AAV Packaging - HT Grade

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:

Serotype selection: For in vitro studies, AAV-DJ and AAV6 are common choices. AAV-DJ generally infects over 80% of cultured cells, while AAV6 is particularly effective with blood-derived cells.

Gradient dilution infection: Similar to in vivo experiments, we advise conducting 3-4 AAV gradient dose infections to find the optimal transgene expression level and to ensure the dose is not cytotoxic.

Assay time: Typically, assays should be performed 2-7 days post-infection to assess the effects accurately.

Recombinant AAV (rAAV) is a powerful tool for delivering exogenous genes, and its use in in vivo research is growing rapidly. However, the ideal injection site, rAAV quantity, concentration, and incubation period can vary widely based on the specific experiment and scientific field. Due to this variability, a universal injection protocol for AAV does not exist, so researchers need to explore each of these variables independently, guided by their unique research goals.

Serotype selection: For cells cultured in vitro, AAV-DJ and AAV6 are the most common choices. In conventional cell culture, AAV-DJ can infect more than 80% of cells, while AAV6 has the strongest infective potency against blood-derived cells.

Gradient dilution infection: The level of transgene expression driven by rAAV may vary substantially across different genes. We therefore recommend that you perform 3-4 AAV gradient dose injections to determine the ideal gene expression level for each rAAV before performing any formal experiments and to verify that your experimental dose is not cytotoxic.

Assay time: usually 2-7 days after infection.

Serotype selection: If you are unsure which AAV serotype is most suitable for your experiments, we advise that you test the infection rates of 3 or more different serotypes in your experimental system with our rAAV fluorescent reporter constructs.

Gradient dilution infection: The level of transgene expression driven by rAAV may vary substantially across different genes. We therefore recommend that you perform 3-4 AAV gradient dose injections to determine the ideal gene expression level for each rAAV before performing any formal experiments.

Experimental control: We advise the use of a GFP positive control vector of the same serotype and promoter as your experimental vector.

As an effective tool for delivering exogenous genes, rAAV are being used by a rapidly increasing number of in vivo researchers. Nevertheless, the ideal injection site, rAAV quantity, rAAV concentration, and incubation period may vary across experiments and scientific fields. Due to this variability, it is difficult to adopt a universal AAV injection protocol that is applicable for all experiments. Clients must therefore explore each of these variables independently based on their specific goals.

As of now, nine naturally occuring serotypes of human AAV have been discovered (AAV1/2/3/4/5/6/7/8/9) and widely applied in scientific research. AAV10 and AAV11 were first discovered in non-human primates in 2004, and no cross-reactivity was observed between AAV10, AAV11, and AAV2, making them promising candidate vectors. Subsequently, researchers isolated AAV12 and AAV13 from simian adenovirus, with limited research on these serotypes currently. Based on these wild-type AAVs, researchers have developed many AAV mutants, such as AAV-DJ and the PHP series, through various modification strategies.

Due to differences in the spatial structure of capsid proteins among AAV serotypes, there are significant variations in their recognition and binding to cell surface receptors, leading to tropism of different AAV serotypes for different tissues. When selecting serotypes, experimental purposes can refer to AAV serotypes used in peer-reviewed literature. For example, AAV1 and AAV9 are more commonly used in brain research than other wild-type AAV serotypes, while AAV6 exhibits higher lymphocyte selectivity.

There are also many engineered serotypes that have been modified or engineered to enhance specific properties for gene therapy applications. These modifications can include alterations to the capsid proteins to change tissue tropism, improve transduction efficiency, evade immune responses, or increase payload capacity. Engineered AAV serotypes have been developed through various strategies such as directed evolution, rational design, or hybridization of existing serotypes. These engineered serotypes offer enhanced performance and versatility, making them valuable tools for targeted gene delivery in biomedical research and therapeutic applications.

PackGene offers nearly 100 serotypes for our packaging service to assist your research work.

Additionally, the development of AAV mutant serotypes with more tissue specificity and stronger infectivity is crucial for innovation in AAV-mediated gene delivery. PackGene provides comprehensive AAV serotype engineeringg services to offer you a one-stop solution.

However, despite the tissue tropism of wild-type AAVs to some extent, the infection of non-target tissues cannot be completely avoided. In such cases, combining tissue- or cell-specific promoters with serotypes can greatly enhance AAV specificity. PackGene offers various tissue-specific promoters, such as the muscle-specific promoter MHCK7-2 and the liver-specific promoter TBG669. Our piVector Design embed in our online ordering system offers various promoters including universal and tissue specific promoters. You may easily build your vector into our AAV backbones that have been rigorously verified for effective viral packaging.

During AAV titer measurement, our instruments are initially calibrated using the AAV standard product ATCC VR-1816™, a globally recognized reference titer verified by 16 laboratories. Subsequently, we employ SYBR Green qPCR methodology to ascertain titers, achieving values of 1E+13GC/ml or higher. This meticulous approach ensures alignment with prevailing academic standards and prevents inaccurate titration results.

Besides qPCR, we have several other methods available for AAV titer determination:

• Genome titer detection by ddPCR.
• Capsid titer detection by ELISA technology.
• Infectious titer detection by TCID50.

Our AAV products are subjected to standard release testing procedures, including endotoxin assessment using Limulus Amebocyte Lysate (LAL) assay, purity analysis via Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), and titer determination using quantitative Polymerase Chain Reaction (qPCR) or droplet digital PCR(ddPCR). Moreover, we conduct restriction enzyme digestion for the Gene of Interest (GOI) plasmids utilized in packaging. Different grade AAV may include different QC tests as listed here.

In addition, we offer 40+ analytical tests to measure titer, AAV genome integrity, characterization, purity, aggregation, contamination and safty, including TEM, AUC, TCID50, Nanopore deep sequencing and may others. Please refer to our anlytical tests webpage.

You only need to provide 1-4 µg of plasmid. We will handle the plasmid preparation necessary for AAV packaging. You don’t need to purchase an additional plasmid prep service unless you wish to receive more plasmid from us. Please note, the timeline in our quote already includes the plasmid preparation.

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.

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.

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.

What are your requirements for the amount, titer, and packaging of the final deliverable rAAV?

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.

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.