FAQ

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.

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.

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.