FAQ about rAAV
Q: What is AAV?
A: Adeno-Associated Virus (AAV) is a single-stranded DNA virus that is not known to cause any human disease. Wildtype AAV consists of a 4.7kb single-stranded DNA genome and a protein capsid constructed from three subunits named VP1, VP2, and VP3. Both ends of the AAV DNA genome form unique “T”-shaped tertiary structures known as ITRs (terminal inverted terminal repeats). These two ITRs are critical for viral genome replication and are an important signal for triggering viral packaging.
Recombinant adeno-associated viruses (rAAV) are generated by replacing the capsid DNA from the wild-type AAV genome with therapeutic transgenes. The DNA of an rAAV therefore consists a therapeutic transgene that is flanked by the two ITRs, and does not contain any wild-type AAV protein coding sequences. Entirely removing wild-type AAV protein coding sequences maximizes the capacity of the rAAV to carry large therapeutic transgenes while simultaneously also reducing immunogenicity and cytotoxicity.
After infecting cells, the linear DNA genome of rAAVs link head to tail to form a DNA ring. This ring of rAAV DNA can persist within a cell without being degraded as foreign DNA for an extended period. Additionally, it has been shown that this DNA ring holds a very low probability of integration into the host genome. The persistence of genomic material inside of host cells in combination with low integration probability makes rAAV the superior choice for the delivery of genetic payloads in gene therapies, and an excellent carrier for delivering foreign genes in animals for research.
A number of AAV serotypes have been discovered or engineered since the first use of rAAV as a genetic tool, and the newest generation rAAV serotypes have been shown to hold tissue or cell-type specific tropisms. These tropisms provide a degree of infection specificity that can be used as a means to target specific tissues or cell types in basic science expuiriments as well and in gene and cell therapies.
Q: What is the upper limit of rAAV as a gene delivery vector for packaging foreign sequences?
Q: What’s the difference between ssAAV and scAAV?
Q: What do the rAAV titer units GC/ml and VG/ml stand for?
VG/ml stands for Vector Genomes/ml, which indicates the titer of the AAV viral vector and the number of genome copies contained in each milliliter of virus fluid. The dot-blotting method, QC-PCR, and real-time PCR can be used to assess VG/ml. The sample is usually treated with DNase l before being assayed 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 200 serotypes of AAV that have been discovered or designed. These different serotypes are defined by differences in the amino acid sequence and three-dimensional structure of their capsid proteins. Differences in the structure of rAAV capsid proteins across serotypes produces in differences in rAAV recognition and binding of host cell surface receptors. This in turn results in variability in the infections rates of rAAV serotypes across tissues and cell types.
PackGene’s expert technical team is available to help you determine the best serotype to select for your experiments based on the wide literature available on rAAV serotype infection rates and our own internal testing. Nevertheless, for target cell types and tissues without substantial literature, we may recommended the execution of pilot experiments using reporter transgenes to determine the most ideal serotype.
Q: What information will I need to provide to place a custom AAV vector construction order?
A: Custom AAV vector construction projects can be generated in several ways, and our expert technical team is available to help in the design process. There are several questions that you may have readily answered to expedite the design process, they are:
- Do you have a gene template? If so, please provide your gene template to us for verification.
- Do you want to synthesize a gene without a template? If so, please provide the gene number, sequence map, species, and gene length.
- Do you want to generate AAVs forthe manipulation of gene expression using techniques such as RNAi or CRISPR? If so, please provide us with the target gene number, species, and gene length.
- Is there a specific promoter sequence you would like to use?
- Is there a specific fluorescent reporter that you would like to use?
- Will several transgenes need to be co-expressed simultaneously?
- Is your total transgene sequence length <4.4kb?
Q: What information will I need to provide to place a custom AAV packaging service?
A: Custom AAV packaging projects can be generated in several ways, and our expert technical team is available to help in the design process. There are several questions that you may have readily answered to expedite the design process, they are:
- Do you know which serotype you would like youuse for your project? Our expert team is available to help guide your selectionif you would like, but you may alternatively find your perfect serotype by looking toward the literature within your field.
- Are you confident that your preferred serotype is capable of infecting the cells you plan to use for your expiriments? If not, we offer fluorescent control test kits for screening various serotypes. These can be used to definethe infectivity of a given serotype in your cells, or to determine the optimal serotype for your experiments.
- Do you plan to provide your own plasmid for packaging? If so, please make sure that you to provide a vector map and full sequence. Additionally, it is best to confirm ITR spacing and to make sure that the plasmid has been fully sequenced to avoid complications associated with common mutation driven by the presence of ITRs.
- What are the amount, titer, and packaging requirements for your final deliverable rAAV?
Q: What are the AAV purification methods and QC testing indicators?
A: PackGene applies iodixanol density gradient centrifugation for purification. Purified AAV undergo a set of standard assays before shipment. In addition, and we also offer several optional QC assays as addon services.
- Purity: SDS-PAGE Coomassie blue staining
- Titer: PackGene applies ATCC standard for calibration and SYBR Green QPCR for titer detection. We guarantee that the fast service titer reaches 1E+13GC/ml.
- Endotoxin Level: endotoxin lower than 10EU/ml
- TEM electron microscope to test for empty/full shell ratio below 30%
- HPLC purity assay
- Mass spectroscropy assay
- ddPCR titer assay without standard calibration