Quantification of Adeno-Associated Virus with Safe Nucleic Acid Dyes

Abstract

<jats:title>Abstract</jats:title><jats:p>Adeno-associated virus (AAV) is the most commonly used viral vector for both biological and gene therapeutic applications<jats:sup>1</jats:sup>. Although many methods have been developed to measure quantity attributes of AAV, they are often technically challenging and time consuming. Here we report a method to titer AAV with GelGreen® dye, a safe green fluorescence nucleic acid dye recently engineered by Biotium company (Fremont, CA). This method, hereinafter referred to as GelGreen method, provides a fast (~ 30 minutes) and reliable strategy for AAV titration. To validate GelGreen method, we measured genome titer of an AAV reference material AAV8RSM and compared our titration results with those determined by Reference Material Working Group (ARMWG). We showed that GelGreen results and capsid Elisa results are comparable to each other. We also showed that GelRed® dye, a red fluorescence dye from Biotium, can be used to directly “visualize” AAV genome titer on a conventional gel imager, presenting an especially direct approach to estimate viral quantity. In summary, we described a technique to titer AAV by using new generation of safe DNA dyes. This technique is simple, safe, reliable and cost-efficient. It has potential to be broadly applied for quantifying and normalizing AAV viral vectors.</jats:p>

Multiple-Monitor HPLC Assays for Rapid Process Development, In-Process Monitoring, and Validation of AAV Production and Purification

“…Measurement of light scattering intensity at a single angle is sufficient for capsid detection in crude and purified samples. Measurement of light scatter at multiple angles is necessary for accurate determination of particle size and mass in highly purified samples [ 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 ].…”

“…The ability of intercalating dyes to pass through lipid membranes makes them natural candidates to evaluate titers of lipid enveloped viruses [ 42 , 43 , 44 , 45 ], but they are apparently not able to enter AAV capsids [ 47 ]. Figure 3 and Figure 4 confirm the inability of Picogreen to detect AAV-encapsidated DNA but reveal DNA associated with empty capsid exteriors.…”

“…They are reported to do so with adenovirus [ 46 ] but not with AAV [ 47 ]. Results can be obtained quickly, but co-stained host cell DNA in the form of degraded chromatin inflates the fluorescence signal [ 6 , 18 , 43 , 44 , 45 , 46 , 47 ]. Chromatin interference can be factored out by measuring dye fluorescence before and after capsid lysis but with a net result of low sensitivity [ 47 ].…”

“…Results can be obtained quickly, but co-stained host cell DNA in the form of degraded chromatin inflates the fluorescence signal [ 6 , 18 , 43 , 44 , 45 , 46 , 47 ]. Chromatin interference can be factored out by measuring dye fluorescence before and after capsid lysis but with a net result of low sensitivity [ 47 ]. HPLC reduces chromatin interference by separating the virus from most of the chromatin and increases sensitivity by concentrating the virus [ 6 , 44 , 45 ].…”

Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies

“…106 DyeBAs Direct quantification of the genome titer from at least crudely purified samples is possible using FS signals from dye-based binding assays (DyeBAs). The approach is simple and fast, requiring neither special instrumentation nor genome-dependent probes to yield results within 30 min-3 h. 59,60 The method is precise, with repeatability and intermediate precision reported within 4%-17% CV and 7%-16% CV, respectively. 59,60 Similar to PCR-based methods, DyeBA requires the removal of non-encapsidated DNA and capsid lysis during sample preparation.…”