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 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:
- Do you have a transgene template?
- If so, please provide your gene template to us for verification.
- If so, please provide the gene number, sequence map, host species, and gene length.
- If so please provide us with the target gene number, host species, and gene length.
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:
- 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.
- 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.
- 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.
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.
Standard 1.Purity: SDS-PAGE Coomassie blue staining. 2.Titer: PackGene applies 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.
Optional 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.
Q: Are pH measurements required, and is a large amount of sample wasted to carry out pH measurements?
A: Measurement of pH is a mandatory for the release of rAAV Fast Service deliverables. A micro pH electrode may be used to save sample and thus the required sample volume to perform pH measurements is only ~15uL-100uL.
Q: What is loading?
A: In accordance with the Pharmacopoeia General Rules 0942, we use the minimum filling quantity inspection method for detecting sample loading quantity.
Q: How to interpret A260/A280 value?
A: A260/A280 is the ratio of sample absorbance measured at wavelengths of 260nm and 280nm. This measure is commonly thought to represent the ratio of DNA to protein in a sample. For rAAV, A260/A280 can used as a measure of the full to empty shell rate and to identify protein contamination. Low A260/A280 levels may suggest that the empty shell rate is high. Alternatively, high A260/A280 may suggest that the sample has been contaminated with proteins that are not incorporated into the AAV capsid shell. The greatest advantages of this measure are its convenience and speed.
Q: What tests are performed to differentiate rAAV capsid proteins from specific protein impurities?
A: SDS-PAGE is used to identify rAAV capsid proteins. In addition, SDS-PAGE can be used to directly identify specific protein impurities including the presence of host proteins, BSA, or degraded AAV capsid proteins.
Q: How is rAAV infection titer measured, given that rAAV does not integrate the host genome?
A: The current standard for determining infection titer is TCID50 (Median Tissue Culture Infectious Dose). In this assay we test the infection rates of rAAV sample serial dilutions in H5 cells. This initial test is then followed by qPCR detection of the rAAV genome.
Q: If I have determined that a rAAV DNA vector reliably drives transgene expression in host cells prior to rAAV packaging, can I assume that this DNA vector will also drive transgene expression after it is packaged into an rAAV that is then used to infect cells?
A: Successful transgene expression via rAAV infection relies on several factors beyond the functionality of the DNA vector. We therefore recommend that independent validation of transgene expression is performed for all packaged rAAV.
Q: What does rcAAV refer to, and are there regulations regarding rcAAV content in GMP rAAV samples?
A: The term rcAAV stands for replication-competent AAV. In most cases rAAVs are designed to be replication incompetent. Thus, rAAV samples should not contain rcAAV and regulations require negligible replication capability for GMP rAAV samples.
Q: What is the difference between visible foreign matter and insoluble particles?
A: Visible foreign matter can be identified by visual inspection. According to pharmacopoeia general rule 0904, visible foreign matter in injections, ophthalmic liquid preparations, and sterile APIs can be visually observed under specified conditions . Visible foreign matter is usually derived from insoluble particles of a size or length is greater than 50μm.
Insoluble particles between 10μm and 50μm cannot be seen with the unaided eye and must therefore be detected by instrumentation. Insoluble particles identification is commonly carried out with an insoluble particle analyzer. According to pharmacopoeia general rule 0903, evaluation of the size and quantity of insoluble particles within materials to be delivered by intravenous injection (solution injection, sterile powder for injection, concentrated solution for injection) can be made according to the project nature.
Q: If I have successfully carried out several in vivo experiments in mice may I assume that mycoplasma and bacterial endotoxin levels are within acceptable ranges and forgo direct testing for these contaminants?
A: Reagents used for Gene Therapies should abide by all domestic regulations in the “Guiding Principles for Quality Control of Human Recombinant DNA Products” by the EDC. Under these standards Mycoplasma and endotoxin testing is required.
Q: What is the source of PackGene cell bank?
A: PackGene’s h293 cell bank is officially authorized for commercial use.
Q: What is the AAV output for a single batch fermentation?
A: PackGene offers single batch fermentation at several volumes, including: 2L, 7L, 25L, 50L, and 200L. AAV yields for each of these production volumes varies across AAV serotypes. As an example, AAV9 is a medium to high-yielding serotype, and expected yields for AAV9 are as follows:
|Expected yield for AAV9|
Q: How are GC/ml and vg/ml related to one another, and how does PackGene determine GC/ml for AAV products?
A: The terms genome copies per ml (GC/ml) and viral genomes per ml (vg/ml) are interchangeable and equal in most cases. At PackGene we may test GC by both qPCR and ddPCR. Testing by qPCR involves the use of a calibration standard while ddPCR may use optional reference products. Measurements by qPCR are more likely to be influenced by inter-lab and inter-operator variables, and ddPCR generally shows lower %RSD precision. Typically, GC is determined by qPCR during process exploration phase and for intermediate products while the GC of final products is more often determined by ddPCR.
Q: Do specific primers need to be designed to determine GC/ml for a custom AAVs?
A: Genome copies per ml (GC/ml) can be determined using primers directed at common vector elements during the early development stage, and thus we use common features such polyA segments or ITRs to determine GC/ml during this stage. However, it is recommended that specific primers targeted at AAV vector transgenes are used for testing the GC of GMP products. Our final fast service titer test is designed to use transgenes specific primers.
Q: What method is used to determine the empty shell rate for AAV samples?
A: Viral empty shell rate can be determined using several techniques including anion chromatography HPLC, Analytical Ultracentrifugation (AUC), Transmission Electron Microscopy (TEM), CyroTEM, or VG Titer/Capsid titer. AUC, TEM and CyroTEM are typically not suitable for quantitative quality control determinations and thus PackGene’s standard method for empty shell rate determination is anion chromatography HPLC. PackGene can provide additional CyroTEM and AUC analytical services to serve as a secondary verification of the results derived from anion chromatography HPLC.
Q: What is the expected empty shell rate PackGene AAV Fast Services?
A: The expected empty shell rate for our AAV Fast Services varies across AAV serotypes. As one example, the expected empty shell rate for AAV9 generated through our AAV Fast Service is lower than 10%.
Q: Is verification of cell banks in accordance with the Chinese Pharmacopoeia 2015 Three General Principles “The Preparation and Verification Regulations of Animal Cell Matrix for the Verification of Biological Products”?
Q: HIV, HBV, HCV testing, pyrogen-free tests, and wild virus tests are not in the standard quality control services list. Do these tests need to be applied?
A: The harvest liquid generated through PackGene’s AAV fast service will be broadly tested for exogenous viral elements as necessary following an evaluation of project characteristics. Both the original harvest liquid and final deliverables for the AAV Fast service will be devoid of such exogenous viral elements.
Q: What does PackGene consider acceptable error or deviation ranges for standard QC analysis methods?
A: The %RSD of general biochemical methods is usually 15%-25%, and the accepted standard of ddPCR is 10%. In most cases the %RSD for PackGene’s QC ddPCR verification data is lower than 5%, and the %RSD of TCID50 is lower than 10%.
Q: Are there any requirements for the number of samples sent in batches?
A: It is recommended that the sample volume of a single commissioned express delivery is not less than 50uL to avoid the effects of freezing and thawing, evaporation, and tube wall adhesion. The recommended sample delivery volume for genome titer, plasmid DNA residue, rcAAV, etc. is more than 10ul. The recommended sample delivery volume for infection titer is more than 20ul, and the sample delivery amount for empty shell rate testing is 5E+13vg.