Cell and gene therapy I Cell therapy I Gene therapy

Cell and gene therapy with digital PCR
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from research to bioprocessing & QC

The potential of cell and gene therapies to either prevent disease progression or provide a cure offers a great clinical benefit over existing therapies which are often geared toward the treatment of symptoms.

Gene therapy involves the transfer of genetic material, usually in a carrier or vector, and the uptake of the gene into the appropriate cells of the body. This can include the insertion of a new copy of a gene, gene editing using e.g., CRISPR technology, and gene silencing. Gene therapy is usually performed by viral-mediated gene transfer with Adeno-Associated Virus (AAV) or lentivirus or alternatively by using plasmids. Cell therapy involves the transfer of cells with the relevant function into the patient. The cells are modified outside the body before being injected into the patient, and can come from the patient (autologous cells) or a donor (allogeneic cells). Examples of cell therapy include CAR T-cells.

cell and gene editing, genetic editing and gene research, in vitro

How to ensure the quality and reproducibility of your therapeutic product

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Over the past decade, cell and gene therapy research has accelerated and there have been several approvals for various disorders, including lymphomas and leukemias, spinal muscular dystrophy and inherited retinal disease. These recent successes are partly due to the rapid advancements in vector biology and drug delivery technology. Nonetheless, these therapies are inherently complex with a large degree of variability. Developing safe and effective cell and gene therapies requires quality control at all stages of the development process.

The superior accuracy and precision offered by digital PCR enables you to ensure the quality and reproducibility of your therapeutic product. Not only can you reliably and precisely determine plasmid quality, viral titer and vector copy number, but you can also perform robust contaminant testing, for example, to detect mycoplasma and residual host cell DNA.

Cell and gene therapy events

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Quality control in cell and gene therapy

We understand how important it is to establish quality control testing for cell and gene therapy products. With recent advances in digital PCR it is increasingly becoming the technology of choice to implement in robust manufacturing and quality control processes.

We took digital PCR one step further with the QIAcuity Digital PCR System, a fully-integrated system based on nanoplate technology which promises a faster time to result, higher throughput, multiplexing and scalability.

A comprehensive assay portfolio integrates seamlessly with the QIAcuity instruments, nanoplates and kits to offer the flexibility and accuracy required for your cell and gene therapy applications.

The system is coupled to a feature-rich QIAcuity Security Edition Software enabling labs to adhere to 21 CFR Part 11 compliance requirements in a GMP setting.

We also guarantee Installation Qualification (IQ) and annual Operational Qualification (OQ) services, 24/7 technical and scientific support and repairs performed by certified professionals ensuring you reliable quality control.

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Cell and gene therapy workflows

Gene therapy

AAV viral titer quantification

Gene therapy using AAV could lead to substantial changes in the treatment of monogenic disorders and perhaps even cures. AAV vectors have a smaller packaging size (~5 kb) so are useful for the delivery of small genes. They are non-integrating and hence have relatively low systemic toxicity and they cannot propagate without a helper virus which makes them safe for clinical use.

Accurate quantitation and characterization of AAV are critical for precise patient dosing. qPCR is a widely used method for AAV titer quantitation. However, the technique is limited by the presence of PCR inhibitors that can hinder amplification and introduce variability. Selecting a standard that amplifies similarly to the AAV vector and maintains a stable standard concentration over time is also challenging.

Digital PCR, conversely, due to partitioning and end-point measurement, demonstrates improved amplification efficiency and higher tolerance to inhibitors than qPCR.

We have developed a new viral vector genome quantification workflow from capsid lysis to titer determination, including singleplex and multiplex assays for quantifying and qualifying AAVs using the QIAcuity Digital PCR System.

The modern approach

It is possible to accelerate gene therapy manufacturing using a faster, scalable plate-based dPCR method.

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Resources for gene therapy applications

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Contamination control

Residual host-cell DNA (HCD) carryover poses major safety concerns when manufacturing protein and vaccine therapeutics. Risks include oncogenicity, infectivity and immunogenicity caused by your cell or gene therapy product. Therefore, levels of HCD must not exceed those established by regulatory agencies such as the U.S. Food and Drug Administration and the World Health Organization.

In comparison to qPCR, digital PCR provides higher sensitivity of detection at a lower template input range, enabling more robust applications. A multicopy target assay ensures that results are not affected by the fragmentation level of residual DNA (resDNA), a problem with qPCR.

Moreover, contaminating DNA in your PCR reagents can be a significant source of background and false positives. Ultra Clean Production enhances the specificity and efficiency of probe-based digital PCR to provide accurate, singleplex, or up to 5-plex analysis. Dedicated processes are implemented to enable Ultra Clean Production of the master mix, which minimizes contaminating DNA background and GC bias and gives you absolute certainty in your PCR reagents, making it an ideal choice for contamination-free, microbial applications as well as quality control applications, like residual DNA testing.

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How to de-risk cell and gene therapies

Explore how well-designed digital PCR assays are taking quality control to the next level, enabling safer biomanufacturing.

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Cell therapy

CAR T-cells

Chimeric antigen receptor (CAR) T-cells are rapidly emerging as a promising treatment for cancers and immune disorders. T-cells are genetically modified with CARs containing antibody-based recognition domains against cell-surface antigens on the target cell type.

T-cells, and more recently NK cells, can be genetically manipulated using lentiviruses and retroviruses. Viral vectors have a high transduction efficiency, but they suffer from immunogenicity and cellular toxicity.

CAR T cell immunotherapy, chimeric antigen receptor T cell immunotherapy

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CAR T-cell development

Current research efforts focus on using CRISPR editing for CAR T-cell engineering for reproducible, safe and potent CAR T-cell products. The current manufacturing process of CAR T-cells requires dedicated equipment and significant technical expertise, so there is an urgent need to develop a safe yet simple approach to CAR T-cell development. In vivo reprogramming of CAR T-cells using nanotechnology to encapsulate CAR-expressing plasmids provides an alternative approach. Viral vectors such as lentiviruses have also been tested for the in vivo generation of CAR T-cells.

We offer a range of products for the quantification of viral titers and viral vectors, detection of residual host cell DNA and solutions for CRISPR-mediated editing for CAR T-cell engineering.

Lentivirus production

One of the most versatile gene transfer methods for T-cell therapies uses recombinant lentiviral vectors since they can transduce both dividing and nondividing cells. Effective and reproducible cell therapies require sensitive and precise methods for quality control of lentivirus production. Traditional qPCR quantification of nucleic acids may be affected by PCR inhibitors and additionally require a reference standard for quantification. Conversely, the QIAcuity Digital PCR System enables fast, reproducible and absolute quantification of viral vectors and viral titer without a reference standard, ensuring quality and reproducibility in cell therapy research. The technology also has a greater tolerance for inhibitors than qPCR.

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Accelerate your data-driven cell and gene therapies

From data analysis to databases, from gene expression to genome assembly, from interpretation to integration – QIAGEN Digital Insights has the breadth and quality of bioinformatics tools you need to drive meaningful insights and fuel cell and gene therapy discoveries.

References

Lindsey A. Murphy et al. (2022). Digital polymerase chain reaction strategies for accurate and precise detection of vector copy number in chimeric antigen receptor T-cell products. Cytotherapy.https://doi.org/10.1016/j.jcyt.2022.09.004

Lindsey A. Murphy et al. (2021). Detection of Vector Copy Number in Bicistronic CD19xCD22 CAR T Cell Products with Digital PCR. Blood. 138 (1): 4001.https://doi.org/10.1182/blood-2021-153889

Cell and gene therapy

Cell and gene therapy with digital PCR – from research to bioprocessing & QC