Communication
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A Lac Repressor Inducible Baculovirus Expression Vector for Controlling Adeno-Associated Virus Capsid Ratios
Version 1
: Received: 19 November 2023 / Approved: 20 November 2023 / Online: 20 November 2023 (05:39:52 CET)
A peer-reviewed article of this Preprint also exists.
Slack, J.; Nguyen, C.; Ibe-Enwo, A. A Lac Repressor-Inducible Baculovirus Expression Vector for Controlling Adeno-Associated Virus Capsid Ratios. Viruses 2024, 16, 51. Slack, J.; Nguyen, C.; Ibe-Enwo, A. A Lac Repressor-Inducible Baculovirus Expression Vector for Controlling Adeno-Associated Virus Capsid Ratios. Viruses 2024, 16, 51.
Abstract
Abstract: The baculovirus expression vector (BEV) system is an efficient, cost effective and scalable method to produce recombinant adeno-associated virus (rAAV) gene therapy vectors. Most BEV designs emulate the wild type AAV transcriptome and translate the AAV capsid proteins, VP1, VP2 and VP3 from a single mRNA transcript with three overlapping open reading frames (ORFs). Non-canonical translation initiation codons for VP1 and VP2 reduce their abundances relative to VP3. Changing capsid ratios to improve rAAV vector efficacy requires theoretical modification of translational context. We have made a Lac repressor inducible system to empirically regulate expression of VP1 and VP2 proteins relative to VP3 in the context of the BEV. We demonstrate the use of this system to tune the abundance, titer and potency of a neurospecific rAAV9 serotype derivative. VP1:VP2:VP3 ratios of 1:1:8 gave optimal potency for this rAAV. It was discovered that ratios of capsid proteins expressed were different than ratios that ultimately were in purified capsids. Over expressed VP1 did not become incorporated into capsids while over expressed VP2 did. Overabundance of VP2 correlated with reduced rAAV titers. This work demonstrates a novel technology for controlling production of rAAV in the BEV system and shows a new perspective on the biology of rAAV capsid assembly.
Keywords
baculovirus expression vector; inducible expression; rAAV; lac repressor
Subject
Biology and Life Sciences, Virology
Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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