Biology subjects

Sinskey, A. J.

Publications and source records attributed to Sinskey, A. J..

4 recordsLinked to original sources

bioRxiv2025

Continuous Production of Recombinant Adeno-Associated Virus in the Insect Cell/Baculovirus Expression Vector System

Continuous production processes may offer significant advantages for biotherapeutic manufacturing, including increased productivity, consistent product quality, reduced facility footprint, and decreased process turnaround time. Despite these benefits, the in-situ formation of defective recombinant baculovirus expression vectors (BEVs) hinders the continuous manufacturing of recombinant adeno-associated viruses (rAAV) in the baculovirus expression vector system. This study investigates an approach of reducing defective viruses through the infusion of standard recombinant baculovirus (rBV) and the compartmentalization of early- and late-stage infected cells, resulting in stable rAAV production. In this study, rAAVs were continuously produced in a series of cascading reactors, comprising a feeder reactor, an infection reactor, and a production reactor. Residence times and transfer rates across the three reactors were optimized based on the production kinetics of rBV and rAAV derived from our mechanistic model. The majority of rBV was produced within the production reactor, thereby reducing the impact of defective viruses in the infection reactor, enabling continuous rAAV production. This study demonstrates the successful implementation of a continuous rAAV production process, yielding over 5x1010 vg/mL per day for 4 weeks. This work represents the first reported continuous rAAV production process utilizing the Sf9/BEVS platform and establishes engineering know-how for overcoming manufacturing challenges associated with rAAV-based gene therapies.

bioengineering↗

bioRxiv2023

Mechanistic Modeling Explains the Production Dynamics of Recombinant Adeno-Associated Virus with the Baculovirus Expression Vector System

The demand for recombinant adeno-associated virus (rAAV) for gene therapy is expected to soon exceed current manufacturing capabilities, considering the expanding number of approved products and of pre-clinical and clinical stage studies. Current rAAV manufacturing processes have less-than-desired yields and produce a significant amount of empty capsids. Recently, FDA approved the first rAAV-based gene therapy product manufactured in the baculovirus expression vector system (BEVS). The BEVS technology, based on an invertebrate cell line derived from Spodoptera frugiperda, demonstrated scalable production of high volumetric titers of full capsids. In this work, we develop a mechanistic model describing the key extracellular and intracellular phenomena occurring during baculovirus infection and rAAV virion maturation in the BEVS. The predictions of the model show good agreement with experimental measurements reported in the literature on rAAV manufacturing in the BEVS, including for TwoBac, ThreeBac, and OneBac constructs. The model is successfully validated against measured concentrations of structural and non-structural protein components, and of vector genome. We carry out a model-based analysis of the process, to provide insights on potential bottlenecks that limit the formation of full capsids. The analysis suggests that vector genome amplification is the limiting step for rAAV production in TwoBac. In turn, vector genome amplification is limited by low Rep78 levels. For ThreeBac, low vector genome amplification dictated by Rep78 limitation appears even more severe than in TwoBac. Transgene expression in the insect cell during rAAV manufacturing is also found to negatively influence the final rAAV production yields.

molecular biology↗

bioRxiv2021

Weighing the DNA content of Adeno-Associated Virus vectors with zeptogram precision using nanomechanical resonators

Quantifying the composition of viral vectors used in vaccine development and gene therapy is critical for assessing their functionality. Adeno-Associated Virus (AAV) vectors, which are the most widely used viral vectors for in-vivo gene therapy, are typically characterized using PCR, ELISA, and Analytical Ultracentrifugation which require laborious protocols or hours of turnaround time. Emerging methods such as Charge-Detection Mass Spectroscopy, Static Light Scattering, and Mass Photometry offer turnaround times of minutes for measuring AAV mass, but mostly require purified AAV-based reference materials for calibration. Here, we demonstrate a method for using Suspended Nanomechanical Resonators (SNR) to directly measure both AAV mass and aggregation from a few microliters of sample within minutes. We achieve a resolution near 10 zeptograms which corresponds to 1% of the genome holding capacity of the AAV capsid. Our results show the potential of our method for providing real-time quality control of viral vectors during biomanufacturing.

bioengineering↗

bioRxiv2020

Constructing an ethanol utilization pathway in Escherichia coli to produce acetyl-CoA derived compounds

Engineering microbes to utilize non-conventional substrates could create short and efficient pathways to convert substrate into product. In this study, we designed and constructed a two-step heterologous ethanol utilization pathway (EUP) in Escherichia coli by using acetaldehyde dehydrogenase (encoded by ada) from Dickeya zeae and alcohol dehydrogenase (encoded by adh2) from Saccharomyces cerevisiae. This EUP can convert ethanol into acetyl-CoA without ATP consumption, and generate two molecules of NADH per molecule of ethanol. We optimized the expression of these two genes and found that ethanol consumption could be improved by expressing them in a specific order (ada-adh2) with a constitutive promoter (PgyrA). The engineered E. coli strain with EUP consumed approximately 8 g/L of ethanol in 96 hours when it was used as sole carbon source. Subsequently, we combined EUP with the biosynthesis of polyhydroxybutyrate (PHB), a biodegradable polymer derived from acetyl-CoA. The engineered E. coli strain carrying EUP and PHB biosynthetic pathway produced 1.1 g/L of PHB from 10 g/L of ethanol and 1 g/L of aspartate family amino acids in 96 hours. We also engineered E. coli strain to produced 24 mg/L of prenol from 10 g/L of ethanol in 48 hours, supporting the feasibility of converting ethanol into different classes of acetyl-CoA derived compounds. HighlightsO_LIEngineered Escherichia coli strains to grow on ethanol as sole carbon source C_LIO_LIDemonstrated that ethanol was converted into acetyl-CoA (AcCoA) through two pathways (acetaldehyde-acetate-AcCoA and acetaldehyde-AcCoA) C_LIO_LIConverted ethanol into two acetyl-CoA derived products with low structural similarity (polyhydroxybutyrate and prenol) C_LIO_LIDiscovered that supplementation of the aspartate family amino acids can substantially improve cell growth on ethanol C_LI

bioengineering↗