Validation of qPCR Assays for Gene Therapy

Though the modern world has progressed a lot in terms of medical interventions and biomedicine, there still are a vast number of diseases and disorders that demand a rather unconventional approach to treatment. Gene therapy has evolved as a technique that aims to address some of these diseases by introducing into a patient a normal copy of one or more defective genes responsible for the patient’s disease. By repairing the damaged gene responsible for the disease, gene therapy aims to introduce a normal copy of the gene into cells containing the damaged version, eventually enabling the cells to produce the normal protein.

Gene therapy uses a vector formulation that contains a genetically engineered construct that is introduced to the host via injection. A high level of specificity is ensured for the constructs such that only the target cells allow the gene incorporation. Adeno-associated virus (AAV) vectors are the most common delivery systems that have been used so far, however other systems like replication-competent viral vectors and microbial vectors have also been used. When properly designed and executed with the right approach, these vectors have the ability to treat a wide range of diseases such as cancer, cystic fibrosis, diabetes, hemophilia, and even AIDS. Gene therapy products mediate their effects by transcription or translation of the genetic material (or sometimes by specifically altering human genetic sequences). The ones that are in current clinical research generally exert their effects on somatic cells, in a site/type-specific manner and hence cannot be passed onto offspring.

Like any other novel biological tool intended for clinical and therapeutic usage, gene therapy products need to be extensively scrutinized for the safety and efficacy of the therapeutic. Agencies like FDA and EMA have stressed the necessity of understanding this class of therapeutics by utilizing available technologies that would provide the best characterization of their safety and efficacy profiles. 

Quantitative polymerase chain reaction (qPCR) assay is one of the most commonly used assays that has been widely accepted and utilized for characterizing, for example, the biodistribution and shedding of components that make up gene therapies like viral vectors and transgenes. Since gene therapy has vectors and vector-derived gene expression as its core components, qPCR offers superior sensitivity and specificity toward the detection of administered nucleic acid sequences at a wide dynamic range. Though there may be differences among scientists on what criteria to be set for using validated qPCR assays, there are recommendations on best practices on how to develop and what performance characteristics to validate for these assays.

When it comes to using qPCR assays, designing appropriate primers and probes sets is critical. These primer and probe sets are then evaluated to choose the set with optimal performance and are then used for continued development and optimization of the assay. When choosing between probe-based qPCR versus dye-based qPCR such as SYBR Green, specificity and sensitivity is the factor to be considered. Probe-based assays are more reliable as they have a lesser chance of false-positive signaling than the dye-based qPCR. This shortcoming of dye-based qPCR can be mitigated to some extent by careful primer design. 

For a large number of samples, probe-based qPCR can be used for multiplexing. For this, probes with different fluorophores are combined within the same reaction setup to detect distinct target sequences. The dual benefit of this approach is to reduce the amount of sample needed as well as reagent cost per run. It is best practice to develop and optimize each individual assay to its peak performance before combining it in a multiplexed assay. Early in method development, it is important to define the design specifications of the assay that will qualify it for its intended use. Prior to moving into validation, it is important to have a good understanding of the assay’s performance including a preliminary assessment of the assay specificity, range, sensitivity, reproducibility, and robustness.

 Validation of qPCR Assays

Since qPCR is an extremely sensitive assay, it can detect even a single copy of target DNA, but the workstations used for different assays must be separated and kept contamination-free. Often nucleic acid extractions and templating are performed in completely separate rooms or in boxed enclosures to reduce the risk of introducing contamination at any step in the process.  

There are different extraction and purification techniques available for nucleic acids: DNA, RNA, or both. While validating qPCR assays, the nucleic acid extraction and purification technique should already be determined in method development. After the nucleic acid is extracted it needs to be quantified and characterized in order to understand the concentration, purity, and integrity prior to use in the qPCR assay. 

To demonstrate the specificity of a qPCR assay, early on in method development, the primer and probes can be evaluated for specificity in silico by utilizing BLAST programs. In addition, gel electrophoresis can be used to confirm that the amplicons generated are of expected sizes. Another method to demonstrate specificity of the target is to ensure no amplification in the assay when a non-specific target is spiked into the reaction. . Throughout the intended dynamic range, the linearity of the assay should be demonstrated and the sample concentration is represented as a function of the obtained response. This is assessed by evaluating a calibration curve that is ideally made up of eight non-zero calibrators that span 6-8 orders of magnitude or a 3-4 log range. The curve is plotted with a semi log-linear curve fit. Linearity is evaluated using the R2 value and efficiency is calculated using the slope of the curve. 

At least three levels of positive controls with varying concentrations can be used to evaluate assay reproducibility within and between runs. In addition, negative controls such as NTC (no template control) samples are used to ensure no observed amplification and specificity of the assay. The positive controls should demonstrate precision and accuracy of the assay that supports its intended use.

Limit of detection (LOD) and limit of quantification (LOQ) of a qPCR assay is generally established empirically by analyzing multiple replicate dilutions of the template. LOD represents the concentration that can be detected in 95% of the replicates whereas LOQ is the minimum number of target sequences that can be detected and quantified with accuracy and reproducibility. Some important factors that influence these limits are assay performance, repeatability, pipetting errors, reagent quality and stability, and reaction homogeneity. Other performance characteristics such as matrix interference, recovery, and stability are also included in qPCR validations. 

There is a lack of regulatory guidance on how to validate qPCR assays, however current recommendations include fit-for-purpose validations to determine how to best characterize and validate the assays for their intended use. 

qPCR is a valuable tool that can be used to further validate and test the efficacy of any therapeutic, including viral vectors for gene therapy. Its ability to gather large amounts of data from a small sample size allows for a high level of insight which is vital for the validation of therapeutic potential. The sensitivity of qPCR is both a good aspect of the assay because of the specificity of the data acquired but also could make for some difficulties when validating and establishing the qPCR primers and thermocycler settings. Because of this, partnering with organizations with qPCR expertise, such as BioAgilytix, would not only increase the speed at which the qPCR data is acquired but also the accuracy. Increased speed and accuracy of the data is necessary for the further development and efficacy of any therapeutic.  

Partner With BioAgilytix

BioAgilytix leverages real-time PCR (also known as quantitative PCR or qPCR) to deliver genomics services supporting the preclinical and clinical phases of drug development. Our molecular suites are equipped with the latest generation of qPCR equipment, including Applied Biosystems ViiA 7, QuantStudio 7 and BioRad ddPCR systems. Contact BioAgilytix today with any questions or requests, or for information on our expertise in gene therapy support, tumor/immune biomarkers or CAR T-cell therapy programs. 


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