PackGene’s Novel Dual-Plasmid System: Advancing AAV Manufacturing Efficiency and Safety

The rapid rise of gene therapy has made adeno-associated virus (AAV) vectors a leading choice for delivering therapeutic genes, thanks to their ability to safely provide long-term gene expression. However, manufacturing remains a major bottleneck. The immense demand for high-dose therapies, particularly for conditions like neuromuscular diseases, is straining current production platforms and creating an urgent need for more scalable and cost-effective solutions.

The traditional triple-plasmid system

For over two decades, the triple-plasmid transfection system has been the workhorse for AAV production in mammalian cells. In this approach, HEK293 cells are transiently transfected with:

1. A transfer plasmid carrying the therapeutic gene cassette flanked by ITRs.
2. A rep/cap plasmid providing essential replication and capsid proteins.
3. A helper plasmid supplying adenoviral helper genes necessary to complete the viral life cycle.

This system is reliable, adaptable to different serotypes, and has proven scalable into hundreds of liters of culture. However, it carries several drawbacks:

• High cost: Manufacturing three GMP-grade plasmids at large scale is expensive.
• Complexity: Increased regulatory complexities, and potential for batch-to-batch variability due to the need for precise optimization of all three plasmid ratios.
• Impurities and replication-competent AAV (rcAAV) risk: Overlaps in sequence homology can lead to replication-competent AAV formation.
• Limited efficiency: Yields often plateau below optimal levels, constraining supply for high-dose therapies.

PackGene’s innovation: the TP011 dual-plasmid system

In response to these limitations, dual-plasmid systems were pioneered, merging Rep/Cap and helper functions into a single transpackaging (TP) plasmid alongside the GOI plasmid. Early designs, such as those described by Grimm et al. (1) and Tang et al. (2), demonstrated cost reductions by simplifying transfection and reducing plasmid requirements. However, initial iterations suffered from low productivity, quality inconsistencies, and elevated rcAAV risks due to promoter overlaps or sequence instabilities.

PackGene Biotech’s TP011 dual-plasmid system exemplifies this next-generation evolution, incorporating a proprietary non-coding regulatory element (RE) in place of the traditional p5 promoter to enhance expression control, minimize recombination hotspots, and elevate productivity. Integrated with PackGene’s π-Alpha 293 high-yield platform—which features an optimized cell line for 10-fold yield increases—and systematic optimizations like transfection reagent screening and enhancers, TP011 addresses key bottlenecks. This article delves into the design, optimization, and performance of TP011, drawing comparisons to triple-plasmid systems and highlighting its potential to lower costs, improve quality, and accelerating AAV manufacturing for gene therapies from preclinical stages to commercial production. By bridging historical foundations with cutting-edge innovations, TP011 positions itself as a scalable, cost-effective alternative to traditional gene therapy manufacturing, designed to meet the growing demand that is projected to exceed current capacities.

1. Design of TP011 dual-plasmid system

The TP011 dual-plasmid system was specifically engineered to minimize the risk of generating rcAAV, a critical safety concern in recombinant AAV (rAAV) production. The system consists of two plasmids: (1) the gene of interest (GOI) or transgene plasmid, containing the transgene (e.g., EGFP+Gluc or disease-relevant genes) flanked by ITRs for efficient packaging; and (2) the TP011 plasmid, a novel RepCap (RC) construct embedded with a proprietary regulatory element and essential helper functions. When co-transfected into a high-yield monoclonal suspension cell line via the dual-plasmid transfection system, and combined with advanced upstream process optimization, the platform enables scalable production from 3L to 200L bioreactor volumes (Figure 1). This end-to-end solution ensures safer, more efficient, and cost-effective AAV manufacturing for both preclinical and GMP applications.

    Figure 1. PackGene high-yield dual plasmid AAV production platform

The TP011 plasmid was thoughtfully engineered to consolidate all essential elements required for high-yield AAV manufacturing, while proactively addressing key safety concerns:

• Rep & Cap genes for replication and capsid formation.
• Adenoviral helper functions (E2A, E4, VA RNA) to support packaging.
• A non-coding RE strategically inserted to disrupt potential recombination events that lead to rcAAV formation.

Figure 2 illustrates the plasmid architecture comparison between the traditional packaging plasmid TP007 and the redesigned TP011. In the TP007 configuration, the p5 promoter is positioned upstream of the Rep and Cap genes, and the E2A/E4/VA RNA sequences are co-located on the same plasmid. This layout poses a risk of homologous recombination events that may lead to the formation of rcAAV during packaging, particularly due to the presence of transcriptionally active sequences.

To address this issue, TP011 introduces two key design modifications. First, the orientation of the p5 promoter is reversed, distancing it from the Rep gene. Second, a non-coding RE is strategically inserted between the Rep&Cap and adenoviral helper sequences (E2A, E4, VA), effectively disrupting potential recombination hotspots. This spatial separation and functional decoupling of the replication and helper elements significantly reduce the likelihood of generating rcAAV during vector production.

The effectiveness of this design was confirmed using Rep2-specific qPCR assays to detect rcAAV contamination. As shown in Figure 2, the traditional TP007 plasmid yielded a detection level of >1 rcAAV per 10⁸ vg, indicating a positive rcAAV signal, whereas the TP011 plasmid produced <1 rcAAV per 10⁸ vg, effectively below the detection threshold and categorized as negative for rcAAV. This data clearly demonstrates that the TP011 design successfully suppresses rcAAV formation during AAV manufacturing.

Figure 2. Design of packaging plasmid in the dual-plasmid system that minimizes rcAAV

2. Systematic Process Optimization: From Screening to DoE

To maximize the yield and quality of our vectors, we performed a stepwise optimization of our production process. Our first step involved a high-throughput screen of several transfection reagents (TRs) in HEK293 cultures. We identified TR05 as the best option, offering a favorable balance of high efficiency, cell viability, and cost-effectiveness (Figure 3A). In a subsequent step, we introduced a small molecule enhancer, ProTF, to the process. This enhancer works by improving plasmid nuclear import and gene expression, and its incorporation was shown to significantly increase the titers of AAV9-Gluc-EGFP (Figure 3B).

We also employed a Box-Behnken design (BBD) DoE experiment to optimize key manufacturing parameters: viable cell density (VCD), plasmid DNA amount, and the amount of TR. Our goal was to create a stable and highly efficient process that would enhance productivity while minimizing impurities.

Through contour plots and ANOVA analyses, we identified an optimal range for these parameters. This optimized process consistently yielded over 5.0×1e11 vg/mL lysate for benchmark GOI (like AAV2-EA0218K), demonstrating a robust production method with minimal variability (coefficient of variation <10%) (Figure 3C-D).

Figure 3. Systematic DoE approach drives enhanced AAV yields

3. TP011 Dual-Plasmid System Enhances Yield While Reducing Host Cell DNA Impurities

The TP011 dual-system, developed with the optimized TR05 transfection reagent, demonstrates a clear advantage over the conventional triple-system in terms of AAV production efficiency, product quality, and impurity reduction. As shown in the data summary and analytical panels (Figure 4A), the dual-system consistently yielded significantly higher viral genome titers in both lysate and affinity chromatography (AC)-purified samples. Specifically, the lysate genome titers reached 7.71 × 10¹¹ and 7.86 × 10¹¹ vg/mL for dual-system runs, while the triple-system only achieved 2.50 × 10¹¹ and 2.57 × 10¹¹ vg/mL, indicating a roughly 3-fold increase in productivity. Similarly, the AC-purified titers from the dual system reached 4.62 × 10¹² and 4.82 × 10¹² vg/mL, compared to 1.55 × 10¹² and 1.53 × 10¹² vg/mL with the triple system, again confirming the enhanced yield performance of the dual setup.

Importantly, this increase in yield did not come at the cost of impurity levels. In fact, the TP011 dual-system exhibited significantly lower residual host cell DNA (HCD) content after purification. Residual HCD levels were measured at 39.61 ng/E13vg and 42.12 ng/E13vg for the dual system, whereas the triple system showed substantially higher levels of 116.13 ng/E13vg and 133.99 ng/E13vg, suggesting a ~3-fold reduction in HCD contaminants (Figure 4B). Lower HCD is a critical quality attribute for gene therapy vectors, as it reduces the risk of immunogenicity and improves overall safety profiles.

As for residual plasmid DNA (pDNA), the levels were comparable between the systems, suggesting that the switch to a dual-system did not adversely affect pDNA removal efficiency, maintaining consistent purification outcomes.

Figure 4D further supports the product quality analysis by demonstrating comparable genome integrity between the dual and triple systems via capillary electrophoresis (CE) analysis, reinforcing that the improved yield and impurity reduction did not compromise vector genome quality or integrity.

Collectively, the data highlights the TP011 dual-system as a superior AAV production platform, offering a substantial increase in viral yield along with marked reduction in HCD impurity, while maintaining plasmid DNA clearance and genome integrity comparable to the traditional triple-system. These attributes make the TP011 dual-system a highly promising approach for scalable, high-quality AAV manufacturing suitable for both research and clinical applications.

Figure 4. TP011-dual system with higher yield and lower HCD residue

4. Dual-Plasmid System Enhanced AAV Yields Across Multiple Serotypes and GOIs

The TP011 dual-plasmid system demonstrated consistently higher AAV productivity across multiple serotypes and therapeutic GOIs compared to the traditional triple-plasmid system. As shown in Figure 5A, dual-TP011 achieved superior lysate genome titers in AAV2, AAV5, and AAV8 serotypes carrying different GOIs, with particularly strong performance in scAAV2-GOI-2, where titers exceeded 1.2 × 10¹² vg/mL, significantly higher than the triple system. Similar improvements were observed in ssAAV2-GOI-1 and ssAAV5-GOI-4.

To further optimize the Dual-TP011 system, we evaluated its performance using three custom RC plasmids bearing different GOIs, specifically GOI-5, GOI-6, and GOI-7. These GOI-specific RC plasmids are known to yield suboptimal AAV titers when used with the conventional triple plasmid system. To overcome this limitation, we incorporated the piVector backbone into the TP011 plasmid and applied a dual-plasmid production format. As shown in Figure 5B, this Dual-TP011–piVector system led to marked improvements in lysate genome titers, with GOI-5 achieving over a 10-fold increase in productivity compared to the triple system. Similarly, GOI-6 and GOI-7 also exhibited notable gains.

These findings highlight the robustness and versatility of the Dual-TP011 platform across multiple serotypes and GOIs, offering meaningful yield enhancements and supporting its suitability for scalable therapeutic vector manufacturing. Furthermore, these results highlight the synergistic value of plasmid engineering and backbone optimization—particularly for custom RepCap constructs with historically low productivity in the triple plasmid system—firmly establishing the Dual-TP011–piVector platform as a superior, cost-effective, and safe solution for high-efficiency rAAV production.

Figure 5. High productivity in different AAV serotypes with different GOIs and TP011 modification

5.Efficient scAAV9 Manufacturing with the TP011 Dual-Plasmid Platform

To further validate the robustness of the Dual-TP011 platform, we assessed its scalability using Ambr250 bioreactors, aiming to confirm direct process transferability to larger-scale production systems. The experiments were performed using scAAV9-GOI-2. As shown in Figure 6A, the Dual-TP011 system achieved comparable lysate genome titers to the traditional triple plasmid system, with yields of 5.39 × 10¹¹ vg/mL and 6.06 × 10¹¹ vg/mL, respectively.

Analysis of downstream (DS) impurities revealed similar profiles between the two systems. Residual HCD was slightly higher in the dual system compared to the triple system (Figure 6B), while residual pDNA was lower in the dual system (118.52 ng/E13vg) compared to the triple system (133.99 ng/E13vg) (Figure 6C). Importantly, Analytical Ultracentrifugation (AUC) confirmed consistent product quality across systems. The dual system exhibited a dominant scAAV peak with reduced empty capsid levels (1.50%), while the triple system showed a slightly lower empty ratio (0.85%) but a more pronounced ssAAV peak (Figures 6D–E).

Overall, these results indicate that the TP011 dual system delivers equivalent vector yield and product quality compared with the triple system, while benefiting from the use of a lower-cost TR05 transfection reagent. This not only ensures robust productivity and impurity clearance but also provides a cost-effective solution for GMP-grade plasmid manufacturing, further reducing the overall cost of AAV production.

Figure 6. TP011 dual-plasmid platform demonstrates high performance in Scale-up AAV production

Conclusion

As the field of gene therapy accelerates, the demand for scalable, efficient, and high-quality AAV manufacturing platforms has never been greater. While the traditional triple-plasmid system has long served as the industry standard, its limitations in cost, complexity, scalability, and safety—particularly regarding the generation of replication-competent AAV (rcAAV)—pose significant challenges as programs move from early research to commercial scale.

PackGene’s TP011 dual-plasmid platform represents a next-generation solution to these challenges. By combining Rep/Cap and adenoviral helper functions into a single plasmid and incorporating a proprietary non-coding regulatory element to minimize recombination risk, TP011 achieves both enhanced safety and higher productivity. Systematic process optimization—including high-throughput transfection reagent screening, the use of small molecule enhancers, and statistical design of experiments (DoE)—has further elevated the platform’s performance, enabling robust, reproducible yields.

Critically, TP011 also reduces residual HCD by approximately threefold compared to the triple system, without compromising genome integrity or plasmid clearance. The platform demonstrates broad applicability across AAV serotypes and diverse GOIs, including low-yield constructs, and has proven its scalability in Ambr 250 bioreactors, showing comparable titers and impurity profiles to the triple system. Incorporating the piVector backbone into TP011 further enhances productivity, particularly for challenging custom RC constructs that show poor performance in the triple plasmid system, with certain GOIs demonstrating over a 10-fold increase in vector titers.

In summary, the TP011 dual-plasmid system, supported by PackGene’s π-Alpha 293 production platform and advanced process innovations, offers a cost-effective, scalable, and high-performance manufacturing solution for rAAV vectors. It not only addresses the limitations of the triple-plasmid system but also positions itself as a commercially viable platform for gene therapy production, capable of meeting the growing demands of the gene therapy pipeline.

Reference:

1.Grimm D, et al. 2003. Helper virus-free, optically controllable, and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6. Mol Ther 7:839–850.

2.Tang Q, et al. 2020. Two-plasmid packaging system for recombinant adeno-associated virus. Biores Open Access 9:219–228.