HiFi sequencing advantages in gene therapy
How can HiFi sequencing help with your gene therapy research?
- Rapidly accelerate the engineering and discovery of novel capsids with desired characteristics such as tissue specificity through full-length AAV genome or cap gene sequencing.
- Monitor and improve AAV manufacturing by characterizing AAV vectors and their impurities with high accuracy.
- Assess packaged impurity sequence profiles like identity, size distribution, and relative abundance.
- Profile payload sequence length and integrity.
- Characterize ITR rearrangements and integrity.
- Confidently evaluate and optimize your AAV design.
- Identify payload sequence truncation hotspots.
- Verify whether a target construct or gene of interest is correctly expressed and spliced.
- Explore potential risks by assessing host genome integration events.
Advantages of highly accurate HiFi reads for verifying AAV constructs
PacBio’s AAV end-to-end workflow and flexible protocol allow scientists to comprehensively profile packaged genomes as individual intact molecules to identify potential contaminants that can affect the safety, efficacy, and cost-effectiveness of AAV vector gene therapy.
- Discover novel potential capsid constructs
- Conduct vector quality control experiments
- Assess the safety of resulting constructs
Sample datasets: Gene therapy vectors
APPLICATION Brief
Accelerate discovery and comprehensively assess AAV vector quality with HiFi reads
With Single Molecule, Real-Time (SMRT) sequencing on the Sequel IIe and Revio systems, you can sequence AAV genome populations to identify truncation, mutation, and host integration events. The AAV workflow from PacBio® accommodates both scAAV and ssAAV constructs with easy on-instrument HiFi read generation and available bioinformatic tools.
Common questions about gene therapy
1Berns, K. I., & Muzyczka, N. (2017). AAV: An Overview of Unanswered Questions. Human gene therapy, 28(4), 308–313.
2Meier AF, Fraefel C, Seyffert M. The Interplay between Adeno-Associated Virus and Its Helper Viruses. Viruses. 2020; 12(6):662.
High accuracy and complete visibility are critical to the success of novel vector discovery, vector design, and manufacturing quality control for gene therapy products. HiFi sequencing is the ideal solution due to its ability to sequence full-length AAV genomes at high accuracy.
With HiFi, you can
- Sequence the entire AAV molecule to discover novel capsid constructs with the potential to improve tissue-specific delivery1
- Identify structural variants, truncations, chimera, even human host material that can affect the safety and integrity of the therapeutic2
1Reference: Structural characterization of a novel human adeno-associated virus capsid with neurotropic properties (nature.com) NATURE COMMUNICATIONS | (2020) 11:3279 | https://doi.org/10.1038/s41467-020-17047-1 | www.nature.com/naturecommunications.
2Reference: Tai, P.W. et al. (2018) Adeno-associated virus genome population sequencing achieves full vector genome resolution and reveals human-vector chimeras. Molecular Therapy. 9:130–141.
Video
Highly accurate AAV sequencing with an end-to-end workflow
PacBio’s AAV end-to-end workflow and flexible protocol allow scientists to comprehensively profile packaged genomes as individual intact molecules to identify potential contaminants that can affect the safety, efficacy, and cost-effectiveness of AAV vector gene therapy.
Brochure
HiFi solutions for cell and gene therapy research
Explore how HiFi sequencing can be used to support your gene and cell therapy research
Customer spotlight: NewBiologix
The NewBiologix poster from the ESGCT meeting in 2024 showcased their work on “an integrated platform for the analysis and quality control of rAAV vectors based on long-read sequencing”.
Highlights:
- Newbiologix includes a second strand synthesis step in their library preparation protocol.
- Two PacBio library preparation methods (thermal annealing and second strand synthesis using BST polymerase) are compared with Nanopore sequencing.
- Compared to ONT, PacBio stands out with its ability to generate higher quality reads, higher detection rate of non-rAAV sequences, full-vector reads and high-fidelity mutation calling on the vector.
AAV capsid discovery and design - novel sequencing approaches
Irvin Garza (UTSW) talks about how he is using domain shuffling and long amplicon sequencing to create novel AAV capsids that have specificity for desired target tissues
Library preparation and HiFi sequencing applications for AAV
Edward Oakeley (Novartis) talks about how he improved on the PacBio AAV protocol with a natural complement formation/second strand synthesis step. A similar protocol can be found here
Optimizing and confirming AAV designs with PacBio sequencing
Elizabeth Tseng (PacBio) demonstrates how to generate and analyze full-length AAV sequencing data. Hear how industry leaders from Homology Medicines and AavantiBio discuss how, when, and where they use PacBio full-length AAV sequencing to improve their overall gene therapy research program.
Resources
Explore the AAV resource hub
PacBio and Form Bio have convened a group of leading experts to form an AAV working group dedicated to developing and promoting standardized nomenclature and reporting practices to foster consistency and reliability in AAV research and development. The resulting materials are available on a new AAV resource hub at aav.formbio.com and include an AAV data dictionary, open source tools, and more
PacBio Compatible Partner
Analyze your HiFi AAV sequencing data quickly and easily
Form Bio, the provider of advanced computational life sciences technology, has joined the PacBio Compatible program. We collaborated with Form Bio on an AAV data dictionary defining the most commonly used terms.
Selected publications
FEATURED LONG-READ SEQUENCING SYSTEMS
PacBio long-read sequencers unlock the tools needed for novel gene therapy approaches.