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The major drawback of the Baculovirus/Sf9 system for recombinant adeno-associated viral (rAAV) manufacturing is that most of the Bac-derived rAAV vector serotypes, with few exceptions, demonstrate altered capsid compositions and lower biological potencies. Here, we describe a new insect cell-based production platform utilizing attenuated Kozak sequence and a leaky ribosome scanning to achieve a serotype-specific modulation of AAV capsid proteins stoichiometry. By way of example, rAAV5 and rAAV9 were produced and comprehensively characterized side by side with HEK293-derived vectors. A mass spectrometry analysis documented a 3-fold increase in both viral protein (VP)1 and VP2 capsid protein content compared with human cell-derived vectors. Furthermore, we conducted an extensive analysis of encapsidated single-stranded viral DNA using next-generation sequencing and show a 6-fold reduction in collaterally packaged contaminating DNA for rAAV5 produced in insect cells. Consequently, the re-designed rAAVs demonstrated significantly higher biological potencies, even in a comparison with HEK293-manufactured rAAVs mediating, in the case of rAAV5, 4-fold higher transduction of brain tissues in mice. Thus, the described system yields rAAV vectors of superior infectivity and higher genetic identity providing a scalable platform for good manufacturing practice (GMP)-grade vector production.

Crossing the blood-brain and the blood-cerebrospinal fluid barriers are one of the fundamental challenges in the development of new therapeutic molecules for brain disorders because they prevent entry of most drugs from the blood into the brain. However, some large molecules, like the protein transferrin, cross these barriers using a specific receptor that transports them into the brain. Mimicking nature, we engineered a receptor/ligand system to cross the brain barrier by combining the human transferrin receptor with the cohesin domain from Clostridium thermocellum, and we tested it in the choroid plexus of the mouse brain with a dockerin ligand. By expressing our receptor in choroidal ependymocytes, which are part of the blood-cerebrospinal fluid barrier, we found that our systemically administrated ligand was able to bind to the receptor and accumulate in ependymocytes, where some of the ligand was transported from the blood side to the brain side.