Recombinant AAV genome size effect on viral vector production, purification, and thermostability

Abstract

Adeno-associated virus (AAV) has shown great promise as a viral vector for gene therapy in clinical applications. The present work studied the effect of genome size on AAV production, purification, and thermostability by producing AAV2-GFP using suspension-adapted HEK293 cells via triple transfection using AAV plasmids containing the same GFP transgene with DNA stuffers for variable-size AAV genomes consisting of 1.9, 3.4, and 4.9 kb (ITR to ITR). Production was performed at the small and large shake flask scales and the results showed that the 4.9 kb GFP genome had significantly reduced encapsidation compared to other genomes. The large shake flask productions were purified by AEX chromatography, and the results suggest that the triple transfection condition significantly affects the AEX retention time and resolution between the full and empty capsid peaks. Charge detection-mass spectrometry was performed on all AEX full-capsid peak samples showing a wide distribution of empty, partial, full length, and copackaged DNA in the capsids. The AEX-purified samples were then analyzed by differential scanning fluorimetry, and the results suggest that sample formulation may improve the thermostability of AAV genome ejection melting temperature regardless of the packaged genome content.

Keywords: adeno-associated virus; anion exchange chromatography; capsid melting temperature; capsid titer; charge detection-mass spectroscopy; differential scanning fluorimetry; gene therapy; genome ejection melting temperature; triple transfection; viral genome titer.

Graphical abstract

Figure 1

GFP genome size effect on AAV production and encapsidation (A) One-way means and ANOVA analysis of the viral genome titer (vg/mL) by GFP genome size for study 1. A Student’s t test comparing each pair of GFP genome sizes is shown at right. The vg titer production of the 1.9 and 3.4 kb genomes are not significantly different (p < 0.2599) but are both significantly different from the 4.9 kb genome (p < 0.0001). (B) One-way means and ANOVA analysis of the purified vg/cp ratio (%) by GFP genome size for study 2. A Student’s t test comparing each pair of GFP genome sizes at right. The purified vg/cp ratio (%) of the 1.9 and 3.4 kb genomes are not significantly different from each other (p < 0.5133) but are each significantly different from the 4.9 kb genome (p < 0.015 and p < 0.046 respectively).

Figure 2

AEX chromatograms of AAV2-GFP purifications (A) Transfection condition 1 AAV2-GFP 1.9 kb. (B) Transfection condition 1 AAV2-GFP 3.4 kb. (C) Transfection condition 1 AAV2-GFP 4.9 kb. (D) Transfection condition 2 AAV2-GFP 1.9 kb. (E) Transfection condition 2 AAV2-GFP 3.4 kb. (F) Transfection condition 2 AAV2-GFP 4.9 kb. (G) Transfection condition 3 AAV2-GFP 1.9 kb. (H) Transfection condition 3 AAV2-GFP 3.4 kb. (I) Transfection condition 3 AAV2-GFP 4.9 kb.

Figure 3

CD-MS mass histogram results for AAV2-GFP AEX chromatography full capsid peaks (A) Transfection condition 1 AAV2-GFP 1.9 kb. (B) Transfection condition 1 AAV2-GFP 3.4 kb. (C) Transfection condition 1 AAV2-GFP 4.9 kb. (D) Transfection condition 2 AAV2-GFP 1.9 kb. (E) Transfection condition 2 AAV2-GFP 3.4 kb. (F) Transfection condition 2 AAV2-GFP 4.9 kb. (G) Transfection condition 3 AAV2-GFP 1.9 kb. (H) Transfection condition 3 AAV2-GFP 3.4 kb. (I) Transfection condition 3 AAV2-GFP 4.9 kb.

Figure 4

Genome ejection melting curves and SLS 473 intensity curve for AEX full and empty capsid peak samples (A) The genome ejection melting curve (blue) based on fluorescent intensity of SYBR Gold bound to DNA for condition 1 AAV2-GFP AEX full capsid peak 1.9 kb sample. The differential (red) is graphed as well to show the maxima, which would be the melting temperature, T_m. T_{m1, avg.} = 59.83°C and T_{m2, avg.} = 69.53°C. (B) The SLS 473 intensity curve (blue) showing aggregation of the condition 1 AAV2-GFP AEX full capsid peak 1.9 kb sample. T_{agg., avg.} = 71.78°C. (C) The genome ejection melting curve (yellow) based on fluorescent intensity of SYBR Gold bound to DNA for condition 1 AAV2-GFP AEX empty capsid peak 1.9 kb sample. The differential (red) is graphed as well to show the maxima, which would be the melting temperature, T_m. T_{m, avg.} = 71.93°C. (D) The SLS473 intensity curve (yellow) showing aggregation of the condition 1 AAV2-GFP AEX empty capsid peak 1.9-kb sample. T_{agg., avg.} = 69.99°C.