AAV Vector Design & Construction

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.

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.

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?

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?