With its discover in 1960, Adeno Associated Virus (AAV) has become a revolutionary viral
vector in gene delivery.
Its low pathogenecity and nearly negligible immune response in mammalian organisms are
the attributes that have made AAV the ideal delivery tool for gene therapy.
Over the years, many modifications have been made to both the production protocol and the
virion itself to improve its production efficiency and increase its potential applications.
For additional background on the discovery, biology and features of AAV, please take a
look at our Adeno-Associated Virus - An introduction video.
The techniques and methods required to produce recombinant AAV will be outlined in this section,
as well as all possible modifications that can be performed on the recombinant AAV.
The development of the recombinant AAV viral vector was first described in 1984, using
a human packaging cell line and a two plasmid transfection system.
It involves 1) a vector plasmid containing the transgene enclosed by inverted terminal
repeats, or the ITRs and 2) a complement plasmid having the necessary Rep and Cap Genes of
AAV.
Since AAV requires the presence of a helper virus to replicate, the packaging cell line
is also infected with adenovirus at the time of plasmid transfection.
Once recombinant AAV is made, the contaminating adenovirus is usually destroyed by heat inactivation
. In these early attempts, the AAV preparations resulted in mixtures that were contaminated
with wild-type inactivated adenovirus particles, thus hindering their potential use.
In the modern system, generation of rAAV vectors requires four key components: 1) a cassette
containing the transgene enclosed by two inverted terminal repeats (ITR); 2) the plasmids containing
the AAV Rep and Cap genes required for capsid formation and replication; 3) the necessary
Adenovirus helper genes provided in plasmid format; and 4) a viral packaging cell line
The AAV genome is only 4.7 kb in length and therefore restricts the size of the possible
transgene that can be inserted into the rAAV cassette.
The size restrictions of the AAV transgene have been demonstrated in experimental settings,
and any transgene that exceeds 4.7 kb in length fails to effectively transduce, or express
its transgene in the long term.
The transgene in the cassette also needs to be flanked by two ITRs, which are necessary
for the packaging of the rAAV into the capsid.
The original AAV genes, Rep and Cap, are provided on a separate plasmid in-trans (2).
Without the presence of the ITR regions on this plasmid, the Rep and Cap genes will not
be packaged into the viral capsid during production and will remain within the parent cell for
the duration of the production.
The required adenovirus helper genes used in rAAV production are provided on yet another
separate plasmid.
The identified helper genes from adenovirus are: E1a, E1b55k, E4orf6, E2a, and VA RNA.
The E4orf6, E2a, and VA RNA genes are provided on a separate plasmid that is transfected
with the other rAAV components while the E1a and E1b55k are generally expressed by the
packaging cell line itself The most commonly used cell line in rAAV generation
is the HEK293 cells, which express the Adenovirus helper genes E1a and E1b55k.
Introducing the other adenoviral helper genes in plasmid form into the HEK293 cells ensures
that expression of the transgene harboring rAAV stays constant throughout the process.
AAV packaging cell lines are available for this purpose and can be used to streamline
this process and reduce the number of reagents that are potentially needed to generate packaged
rAAV.
A detailed packaging protocol, showing a step by step method for the production of AAV is
outlined in our knowledge base (in the link provided below).
Briefly, HEK293 packaging cell line is prepared the day before transfection with 70% confluency
on the day of transfection.
Follow the manufacturer’s protocol to perform 3-plasmid co transfection.
Forty-eight hours post transfection, collect the cells using a cell scraper and subject
the cell pellet to 3 freeze-thaw cycles.
The freeze-thaw cycles should be performed using dry ice/ethanol bath and a 37 degrees
water bath.
After the freeze-thaw cycles, the recombinant AAV is released into the supernatant and can
be used immediately for transduction experiments or be subjected to further purification using
iodixanol gradient.
As was mentioned previously, other changes have been performed by scientists to rAAV
vectors to make them more effective, such as capsid modification to change its tropism
towards certain tissue types.
These modifications include: Transcapsidation: the process of packaging
the ITR of one serotype of AAV into the capsid of a different serotype
Adsorption of Receptor Ligands: the addition of foreign peptides to the surface of the
rAAV capsid Mosaic Capsid: a modification involving the
packaging of the AAV genome or rAAV transgene into an AAV capsid made up of a mixture of
unmodified capsid proteins from two separate serotypes
And; Chimeric capsids: which are packaged capsids that have a foreign protein sequence
inserted into the Open Reading Frame of the capsid gene
abm offers a library of customizable AAV constructs, containing most human, mouse and rat genes.
To access our AAV products, go to www.abmgood.com, and from there click on the AAV link.
This link will take you to our AAV product page, which has a description of our AAV products,
as well as a detailed table outlining the tropism of different subtypes of AAV.
Towards the bottom of this page, you will find the available promoter choices.
Select an appropriate promoter and search for your gene of interest by name or accession
number.
Alternatively, you can browse through the gene list provided at the bottom of the page.
At the top of the search result, different promoters can be chosen.
Depending on the size of your gene of interest, the promoter choices may be limited.
The table below details all available constructs of the gene, and shows the available serotypes
as well as what species the gene originated from.
Having difficulty finding your gene of interest?
Try our AAV custom service option.
From our home page, click on custom services, and from there to “Custom Adeno-Associated
Virus (AAV) Service”.
From this page, you can select the method of providing your gene of interest, the titer
of AAV you would like to receive, and the serotype of the AAV virion.
This will tailor the list of available services to your specific needs.
Please leave your Questions and comments below and we will answer them as soon as possible.
For more information please visit our website.
Thank you for Watching!