Near-perfect infectivity of wild-type AAV as benchmark for infectivity of recombinant AAV vectors

Abstract

Viral vectors derived from adeno-associated viruses (AAVs) are widely used for gene transfer both in vitro and in vivo. The increasing use of AAV as a gene transfer vector, as well as recently shown immunological complications in clinical trials, highlight the necessity to define the specific activity of vector preparations beyond current standards. In this report, we determined the infectious, physical and genome-containing particle titers of several wild-type AAV type 2 (wtAAV2) and recombinant AAV type 2 (rAAV2) preparations that were produced and purified by standard methods. We found that the infectivity of wtAAV2 approaches a physical-to-infectious particle ratio of one. This near-perfect physical-to-infectious particle ratio defines a 'ceiling' for the theoretically achievable quality of recombinant AAV vectors. In comparison, for rAAV2, only approximately 50 out of 100 viral particles contained a genome and, more strikingly, only approximately 1 of the 100 viral particles was infectious. Our findings suggest that current strategies for rAAV vector design, production and/or purification should be amenable to improvements. Ultimately, this could result in the generation of near-perfect vector particles, a prospect with significant implications for gene therapy.

Figure 1. Determination of the infectious unit titer in wtAAV2a and rAAV2-GFPa and the transducing unit titer in rAAV-GFPa

(a, b) wtAAV2a and rAAV2-GFPa were titrated by the infectious center assay (ICA). Briefly, HeLa or C12 cells were infected in serial dilution with wtAAV2a or rAAV2-GFPa, respectively. The cells were co-infected with Adenovirus type 5 (Ad5), harvested 40 hours post-infection and transferred to nylon membranes. The membranes were hybridized to a rep-specific probe for wtAAV2a or a CMV-promoter-specific probe for rAAV2-GFPa. The titer was calculated by multiplying the number of positive cells with the dilution factor and dividing it with the volume of virus (in ml) used for the infection. The dilution factors are as indicated. Cells = HeLa or C12 cells that have not been infected with either AAV or Ad5, Ad5 = cells infected only with Ad5. (c) Determination of the transducing units (tu) titer of rAAV2-GFPa. C12 cells were infected with rAAV2-GFPa in serial dilutions and co-infected with Ad5. 40 hours post-infection the cells were analyzed by flow cytometry for GFP expression. The titer was calculated from the two dilutions within a linear range for the percentage of GFP expressing cells. All experiments were done three times, n=3, each in triplicate. The data is represented as mean±SEM.

Figure 2. Determination of full and empty viral particles by electron microscopy

Virus was applied to formvar/carbon copper grids, dried, washed and stained with 2% uranyl acetate. Grids were analyzed at 50k magnification with a transmission electron microscope. For each sample three fields, n=3, containing approximately 200 particles were counted. Percentages are represented as mean±SEM, scale bar=200nm