Improved Recombinant Adeno-Associated Viral Vector Production via Molecular Evolution of the Viral Rep Protein

Abstract

In the dynamic field of gene therapy, recombinant adeno-associated viruses (rAAVs) have become leading viral vectors due to their safety, long-term expression, and wide-ranging cell and tissue tropism. With numerous FDA approvals and commercial products underscoring their potential, there is a critical need for efficient production processes to achieve high vector titers and quality. A major challenge in rAAV production is the efficient packaging of the genome into the viral capsid, with empty or partially filled capsids often representing over 90% of the produced material. To tackle this issue, we engineered the replication and packaging proteins of an AAV (Rep) to boost their functionality and improve vector titers. We subjected a complex Rep library derived from the AAV serotypes 1-13 to directed evolution in an AAV producer cell line. After each round of selection, single clones were analyzed, showing enrichment of specific hybrid Rep domains. Comparative analysis of these selected clones revealed considerable differences in their ability to package AAV2-based viral genomes, with hybrid Rep proteins achieving up to a 2.5-fold increase in packaging efficiency compared to their parental counterparts. These results suggest that optimizing rep gene variants through directed evolution is an effective strategy to enhance rAAV production efficiency.

Keywords: AAV; directed evolution; protein engineering; rAAV production; replication proteins.

Figure 1

Test of different AAV Rep proteins in rAAV2 production. (A) Schematic representation of the cloning strategy for natural rep variants 1 to 13 or chimeric rep ORFs. The cloning procedure included a unique stretch between rep and cap in each AAV genome. (B) Fold change in viral genomes per ml cell culture lysate (vg/mL). Adherent HEK293T cells were transfected with a rAAV-GFP reporter, an adenohelper plasmid and one plasmid of the indicated Rep variants. AAV2 Cap was co-expressed from the RepX plasmids as shown in panel (A). rAAV-GFP titers acquired with Rep2 were averaged and set to 1, n = 3. (C) Fold change in total capsids per ml cell culture lysate (vp/mL). HEK293T cells were transfected as described in panel (B). vp/mL titers acquired with Rep2 were averaged and set to 1, n = 2. (D) Fold change in packaging rate calculated from panels (B,C). Rates acquired with Rep2 were averaged and set to 1, n = 2. All data were consistently normalized to the Rep2 variant, as indicated by the red bar.

Figure 2

Generation of a chimeric rep library using DNA family shuffling. (A) Steps involved in DNA family shuffling. A pool of PCR-amplified rep ORFs undergoes a DNaseI digest under conditions indicated in respective agarose gel electrophoresis images. This involves varying times of incubation (30, 60, or 90 s) and DNaseI concentration (stock = 1 U/µL or 1:10 dilution). Fragments in the indicated range (100–1000 bp) are extracted from the gel and serve as input for a primerless, homology-based PCR. Finally, complete ORFs were amplified with a rescue PCR using primers binding outside of the shuffled region. (B) Percentage homology matrix of AAV rep ORFs 1–13. The # symbols in the figure mark the diagonal where each Rep variant is compared to itself, indicating 100% similarity (C) Hybrid rep library generated in this work, later denoted as library 0. Each line represents one clone from the 5′ to the 3′ end. Color blocks indicate the underlying parental rep sequences as shown by the color code at the bottom. “Maximum traces” were generated as described in Figure S3 and were used to build a graphical composition summary of each chimera. White annotated regions could not be exclusively assigned to one rep reference sequence.

Figure 3

Directed evolution of Rep proteins for rAAV production. (A) Schematic representation of the directed evolution approach used. Suspension HEK293 cells were transfected with the chimeric rep library and an adenohelper plasmid for wild-type AAV2 production. Cells were lysed to recover AAV2 particles packaged with the chimeric rep ORFs. Primers binding outside of the shuffled region were used to PCR amplify the hybrid regions. Finally, chimeric rep ORFs were cloned into an acceptor plasmid with AAV2 cap for clonal assessment and initiation of second selection round. (B) Chimeric Rep/Cap plasmid amounts and corresponding genomic titers as fold changes. Titers obtained with the lowest DNA amount in round 1 (0.25 ng) were set to 1. (C) Clonal composition of rep hybrids after each selection round. Each line corresponds to one clone from the 5′ to 3′ end. Color blocks represent underlying rep reference sequences, as indicated with the color code at the bottom. R0 = primary, pre-selected library. R1/R2 = selection rounds 1 and 2.

Figure 4

Functional validation of single rep hybrids. (A) High-throughput screening of Rep variants in a micro-scale setting (24 deep-well). Data from the indicated analytical measurements are represented as a heat map with Rep2-values set to 100% (white) and the highest limit corresponding to 500% of Rep2 levels (red); n = 2–3 biological replicates. Genomic titers ranged from 1 × 10⁹ to 2 × 10¹⁰ vg/mL, with values below 5 × 10⁸ vg/mL classified as negative. Capsid titers ranged from 1 × 10¹⁰ to 5 × 10¹¹ vp/mL. (B–F) Testing of selected Rep candidates in mini-scale (30 mL shake flasks; n = 2–3 biological replicates). Indicated parameters were assessed: (i) genomic titer (panel (A); genomic titers ranged from 6 × 10⁹ to 2 × 10¹¹ vg/mL); (ii) capsid titer (panel (B); capsid titers ranged from 2 × 10¹¹ to 8 × 10¹² vp/mL; (iii) packaging rate (ratio of (A) to (B)); (iv) functionality of resulting AAV vectors (% of GFP positive cells) after transduction with crude cell lysates (equal volumes were applied (1:10 dilution of stock)); and (v) cell viability at day of harvest. (G) Overview of the clonal composition of selected rep hybrids (5′ to 3′ end). Color blocks represent underlying rep reference sequences, as indicated with the color code at the bottom. The red bar in all images highlights the Rep2 wildtype variant.

Figure 5

(A–C): Production of rAAV-GFP with the indicated Rep variants or Rep2 control in the AMBR15 system. Assessed parameters: (i) genomic titer (panel (A)), (ii) capsid titer (panel (B)), (iii) packaging rate (ratio of (A) to (B)). n = 3 biological replicates. The red bar in all images highlights the Rep2 wildtype variant. (D) Western blot analysis using the JESS Simple Western™ system. Cell lysates from the indicated rAAV productions in the AMBR250 system were analyzed for capsid protein expression (VP1-VP3; top) or Replication protein (Rep) expression (bottom). A semi-quantitative analysis is provided in Tables S2 and S3 TTG = therapeutic transgene. (E) Cryo-EM analysis of the indicated rAAV productions in AMBR250 purified using small-scale AAVX columns. For each sample, a representative image is shown. Two zoom-in areas show examples of typical empty (E) and full (F) particles. Scale bars = 200 nm.