scAAV8-CAG-GFP

The effectiveness of adeno-associated virus (AAV)-based gene therapy is often limited by the presence of AAV neutralizing antibodies (NAbs). Existing assays are inconsistent across laboratories and cell-dependent, challenging their general applicability. Here, researchers present the AAV homology-directed repair (HDR) assay, which leverages the CRISPR-Cas9 system to provide a precise and sensitive method for detecting AAV NAbs. The assay employs a promoter-less AAV HDR vector integrated into electroporated cells, facilitating stable expression of a quantifiable fluorescent reporter gene and subsequent NAb titer assessment. Comparative evaluations demonstrated that the AAV-HDR approach outperformed the conventional AAV overexpression (AAV-OE) assay in terms of sensitivity and consistency. Crucially, it produced consistent results across a variety of cell lines, suggesting its potential to become a universal standard for NAb titer measurement. Researchers further validated the AAV-HDR titration assay by comparing it to the established NT50 assay. Notably, the AAV-HDR approach showed strong correlation with both the AAV-OE assay and the NT50 NAb titer values and demonstrated higher efficacy in identifying low-titer antibodies compared with the NT50 approach.

Given the growing promise of AAV8-based liver gene therapy, the researchers focused their research on AAV8 vectors. Unlike AAV8 vectors, self-complementary AAV (scAAV) vectors are known for their superior in vitro transduction efficiency. Unlike single-stranded AAV vectors, they have the advantage of not requiring the synthesis of the second strand before initiating transcription. The researchers used the scAAV8-CAG-GFP vector to introduce it into the K562 leukemia cell line (Figure 1A). FACS analysis of K562 cells after transduction at increasing MOIs showed a gradual increase in GFP expression from 7% to more than 80% (Figures 1B, C). To describe the relationship between MOI and GFP%, a standard curve was constructed. Notably, both binomial and logistic regressions showed robust agreement between GFP% and AAV virion counts. Given the slightly higher R2 value of the binomial regression (over 0.99), it was chosen for subsequent evaluation (Figure 1D). These data highlight the effectiveness of AAV8 vectors in transducing K562 cells and the positive correlation between MOI and transduction efficiency.

Figure 1. Correlation of AAV vector MOI with GFP expression post-transduction. (Li G, et al., 2024)