The final product of recombinant adeno-associated virus (rAAV) vector production is usually a mixture of full and empty capsids, where, in addition to the desired recombinant genome, full viral capsids may also contain impurities such as residual plasmids, host cell DNA, and genomic fragments. For a safe and effective drug, the amount of complete recombinant genomes is crucial. Research has shown that this goal can be achieved by optimizing various steps of the preparatory processes in rAAV vector production. This thesis presents an approach to obtaining a higher quantity of higher quality product based on currently available literature. In addition to several quality control points and suggestions for using some newer technologies such as ddPCR and monolithic chromatography, a possibility of altering the plasmid sequence itself is proposed. Some studies have shown that the use of Rep and Cap genes from the same serotype increases product quality. Additionally, the use of an extra sequence, "spacer," reduces the amount of undesired genome parts packaged. The use of a tetracycline-inducible promoter is also proposed to regulate the timing of viral capsid production. Finally, a protocol for improved rAAV production is proposed. The proposed protocol thus includes a comprehensive approach to optimizing production, based on the latest research and technologies, and provides a foundation for practical research in the future.
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