Adeno-associated virus (AAV) is a non-enveloped virus with an icosahedral structure and a 4.7kb linear single-stranded DNA genome. AAV has the advantages of a wide host range, high safety, low immunogenicity, and long-term stable expression. It is an ideal gene therapy tool and is widely used in basic research and clinical trials in various fields.
AAV was produced and packaged in 293T cells using a three-plasmid co-transfection Helper-free system, and a complete purification and quality control method was established. The produced AAV particles can be directly used in various in vivo and in vitro experiments.
Advantages of adeno-associated virus:
1. Wide host range: can infect dividing cells and non-dividing cells; multiple subtypes have multiple tissue tropisms and can be used for targeted injections in the nervous system, myocardium, liver, eyes, muscles, etc.
2. Good diffusibility: small size, high titer, far superior diffusibility than adenovirus and lentivirus, can cross the blood-brain barrier, and is an excellent tool for infecting neurons and glial cells.
3. High safety factor: So far, wild-type AAV has not been found to be pathogenic to humans; ITR and rep/cap genes are expressed by independent plasmids, further eliminating the risk of infection.
4. Low immunogenicity: The genome of only 4.7kb is convenient for recombinant DNA technology operation, and local high-dose use in animal experiments rarely causes nonspecific reactions and immunosuppression.
5. Long-term stable expression: AAV infection can mediate low-frequency (<2%) directional integration of exogenous genes. Most recombinant AAVs will not be integrated into the genome after infecting cells, but will form companion bodies in host cells. Therefore, AAV can mediate long-term and stable replication and transcription of exogenous genes.
6. Good stability: AAV will not be inactivated at 60°C and can resist treatment with a variety of organic solvents (such as chloroform)
Different AAV subtypes have different tissue affinity. We recommend selecting AAV subtypes with corresponding affinity for construction according to the organs/cells targeted by the experiment, which can effectively improve the infection efficiency.
Application prospects:
1. Gene therapy: AAV, as a vector in gene therapy, can carry healthy genes into the patient's cells to repair or replace defective genes.
2. Vaccine development: AAV is also used in vaccine development. For example, AAV vectors can deliver antigen genes into host cells to stimulate immune responses and prevent certain diseases.
3. Neuroscience research: AAV can efficiently deliver genes to nerve cells and can be used in gene editing technologies such as CRISPR to specifically change gene expression in the nervous system.
4. Cancer treatment: AAV is also used to deliver anti-cancer genes to cancer cells to promote the immune system to recognize and eliminate cancer cells.
Service/experimental process:
1. Communication of the scheme
2. Gene cloning: construct the target gene cDNA (Gene of Interest) into our proprietary AAV transfer plasmid and sequence verification
3. Virus packaging: co-transfection of three plasmids (transfer plasmid + virus packaging plasmid + auxiliary plasmid) into 293T cells
4. Virus collection: harvest the virus in the culture supernatant and cell pellet after 72h incubation
5. Purification and concentration: iodixanol gradient centrifugation, efficient separation of empty capsid virus and cell impurities, enrichment of infectious virus to more than 90%; ultrafiltration concentration
6. Quality control:
- Multiple restriction enzyme digestion plasmid identification
- qPCR detection of virus titer
- SDS-PAGE/Coomassie brilliant blue determination of purity
- Cell transduction detection (with fluorescent label)
- HPLC/endotoxin detection/microbial detection (optional for high-purity AAV)
Deliverables:
Customized AAV virus particles
Control virus (can be used for transduction test)
Detailed experimental report
References
https://resources.amsbio.com/Catalog/Viral%20Delivery%20systems%200522.pdf