2) Adeno Associated Virus (AAV) – Production and Modification of AAV

2) Adeno Associated Virus (AAV) – Production and Modification of AAV

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!


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