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FAQ about rAAV


Q: What is an 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 capsid proteins 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 (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 protein DNA from the wild-type AAV genome with transgenes expression cassettes. The DNA of an rAAV therefore consists of a transgene expression cassette that is flanked by two ITRs and does not contain any wild-type AAV capsid protein coding sequences. Entirely removing wild-type AAV protein coding sequences maximizes the capacity of the rAAV to carry large therapeutic transgenes while simultaneously 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 an excellent carrier for delivering foreign genes in animals for research and the superior choice for the delivery of genetic payloads in gene therapies.

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 experiments as well and in gene and cell therapies.

Q: What is the maximum size of an rAAV transgene?

A: The upper limit of rAAV genome packaging is ~ 5Kb including the required 145bp ITR sequences at either end. Thus, rAAV accommodate a ~4.5Kb transgene expression cassette. An rAAV transgene expression cassette usually includes a promoter, a gene of interest, and a terminator signal. PackGene’s K104 vector has been designed to maximize gene of interest capacity by integrating the smallest available mammalian promoter region in miniCMV (180 bp) and terminator region (50 bp). PackGene’s K104 vector can thus accept a gene of interest up to 4.4kb in length.

Q: What’s the difference between ssAAV and dsAAV?

A: Both wild-type and many rAAV hold a single stranded DNA genome (ssAAV), but rAAV have also been engineered to house a double stranded DNA genome (dsAAV). Transgene expression following infection with ssAAV requires that a second strand of DNA is synthesized within the host cell to convert the ssAAV genome into double stranded DNA. This process results in peak transgene expression ~7 days after infection with ssAAV. The use of dsAAV circumvents this initial step, and thus the time required to achieve peak expression of transgenes is reduced from ~7 days down to ~2-3 days. In addition, it has been shown that the infection efficacy of dsAAV is between 6- and 15-fold higher than ssAAV. Effective titer and production costs associated with dsAAV are thus much lower than ssAAV.

Q: What do the rAAV titer units GC/ml and VG/ml stand for?

A: GC stands for genomic copies and identifies the number of gene copies in a solution. GC/ml stands for the total number of genome copies contained in the viral particles per ml of fluid. PackGene applies SYBR green fluorescent quantitative PCR to detect the titer of AAV with ATCC standard calibration and detection via specific PCR primers aimed at the rAAV ITR region. PackGene’s AAV Fast Services provides an AAV solution deliverable with at least 1E+13 GC/ml.

VG/ml stands for Vector Genomes/ml and identifies both 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. Industry standards dictate that samples are treated with DNase before being assayed for VG/ml, and thus viral titer in VG/ml is commonly equivalent to GC/ml and represents the total number of genome copies per milliliter of virus fluid.

Q: What are rAAV serotypes , and what steps should be taken to ensure that the best serotype is chosen?

A: Different AAV serotypes are defined by differences in the amino acid sequence and three-dimensional structure of their capsid proteins, and more than 200 AAV serotypes of have been discovered or designed. Serotype specific differences in rAAV capsid proteins correspond with variations in cell surface receptor recognition and binding. This, in turn, results in variations in the infection rate of rAAV serotypes across tissues and cell types.

PackGene’s expert technical team is available to help you determine the optimum serotype for your experiments based on the literature regarding rAAV serotype infection rates and our own internal testing. Nevertheless, for target cell types or tissues without substantial literature available, 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 rAAV 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 prepare answers for to expedite the design process, they are:

  1. Do you have a transgene template?
    • If so, please provide your gene template to us for verification.
  2. Do you want to overexpress a gene without a template?
    • If so, please provide the gene number, sequence map, host species, and gene length.
  3. Do you want to generate rAAVs for the manipulation of gene expression using techniques such as RNAi or CRISPR?
    • If so please provide us with the target gene number, host species, and gene length.
  4. Is there a specific promoter sequence you would like to use?
  5. Is there a specific fluorescent tag or reporter that you would like to use?
  6. Will several transgenes need to be co-expressed simultaneously?
  7. Is your total transgene sequence length <4.4kb?

Q: What information will I need to provide to place a custom rAAV packaging order?

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 prepare answers for to expedite the design process, they are:

  1. Do you know which serotype you would like you use for your project?
    • Our expert team is available to help guide your selection if you would like, but you may alternatively find your ideal serotype by looking toward the literature within your field.
  2. Are you confident that your preferred serotype is capable of infecting the cells that you plan to use for your experiments?
    • If not, we offer fluorescent control test kits for screening various serotypes. These can be used to define the infectivity of a given serotype in your cells, or to determine the optimal serotype for your experiments.
  3. 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 mutations that can be driven by the presence of ITRs.
  4. What are your requirements for the amount, titer, and packaging of the final deliverable rAAV?

Q: What rAAV purification methods does PackGene use?

A: PackGene applies iodixanol density gradient centrifugation for purification.

Q: What QC testing does PackGene perform on rAAV deliverable?

A: Purified rAAV undergo a set of standard assays before shipment. In addition, and we also offer several optional QC assays as addon services.

1.Purity: SDS-PAGE Coomassie blue staining.
2.Titer: PackGene applies ATCC standards for calibration and SYBR Green QPCR for rAAV titer detection. We guarantee that rAAV deliverables for our fast service will reach titers of at least 1E+13GC/ml.
3.Endotoxin levels: Endotoxin levels in rAAV deliverables for our fast service will be lower than 10EU/ml.

1.TEM electron microscopy to test empty/full shell ratio.
2.HPLC as an additional purity assay.
3.Mass spectroscopy as an additional purity assay.
ddPCR titer as an additional titer assay without standard calibration.