The emerging fields of cell and gene therapy hint at a future with treatments and cures for diseases that, with any luck, will become diseases of the past. With such high stakes, the clinical safety and efficacy of those treatments are equally as important as the innovation required to invent them.
Having accurate and complete information to support the research that can help fuel the development of these therapeutic tools is crucial. Luckily, PacBio HiFi sequencing unites the advantages of short- and long-read technologies at Sanger-level accuracy for gene and cell therapy research.
Keep reading to see how HiFi sequencing is powering solutions in cell and gene therapy research.
For gene and cell therapy research, accuracy matters
When it comes to your cell and gene therapy research, why compromise with technologies that provide limited information? Failures and inaccuracies can be costly—not to mention dangerous. SMRT sequencing, the technology underlying HiFi reads, uses millions of zero-mode waveguides to generate HiFi reads with 99.9% accuracy. That’s accuracy you can trust for life-changing therapeutic research.
HiFi sequencing in gene therapy research
Adeno-associated virus (AAV) is one of the most actively investigated gene therapy vehicles today, with two AAV-based therapies receiving FDA approval in 2023 and undoubtedly more on the way. Given the clinical success of AAV-based gene therapies, the design of such AAV vectors is becoming increasingly important, and in turn, increasingly scrutinized. That’s where HiFi sequencing comes in.
The success of AAV-based gene therapies largely depends on the AAV vector design and its evaluation. Traditional short-read methods can partially cover the target construct sequence, but struggle with inverted terminal repeats (ITR), resulting in ambiguous data and incomplete coverage. In contrast, HiFi sequencing achieves single-molecule resolution covering the full length of the target construct, giving you the ability to uncover hidden issues that could affect gene transfer efficacy and safety. This accuracy translates into optimized AAV designs, higher chances of AAV success, and reduced risk of costly failures.
HiFi sequencing can be used to discover novel AAV vectors, improve vector design, and evaluate designs by identifying truncation, impurity, and host integration events.
Read more about using HiFi sequencing for AAV discovery and design through production.
HiFi sequencing in gene editing research
Scientists are only beginning to harness the potential of CRISPR-Cas9 gene editing technology to treat genetic disorders. Unknown off-target effects remain one of the most important safety concerns of using this new technology. Because off-target effects could be lurking anywhere in the genome, being able to detect and accurately measure these effects is vital.
Using highly accurate HiFi reads, you can verify gene editing and insertion strategies while pinpointing if and where off-target effects may occur. HiFi sequencing is one of the only known methods that can accurately detect edits of different lengths, ranging from single bases to several kb. It can also capture almost all editing outcomes at base-level accuracy, so you can confirm both on- and off-target effects that may be overlooked by other methods.
HiFi sequencing in cell therapy research
The promise of cell therapy research often relies on master cell banks and patient or donor cells. Use HiFi sequencing to confirm the genomic integrity of these cells in your research. Unlike other sequencing technologies, HiFi reads can detect virtually any type of variant, including SNVs, indels, SVs, and even 5mC methylation, while simultaneously phasing genomes in the same sequencing run. Accelerate your discovery and development with more accurate results and shorter run times compared to other technologies.
HiFi sequencing in mRNA vaccine research
Following the COVID-19 pandemic, mRNA vaccines have emerged as star players in the fight against infectious diseases. Research into mRNA vaccines continues to grow, with potential applications in cancer treatment as well.
Translating mRNA hype into real mRNA vaccine outcomes requires accurate information about the stability and efficacy of those vaccines. That means characterizing mRNA integrity and stability predictors, such as the length of its poly(A) tail.
PacBio HiFi sequencing allows you to sequence full-length mRNA to determine size and isoform distribution with high accuracy. Unlike other methods, HiFi sequencing can accurately sequence through long homopolymers, such as poly(A) tails, allowing you to understand and characterize these determinants of vaccine stability.
PacBio offers a toolbox of sequencing methods
HiFi reads benefit multiple sequencing approaches, offering a menu of options that can flex and grow as your research does. This sequencing toolbox includes whole genome sequencing (WGS), plasmid sequencing, several targeted sequencing methods, and RNA sequencing.
| Method | The HiFi advantage |
|---|---|
| Whole genome sequencing | Confidently sequence through high-GC and other difficult regions for even coverage across the genome, resulting in full genomes with extraordinary accuracy, including DNA methylation and phasing. |
| Plasmid sequencing | Capture and verify the full length of your plasmid—including backbone, promoters, resistance genes, and gene inserts—with Sanger-level accuracy. |
| Targeted sequencing | Targeted sequencing Remove the limits on your sequencing imposed by other technologies. Sequence genes, inserts, or large libraries at full length with HiFi reads. |
| Amplification-free target enrichment | Enrich for your target without the errors and biases introduced by PCR amplification, while sequencing through difficult regions such as high GC, long indels, and more. Accurately measure the length of tandem repeats and analyze on- and off-target editing outcomes. |
| Amplicon sequencing | Use sequencing of long amplicons (up to several kb) to easily sequence full-length genes or target regions of interest. |
| Hybrid capture | Evaluate vector integration sites and reveal potentially resulting concatemers, inversions, and regions of genomic instability. |
| RNA sequencing | Characterize isoform-specific expression, identify potential novel targets, and sequence full-length mRNA transcripts for an unambiguous view of the transcriptome. |
See HiFi sequencing in action
Curious about how to apply PacBio sequencing in the real world of cell and gene therapy research? Hear three stories from scientists like you who are using HiFi sequencing in AAV research applications.
In the first example, researchers at Homology Medicines and AavantiBio discuss how they use PacBio sequencing to optimize and confirm AAV vector design in their gene therapy research programs:
“If you want to look at the sequence of a whole AAV or the sequence of an integrated AAV in the genome, you need to have long reads. You need to be able to span that long distance. With ITRs, you simply can’t see them with other technologies. With PacBio, you can.”
—Dr. John Thompson, former VP of Genomics and Computational Biology at Homology Medicine
In the webinar below, Irvin Garza discusses the practical applications of HiFi sequencing in creating novel AAV capsids. In a recently published paper, Garza and his team used domain shuffling to create highly diverse capsid libraries. They then used PacBio SMRT sequencing for high-throughput screening of the AAV capsid variants. “The high sensitivity of PacBio long-read sequencing is what enabled us to read the full 2.2 kb Cap gene within our studies,” Garza says.
“PacBio is super helpful in every single question that I’ve had, from the bioinformatics to the actual library construction or even knowing how many SMRT Cells I need to run this many samples.”
—Irvin Garza, graduate student in the Gray Lab and Bailey Lab at UT Southwestern
In the third example, Dr. Edward J. Oakeley discusses how his team at Novartis has been using PacBio technology to sequence AAV. He explains why it’s necessary to use AAV mode in PacBio SMRT Link software to get high-quality data out of their experiments.
“PacBio data is awesome for AAV sequencing.”
—Dr. Edward Oakeley, former Associate Director of Genomics at Novartis