Accurate Quantification and Characterization of Adeno-Associated Viral Vectors.

David Dobnik,Maja Leskovec,Tjaša Jakomin,Nejc Košir,Stephen M Kaminsky,Polona Kogovšek,Magda Tušek Žnidarič,Hyunmi Lee,Janet Mostrom,Maja Ravnikar

doi:10.3389/fmicb.2019.01570

David Dobnik, Maja Leskovec + Show 8 more

Open Access

https://doi.org/10.3389/fmicb.2019.01570

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Abstract

One of the main challenges in the gene therapy viral vector development is to establish an optimized process for its large scale production. This requires optimization for upstream and downstream processes as well as methods that enable the step-by step analytical characterization of the virus, the results of which inform the iterative refinement of production for yield, purity and potency. The biggest problem here is a plethora of viral vector formulations, many of which interfere with analytical techniques. We took adeno-associated virus (AAV) as an example and showed benefits of combined use of molecular methods and transmission electron microscopy (TEM) for viral vectors’ characterization and quantification. Results of the analyses showed that droplet digital PCR (ddPCR) performs better than quantitative real-time PCR (qPCR), in terms of robustness and assay variance, and this was especially relevant for partially purified (in-process) samples. Moreover, we demonstrate the importance of sample preparation prior to PCR analysis. We evaluated viral structure, presence of aggregates and impurities with TEM analysis and found that these impacted the differences in viral titers observed by qPCR and ddPCR and could be altered by sample preparation. These results serve as a guide for the establishment of the analytical methods required to provide measures of identity and purity for AAV viral vectors.

Highlights

Adeno associated virus (AAV) is an important viral vector for gene therapy
AAVrh.10 vectors used in the study were composed of the Rhesus serotype 10 capsid proteins and the vector genome containing the AAV2 5 inverted terminal repeat (ITR), the AAV2 packaging signal (C), CMV enhancer, chicken β-actin promoter and splice donor and rabbit β-globin intron with splice acceptor, the cDNA for the transgene followed by the rabbit β -globin polyA signal, and the AAV2 3 ITR
We evaluated the effect of different options for pretreatments of samples prior to quantitative real-time PCR (qPCR) and droplet digital PCR (ddPCR), namely DNase digestion only, proteinase K treatment only, DNase digestion followed with proteinase K and no treatment (Table 1). qPCR results were evaluated individually for two dilutions of the initial sample (10-fold difference; high and low concentration)

Summary

Introduction

Adeno associated virus (AAV) is an important viral vector for gene therapy. It is useful due to its vast tropism, minimal immunogenicity, lack of association with any disease, and the capacity to achieve efficient and persistent gene transfer. AAV has limited packaging capability with about 5 kb of single stranded DNA. It would be the ideal gene transfer vector, if all cell types and tissues would be susceptible to AAV infection (Zinn and Vandenberghe, 2014). The first European approval of AAV-based gene therapy revealed the need for more efficient AAV vector manufacturing and downstream processing as the cost, among other reasons led to the product’s rapid commercial demise (Ai et al, 2017). Clinical dosing of recombinant AAV (rAAV) therapeutics are usually based on vector genome (vg) titer per mL, requiring availability of accurate quality control methods (D’Costa et al, 2016)

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