Long-read sequencing myths: debunked.
Part 6 — cell & gene therapy

 

Cell and gene therapy research is crucial for biopharma development, with HiFi long-read sequencing significantly enhancing many sequencing applications throughout the process. Even though AAV sequencing is one of the newest applications of genomics, it is also one of the most promising in disease research today. Adeno-associated virus (AAV) ranks among the most actively experimented upon vehicles for gene therapy.¹ Gene therapies using AAV and cell therapies like CAR-T hold the potential to cure previously incurable diseases. CAR-T cell cancer treatments, in particular, are showing great promise in combating this devastating illness. In 2023, the FDA approved several new AAV-based therapies and cell-based gene therapies for treating Duchenne muscular dystrophy, severe hemophilia A, and sickle cell disease.^2,3,4

The design of AAV vectors has consequences on gene therapy research, which could be the last stand in the fight against these diseases, and maybe many more. Highly accurate long-read sequencing supports the investigation into design, validation, and optimization of potential gene therapies using such viral vectors.

This is the final episode in our six-part myth-busting series. Today, we’re debunking common misconceptions about PacBio HiFi sequencing in cell and gene therapy research.

---

Myth #1:

PacBio HiFi sequencing is too expensive to use when developing and assessing or optimizing gene therapy product design, efficacy and potential safety.

Fact:

This statement is misleading.

---

Vector design plays a crucial role in gene therapy development success. In cell and gene therapy, where safety is paramount and R&D takes notoriously long, unexpected errors can quickly derail years of work, potentially delaying lifesaving therapies for those who need it. Fully—and accurately—characterizing your AAV product can mean reducing the risk of extremely costly failures during clinical trials.

Understanding the full extent of on- and off-target editing, vector or construct integration, and insertional mutagenesis are key components of validating AAV product design and ensuring its manufacturability. Using the exceptional accuracy and lengths of HiFi reads means that you can be confident in your product designs and avoid surprises down the line that require you to go back to the drawing board.

For more, read our best practices for gene therapy product characterization using HiFi sequencing with Dr. Claire Aldridge at Form Bio.

---

Myth #2:

PacBio long reads are only good for de novo genome assemblies.

Fact:

This statement is incomplete.

---

HiFi reads are good for assembling genomes, it’s true, but they can do so much more.

Count cell and gene therapy research are among the many applications you can do with HiFi sequencing. Whether it’s full-length AAV sequencing, gene editing assessments, plasmid or amplicon library screening, or vector integration, the winning combination of >20 kb reads and 99.9% accuracy with HiFi allows you to detect variants or events that short-read sequencing would miss.

You can use HiFi sequencing for every research stage of AAV gene therapy development:

• Discover AAV vectors: discover novel capsids with targeted sequencing
• Optimize AAV vector design: improve designs by observing the frequency of truncations, fragmentation, and other non-full-length anomalies
• Confirm mRNA transcripts: quantify isoforms with full-length isoform sequencing
• Study host integration: understand the frequency of these events, to ensure the potential safety and efficacy of your product
• Ensure quality in AAV production: compare vectors and unresolved genomes and assess vector preps produced by different platforms

Read more about what you can do with highly accurate HiFi reads for AAV sequencing.

And what about gene editing? HiFi sequencing can power your gene editing research by enabling you to:

• Sequence beyond your target to fully understand the extent of CRISPR-Cas9 editing
• Assess indels and other mutations that result from gene insertion at a CRISPR target locus
• Detect rare off-target mutations
• Understand the effects of haplotype and SNVs on gene editing in cases of allele-specific Cas9 cleavage
• Avoid PCR biases and limitations by using amplification-free approaches

Read more about what you can do with highly accurate HiFi reads for gene editing research.

---

Myth #3:

Accuracy isn’t that important for characterizing AAV impurities, such as partial genomes.

Fact:

This statement is false.

---

Accuracy is imperative in all areas of science, and the stakes are arguably even higher in cell and gene therapy research, where clinical trials, patient outcomes, and life-changing therapeutics are on the line.

With highly accurate HiFi long reads, you can assess your AAV for impurities, like partial genomes, while at the same time getting critical information about sequence identity. PacBio HiFi sequencing unites the advantages of long reads with Sanger-level accuracy. This means you can monitor and improve AAV discovery and manufacturing with a single technology, reducing the risk of costly failures, which can set development timelines back by months or even years.

Missing crucial information about the quality of your vector can impact the effectiveness of your design. Hear how biopharmaceutical researchers at Homology Medicines are using highly accurate HiFi reads to discover novel vectors and improve their vector designs in this on-demand webinar.
 

---

Myth #4:

Pairing nanopore with short-read sequencing is the easiest way to characterize AAVs from inverted tandem repeat (ITR) to ITR.

Fact:

This statement is short-sighted.

---

Neither ONT nor short-reads can fully resolve ITRs and additional bioinformatics alignment work is necessary when using short reads. Why run multiple assays when you can do it better in one? Highly accurate long-read sequencing combines the accuracy and read length that you need in one experiment. Never again throw away reads because accuracy is too low or because they are too short to align. In addition, long-read sequencing with HiFi reads support a broad menu of applications that are critical for cell and gene therapy product design and development, including:

• AAV sequencing
• Amplicon-based construct screening
• Gene editing and on- and off-target assessment
• Full-length plasmid sequencing
• Vector integration
• RNA sequencing
• Whole-genome sequencing

---

Myth #5:

AAV is complex and difficult to sequence using PacBio HiFi sequencing.

Fact:

This statement is incorrect.

---

PacBio and our partners at Form Bio offer an end-to-end workflow for AAV sequencing and data analysis, in an all-in-one solution to optimize your AAV vector designs. This workflow accommodates both ssAAV and scAAV sequencing using the same protocol. Form Bio workflows are certified PacBio compatible and provide analysis software to help you analyze and visualize your AAV data, so you can save time and resources.

With this protocol, you can use HiFi reads to:

• Sequence tissues for novel AAV vector discovery
• Improve vector design
• Identify impurities, truncation events, and host integration events

 

 
See how HiFi sequencing makes it easy to sequence AAV genome populations to identify truncation, mutation, and host integration events.

---

Forget the old rulebook and start making tomorrow’s genomic discoveries, today

 
As we’ve shown here — and throughout our myth-busting series — PacBio HiFi sequencing can benefit almost any genomics application by virtue of its long read lengths and exceptional accuracy. Leave those dated misconceptions in the past and start using the power of HiFi sequencing to fuel tomorrow’s groundbreaking discoveries.

Did you miss the other installments in our myth-busting series? Don’t worry, you can catch up here:

• Part 1 – HiFi sequencing
• Part 2 – human genomics
• Part 3 – cancer genomics
• Part 4 – plant and animal genomics
• Part 5 – microbiology

Are there any other myths about long-read sequencing that you want busted? Let us know! Speak with a PacBio scientist to find out what you can do with HiFi sequencing.
 

See how HiFi sequencing is a fit for your gene therapy research

Learn more
 

References

---