A new spatial biology is in sight—literally. It is a more vivid, more detailed, and ultimately more informative spatial biology. It can distinguish between cell types that were once indistinguishable, and it can do so while capturing their spatial context. It can even pinpoint the subcellular locations of individual molecules. And it can accomplish these tasks with unprecedented precision because technology is now available that opens a new dimension beyond the usual three spatial dimensions. This new dimension may be called the “plex” dimension.

Plex refers to the number of fluorescence markers that are used with microscopy and other cell analysis platforms. Conventional platforms may accommodate just a handful of markers, constraining investigations of complex biological phenomena. But such investigations may require many markers.

Unfortunately, using more and more markers—and thereby shifting from low-plex to high-plex spatial biology—has been too difficult for most laboratories. They’ve balked at the need for special expertise, complicated workflows, and instrument upgrades. Fortunately, these difficulties can be overcome with new multiplex imaging technologies. For example, there are antibody panels that are compatible with streamlined workflows and automated imaging systems.

To learn more about these technologies, consult the articles in this eBook—especially the article describing organ mapping antibody panels. Also, be sure to read the articles that describe the kinds of spatial biology applications that are bound to become more common as high-plex technology becomes more accessible. Indeed, this technology is democratizing spatial biology.

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The rapid qPCR assay described in this tech note is a cell-based technology developed by SK pharmteco to monitor the emergence of rcAAVs. The company has also developed technologies to mitigate the risk of rcAAV formation. Together, these technologies can help manufacturers of gene therapies sustain high-throughput operations, improve quality control, and enhance the safety and efficacy of their products.

The production of recombinant AAVs involves the co-transfection of three plasmids into HEK293 cells: the rep-cap plasmid, the adenovirus helper plasmid, and the AAV genome plasmid. The rep-cap plasmid carries the AAV replication (rep) and capsid (cap) genes essential for AAV genome replication and capsid formation. The adenovirus helper plasmid provides essential helper functions for AAV replication and packaging, whereas the AAV genome plasmid contains the therapeutic gene flanked by inverted terminal repeats (ITRs). The triple-transfection method is efficient and scalable, enabling the production of various AAV serotypes by altering the cap gene within the rep-cap plasmid.

However, during recombinant AAV production, nonhomologous recombination between the AAV vector and packaging DNA can lead to the formation of rcAAVs. These replication-competent vectors, if not managed properly, can replicate in the presence of a helper virus, potentially leading to unintended infection and replication within host cells. The presence of rcAAVs in clinical-grade AAV vector preparations is not just a concern, but a significant and pressing issue, as their behavior in the host post-administration, especially in the presence of natural helper viruses, is not fully understood.

Advanced detection methods

To address the challenge of rcAAVs, stringent quality control measures and advanced diagnostic techniques are required. Traditional methods for detecting rcAAVs, such as PCR-based assays, are effective but labor intensive and unsuitable for high-throughput commercial manufacturing. Recent advancements include the use of single-molecule, real-time sequencing and AAV genome population sequencing to detect and characterize rcAAVs. These technologies offer high sensitivity and specificity, and they are capable of identifying diverse recombination events leading to rcAAV formation.

At SK pharmteco, a cell-based qPCR assay has been developed to monitor the emergence of rcAAVs. This assay uses rep2-specific primers/probes, followed by confirmation with cap gene–specific primers/probes, to detect rcAAV events at a limit of detection of 10 infectious units. The qPCR assay is integrated into the manufacturing process, enabling timely detection and mitigation of rcAAV formation to ensure patient safety.

The application of the cell-based qPCR assay at SK pharmteco has proven robust and reliable. Transient transfection, involving multiple steps—such as cell expansion, plasmid DNA-transfection reagent complex formation, and bioreactor transfer—leads to the generation of viral particles (Figure 1).

The qPCR assay monitors for rcAAV emergence through continuous rounds of propagation of harvest lysates in the presence of adenovirus (Figure 2). This method amplifies AAV genomes containing ITR–rep-cap–ITR sequences, increasing progeny virus through subsequent rounds of infection. Extensive testing validated the assay’s specificity and sensitivity. Specificity acceptance criteria ensured accurate identification of target sequences, with positive amplification detected only in positive controls and test particles (Table 1).

The assay’s ability to detect the lowest concentration of rAAV2 confirmed the detection limit, ensuring high sensitivity. The linearity of the assay was evaluated through quantitative PCR standard curves, verifying accuracy across a range of input concentrations.

A bridging study was also conducted to evaluate the assay’s applicability to other AAV serotypes. The study confirmed that the assay design suits various AAV serotypes, demonstrating its broad utility in viral gene therapy applications. Primers and probes for AAV2 were validated, ensuring reliable detection and quantification of rcAAV2. Compatibility with different adenovirus preparations further enhanced the assay’s flexibility and practicality.

Importance of results for gene therapy production

The ability to reliably detect and mitigate rcAAV formation is crucial for the safety and efficacy of gene therapy products. The presence of rcAAVs poses significant risks, including potential unintended replication and infection within the host. Advanced detection technologies, such as the cell-based qPCR assay, provide a robust solution to monitor and control rcAAV emergence during manufacturing. Ensuring the absence of rcAAVs in clinical-grade AAV vector preparations enhances the safety profile of gene therapy vectors, reducing the risk of adverse events in patients.

The scalability and high-throughput capability of the qPCR assay make it suitable for commercial manufacturing settings. This ensures that quality control measures can be consistently applied across large production volumes, maintaining the integrity and safety of gene therapy products.

In addition to advanced detection methods, SK pharmteco has implemented several strategies to mitigate the risk of rcAAV formation. Promoter rearrangement is one strategy, where the P5 promoter is positioned 3′ of the cap gene in the vector design. This selective promoter activity prevents unintended gene expression that could contribute to rcAAV formation. Incorporation of introns to exceed AAV’s packaging limit is another strategy, ensuring that intact contigs cannot be packaged, thus reducing rcAAV emergence.

Observational data revealed that configuring test article constructs with the P5 promoter positioned upstream of essential replication and capsid genes led to a higher risk of rcAAV formation. Conversely, positioning the P5 promoter 3′ of these genes significantly reduced this risk, supporting the influence of promoter placement on vector safety. These mitigation strategies, combined with advanced detection methods, provide a comprehensive approach to ensuring the safety and efficacy of rAAV-based therapies.

Future directions

The ongoing development and refinement of the assay design focuses on enhancing sensitivity and accuracy. Integrating adenovirus 5 (Ad5) earlier in the protocol and optimizing freeze/thaw cycles are potential adjustments to improve assay performance. Shifting from qPCR to droplet digital PCR for endpoint analysis could offer superior precision and sensitivity, which would be particularly useful in detecting low levels of target DNA.

Future studies will involve pilot testing these adjustments, systematic analysis of freeze/thaw cycle effects, and controlled experiments to confirm the benefits of early Ad5 addition. Further research will explore alternative promoter and gene configurations to robustly prevent rcAAV formation without compromising vector efficacy. These efforts aim to enhance the capability to safely and effectively utilize rAAV vectors in clinical settings.

At SK pharmteco, Oxana M. Tsygankova, PhD, serves as senior scientist; Lana Sweet, director of virology, cell and gene therapy; Dana Cipriano, global head of testing and analytics; and Brian Tomkowicz, PhD, head of viral vector R&D.

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Perhaps the greatest challenge facing cell therapies for this purpose lies in the immunosuppressive mechanisms found in the tumor microenvironment. This is harsh biological territory, with high interstitial pressure, reduced oxygen tension, and a barrage of immunosuppressive proteins. Studies show that therapeutic cells exposed to this environment suffer from depletion, exhaustion, and mitochondrial dysfunction.

Thus, there is a critical need for manufacturing processes to generate more potent cell populations, both by producing higher proportions of antigen-targeted cells (for example, chimeric antigen receptor T cells, or CAR T cells) and by boosting the cytotoxic potential of those cells.

New evidence indicates that cell therapies can be “rewired” metabolically to overcome these challenges, leading to treatments that could be far more effective against a range of solid tumors. The idea is simple: instead of growing therapeutic cells in conventional culture conditions designed to keep cells as happy as possible, why not acclimate them under conditions mimicking that of the tumor microenvironment they will have to face in vivo?

The cells that survive and expand in vitro under these harsh conditions should be far more likely to remain effective when they reach the tumor microenvironment. Results are already showing that metabolic rewiring can create higher-yielding, effective cell therapies.

Feasible approach

This approach is feasible for any scientist developing cell therapies. For the work described below, teams at Xcell Biosciences and Labcorp used the AVATAR system to compare the performance and effectiveness of CAR T cells grown in conventional culture conditions versus conditions more closely mirroring the tumor microenvironment.

Cells were prepared according to established cell therapy manufacturing protocols and cultured under a range of O2 and pressure levels. We first assessed three different culture environments: standard culture conditions with no change to oxygen or pressure in a conventional CO2 incubator (20.5% O2 with 0 pounds per square inch (PSI)); the AVATAR incubator set to pressure and oxygen levels replicating the arterial vasculature system at 15% O2 with pressure at 2 PSI; and the AVATAR incubator at 15% O2 and pressure at 5 PSI. All incubation conditions were maintained at 5% CO2 and 37°C.

CD3 T cells were thawed alongside soluble anti-CD3 and cultured for two days. All three cell populations were then transduced with a lentivirus designed for CD19 CAR expression and returned to their incubator conditions to grow for 10 more days. CD19 CAR expression was evaluated with flow cytometry, and cytotoxic activity was assessed with a targeted killing assay.

These in vitro experiments demonstrated that culturing with adjusted O2 and pressure levels had no detrimental effect on the therapeutic cells, and these cells actually outperformed cells grown in standard incubator conditions (Figure 1). For example, we found that both cell populations grown with modified oxygen and pressure settings had higher proportions of CAR T cells following lentiviral transduction compared to the conventional incubator population. Cells transduced in the conventional CO2 incubator conditions yielded 10% to 20% CD19 CAR T cells, while those transduced in high-pressure conditions yielded as much as 40% of the desired therapeutic cells.

By the end of the expansion process, cell yield was more than twice as high in the two groups expanded at 15% O2 and at 2 or 5 PSI as compared to the standard incubator cells.
Potency tested

The potency of CD19 CAR T cells manufactured in the three culture environments was next tested in vitro with a targeted NALM6 cytotoxicity assay. For this work, we co-cultured T cells with NALM6, CD19-expressing target cells, for 48 hours, and measured lysis rates by flow cytometry. As expected, CD19 CAR–specific killing was exhibited in a dose-dependent manner, and cells grown in the AVATAR system showed no impairments to their cytotoxic function.

Cytotoxicity was confirmed in a mouse model of B-cell acute lymphoblastic leukemia. CD19 CAR T cells were administered to mice that had been inoculated with NALM6 cells intravenously. For five weeks, we regularly measured tumor growth with bioluminescence imaging, and we collected blood samples to identify circulating T cells and assess their phenotype using flow cytometry.

Results showed that mice treated with the two AVATAR cell populations had good tumor control outcomes, confirming that the modified culture conditions do not impair cancer-fighting ability in vivo (Figure 2). We also examined blood samples for T cells using Labcorp’s Custom Expanded Persistence T Memory Panel. The animals exhibited persistence of the therapeutic cells in relevant organs, and the CAR T cells maintained their phenotype with strong central memory and effector memory populations.

Finally, we performed experiments on CD19 CAR T cells grown in conditions of even more acute hypoxia, with conditions of 5%, 10%, and 15% oxygen, while maintaining 5 PSI, and compared them to cells grown in the conventional CO2 incubator. We again evaluated the cells’ potency in vitro with a targeted NALM6 cytotoxic assay, but importantly, tested the ability of CD19 CAR T cells to kill for extended time periods at low effector-to-target cell ratios (Figure 3).

The improved cytotoxic function of CD19 CAR T cells grown under lower oxygen conditions is currently being confirmed in further in vivo experiments. While this work is being performed with a mouse model for hematological cancer, follow-up studies using this same approach for solid tumors are already underway.

All of this work was completed with well-established protocols for cell therapy development, with no special techniques required for the cells expanded in the AVATAR system. In the future, a more automated workflow will be enabled with the upcoming AVATAR Foundry system, which has been designed specifically for the manufacture of cell therapies. This would allow clinical research teams to incorporate hyperbaric pressure and oxygen control into the manufacturing process more easily, accelerating the process of discovering the optimal environmental conditions to enhance the potency and yield of each therapy candidate.

The GMP-compliant AVATAR Foundry system is currently available to scientists through a beta access program. To see more of this data, view a poster (“Metabolic reprogramming enhances expansion and potency of CAR-T cells”) that Xcell Biosciences and Labcorp scientists presented at the American Association for Cancer Research Annual Meeting in 2024.

James Lim, PhD, is a co-founder and the CSO of Xcell Biosciences. Scott Wise, MS, is executive director of preclinical oncology at Labcorp.

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HEMCO

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Particle Measuring Systems

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Shimadzu

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Beckman Coulter Life Sciences

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Cytomos

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Thermo Fisher Scientific

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One area for improvement is the transfection process used in the upstream manufacturing of AAV gene therapies. The TransIT-VirusGEN^® transfection solution includes both polyamine-containing polymers and lipids that help overcome barriers during transfection, enabling high transfection efficiency and low cellular toxicity. The polymer facilitates nucleic acid condensation, binding, and uptake by the cells, while the lipid promotes endosomal escape.

The RevIT AAV Enhancer can be used in conjunction with both the VirusGEN solution and conventional polymeric transfection reagents to produce 2–4X higher genome titers in suspension HEK 293 cells. The enhancer is simple to use, easily integrates into existing workflows, and produces high-quality titers across a range of AAV serotypes and cell growth media. When combined with the TransIT-VirusGEN solution, the enhancer enables use of lower amounts of plasmid DNA (pDNA), which represents a key cost-saving opportunity.

Improved genome titers across multiple AAV serotypes

The efficacy of RevIT AAV Enhancer was assessed across multiple serotypes (AAV2, AAV5, AAV8, and AAV9) in 293-VP 2.0 cells (Thermo Fisher) using TransIT-VirusGEN and the single-component polymeric transfection reagents (Figure 1). In all serotypes, use of the RevIT AAV Enhancer in conjunction with TransIT-VirusGEN increased genome titers 1.7- to 2.4-fold compared to the TransIT-VirusGEN control. The RevIT AAV Enhancer plus TransIT-VirusGEN condition also delivered up to 6-fold higher genome titers and 2.9-fold higher percent full capsids. RevIT AAV Enhancer increased genome titers 1.7- to 2.2-fold with other transfection reagents compared to their respective controls, demonstrating broad applicability.

The enhancer was also tested in AAV2, AAV5, AAV8, and AAV9 serotypes using 293-VP 2.0 cells grown in either Viral Production Medium (Thermo Fisher) or BalanCD HEK 293 Medium (Irvine Scientific). There was little to no difference in performance across these growth media formulations (Figure 2). Together, these data demonstrate broad serotype and growth medium compatibility when using RevIT AAV Enhancer in conjunction with TransIT-VirusGEN and single-component polymeric transfection reagents.

Cost savings achieved with lower pDNA doses

Protocols for commonly used single-component polymeric transfection reagents recommend pDNA doses of 1 μg/106 cells (for example, 3 μg/mL at a density of 3 × 106 cells/mL) for triple-transfection-mediated AAV production in suspension cells. In contrast, the TransIT-VirusGEN protocol recommends a pDNA dose of 2 μg/mL of cell culture regardless of cell density, which represents a 33% decrease in pDNA usage. Scaling pDNA dosage by cell density does not improve viral titers, even when RevIT AAV Enhancer is employed, suggesting that maintaining a pDNA dose of 2 μg/mL cell culture with TransIT-VirusGEN can reduce the usage of valuable pDNA and save on manufacturing costs.

Lower amounts of pDNA with RevIT AAV Enhancer were tested, demonstrating that the enhancer allowed for a decrease in pDNA doses to as low as 0.75 μg/mL in some serotypes while still maintaining high genome titers (Figure 3). This allows for up to a 75% decrease in pDNA usage compared to a traditional DNA dose method of 3 μg/mL pDNA for a density of 3 × 106 cells/mL. Decreasing pDNA doses also led to a higher percentage of full capsids, demonstrating that drastic cost savings and higher quality AAV can be achieved using lower pDNA doses in conjunction with RevIT AAV Enhancer.

A proven strategy to increase titers and decrease costs

Use of the RevIT AAV Enhancer substantially increases AAV genome titers across multiple serotypes and transfection platforms, including the TransIT-VirusGEN transfection reagent and polymeric transfection reagents. Simple optimization allows for fast and easy integration of RevIT AAV Enhancer into existing AAV manufacturing workflows to increase titers by 2–4X. The enhancer also enables reductions in the amount of pDNA required during the transfection process, leading to considerable cost savings in AAV-based gene therapy manufacturing.

Download our white paper on use of the RevIT AAV Enhancer for additional studies and
details on materials and methods:(https://www.mirusbio.com/content-download-revit-aav-enhancer-white-paper/).

Becky Reese, PhD, is senior scientist, Jennifer Swanson is R&D associate scientist III, Austin Storck is associate scientist III , and Laura Juckem, PhD, is vice president of research and development at Mirus Bio.

References

1. Au HK, Isalan M, Mielcarek M. Gene therapy advances: A Meta-Analysis of AAV Usage in Clinical Settings. Front. Med. (Lausanne) 2022; 8.

2. Clément N, Grieger JC. Manufacturing of recombinant adeno-associated viral vectors for clinical trials. Mol. Ther. Methods Clin. Dev. 2016; 3: 16002. 

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