Until about 2016, the majority of vectors used in gene therapy were Adenovirus and Retrovirus vectors. In recent years, a change toward Lentivirus and Adeno-associated virus (AAV) vectors has occurred. This is reflected when looking at the numbers in current clinical trials underway in the gene therapy field.
Why is AAV becoming more important?
- AAV is not pathogenic – AAV is actually integrated in many human bodies whom are asymptomatic.
- AAV is replication defective – it does not replicate on its own. AAV only replicates in the presence of a helper virus such as Adenovirus or Herpes Simplex virus.
- It is small (22 nm diameter) and stable – from a production standpoint, it is easy to purify.
This combination of favorable traits has increased the popularity of AAV and it is quickly becoming the vector of choice for in vivo gene therapy. However, due to current production processes, production cannot keep up with market needs. This production gap leaves many late phase clinical trials short on supply.
Current production process challenges:
- Difficulty in scale-up – the process relies on transient transfection of substrate-dependent cells.
- Cells must attach to the culture vessels in order to grow – limited by the available surface when scaling-up.
- Instead production must scale-out – increase the number of production vessels or facilities.
- Reproducibility challenges – since the process is difficult to control, bringing in the required DNA for vector production in each batch creates a reproducibility challenge.
- High cost of reagents – in particular, plasmid DNA must be of high quality (possibly GMP quality in later phases).
Current AAV Vector Generation
What do AAV vectors look like?
The wildtype AAV particle consists of a capsid containing three different proteins. The capsid contains the small single stranded viral genome that encodes for the rep genes (required for AAV replication) and the cap genes (which form the capsid).
In order to replicate, AAV must be in the presence of helper functions. In nature, these are brought in by HSV or AV infection. To commercially produce and use AAV as a viral vector, it must be taken apart and the required components must be placed onto different plasmids.
- The first plasmid required will contain an AAV genome in which the rep and cap genes have been replaced with the gene of interest (normally the therapeutic gene used for treatment).
- The second plasmid required contains the rep and cap genes which will support the formation of the viral particles.
- The third plasmid contains the helper functions that assist in replication.
- Again, the first plasmid required will contain an AAV genome in which the rep and cap genes have been replaced with the gene of interest (normally the therapeutic gene used for treatment).
- However, in a two-plasmid system, the two latter plasmids are combined into one plasmid containing the rep and cap genes as well as helper functions.
AAV Vector Generation
To generate AAV, the three plasmids need to be co-transfected transiently to produce cell lines which then result in the formation of AAV vector particles. The new vector particles consist of the capsid carrying AAV genome which has the gene of interest integrated. This vector can then be administered to the patient to transfer the therapeutic genes to the target cells of the patient.
About the Author
Robert is a Validation Scientist with 10 years of experience in pharmaceutical operations ranging from small to large scale viral and bacterial production in processing and laboratory areas.
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