FAQ of rAAV
Q: What is AAV?
A: Adeno-Associated Virus (AAV) is a single-stranded DNA virus. The current scientific consensus is that AAV does not cause any human diseases. AAV consists of a protein capsid and a 4.7kb single-stranded DNA genome. The protein capsid consists of three subunits, namely VP1, VP2, and VP3. There are two “T”-shaped terminal inverted terminal repeats (ITR) at both ends of the AAV genome. These two ITRs are the starting point of viral DNA replication and the signal that triggers viral packaging.
The recombinant adeno-associated virus (rAAV) carries the same protein as the wild-type AAV. While the genome encoding viral protein in the capsid is completely deleted and replaced by therapeutic transgene, leaving only the ITRs that play the role in guiding the replication of the genome and the assembly of viral vectors. By deleting the encoding genome of wild-type AAV, we can maximize the capacity of the recombinant AAV to carry therapeutic transgene and also reduce immunogenicity and cytotoxicity when transgene is delivered in vivo.
After infecting cells, rAAV itself will link head to tail to form a ring, and can exist for a long time without being degraded by the cell system as foreign DNA. Therefore, it can be retained in mammalian organs or tissues for a long time, and shows a very low probability of integrating the genome. This feature makes AAV the priority choice for gene therapy, and it has also become an excellent carrier for delivering foreign genes in animals among scientific research.
As a new generation gene therapy vector, AAV shows another important feature in its specificity. Thanks to the different serotypes of AAV, each serotype has a various ability to recognize and infect different organs, and with the appropriate injection method, better results will be achieved. AAV is strongly recommended for animal experiments.
Q: What is the upper limit of rAAV as a gene delivery vector for packaging foreign sequences?
A: The upper limit of rAAV genome packaging is about 5Kb, except for the 145bp of ITR sequences at both ends, it can accommodate 4.5Kb of foreign sequence, including promoter, foreign gene and termination signal. The K104 vector produced by PackGene consists of the smallest promoter miniCMV (the core region of CMV, 180 bp) and terminator (50 bp), which can express genes with a length of about 4.4kb and it is the limit of gene length that can be packaged.
Q: what’s the difference between scAAV and ssAAV?
A: The expression level of double-stranded AAV (scAAV) can reach its peak 2~3 days after infection, while it takes about 7 days for the expression of single-stranded AAV (ssAAV) to reach the peak after infection. ssAAV has to become scAAV before it transcribes and translate foreign genes and this is a slow process. The infection efficiency of scAAV is 6-15 times higher than that of ssAAV. With the same titer, production scale of scAAV needs to be larger than that of ssAAV, so the packaging price of scAAV is much higher.
Q: what does rAAV titer unit GC/ml stand for?
A: GC stands for genomic copies, the number of gene copies. GC/ml stands for the total number of genome copies contained in the viral particles per ml of viral fluid. PackGene apply SYBR green fluorescent quantitative PCR to detect the titer of AAV and calibrate with the ATCC standards, and the specific PCR primers are aimed at the ITR region. PackGene will provide AAV products with 1E+13 GC/ml or above.
VG/ml stands for Vector Genomes/ml, which also indicates the titer of AAV viral vector and the number of genome copies contained in each milliliter of virus fluid. Dot-blotting method, QC-PCR, real time PCR method are usually applied for assay. The sample is usually treated with DNasel before assay, so the titer generally refers to the total number of genome copies contained in the viral particle per milliliter of virus fluid.
Q: What are the serotypes of rAAV? How to choose the right serotype?
A: There are more than 10 serotypes of AAV that have been discovered or design (AAV1/2/3/4/5/6/7/8/9/PHP.eB/PHP.S/rh10/DJ2-retro). Due to the difference in amino acid sequence and spatial structure of the capsid protein, the recognition and binding of cell surface receptors vary, resulting in different infection efficiency.
The infection efficiency of AAV serotypes of various tissues reported in the paper might be different. For target tissues with less literature data, it is recommended that researchers apply pre-experiment to determine the most ideal serotype. This will also be one of the heat experimental innovations.
Generally, AAV-DJ can infect over 80% cell types and AAV-6 is suitable for blood-derived cells.
Q: What kind of information should be known about customized service (AAV vector construction)?
A: 1. Do you have a gene template? Please provide your gene template to us after verification.
2. If you want to synthesize a gene without template, please provide the gene number, sequence map, species, and gene length.
3. You may consider which promoter is used, what kind of label or fluorescence is added and whether co-expression of several genes is required.
4. Please pay attention to the capacity between ITR in AAV vector.
5. As for shRNA, it’s recommends that customers choose three targets, or choose a verified target. If the customer order for a shRNA design, we would not guarantee the infection efficiency. We also recommend that customers choose the best infection target with in-vitro pre-experiment before AAV virus packaging.
Q: What kind of information should be known about customized service (rAAV packaging service)?
A: 1. Different serotypes should be chosen according to infection site. You might refer to related paper or ask for technical support from PackGene if you are not sure about the serotype. We also provide fluorescent control test kit of different serotypes for screening, which can help determine the suitable serotype.
2. For those customers who provide plasmid for packaging, please make sure that you also provide vector map sequence, confirm your demand for an AAV vector, pay attention to the ITR spacing and make sure that the plasmid has been sequenced and verified.
3. What kind of AAV virus packaging information belongs to? Type A/B/C/D.
4. Confirm the total amount, titer and packaging requirements of rAAV.
Q: What are the AAV purification methods and QC testing indicators?
A: PackGene apply iodixanol density gradient centrifugation for purification.
PackGene QC assay indicators:
1. Purity: SDS-PAGE Coomassie blue staining
2. Titer: PackGene apply ATCC standard for calibration and SYBR Green QPCR for titer detection. We guarantee that the product titer reaches 1E+13GC/ml.
3. Endotoxin testing: endotoxin lower than 10EU/ml
4. TEM electron microscope: empty/full shell ratio below 30% (optional)
5. HPLC purity assay (optional)
6. Mass spectrometer assay (optional)
7. ddPCR titer assay without standard calibration (optional)