Adeno-associated virus (AAV) was discovered in 1965, as a contaminant of adenovirus (Ad) preparations, hence the name. It is replication-defective, non-enveloped small viruses (20nm) with a genome of single stranded DNA, from the parvovirus family. As 80-90% of humans are sero-positive with AAV2, most people treated with AAVs are non-pathogenic. Recombinant AAVs can infect both dividing and non-dividing cells and persist in an extrachromosomal state without integrating into the genome of the host cell.
The advantages of AAV compared to other viral vectors
● Long-term gene expression.
rAAV genomes do not integrate into the
host cellular genome, it
persist in the nucleus in episomal forms. As cells replicate and divide, these episome
will be lost. However, It can be expressed continuously for more than 6 months in tissues where cell division is not vigorous.
● Strong diffusivity. Due to small size and higher titer, rAAV has much higher diffusivity than adenovirus and lentivirus. And AAV can cross the blood-brain barrier, it is an ideal tool for neuron and glial cell infection.
● Specific expression can be achieved. AAV has a wide variety of promoters and serotypes, This enables AAV to recognize and infect different organs and cells, which make AAV as the first choice for animal experiments.
● High safety. AAV has not been found to cause disease to humans, and it is the safest viral vector approved by the FDA in US that can be directly used in human for gene therapy.
● low immunogenicity: When AAV is used to infect muscle, brain, eye and many others with local high dose, it is not easy to cause immune response;
● High stability. rAAV virus can be stored for 1 week at 4°C, and it is resistant to some reagents such as chloroform.
Viral vector |
AAV |
LV |
Ad |
Genome |
ssDNA |
ssRNA (+) |
dsDNA |
Coat |
Naked |
Enveloped |
Naked |
Type |
Non-integrating |
Integrating |
Non-integrating |
Infection |
Dividing and non-dividing cells |
Dividing and non-dividing cells |
Dividing and non-dividing cells |
Packaging
Capacity |
4.9kb |
6kb |
7.5kb |
Transgene
expression |
Potentially long-lasting |
Long-lasting |
Transient |
Immune
Response |
Very Low |
Low |
High |
Titer |
Up to 1012-13v.g/ml |
Up to 109TU/ml |
Up to 1012pfu/ml |
Expression abundance |
High-level expression |
Moderate to high level expression |
High-level expression |
Safety |
No pathogenicity has been found yet,
Has been approved by the EU and FDA,
Used as a carrier for gene therapy drugs |
No pathogenicity has been found yet,
It has been used in CAR-T therapy in the human |
May cause some coughing and runny nose |
Packaging service
Process of AAV packaging
AAV virus packaging.(Melissa A. Kotterman.Nature Reviews et al.2014.)
Step1: Clone the foreign gene into a suitable viral vector.
Step2: The recombinant expression plasmid is co-transfected into the AAV-293 cells with pHelper (carrying adenovirus derived genes) and pAAV-RC (carrying AAV replication and capsid genes), which supply the trans-acting factors required for AAV replication and packaging in the AAV-293 cells. Recombinant AAV is assembled in packaging cells 2 to 3 days after transfection.
Step3: Recombinant AAV viral particles are prepared from infected AAV-293 cells and may then be used to infect a variety of mammalian cells. Generally, AAV viral particles are enriched in packaging cells, so collecting cells and then lysing them to release AAV particles into the supernatant can recover most of the AAV viral particles.
Step4: Concentrate and purify the
virus-
containing supernatant above. The original supernatant contains many intracellular protein molecules and fragments. Purified virus is required for animal experiments, otherwise the required dose will not be achieved and side effects will be caused.
Step5: Quantitative PCR (qPCR) is one of the common methods to quantify the AAV vector titer. This method can obtain the physical titer of the AAV genome packaged in the viral particles. The infection titer of AAV varies greatly due to the infected cells, AAV coat protein and test conditions, and the experimental data in vitro cannot reflect the infection in vivo. Therefore, the physical titer obtained by qPCR is a more objective value.
How do I obtain virus packaging services?
BrainVTA provide professional,convenient and R & D products experienced service of AAV, ranging from small-scale to the large-scale, which help scientist accelerate the research process in circuit tracing, mechanisms of disease and treatment of disease.
So far, we have nearly 1000 pre-made AAV products with different serotypes and promoters. Meanwhile, we have offered 2000 customized AAV services for our clients with the following serotypes: AAV-1, AAV-2, AAV-5, AAV-6, AAV-8, AAV-DJ, AAV-PHP.eB, AAV-retro, AAV-anc80 and AAV-9. Artificially designed serotypes and vectors also can be produced on a case-by-case basis.
Please click
Pre-Made AAVs for the in stock AAVs.
By clicking
here for Custom-Made AAVs service
.
Virus serotypes
BrainVTA currently offers the following serotypes: 1, 2, 5, 6, 8, 9, rh10, DJ, DJ8, PHP.B, PHP.eB, PHP.S, retro, pan, anc80. Novel peptide modified serotypes can also be produced on a case-by-case basis. Tissue specificity is determined by the serotype. If you Don't know which AAV serotype to use, see the following table to explore available AAV serotype to meet the efficient delivery of gene into the cells or tissues of interest.
Serotype |
Tropism |
AAV1 |
Muscle, heart, CNS, RPE, lung, skeletal muscle |
AAV2 |
In vitro, CNS, RPE, muscle, liver,CNS |
AAV5 |
Lung, RPE, CNS, pancreas, adipose, liver |
AAV6 |
Muscle, lung, heart |
AAV8 |
Liver, muscle, RPE, CNS, adipose, pancreas |
AAV-pan |
Pancreas |
AAV9 |
Lung, liver, muscle, heart, CNS, adipose, BBB |
AAV.rh10 |
CNS, BBB, pleura |
AAV-Rec2 |
Adipose |
AAV.DJ |
In vitro, liver, heart, kidney |
AAV2/RETRO |
Retro |
AAV.PHP.S |
DRG, heart, colon |
AAV.PHP.B |
BBB |
AAV.PHP.eB |
BBB |
Tips:
● If you are not sure which serotype to choose, you can try AAV9 .
● If you use AAV for the first time, you can use the Rainbow Colors AAV for pre-experiment. By which you can compare the infection effects of different serotypes on the target tissue, and explore the best injection method, injection site, virus dosage, etc., thereby getting more ideal experimental results.
Rainbow Colors AAV
Rainbow Colors AAV is a set product that combines common serotypes suitable for different organizations.
Rainbow Colors AAV |
Serotype |
Volume |
Eyes/other organs |
1/2/5/6/8/9/DJ |
10 μL/serotype |
Nervous system |
1/9/Retro/PHP. eB |
10 μL/serotype |
Liver/Kidney |
2/8/9/DJ |
100 μL/serotype |
Heart/Vascular/Muscle |
1/6/8/9 |
100 μL/serotype |
*AAV1 is used for anterograde tracer.
AAV9 is used as anterograde tracer.
AAVRetro is used for retrograde tracer.
AAV PHP.eB is suitable for crossing the blood-brain barrier.
Tissue-specific promoters
BrainVTA offers various tissue-specific promoters. Listed below are the most commonly chosen promoters. Don' see the promoter of interest,
Please inquire.
NO. |
Name |
Characteristics |
Size(bp) |
Strong promoters |
1 |
CMV |
Ubiquitous |
584 |
2 |
CAG |
Ubiquitous |
1678 |
3 |
CBA( Cbh ) |
Ubiquitous |
793 |
4 |
n Ef1α |
Short Ef1α,Ubiquitous |
493 |
5 |
Ef1α |
Ubiquitous |
1179 |
6 |
hUbC |
Ubiquitous |
1130 |
Inducible promoters |
1 |
TRE3G |
Tetracycline(Tet)-inducible |
350 |
Activity–dependent promoters |
1 |
C-fos |
Immediate-early gene promoter |
662 |
2 |
E-SARE |
Enhanced synaptic activity–responsive element |
954 |
Neuron-specific promoters |
1 |
Mecp2 |
Truncated Mcep2 neuron specific |
230 |
2 |
hSyn |
Mature neuron specific |
485 |
3 |
CaMKIIa |
Specific expression inexci-tatory neurons in the neocortex and hippocampus |
1293 |
4 |
PV |
GABAergic neuron subtypes |
2396 |
5 |
SST |
GABAergic neuron subtypes |
2597 |
6 |
VGAT |
GABAergic neuron |
1800 |
7 |
mDlX |
GABAergic neuron |
530 |
8 |
TH |
Dopaminergic neuron specific |
299 |
9 |
TPH2 |
Tryptophan hydroxylase promoter |
2042 |
10 |
ChAT |
Cholinergic neuron specific |
1500 |
11 |
GFAP |
Astrocyte specific |
2207 |
12 |
GFAP104 |
Short GFAP |
845 |
13 |
Cx30(GJB6) |
Astrocyte specific |
1505 |
14 |
CX3CR1 |
Microglia specific |
1263 |
15 |
Mash1/Ascl1 |
Neural stem cell specific |
1469 |
16 |
Nestin |
Neural stem cell specific |
1394 |
17 |
L7/Pcp2 |
Purkinje cell |
990 |
18 |
MBP |
Myelin basic protein promoter, efficient transduction of oligodendrocytes |
1300 |
19 |
TRPV1 |
Transient receptor potential cation channel, subfamily V, member 1 |
2073 |
20 |
TRPV2 |
Transient receptor potential cation channel, subfamily V, member 2 |
1700 |
21 |
D1 |
D1 dopamine receptor |
827 |
22 |
D2 |
D2 dopamine receptor |
1211 |
23 |
OT |
Oxytocin promoter |
2612 |
24 |
CRH |
Corticotropin-Releasing Hormone (CRH) promoter |
456 |
25 |
PTH |
Human parathyroid hormone promoter |
925 |
Eye-specific promoters |
1 |
hGRK1 |
Human rhodopsin kinase promoter |
292 |
2 |
CAR |
Cone arrestin gene promoter |
523 |
3 |
Grm6 |
Bipolar cell specific expression |
200 |
4 |
mRHOP |
Rhodopsin gene promoter |
524 |
5 |
ROH |
Rhodopsin gene promoter |
2200 |
6 |
Nrl |
Neural retina leucine zipper gene promoter |
3200 |
7 |
RK |
Rhodopsin kinase promoter |
295 |
Liver-specific promoters |
1 |
ALB |
Albumin enhancer promoter |
1001 |
2 |
TBG |
Serine (or cysteine) peptidase inhibitor, clade A |
410 |
Selection of vectors
BrainVTA provides a variety of AAV vectors, which can manipulate coding and non-coding genes through overexpression, RNAi and other technologies, such as lncRNA, microRNA, and circRNA. For more details, please contact
[email protected].