CRISPR Gene Therapies: Assessing the Success of Gene Edits Using ddPCR

β-thalassemia is one of the most common autosomal recessive diseases in the world and researchers are seeking to produce a gene therapy to treat the condition. Scientists are applying a variety of strategies and techniques to correct the underlying genetic imbalance that causes β-thalassemia, including using CRISPR to correct the mutated gene. The approach is promising, but scientists are still working to improve CRISPR’s editing efficiency, which is still an open question. Consequently, CRISPR’s ability to successfully correct mutations 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 promising yet complex gene editing approach

Roughly 1.5% of the global population carries mutations associated with β-thalassemia, with more than 60,000 new cases diagnosed every year. Unfortunately, scientists do not have a straightforward path towards addressing this condition at the genetic level. Adult hemoglobin is composed of two pairs of globin subunits, α-globin and β-globin, which must be expressed in equal numbers for hemoglobin to develop normally. People with β-thalassemia harbor genetic mutations in the gene for β-globin, HBB, that lead to downregulation of the gene. Free α-globin, then forms toxic precipitates that impair red blood cell development and kill mature red blood cells. As a result, patients experience a wide range of severe symptoms, and the condition often leads to early death.

Some research suggests that deleting the α-globin gene, HBA, may improve outcomes. 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 HBA and replace it with HBB in hopes of restoring the balance between the two hemoglobin subunits.

Performing such an edit is a complex task. First, after designing a guide RNA (gRNA) that locates the gene that needs to be edited, scientists need to introduce it to one’s cells using a viral vector. Then, the gRNA must identify the correct cutting sites flanking the HBA gene, while Cas9 must perform the cuts. The same gRNA must facilitate the insertion of the HBB gene at the same locus. This dual edit approach will not work if CRISPR does not successfully remove HBA and introduce the HBB gene in the same spot. Such a multifaceted edit requires rigorous quality control to ensure CRISPR performs the correct edits in the correct locations. This is where ddPCR technology comes in.

Advantages of ddPCR assays

ddPCR 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, ddPCR assays can detect CRISPR edits via both homology-directed repair (HDR) and nonhomologous end joining (NHEJ). It can also detect excisions and inversions independent of sequence length.

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 contains a strand featuring the target genetic sequence, that DNA will amplify, and the droplet will release a strong fluorescent signal. If a droplet does not contain the target sequence, the droplet will only emit weak fluorescence. By counting the strongly vs weakly fluorescent droplets, one can detect specific sequences with great sensitivity and measure the concentration of the target sequence in the original sample with great accuracy.

Compared to next-generation sequencing, ddPCR is fast, inexpensive, and not labor-intensive, and it can detect rare events without being limited by read depth. ddPCR is also more sensitive and accurate than quantitative PCR (qPCR). While researchers must use a standard curve to interpret qPCR results, exposing the data to amplification bias, ddPCR quantifies genetic variants directly, without a standard curve, and thereby provides an absolute count.

The abovementioned European group that developed the dual editing approach for treating β-thalassemia used ddPCR assays to assess the success of their edits. The researchers first used ddPCR technology to quantify HBA copy number, which correlates 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; the team confirmed 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, these researchers would not have been able to show that their CRISPR strategy has potential for future clinical testing.

Future CRISPR applications

Approximately 30 clinical trials are in planning or underway to study 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 extra care to ensure their therapies are safe and effective. ddPCR technology can 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 cell 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.