Adeno-associated viruses (AAV)

In clinical research, the AAV has shown great potential for gene transfer efficiency. The non-pathogenicity and the long-term expression let it become the most ideal viral vector for gene therapy.

Further reading: The four kinds of viral vectors which most use in gene therapy

How to prepare adeno-associated viruses (AAV) vectors?

The preparation process of adeno-associated virus vector:

Further reading:The 9 best biological buffers for cell culture

After performing the above steps, the preparation of the AAV vector is completed. The water that bears the boat is the same that swallows it up. A slight error in the process of viral vector production may cause cell mutation and disease. In order for the viral vector to have the best repair effect, this seemingly simple process is actually stepped by step. The following are the things you must be known in the integrated vector manufacturing process:

The points of prepare AAV vectors

Plasmid design:

Purification and testing are required in the process of plastid design, construction, and production to reduce or remove possible genetic contaminants and minimize gene homology, and by changing rep/cap the direction of transcription, to minimize the production of replication-competent adeno-associated viruses.

Choose serotype of virus:

Adeno-associated viruses of different serotypes can infect cells of different types. The AAV1-AAV9 was eight known AAV serotypes. The AAV2 is the most common in clinical applications among them.

The tissues or organs that can be infected by AAV serotypes:

Modified virus capsid:

As the adeno-associated virus is usually not pathogenic, patients will not have any symptoms even if they are infected, but antibodies to the virus are already produced in the body. Before using the adeno-associated virus as a vector, it is necessary to test whether the patient already has antibodies, and it is also possible to reduce the immunogenicity of the patient's body to the original capsid by modifying the virus capsid.

Use the helper virus:

Adeno-associated virus needs to provide the function of infection and helper protein through helper virus. Firstly, it uses a susceptible cell line to transfect two helper plastids to prepare it and then uses adenovirus as a helper virus to infect the glands. The virus genome can enter the lysis process to form adeno-associated virus particles. Usually, the supernatant and cell lysate can be collected 48 to 72 hours after infection to purify the viral vector. Please note that the process should be designed to inactivate the helper virus.

Nuclease digestion:

Cell lysis will release a large amount of host cell DNA and RNA, as well as unencapsulated viral DNA, which increases the viscosity of the cell lysate and complicates further purification steps. Therefore, DNA and RNA must be digested with nuclease to remove unnecessary nucleic acids and avoid aggregation.

Zwitterionic surfactants:

In addition to nucleases, some purification methods also use zwitterionic surfactants. CHAPS (CAS 75621-03-3) has several characteristics that make it the best choice for the use of zwitterionic surfactants in the purification process:

Further reading:Why use CHAPS?

Further reading:What is the critical micelle concentration (CMC)?

Further reading: What Bio-buffers and Bio-detergents are suitable for the purification process of viral vectors?

The concept of gene therapy seems simple, but it is not easy to implement. In the face of different diseases and genes, different "customized" designs are also required. There are many difficulties that need to be overcome in the research and development of gene therapy drugs, resulting in high research and development costs behind each gene therapy drug. Many scholars have devoted their whole lives to achieve breakthroughs in gene therapy to benefit the people who are suffering from this disease.

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Reference:

^[1] 一种快速高效分离和纯化重组腺病毒相关病毒的方法和用途: https://patents.google.com/patent/CN1139656C/zh

^[2] Proton NMR based investigation of the effects of temperature and NaCl on micellar properties of CHAPS: https://www.semanticscholar.org/paper/Proton-NMR-based-investigation-of-the-effects-of-on-Qin-Liu/29678d6ce3b6437498748054e51c4b4a920249af

^[3] AAV Purification by Iodixanol Gradient Ultracentrifugation: https://www.addgene.org/protocols/aav-purification-iodixanol-gradient-ultracentrifugation/#:~:text=In%20research%2C%20iodixanol%20is%20used,organelles%2C%20lipoproteins%2C%20and%20macromolecules.&text=The%20iodixanol%20gradient%20in%20this,from%20an%20impure%20AAV%20preparation.

^[4] Removal of Endotoxin from rAAV Samples Using a Simple Detergent-Based Protocol : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6804492/

^[5] Effective and Accurate Gene Silencing by a Recombinant AAV-Compatible MicroRNA Scaffold: https://pubmed.ncbi.nlm.nih.gov/31843447/

^[6] Streamlined large-scale production of recombinant adeno-associated virus (rAAV) vectors: https://pubmed.ncbi.nlm.nih.gov/11883083/

^[7] Proton NMR based investigation of the effects of temperature and NaCl on micellar properties of CHAPS: https://pubmed.ncbi.nlm.nih.gov/21322620/

^[8] Production of adeno-associated virus vectors for in vitro and in vivo applications: https://www.nature.com/articles/s41598-019-49624-w

^[9] 藉Recent advances in recombinant adeno-associated virus vector production: https://www.sciencedirect.com/science/article/pii/S0085253815482031

^[10] Downstream Manufacturing of Gene Therapy Vectors: https://downstreamcolumn.com/downstream-manufacturing-gene-therapy-vectors/