Enhancing rAAV Production through Directed Evolution and High-throughput Screening of Rep Protein

doi:10.13560/j.cnki.biotech.bull.1985.2026-0320

Abstract

Abstract:

Objective This study aimed to perform random mutagenesis of the Rep-coding gene, construct a mutant library, and establish a high-throughput screening platform for the identification of Rep variants capable of enhancing rAAV production, and then elucidate their mechanism of action. Method A Rep mutant library was constructed by error-prone PCR. HEK293 cells cultured in baffled 24-well deep-well plates were subjected to triple-plasmid transfection. Using rAAV genome titer as the screening criterion, culture wells with yields close to or exceeding 80% of the control group were selected as candidate mutant groups. Rep mutants from candidate groups were further validated in shake-flask cultures to obtain beneficial Rep variants. The underlying mechanisms of the enhanced rAAV production were investigated by analyzing rep/cap gene transcription, Rep/Cap protein expression, and rAAV transduction efficiency, as well as by predicting the Rep78 structure using AlphaFold3 and locating the mutation sites. Result Through high-throughput screening and shake-flask verification, we identified two beneficial Rep protein mutants, P39L and Q202H, which enhanced rAAV yield to 2.02-fold and 1.87-fold that of the control, respectively. Saturation mutagenesis at positions P39 and Q202 of the Rep protein did not further improve rAAV yield. Further analysis indicated that P39L and Q202H did not significantly affect Rep/Cap expression levels or rAAV transduction efficiency. Structural localization indicated that P39 is located in the origin‑binding domain (OBD), while Q202 is situated in the OBD-HD (homeodomain) transition region, in proximity to the OBD. Conclusion This study established a directed evolution and high-throughput screening platform for Rep protein and identified two beneficial Rep mutants, P39L and Q202H, which enhanced rAAV yield to 2.02-fold and 1.87-fold that of the control, respectively. OBD and its nearby regions are important sensitive areas affecting rAAV yield.

Key words: recombinant adeno-associated virus, Rep protein, random mutagenesis, high-throughput screening, viral yield

SONG Zi-chuan, REN He, MO Wan-ting, NIE Jian-qi, BAI Zhong-hu. Enhancing rAAV Production through Directed Evolution and High-throughput Screening of Rep Protein[J]. Biotechnology Bulletin, 2026, 42(11): 309-317.

Figures/Tables 5

Fig. 1 Schematic workflow of high-throughput screening and functional analysis of Rep protein mutants

Fig. 2 High-throughput screening and shake-flask validation of Rep protein mutantsA, B: rAAV yields of candidate mutant groups. C: Shake-flask validation results of single-point mutants from candidate mutant groups. D: Shake-flask validation results of multi-point combination mutants from candidate mutant groups. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively; ns indicates no significant difference. The same below

Fig. 3 rAAV yields of saturation mutants at P39 and Q202 sitesA: rAAV yields of saturation mutants at the P39 site. B: rAAV yields of saturation mutants at the Q202 site

Fig. 4 Effects of P39L and Q202H mutations on rep/cap gene transcription, protein expression and rAAV transduction efficiencyA: rep gene transcription levels at 24 h and 72 h post-transfection. B: Rep78/68 gene transcription levels at 24 h and 72 h post-transfection. C: cap gene transcription levels at 24 h and 72 h post-transfection. D: Western blot analysis of Rep78/68, Rep52/40, and VP1/2/3 protein expression at 72 h post-transfection. E: Percentage of EGFP-positive cells detected by flow cytometry at 48 h post‑infection. F: Representative fluorescence micrographs showing EGFP expression at 48 h post-transduction. Scale bars, 100 μm

Fig. 5 AlphaFold3‑predicted structure of Rep78 protein and the localization of P39 and Q202A: Predicted structure of Rep78 protein with annotation of major functional regions. B: Spatial localization of P39 in the OBD region. C: Spatial localization of Q202 in the OBD-HD transition region

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