Frequent Asked Questions (FAQs)
Based on our interaction with customers and
researchers who are interested in using AAV in their work, we put
together this list of questions and answers. The purpose is to give
people short information on some major issues on AAV and its
application, which ought to be regarded as a start point for more
detailed investigation and literature search.
Questions:
General information
- What
are the advantages of using AAV for gene delivery
- How
is recombinant AAV generated
- What
are the restrictions/limitations of AAV
- How
AAV is measured or, the difference between particle number,
genome copy, infectious titer, and MOI
- What
are the differences between AAV serotypes
- How
can I express siRNA with AAV
- What
is double stand (ds)-AAV
- What
do I need to provide for custom AAV production
- How
long does it take to generate AAV
- What
do I receive as pre-made or custom-made AAV
- What
else can you do to characterize my AAV vector
Questions: Application
- How
much vector do I need in my experiment
- How
do I infect in vitro cultured cells
- Should
I avoid serum when infecting cells with AAV
- How
long do I need to wait to see expression
- Protocol
for in vivo AAV infection
- Where
do AAV vectors go after intravenous injection
- Can
AAV elicit immune response in animals
Questions: Safety
- Can
I use AAV in my lab
- How
to inactivate/decontaminate AAV
- How
stable is AAV, and how can I handle and store them
1. What are the advantages of
using AAV for gene delivery?
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Adeno-associated virus (AAV) is
a DNA virus with single strand genome of 5000 bases. Replication of
AAV genome is dependent on co-infection of other
viruses such as adenovirus or HSV. Wild type AAV has not been
reported to cause any diseases. Together with its replication
defective nature, AAV has good safety profile to be used in gene
transfer in vivo, and as potential gene therapy vehicles.
AAV is capable of
infecting non-dividing cells and integrating viral DNA into host genome
for long-term expression.
Compared with other viral
vectors such as adenovirus, AAV elicits very mild immune response in
animal models.
2. How is recombinant AAV
generated?
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Recombinant AAV (rAAV) is
generated by replacing Rep and Cap coding region with exogenous gene
expression cassette. The two ITRs (inverted terminal repeats) on AAV
genome are the only cis-acting elements required for genome
replication and packaging. Rep and Cap genes together with helper
genes from adenovirus are provided from separate plasmids for rAAV
generation. In Applied Viromics, we triple-transfect HEK 293 cells with
AAV shuttle plasmid (ITRs and transgene cassette), pRep-Cap, and
pHELP (adenoviral helper genes).
3. What are the
restrictions/limitations of AAV?
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AAV has limited packaging
capacity of 5000bp. By using shuttle vectors of Applied Viromics,
one can only pack genes ranging 2.2-3.5kb in size.
4. How is AAV measured or, the difference between particle number,
genome copy, infectious titer, and MOI ?
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AAV is quantified by genome copy
(GC) number. These genome-containing particles are functional vectors that
infect target cells. Besides these "full" AAV, empty viral
particles are also produced. Measurement of GC rather than total
particle number (by protein assay) is thus more relevant.
Infectious titer is determined
experimentally by infecting target cells with certain concentrations
of AAV under specific conditions (cell type, growth condition of the
cells, media volume, incubation time, read-out type and assay
details, etc.). AAV vectors made by Applied
Viromics usually have a infectious titer of 1 per 20-50GC. Detail in
infectious titer assay is available.
MOI stands for multiplicity of
infection. Once infectious titer for certain
cell type is determined, MOI is determined by calculating number of
infectious particles per cell. MOI of 1 means in average each cell
is infected by one AAV vector.
Infection is achieved by
delivering viral genome into target cells. However, expression of
detectable levels of proteins from the delivered genes (called
transduction) is very different. For
AAV, getting from infection to transduction is complex because
single strand AAV genome needs to be converted to double strand
before gene expression can occur. This step has been proven to be
rate-limiting. Many researchers measure AAV infectivity by checking
marker gene expression in infected cells. Such method will
significantly under-estimate AAV infectivity. Enhancing agent such
as wild type adenovirus or DNA synthesis inhibitors (hydroxyurea or etoposide) can be used to promote AAV
genome conversion, thus improve the measurable AAV infectious titer.
5. What
are the differences between AAV serotypes?
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Applied Viromics offers AAV serotypes 1 to 6. In
fact more serotypes are isolated. These different serotypes of AAV
have variable sequences in ITRs, Cap and Rep proteins. Variations on
capsid protein confer serotypes of AAV to have different tropism. It offers a good
tool for researchers to target specific cell types or tissues for
infection. Also, neutralizing antibodies against one serotype may
not block infection of another serotype. This provides an opportunity for
multiple infection.
AAV serotypes offered by Applied Viromics all have
AAV2 ITR sequences, and Rep from AAV2. Only Cap sequences are from
their perspective serotypes. Thus one AAV shuttle plasmid can be
used to produce all 6 serotypes by pairing up with different pRep-Cap
plasmids.
6. Can I express siRNA with AAV?
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Yes. Applied Viromics has AAV shuttle plasmids
designed for siRNA expression. You can find detailed information
about these plasmids and contact us for services we offer on siRNA
shuttle plasmid construction.
7. What
is double stand (ds)-AAV?
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They are AAV with self-complementary genome. One
of the two ITRs is mutated to remove DNA excision site. During
replication AAV genome folds on itself to form double strand
structure. This ds-AAV has better expression than normal single strand
genome since unlike the single strand genome, double strand DNA is
ready to be transcribed after AAV infection.
However, capacity is reduced to 2500bp, including ITRs and the whole
expression cassette. Applied Viromics has ds-AAV shuttle
plasmids available. Despite the small capacity, they are very good tools for expressing
small proteins or siRNA.
8.What
do I need to provide for custom AAV production?
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We have pre-made AAV which can be ordered from our
website.
For custom-made AAV, you can construct your own AAV
shuttle plasmid with your expression cassette, and we package AAV vector for you.
If you do not have AAV shuttle plasmid prepared,
you can provide us with your gene of interest in a plasmid or as PCR
fragment. We
will clone the gene into one of our shuttle plasmid. Then we produce
AAV vector for you.
If you do not have the gene with you, we can clone
the gene from a library, construct shuttle plasmid, and produce AAV
vector for you.
In terms of the gene of interest, we would like to
check with you about its size (not to exceed packaging limit), and
its toxicity to HEK 293 cells. Applied Viromics will always be working
together with our customers to come up with the best solution in
terms of shuttle plasmid design. Please contact us for details.
9. How
long does it take to generate AAV?
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For a standard batch production, it takes 3 weeks to get the AAV produced, purified, and assayed for
delivery.
On top of this time line, additional steps will
take extra time:
--Constructing and producing
AAV shuttle plasmid: 1 - 2 weeks;
--Clone gene from
library:
2 - 3 weeks.
10. What
else can you do to characterize my AAV vector?
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We routinely perform ID test on AAV shuttle
plasmid by restriction enzyme digestion, and picogreen assay on AAV
genome titer. Additional tests are available upon request (with
extra charge):
--SDS-PAGE and silver staining
assay
--endotoxin assay
11. How
much vector do I need in my experiment?
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For in vitro assay, infectious titer on specific
cell line needs to be determined. Using 293 as an example,
5000GC/cell is sufficient to achieve >90% transduction. Thus 1e11
GC of AAV is capable of infecting 20 million cells.
Vector demand for in vivo study varies on experimental
design. Generally speaking, 1e12 GC per kg body weight is commonly
used to infect mice by intravenous injection. Taken the average
weight of a mouse at 25g, 1e12 GC AAV is sufficient to infect 40
mice.
12. How do I infect in vitro cultured cells?
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See protocol for detail.
13. Should
I avoid serum when infecting cells with AAV?
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For in vitro study, fetal bovine serum and calf
serum are not found to interfere with AAV infection. 44% of human population
have antibodies against AAV. If you need
to use human serum in your study, it is recommended that you test
for titer of neutralizing antibody. Please contact us for detail of
our assay service.
14. How
long do I need to wait to see expression?
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In cell culture, transgene expression is readily
detectable 24hrs post-infection, while we recommend to wait 48hrs.
In vivo study takes longer. As reported,
short-term expression is detectable after 1 week of viral delivery,
while long-term gene expression can last more than 1 year.
15. Protocol
for in vivo AAV infection
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This protocol is a generic version of delivering
AAV by intravenous injection to mice. The purpose of preparing this
protocol is to give reader a general idea about in vivo AAV
infection.
For your own study, literature search on similar
work, regulations of your animal use committee, as well as your
specific experimental plan will shape the protocol in more detail.
16. Where
do AAV vectors go after intravenous injection?
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In vivo distribution of AAV vectors varies by
their serotype, route of administration, dose, and time of assay.
For tail-vein injection of AAV2, organs that see
most of the AAV vectors are lung and liver. Although many other
organs and body parts have detectable AAV levels, relative amount is
very low.
For getting AAV to specific tissues or organs,
please refer to section of AAV targeting.
17. Can
AAV elicit immune response in animals?
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Except immune-deficient animals, AAV does elicit
immune response when injected to naive animals.
The immune response has both humeral and cellular components. This
prevents the same AAV vectors to be administered repeatedly.
In AAV-positive animals, one can detect
neutralizing antibodies against AAV capsids, preventing AAV
infection. Antibodies are also generated against transgene product,
if the protein is different from endogenous one. T-cell response against
infected cells was also reported.
However, since recombinant AAV does not express
any viral protein, the immune response against AAV seems much mild
compared with what has been observed from adenoviral
infection.
18. Can
I use AAV in my lab?
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The use of AAV requires a lab equipped with
biosafety level two facilities. Please refer to MSDS for detail.
19. How can I inactivate/decontaminate
AAV?
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AAV is readily inactivated by 1% bleach, 70%
ethanol, or other decontaminating products. Please refer to MSDS for
detail.
20. How
stable is AAV, and how can I handle and store them?
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AAV is stable at 4oC for at least a week. Best way
for long term storage is at -80oC.
Common precautions on AAV handling are to avoid
repeated freeze/thaw, avoid making very diluted stock for storage,
and avoid excessive pipetting and transfer of small volume since the
vector may stick to plastics.
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