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Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells
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Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells
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Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells
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Journal Article
Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells
2017
Overview
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.
Kondratov et al. 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. The established system yields rAAV vectors of superior infectivity and higher genetic identity, providing a scalable platform for GMP-grade vector production.
