Assessing the Efficacy of CRISPR Gene Therapies Using ddPCR Technology

Assessing the Success of CRISPR Gene Therapies Using ddPCR Technology Marwan Alsarraj, Global Segment Manager, Life Science Group Currently, nearly 40 clinical trials plan to study whether CRISPR can be used to treat genetic diseases. For instance, Vertex is running a Phase I/II trial for patients with the blood disorder β- thalassemia. The particular CRISPR edit needed to treat β-thalassemia is complicated: it requires the simultaneous deletion of one gene (HBA2) and the correction of another (HBB). Without both edits, the treatment won't work. And if the edits occur in the wrong place, as has been observed to varying degrees in other studies, the therapy can damage healthy genes. In the preclinical work that led to the clinical trial, researchers assessed the success of the CRISPR edits using Bio-Rad's Droplet Digital PCR technology. In this article, Marwan Alsarraj will write a story about the future of CRISPR-based gene therapies and the technical considerations companies must make as they assess their potential safety and efficacy during manufacturing. Assessing the Success of CRISPR Gene Therapies Using ddPCR Technology Although many have never heard of it, β-thalassemia is one of the most common autosomal recessive diseases globally: Scientists have estimated that about 1.5% of the global population carries mutations associated with β-thalassemia, with more than 60,000 new cases diagnosed every year. This serious blood disorder reduces the production of hemoglobin, the ironcontaining protein in red blood cells that plays an essential role in delivering oxygen throughout the body. Therefore, scientists and researchers seek to produce a gene therapy to treat the condition and correct the underlying genetic imbalance that causes β-thalassemia. One of the most promising approaches involves using CRISPR to correct the mutated gene. However, scientists are still working to improve and validate CRISPR's editing efficiency. Furthermore, CRISPR's ability to successfully correct mutation associated with β-thalassemia is still uncertain. Therefore, researchers need to pair CRISPR editing protocols with a quality control tool such as Droplet Digital PCR (ddPCR) that accurately detects the presence of successful CRISPR edits. A complex gene editing solution with promising results When correcting the mutations with β-thalassemia, scientists do not have a straightforward path towards addressing this condition at the genetic level. Adult hemoglobin is composed of two globin subunits, α-globin and β-globin, which must be expressed in equal numbers for hemoglobin to develop correctly. People with β-thalassemia have a genetic mutation in the gene for β-globin, HBB, leading to the gene's downregulation. With unequal quantities of α-globin and β-globin circulating, the free α-globin forms toxic precipitates that impair the development of red blood cells and kill mature red blood cells. As a result, patients can experience a wide range of severe symptoms, including an increased risk of developing blood clots, weakness and fatigue, which often leads to early death. Some research suggests that deleting the α-globin gene, HBA, may improve outcomes. Alternatively, introducing a healthy HBB gene via a lentiviral vector improves patients' clinical outcomes, but only if these patients already express some β-globin. A research group based in France and Italy recently combined these two approaches: they used CRISPR to delete 1 copy of HBA2 and replaced it with HBB in hopes of restoring the balance between two hemoglobin subunits. Performing this kind of dual edit is a complex task. Researchers must first design a guide RNA (gRNA) to locate the gene to be edited. Once the gRNA identifies the correct site containing the HBA gene, Cas9 must perform the cut and facilitate the insertion of the HBB gene in the exact location the HBA gene previously occupied. A dual edit approach only works if CRISPR correctly removes HBA and introduces the HBB in the same spot. Assessing the success of this technique requires rigorous quality control to ensure CRISPR performs the correct edits. This is where ddPCR technology comes into the equation. Advantages of ddPCR assays While researchers commonly use qPCR to assess gene-editing success, this technique can have drawbacks, especially quantification. qPCR can only estimate the transgene copy numbers by relying on a standard curve of serial dilutions to interpret samples. Therefore, the results are less sensitive and can not measure down to one gene per cell. In contrast, Droplet Digital PCR (ddPCR) is well-suited to the task, as it delivers absolute nucleic acid quantification without the aid of a standard curve. DdPCR technology is a highly sensitive tool designed to detect and quantify rare genetic variants, and it can be used to detect outcomes of CRISPR editing. For example, researchers have used ddPCR assays to detect CRISPR edits via homology-directed repair (HDR) and nonhomologous end-joining (NHEJ). ddPCR technology works by partitioning a sample into approximately 20,000 nL-sized droplets and running a separate PCR reaction in each one. Each droplet contains one or a few nucleic acid strands. If a droplet includes a strand with the target genetic sequence, that DNA will amplify, and the droplet will emit a strong fluorescent signal. On the other hand, if a droplet does not contain the target sequence, the droplet will only emit a weak fluorescence signal. By counting the strongly vs. weakly fluorescent droplets, scientists can detect specific sequences with great sensitivity and measure the concentration of the target sequence in the original sample. Compared to next-generation sequencing, ddPCR technology is fast, inexpensive and not laborintensive, and it can detect rare events without being limited by read depth. ddPCR technology is also more sensitive and accurate than quantitative PCR (qPCR). The previously mentioned European group used ddPCR assays to assess the success of their dual editing approach for treating β-thalassemia. The researchers first used ddPCR technology to quantify HBA copy number, which correlated with β-thalassemia severity. They also used it to detect the successful insertion of HBB. In human umbilical cord blood-derived erythroid progenitor (HUDEP-2) cells, the team showed robust insertion of the HBB gene, confirming on-target integration of the gene at 0.8 copies per cell. The team could not have detected this integration using qPCR. Because of the inherent variability in how qPCR results are measured, the technique cannot detect gene copies at concentrations lower than two or three copies per cell. Without ddPCR technology, these researchers would not have shown that their CRISPR strategy has potential for future clinical testing. Future CRISPR Applications Nearly 40 clinical trials are currently investigating whether CRISPR can be used to treat genetic diseases, and regulatory agencies might approve the first CRISPR-based gene therapy in less than a decade. But given the continued challenge of developing a reliable CRISPR editing protocol, biopharmaceutical companies developing CRISPR therapies must take care to ensure their therapies are safe and effective by using tools like ddPCR technology to provide the confidence they need. For example, ddPCR assays can be designed to detect any CRISPR edits by using probes that span the junction between the native genome and the donor sequence. Researchers and biomanufacturers can screen out cell lines containing unsuccessful edits before they even reach patients by analyzing cells lines for specific CRISPR edits. This, in turn, will increase the chance of clinical success for CRISPR-based gene therapies and open the door to a new generation of treatments for difficult-to-treat genetic diseases like β- thalassemia. About the Author Marwan Alsarraj is Global Segment Manager, Life Science Group at Bio-Rad, with more than 20 years of experience developing and commercializing technologies for life science research. He holds an M.S. in Biology from the University of Texas at El Paso. Learn More About ddPCR for CRISPR Quality Control Explore how Droplet Digital PCR enables sensitive, absolute quantification of CRISPR edits across research and manufacturing workflows. Explore ddPCR Solutions