is about 1E+12~1E+13 GC/ml (genome copies per ml).

What does the AAV vector use for in laboratory?
AAV vector can be used to deliver a gene, shRNA to knockdown a gene, knockout gene or correct mutated gene in vitro or in vivo? AAV can also be used to create disease model, validate the result of Genome-Wide Association Studies (GWAS).

How to store recombinant AAV preparation?
It is advisable to be aliquoted upon receipt of rAAV and be stored at -80oC. Once an aliquot is thawed it can be stored at 4oC for up two weeks. Stability studies show rAAV vectors are highly stable at temperatures of 4 0C or less. Avoiding repeated thawing and freezing.

What's the difference between physical and genomic particles?
The rAAV particle that have been successfully packaged with the genome are called genomic particles which has the ability to transduce the cells they come into contact with and are therefore functional and rAAV particles that have been packed without genome are called physical particles that are non-functional.

What are the advantages of gene delivery by rAAV?
rAAV has the capacity to produce high titer virus with broad spectrum of tropism in dividing and non-dividing cells and potential for long-term gene transfer with minimum immnunogenicity.
 


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