Chemically Defined, High-Density Insect Cell-Based Expression System for Scalable AAV Vector Production.

The recombinant adeno-associated virus (AAV) vector is one of the most utilized viral vectors in gene therapy due to its robust, long-term in vivo transgene expression and low toxicity. One major hurdle for clinical AAV applications is large-scale manufacturing. In this regard, the baculovirus-based AAV production system is highly attractive due to its scalability and predictable biosafety. Here, we describe a simple method to improve the baculovirus-based AAV production using the ExpiSf Baculovirus Expression System with a chemically defined medium for suspension culture of high-density ExpiSf9 cells. Baculovirus-infected ExpiSf9 cells produced up to 5 × 1011 genome copies of highly purified AAV vectors per 1 mL of suspension culture, which is up to a 19-fold higher yield than the titers we obtained from the conventional Sf9 cell-based system. When mice were administered the same dose of AAV vectors, we saw comparable transduction efficiency and biodistributions between the vectors made in ExpiSf9 and Sf9 cells. Thus, the ExpiSf Baculovirus Expression System would support facile and scalable AAV manufacturing amenable for preclinical and clinical applications.

The protein kinase PERK/EIF2AK3 regulates proinsulin processing not via protein synthesis but by controlling endoplasmic reticulum chaperones.

Loss-of-function mutations of the protein kinase PERK (EIF2AK3) in humans and mice cause permanent neonatal diabetes and severe proinsulin aggregation in the endoplasmic reticulum (ER), highlighting the essential role of PERK in insulin production in pancreatic ß cells. As PERK is generally known as a translational regulator of the unfolded protein response (UPR), the underlying cause of these ß cell defects has often been attributed to derepression of proinsulin synthesis, resulting in proinsulin overload in the ER. Using high-resolution imaging and standard protein fractionation and immunological methods we have examined the PERK-dependent phenotype more closely. We found that whereas proinsulin aggregation requires new protein synthesis, global protein and proinsulin synthesis are down-regulated in PERK-inhibited cells, strongly arguing against proinsulin overproduction being the root cause of their aberrant ER phenotype. Furthermore, we show that PERK regulates proinsulin proteostasis by modulating ER chaperones, including BiP and ERp72. Transgenic overexpression of BiP and BiP knockdown (KD) both promoted proinsulin aggregation, whereas ERp72 overexpression and knockdown rescued it. These findings underscore the importance of ER chaperones working in concert to achieve control of insulin production and identify a role for PERK in maintaining a functional balance among these chaperones.