Shaping the Future of Medicine with Gene Therapies
Digital PCR helps improve sensitivity and efficiency in viral vector production workflows.
Since the first successful human gene therapy trial in the late 1980s for adenosine deaminase (ADA) deficiency,1 the field has made tremendous progress culminating in several FDA approved and thousands of ongoing gene and cell therapy clinical trials. Thousands of patients hope to defeat their incurable genetic diseases such as cystic fibrosis, Duchenne muscular dystrophy, and inherited metabolic disorders with gene and cell therapies.2
Gene and Cell Therapy Development
Developing Viral Vectors for Therapeutic Use
Gene and cell therapies are life-altering treatments for patients suffering from incurable diseases. In gene therapy, patients receive a viral vector carrying therapeutic genetic material to restore a defective gene’s function by either introducing a healthy gene copy or normalizing the levels of the dysfunctional gene product. In cell therapy, researchers first genetically modify relevant cell types in vitro to express therapeutic proteins before administering them into a patient.2
Both therapies require a gene delivery vehicle to transport therapeutic gene cargo to patient cells. Viruses are naturally adept at infecting human cells and injecting nucleic acids. Gene and cell therapy researchers have exploited modified viruses, such as lentiviruses (LVs) and adeno associated viruses (AAVs), as gene delivery vehicles. These viruses typically encase their genomes into viral capsids. By transfecting several plasmids encoding for viral capsid parts and the transgene of interest into expression cell lines, researchers develop viral vectors for therapeutic use.3,4
Quality Control During Therapy Development
The purity and efficacy of the gene delivery vectors carrying therapeutic transgenes determine a gene or cell therapy’s success. Therefore, researchers employ stringent quality control assessments during a therapy’s development and manufacturing processes. Proper viral vector assembly requires full transgene packaging in the vector capsid.
First, researchers must screen and separate empty or partially assembled vectors from fully assembled vector particles carrying the transgene of interest. Researchers then need to purify any biological impurities from these fully assembled particles.
Lastly, measuring the viral concentration as vector genomes per milliliter allows researchers to establish the correct therapeutic dose. Once they obtain highly pure viral vectors, they monitor the expression, efficacy, and potency of cell and gene therapeutics at both preclinical and clinical stages.3,4
qPCR is a Standard Tool for Characterizing Vectors
Absolute Quantification is Essential in Specific Cell and Gene Therapy Workflows
Quantitative PCR (qPCR) is a standard tool for researchers characterizing vectors, including quantifying viral genome concentration, checking for genomic impurities, assessing transgene fidelity, and in vivo monitoring of transgene transcripts in preclinical and clinical testing.5
However, for cell and gene therapy workflow steps that require absolute quantification, it is important to consider qPCR’s requirement for a standard curve. Researchers must have good quality, standardized controls or reference material to develop a standard curve, which only allows them to determine nucleic acid target or transcript levels. They further need multiple biological replicates to validate their observations.
Digital PCR: A robust tool for absolute quantification
Researchers’ Preferred Methodology in Vector Quality Control Assays
Highly Precise and Sensitive
The advent of digital PCR (dPCR) overcomes many of these challenges and is quickly becoming gene and cell therapy researchers’ preferred methodology in vector quality control assays. In dPCR, a sample gets distributed into thousands of nanoliter-scale reactions. Depending on the number of nucleic acid molecules in each reaction, the assay allows researchers to directly obtain absolute levels of nucleic acid present in the sample. This approach makes dPCR highly precise and sensitive compared to standard quantitative methods.
More Robust and Efficient Process
With dPCR, researchers do not have to develop a standard curve, which makes the process more robust and efficient. Using dPCR, researchers quantify absolute levels of viral vector genomes and measure vector copy numbers integrated into host cell genomes.
Better Consistency, Accuracy, and Precision in Gene & Cell Therapy Workflows
Moreover, dPCR allows researchers to determine vector yield at manufacturing stages and identify transgene transcripts during clinical trials. dPCR’s ability to test vector integrity, point mutations, and residual plasmid or genomic impurities helps improve consistency, accuracy, and precision in gene and cell therapy workflows. At preclinical and clinical stages, dPCR facilitates gene expression analysis and viral particle tissue distribution tracking over time to evaluate a therapy’s efficacy.
Faster and Safer Therapy Manufacturing
With thousands of new gene and cell therapy products in the pipeline, developing effective and safe therapeutics demands robust, innovative solutions at each workflow stage. Viral quantification analyses with dPCR enable faster and safer therapy manufacturing.
- R.Naam, “More than human,” https://www.nytimes.com/2005/07/03/books/chapters/more-than-human.html, accessed on June 8, 2022.
- K. Bulaklak, C.A. Gersbach, “The once and future gene therapy,” Nat Commun, 11:5820,
- “Gene therapy from set up to scale up,” https://assets.thermofisher.com/TFSAssets/BID/brochures/gene-therapy-brochure.pdf, accessed on June 8, 2022.
- M.F. Naso et al., “Adeno-associated virus (AAV) as a vector for gene therapy,” BioDrugs, 31:317–34, 2017.
- “qPCR: The weapon of choice for gene and cell therapy biodistribution,” https://www.lovelacebiomedical.org/qpcr-the-weapon-of-choice-for-gene-and-celltherapy-biodistribution/, accessed on June 8, 2022.