Manufacturing practices for recombinant adeno-associated viruses (AAV) have improved in the last decade through the development of new platforms in conjunction with better production and purification methods. In this review, we discuss the advantages and limitations of the most popular systems and methods employed with mammalian cell platforms. Methods and systems such as transient transfection, packaging and producer cells and adenovirus and herpes simplex virus are described. In terms of best production yields, they are comparable with about 10 -10 vector genomes produced per cell but transient transfection of HEK293 cells is by far the most commonly used. For small-scale productions, AAV can be directly purified from the producing cell lysate by ultracentrifugation on a CsCl or iodixanol-step gradient whereas large-scale purification requires a combination of multiple steps. Micro/macrofiltration (i.e. including tangential flow filtration and/or dead-end filtration) and chromatography based-methods are used for large-scale purification. Purified AAV products must then be quantified and characterized to ensure quality. Recent purification methods and current analytical techniques are reviewed here. Finally, AAV technology is very promising, but manufacturing improvements are still required to meet the needs of affordable, safe and effective AAV vectors essential for licensing of gene therapy clinical protocols.

No abstract

S136lines (KB cells, neuroblastoma, known to overexpress FA receptors, and cell lines (NIH 3T3, HTB and ARPE19) that have a low level of FA receptor expression. Western blot and confocal microscopy were performed to detect the FA protein level and to visualize the intracellular traffi cking of NPs. Results: In line with previous reports, the UV absorbance spectra determined the formation of the conjugations, which were further confi rmed by FT-IR analysis compared with the control folic acid molecules. In terms of CK30 NPs, the intact plasmid DNA came out from the digested NP formulation by trypsinsation and refl ected the integrity of plasmid DNA inside formulation state. We observed a signifi cant number of labelled NPs (red) into the nucleus (labelled with DAPI). Earlier it was observed that CK30PEG compacted NPs can traffi c to the nucleus via nucleolin receptors. Therefore, in our current fi ndings, we can consider that FA conjugation helped the NPs to enter inside the cell via folate receptor mediated endocytosis followed by nucleolin mediated uptake inside the nucleus. When we compared this result with FA-nanoceria-DyLight550 compacted DNA under the same condition, we found a promising result that nanoceria-DyLight550 compacted NPs (labelled red) only showed up inside the cytosol but not in the nucleus (Fig. 1). Therefore, our initial fi nding demonstrates that FA conjugation could boost up the CK30PEG-FA and nanoceria conjugated NPs to cross the cell membrane barriers for targeted delivery and express gene of interest. Conclusions: We explored a smart NP gene delivery system with multifunctionalities. The design of multifunctional NP complexes in this study can be custom-built and functionalized to target different diseases by binding the surface of NPs with a cell-specifi c targeting molecule and therapeutic specifi city. We are now conducting these approaches by using different bio-markers in mouse models in vivo for targeted delivery. Project success will provide attractive tools and templates with broad potential for precision medicine. Fig.1: Cell transfections in KB cells with (A) FA-CK30PEG-DyLight550, and (B) nanoceria compacted plasmid DNA NPs respectively. Images were captured at 63x in Zeiss CLSM 710 sprectral confocal laser scanning microscope.