Custom Adeno-associated Virus Vector Production Service
Overview of Adeno-associated Virus Vector
Adeno-associated virus (AAV) is a frequently utilized viral vector for gene therapy and scientific research. It doesn't display any cytotoxicity or much pathogenicity. The 4.7kb single-stranded DNA of wild-type AAV, a parvovirus family member, is enclosed in a non-enveloped capsid. Rep, Cap, and AAP are the three genes that the viral genome encodes. The rep gene produces proteins that are involved in packing, transcriptional control, and viral genome replication. The viral capsid is made up of the three structural proteins (VP1-3) encoded by the cap gene. The assembly-activating protein (AAP), required for viral assembly, is encoded by the aap gene. The AAV vectors we frequently use are recombinant AAVs in which a transgenic expression cassette has been substituted for the rep, cap, and aap genes. Due to its numerous benefits, including low immunogenicity, long-lasting transgene expression, the absence of adverse events in clinical trials, excellent stability, and a wide variety of cell types to infect, AAVs are one of the most effective vectors in gene therapy. To give consumers access to high-quality AAV viruses, Creative Biolabs has created proprietary ssAAV and scAAV vectors, setted up a recombinant AAV production infrastructure, and implemented stringent quality control requirements.
Production
The triple plasmid system in conjunction with E1-expressing cells (often 293HEK) is the most popular method for producing AAV through transient transfection. In Figure 1, the production process is depicted. The vector genome, which is an expression cassette with a promoter, a gene of interest, and a PolyA signal flanked by ITR, is found on one plasmid (the Transfer plasmid). The second plasmid, known as the Rep-Cap plasmid, encodes both Cap proteins that are unique to the target serotype and Rep proteins that are typically from serotype 2. The minimal set of adenoviral genes (E2, E4, and VARNA) needed to support AAV replication is contained on the third plasmid (Ad helper). Virus particles were captured 72 hours after three plasmids were proportionally transfected into HEK293T packaging cells.
Creative Biolabs can package the following AAV serotypes: AAV1; AAV2; AAV3; AAV4; AAV5; AAV6; AAV7; AAV8; AAV9; AAVrh10; AAVDJ; AAVDJ/8; AAVPHP.eB; AAVPHP.S; AAV2-retro; and AAV2-QuadYF. To address the demands of our customers for large-scale AAV manufacturing, we have also built a baculovirus production system platform. Please get in touch with us for further information and expert services.
Fig.1 Production of AAV by triple transfection.1
Purification
To facilitate pre-clinical AAV gene transfer experiments, high-quality vector preparations are crucial. We often employ PEG to concentrate viral particles for AAV utilized in vitro, with the greatest titer reaching 1012GC/mL, which is more practical. We typically offer CsCl Gradient Ultracentrifugation purification for AAV utilized in vivo. The purity can increase to nearly 90%, and the titer can approach 1013GC/mL. We can also offer other purification techniques including Ion Exchange Chromatography, Affinity Chromatography, and Iodixanol Gradient Ultracentrifugation. Please contact us if youhave other purification needs, we can offer you a tailored purification solution.
Quality Control
The quality analysis services for in vitro use of crude viruses include Mycoplasma identification, Sterility testing, and Titer determination. In addition to the aforementioned assays, we also provide an Endotoxin test and Purity detection for ultra-purified viruses utilized in vivo. We can also offer Full/empty capsid ratio analysis to satisfy the specific research needs of our customers. Creative Biolabs offers premium AAV vector production services to satisfy clients' global demands. Please reach out to us
Reference
- Ayuso, Eduard et al. "Production, purification and characterization of adeno-associated vectors." Current gene therapy vol. 10,6 (2010): 423-36. doi:10.2174/156652310793797685
