Establishment of a Novel Cell Line for the Enhanced Production of Recombinant Adeno-Associated Virus Vectors for Gene Therapy

Abstract

Adeno-associated viral (AAV) vectors show great promise because of their excellent safety profile; however, preexisting immune responses have necessitated the administration of high titer AAV, posing a significant challenge to the advancement of gene therapy involving AAV vectors. Recombinant AAV vectors contain minimum viral proteins necessary for their assembly and gene delivery functions. During the process of AAV assembly and production, AAV vectors acquire, inherently and submissively, various cellular proteins, but the identity of these proteins is poorly characterized. We reason that by identifying host cell proteins inherently associated with AAV vectors we may better understand the contribution of cellular components to AAV vector assembly and, ultimately, may improve the production of AAV vectors for gene therapy. In this study, three serotypes of recombinant AAV, namely AAV2, AAV5, and AAV8, were investigated. We used liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods to identify protein composition in purified AAV vectors, confirmed protein identities using western blotting, and explored the potential function of selected proteins in AAV vector production using small hairpin (shRNA) methods. Using LC-MS/MS, we identified 44 AAVassociated cellular proteins including Y-box binding protein (YB1). We showed for the first time that the establishment of a novel producer cell line by introducing an shRNA sequence down-regulating YB1 resulted in up to 45-and 9-fold increase in physical vector genome titers of AAV2 and AAV8, respectively, and up to 7-fold increase in AAV2 transduction vector genome titers. Our results revealed that YB1 gene knockdown promoted AAV2 rep expression and vector DNA production and reduced the number of empty particles in AAV2 products, suggesting that YB1 plays an important role in AAV vector assembly by competition with adenovirus E2A and AAV capsid proteins for binding to the inverted terminal repeat (ITR) sequence. The significance and implications of our findings in future improvement of AAV production are discussed.

Analytical Ultracentrifugation as an Approach to Characterize Recombinant Adeno-Associated Viral Vectors

“…The C(S) values were determined using the SEDFIT algorithm. 38 To calculate the molar concentrations and percent values for each viral species from the absorbance data, extinction coefficients were determined. Molar absorbance extinction coefficients for both the empty particles ( 260 /capsid = 3.72 x 10 6 ) and genome containing particles ( 260 /capsid = 3x 10 7 ) were calculated using genome size and published formulae.…”

“…38 The molar concentration of both the genome-containing vector and the empty virion were calculated using Beer's Law, and the fractional content of each capsid species was reported as a % of the total. This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.…”

“…In addition, the preparation may contain incomplete portions of the rAAV genome or non-genomic nucleic acid contaminants. As adaptation of the transient transfection protocol to a manufacturing process is challenging, alternate scalable production methods such as those using baculovirus, [25][26][27][28] herpes simplex HSV, 30 or packaging / producer cell lines [31][32][33][34][35][36][37][38] are being considered. However, the production method likely influences the quality of the final rAAV vector product, particularly with respect to the amounts of empty particles, capsids containing fragmented genomes and capsids containing non-genomic nucleic acid contaminants.…”

A rAAV2‐producing yeast screening model to identify host proteins enhancing rAAV DNA replication and vector yield

“…This biological process is not fully understood, but studies suggest that it requires the interplay of Rep52/78 proteins, single‐stranded DNA, and pre‐formed capsids . In mammalian cells, the encapsidation rate of rAAV vectors (determined as capsid‐to‐vg ratio) ranges between 5 and 20, and it is influenced by the cell line molecular design and AAV components . In yeast, rAAV DNA encapsidation is suboptimal, evidenced by the excess of empty capsids and nonencapsidated rAAV DNA generated.…”

“…42 In mammalian cells, the encapsidation rate of rAAV vectors (determined as capsid-to-vg ratio) ranges between 5 and 20, and it is influenced by the cell line molecular design and AAV components. 43,44 In yeast, rAAV DNA encapsidation is suboptimal, evidenced by the excess of empty capsids and nonencapsidated rAAV DNA generated. This model, however, was useful to identify proteins such as HEM4 and TOP2 that exert a positive influence in encapsidation.…”