Multidose transient transfection of human embryonic kidney 293 cells modulates recombinant adeno-associated virus2/5 Rep protein expression and influences the enrichment fraction of filled capsids.

Recombinant adeno-associated virus (rAAV) is a commonly used in vivo gene therapy vector because of its nonpathogenicity, long-term transgene expression, broad tropism, and ability to transduce both dividing and nondividing cells. However, rAAV vector production via transient transfection of mammalian cells typically yields a low fraction of filled-to-total capsids (~1%-30% of total capsids produced). Analysis of our previously developed mechanistic model for rAAV2/5 production attributed these low fill fractions to a poorly coordinated timeline between capsid synthesis and viral DNA replication and the repression of later phase capsid formation by Rep proteins. Here, we extend the model by quantifying the expression dynamics of total Rep proteins and their influence on the key steps of rAAV2/5 production using a multiple dosing transfection of human embryonic kidney 293 (HEK293) cells. We report that the availability of preformed empty capsids and viral DNA copies per cell are not limiting to the capsid-filling reaction. However, optimal expression of Rep proteins (<240 ± 13 ag per cell) enables enrichment of the filled capsid population (>12% of total capsids/cell) upstream. Our analysis suggests increased enrichment of filled capsids via regulating the expression of Rep proteins is possible but at the expense of per cell capsid titer in a triple plasmid transfection. Our study reveals an intrinsic limitation of scaling rAAV2/5 vector genome (vg) production and underscores the need for approaches that allow for regulating the expression of Rep proteins to maximize vg titer per cell upstream.

Analysis of Poly(ethylene terephthalate) degradation kinetics of evolved IsPETase variants using a surface crowding model.

Poly(ethylene terephthalate) (PET) is a major plastic polymer utilized in the single-use and textile industries. The discovery of PET-degrading enzymes (PETases) has led to an increased interest in the biological recycling of PET in addition to mechanical recycling. IsPETase from Ideonella sakaiensis is a candidate catalyst, but little is understood about its structure-function relationships with regards to PET degradation. To understand the effects of mutations on IsPETase productivity, we develop a directed evolution assay to identify mutations beneficial to PET film degradation at 30 °C. IsPETase also displays enzyme concentration-dependent inhibition effects, and surface crowding has been proposed as a causal phenomenon. Based on total internal reflectance fluorescence microscopy and adsorption experiments, IsPETase is likely experiencing crowded conditions on PET films. Molecular dynamics simulations of IsPETase variants reveal a decrease in active site flexibility in free enzymes and reduced probability of productive active site formation in substrate-bound enzymes under crowding. Hence, we develop a surface crowding model to analyze the biochemical effects of three hit mutations (T116P, S238N, S290P) that enhanced ambient temperature activity and/or thermostability. We find that T116P decreases susceptibility to crowding, resulting in higher PET degradation product accumulation despite no change in intrinsic catalytic rate. In conclusion, we show that a macromolecular crowding-based biochemical model can be used to analyze the effects of mutations on properties of PETases and that crowding behavior is a major property to be targeted for enzyme engineering for improved PET degradation.

Mechanistic modeling of viral particle production.

Viral systems such as wild-type viruses, viral vectors, and virus-like particles are essential components of modern biotechnology and medicine. Despite their importance, the commercial-scale production of viral systems remains highly inefficient for multiple reasons. Computational strategies are a promising avenue for improving process development, optimization, and control, but require a mathematical description of the system. This article reviews mechanistic modeling strategies for the production of viral particles, both at the cellular and bioreactor scales. In many cases, techniques and models from adjacent fields such as epidemiology and wild-type viral infection kinetics can be adapted to construct a suitable process model. These process models can then be employed for various purposes such as in-silico testing of novel process operating strategies and/or advanced process control.