Transfection of AAV containing a K340H mutation in Rep.

(A) To evaluate the activity of Rep, a K340H mutation was generated in the genome. Residue K340 directly interfaces with ATP in the helicase region and is required for ATP-dependent helicase activity. Dashed lines are noncovalent interactions. Image generated with Pymol. (B) Data obtained for transfections using the native genome (WT) and a genome harboring the K340H mutation. The data represents the average calculated from 2 biological replicates and 3 technical replicates, with error bars representing ±1 standard deviations. The viral genomes obtained for WT are significantly higher than K340H (ANOVA single-factor, p value = 0.003).

Minimizing cross packaging of DNA in capsids.

(A) Cross packaging was evaluated by mixing ITR-CMV-gfp and ITR-rep2-cap9 prior to transfection into HEK293 cells. After harvesting virus that was produced, multiplexed qPCR was used to quantify each cargo by leveraging primers targeting the CMV promoter in ITR-CMV-gfp and rep in ITR-rep2-cap9. Experiments performed in (B) the absence or (C) presence of dummy DNA. For B and C, total virion formation having each DNA cargo was quantified. For each experiment, the data represents 4 biological and 3 technical replicates, with error bars representing ±1 standard deviation. When transfecting 20 ng of ITR-CMV-gfp and ITR-rep2-cap9 in the presence of herring DNA, the value obtained for ITR-rep2-cap9 was higher than ITR-CMV-gfp (two-sample t-test, p value = 0.12), presenting a ratio of ∼1:18 for CMV:rep. For the 20-ng transfection without herring DNA, the qPCR titer for ITR-rep2-cap9 was also higher than that of ITR-CMV-gfp (two-sample t-test, p value = 0.03), with a CMV:rep ratio of ∼1/12.

Non-synonymous mutant enrichment following selection.

(A) To identify Rep with improved activities, mutants were transfected into HEK293 after mixing with the positive and negative controls at ratios of 18:1:1. This mixture (20 ng) was mixed with helper plasmid (XX6) and herring DNA prior to transfection. Each selection was performed triplicate. (B) The ratio of selected to non-selected DNA was calculated for each tile to establish the enrichment for each possible non-synonymous mutation. Values shown represent the average from the three biological replicate selections performed. The x-axis represents the residue mutated in the primary structure while the y axis represents the amino acid substitution at each position. Function regions of the protein are marked above the heatmap. (C) Violin plots enrichment show the dispersion of enrichment values across each domain for non-synonymous mutations and stop codons. Solid black lines represent the enrichment of the positive and negative controls. Dashed lines represent one standard deviation of the mean. NLS, nuclear localization signal; OD, oligomerization domain; ZFD, zinc-finger domain.

Synonymous mutations enriched by selections.

(A) Silent mutations found near the p19 promoter have 1-3 nucleotide substitutions compared to WT Rep2. (B) The enrichment of AAV variants whose abundances increased dramatically following the selection. These variants had different combinations of synonymous mutations proximal to the p19 promoter. All of these variants presented enrichments that are significantly higher than that of positive control (WT) in the selection. (C) Individual virus preparations were generated for the positive control (WT) and the most enriched variant having synonymous mutations. Following packaging of ITR-CMV-gfp, the titer of synthetic cargo packaged was measured. The value obtained for the mutant and WT were not significantly different (**, p < 0.0001; n.s., not significant). The data represents 3 biological and 3 technical replicates, with error bars representing ±1 standard deviations.