Leading researchers in AAV-based gene therapy development recently presented how they are leveraging PacBio long-read sequencing technology to advance product design and performance.
Building treatments for individuals with genetic diseases represents one of the most challenging and exciting frontiers in biomedical research. At the forefront of the effort to develop solutions for such conditions is a promising technique called gene therapy.
How gene therapy works
Gene therapy involves treating an illness by carefully inserting a piece of corrective genetic material directly into the cells of affected tissue. This genetic material can compensate for a deficiency, correct dysfunctions, and potentially confer new capacities to fight infections or even cancers. Moreover, gene therapy approaches can accomplish this without the use of pharmaceuticals or invasive surgeries. To ensure the success of this promising methodology, confirming the safe delivery of the relevant gene therapeutic is paramount. Bioengineered viruses are the delivery vehicle of choice for this approach, and one variety, known as adeno-associated virus (AAV), holds tremendous promise.
Figure 1. The three phases of AAV-based gene therapy development that are empowered by HiFi sequencing.
We sat down with PacBio scientist Elizabeth Tseng, Ph.D. and doctors Don Selby and Terrance Hanscom of Homology Medicines Inc. to discuss how the unparalleled accuracy and flexibility of HiFi sequencing technology is revolutionizing gene therapy with AVV at every stage of development: from discovery to design and evaluation.
- The incredible length and accuracy of HiFi reads can be leveraged to discover elusive new AAV species and strains that are better suited to the requirements of your therapy design.
- Prep-free DNA base modification detection and the accuracy of HiFi sequencing can be used to ensure that your vectors contain the intended therapeutic. With the power of HiFi reads, you can catch previously invisible errors and pinpoint the areas of your gene therapeutic that may need improvement.
- Full-length RNA isoform sequences obtained with the Iso-Seq method allows you to evaluate whether the potentially therapeutic genes being introduced into a host will be delivering the intended remedy, and if not, can provide the detailed information needed to understand what needs to be revised.
Uncover elusive vectors with state-of-the-art HiFi sequencing tools
Leverage the incredible length and accuracy of HiFi reads to discover elusive new AAV species and strains that are better suited to the requirements of your therapy design.
In the first part of our webinar, Dr. Elizabeth Tseng introduced a compelling overview of how HiFi sequencing can be used to potentially improve AAV gene therapy at each stage of development. She outlined PacBio’s circular consensus sequencing (a.k.a. HiFi sequencing) workflow and how it is able to read the entire ~4.7kb AAV genome many times over (including ITR (Inverted Terminal Repeat) regions and those with high GC content that are missed by competing short-read technologies), to create genome assemblies of unparalleled detail and accuracy.
This incredible level of precision makes it possible for R&D teams to discover new species and even strains of AAV that may be better suited to the requirements of a specific therapy objective than variants such as HEK293 and Sf9 that are currently commonplace. This detailed and accurate insight from HiFi sequencing makes it possible for gene therapy developers to potentially create AAV delivery vectors that are customized for their therapy deliverable.
Experience a revolution in vector development driven by insights from HiFi data
Help ensure that your vectors contain the correct therapeutic gene sequences, catch previously invisible errors, and pinpoint structural areas of your gene therapeutic that may need improvement with prep-free base modification detection and the stunning accuracy of HiFi sequencing.
Building on Dr. Tseng’s talk, Don Selby Ph.D., a molecular physiologist, and principal scientist working on vector design with Homology Medicines, Inc., outlined how HiFi sequencing has been instrumental in drafting and troubleshooting the AAV genetic payloads that are in development at the company. He summarized how the length and superb accuracy of HiFi sequencing is not only helpful in understanding whether the sequence of the gene therapeutic is correct during development, but that HiFi’s methylation detection capability allows his team to troubleshoot gene designs that break after insertion into the AAV delivery vector.
“What you have at the end of the day is a much more efficient design process, so as far as us coming up with [new] vectors and improving efficiency, [HiFi] is a game-changer for us.”
-Don Selby Ph.D., Principal Scientist, Homology Medicines, Inc.
The team first takes the single-stranded fragments of the broken gene and binds them to a full-length, unbroken complementary strand. This concatenated piece of DNA is then repaired with methylated DNA bases. This repaired molecule is then sent for HiFi sequencing with base-modification detection. The HiFi reads that come out of the sequencing process show these methylated DNA bases and exactly where they are in the DNA molecule. This allows the researchers to determine where their therapeutic gene broke and gives them the information necessary to revise their gene design to prevent breakage in further iterations.
Monitor payload performance like never before with the Iso-Seq method
Evaluate whether the potentially therapeutic genes being proposed for introduction into a host will be delivering the intended remedy, and if not, get the detailed information needed to understand what to revise with full-length RNA isoforms.
Even with the design and manufacturing steps of the gene therapeutic and AAV vector completed, the final and most crucial step in development is to verify that the payload is having the intended effects. No other technology can provide the length and accuracy to evaluate payload expression than HiFi sequencing with the Iso-Seq method. That is why Terrance Hanscom of Homology Medicines, Inc. uses HiFi sequencing to research and evaluate complete mRNA isoforms transcribed from experimental therapeutic genes after they have entered a target cell.
“From your [therapeutic] gene you’re going to have several different mRNA isoforms …if you’re looking with short-read technology you [won’t] know which RNA isoform the read came from and this is really important to us, this [problem] is central to safety and efficacy for our patients, PacBio’s Iso-Seq method solution shows us exactly what we are delivering.”
-Terrance Hanscom Ph.D., Homology Medicines, Inc.
This makes HiFi sequencing with the Iso-Seq method an indispensable tool for performance evaluation in vivo. Complete isoform expression data obtained using the Iso-Seq method is a vital part of the AAV-based gene therapy R&D pipeline, making it possible for research and development teams to examine each isoform, improve codon optimization models, and more.
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Optimize every stage of AAV gene therapy development with the power of HiFi reads
HiFi sequencing solutions allow gene therapy researchers to uncover and observe the minutiae of AAV vector design and development like never before. HiFi data makes it possible to discover elusive new AAV species and strains that are better suited to the requirements of your therapy design. Catch previously invisible errors, and pinpoint structural areas that need improvement with prep-free base modification detection and the near-perfect accuracy of HiFi sequencing.