Structural Changes Likely Cause Chemical Differences between Empty and Full AAV Capsids.

Due to the success of adeno associated viruses (AAVs) in treating single-gene diseases, improved manufacturing technology is now needed to meet their demand. The largest challenge is creating a process to separate empty and full capsids. Patients received larger capsid doses than necessary due to the presence of empty capsids. By enabling the better separation of empty and full capsids, patients would receive the greatest therapeutic benefit with the least amount of virus capsids, thus limiting potential side effects from empty capsids. The two most common empty/full separation methods used in downstream processing are ultracentrifugation and anion exchange chromatography. Both processes have limitations, leading to a need for the identification of other structural differences that can be exploited to separate empty and full capsids. Here, we describe four possible theories of the structural changes that occur when AAV capsids envelop a genome. These theories include conformational changes occurring due to either the expansion or contraction of the capsid in the presence of nucleic acids, the constraining of the N-terminus into the five-fold pore when the genome is present, and the increased number of VP3 proteins in full capsids. These theories may reveal structural differences that can be exploited to separate full and empty capsids during manufacturing.

Combined effect of heat and corona charge on molecular delivery to a T cell line in vitro.

With the rapid increase of gene and immunotherapies for treating cancer, there is a need to efficiently transfect cells. Previous studies suggest that electrotransfer can provide a non-viral method for gene delivery. Electrotransfer traditionally relies upon the application of direct current pulses to the cells of interest. Corona charge was investigated in this study as an alternative to traditional methods as a means of creating the electric field necessary to deliver materials via electrotransfer. The goal was to determine if there was an increase in molecular delivery across the membrane of a human T cell line used as a model system. In a novel dish created for the study, the effects of elevated temperatures (37, 40, 43, and 45°C) during the treatment process were also examined in combination with corona charge application. Results showed that treating cells with corona charge at room temperature (~23°C) caused a statistically significant increase in molecular delivery while maintaining viability. Heat alone did not cause a statistically significant effect on molecular delivery. Combined corona charge treatment and heating resulted in a statistically significant increase on molecular delivery compared to controls that were only heated. Combined corona charge treatment and heating to all temperatures when compared to controls treated at room temperature, showed a statistically significant increase in molecular delivery.