Gene therapy + Gene Editing

Accelerate gene therapy research with long read HiFi sequencing

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Gene therapy solutions

Highly accurate HiFi sequencing allows researchers to comprehensively discover, design and confirm their gene therapy approaches.

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Improve vector design with full-length AAV sequencing

Discover, verify, and improve AAV vector design with full-length, highly accurate AAV sequencing.

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Verify expression vectors are expressing desired transcripts

Confirm that gene therapy vectors are expressing the desired transcripts for protein synthesis with full-length isoform sequencing (Iso-Seq method).

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Confirm CRISPR-Cas9 gene editing outcomes

Sequence previously inaccessible regions of the genome to provide base-level resolution of disease-causing repeat expansions.

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ON-DEMAND WEBINAR

HIGHLY ACCURATE HIFI READS FOR DISCOVERY, DESIGN, AND MANUFACTURING OF AAV GENE THERAPY

  • Understand the challenges of AAV sequencing and the unique advantages of using PacBio long reads to address these unmet needs
  • Hear how biopharmaceutical researchers are using highly accurate HiFi reads for vector discovery, design, and quality control
  • Learn about the PacBio AAV solution and analyses

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Benefits of gene therapy and the promise of adeno-associated virus (AAV) vectors

AAV gene therapy can address the underlying genetic problems that cause disease and holds great promise to help improve human health. By increasing our understanding of the genetic components underlying disease, scientists can improve AAV gene therapy and change the course of disease.

PacBio AAV sequencing offers distinct advantage of being able to sequence the entire, intact, ~4.7kb construct with high accuracy. The ability to see full length is important for discovery and for identifying truncated products while high accuracy is important for identifying undesired mutations.

PROFILE Comprehensively profile packaged genomes as a single intact molecule
ASSESS Directly assess vector integrity without extensive preparation
REVEAL Reveal the relative distribution of truncated genomes versus full-length genomes in vector preparation
DETECT Detect reverse-packaged genomes that encompass sequences originating from plasmid backbone
IDENTIFY Identify sequences from packaging helper plasmids
FIND Find host-cell genomic sequences that are chimeric with inverted terminal repeat (ITR) - containing vector sequences
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HiFi sequencing advantages in gene therapy

Common questions about gene therapy

Gene therapy is a method of treating genetic disease by adding, deleting, or correcting genetic material in the cells of the person with a disease with the goal to change the course of disease.

Gene therapy by direct delivery is the process by which cells are extracted from the patient, modified with the therapeutic gene, and injected back into the patient. Alternatively, the delivery method can be cell-based where ex-vivo targeted cells are edited using CRISPR-Cas9 and then directly transferred back into the patient’s body.

Adeno-associated virus (AAV) is a non-enveloped virus approximately 4.7 kb in size and can be engineered to deliver DNA to target cells. Adeno-Associated Virus (AAV) vectors are typically preferred due to low toxicity [2], dependence upon other viruses for replication [3], broad tropism and the ability to infect both dividing and non-dividing cells.

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.

Short reads, qPCR, and less accurate long-read technology can miss important changes that may be present in the AAV sequence, potentially affecting the safety and efficacy of gene therapy products. HiFi sequencing is the ideal solution due to its ability to sequence full length AAV genomes at high accuracy, which allows for detection of fragmentation, mutations, and large structural events.

Gene therapy technique in action

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Gene therapy validation with single-molecule resolution using highly accurate HiFi reads

Discover how gene editing protocols can benefit from long-read sequencing at multiple stages to optimize and enhance vector integrity.

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Highly accurate SMRT sequencing for gene therapy applications

Phillip Tai from the University of Massachusetts Medical School discusses his investigation in the design compatibility of CRISPR-Cas9 components in AAV vectors.

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Researchers reveal impurities in AAV packaged genomes with HiFi sequencing — not detectable by short-read sequencing

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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

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Accurately confirm CRISPR-Cas9 off-target sequencing and analysis

Ida Hoijer of Uppsala University shares her research on developing a long-read sequencing-based method for detection of CRISPR-Cas9 off-target effects, along with a customized analysis pipeline.

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Diversity of unexpected CRISPR-Cas9 edits revealed by SMRT sequencing

Researchers discover CRISPR-Cas9 method results in far more genomic changes than previously thought — including big deletions and rearrangements — was made possible by the use of long-read SMRT sequencing.

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PacBio sequencing reveals AAV integration into human host genome

Using PacBio long-read sequencing, researchers discover heavy rearrangement at integration site which can potentially impact vector integrity and the safety of the gene therapy drug

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APPLICATION BROCHURE

AAV SEQUENCING BEST PRACTICES

Accelerate discovery and comprehensively assess adeno-associated virus (AAV) vector quality with highly accurate long reads (HiFi reads)

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