This guest blog post is authored by Nadia Sellami, PhD, Sr Specialist, Gene and Cell Therapy, PacBio. Dr. Sellami leads the global marketing for gene and cell therapy sequencing applications at PacBio.
The gene therapy field is rapidly growing, with new therapies being approved, and many researchers and companies are working on novel AAV-based therapeutics to address diseases for which there is currently no cure.
Gene therapy research has reached a critical point, where novel therapies demand the utmost precision. The ability to sequence complete AAV genomes potentially enhances vector design and also reveals packaging challenges that were previously invisible. Through HiFi sequencing data, researchers can pinpoint fragmentation and truncation issues, verify the expression of desired transcripts, and identify truncations, impurities, and host integration events.
The synergy between PacBio HiFi sequencing and Form Bio’s AI technology enables research in characterizing and optimizing AAV quality, potentially unveiling previously unseen issues that could significantly impact the safety, efficacy, and manufacturability of gene therapies.
Helping to Design the Best AAV Vector for Optimized Manufacturing Conditions
Part of the gene therapy vector design and characterization process is to accurately sequence the produced AAV vector genomes fully as well as determine the quantity and identity of impurities contained in the vector preparations, such as truncated or chimeric genomes.
These full-length AAV sequencing insights can then be used to optimize the vector design to minimize any unintended impurities. Form Bio AI tools used in conjunction with well-characterized PacBio HiFi data enable researchers to discover new, optimized derivatives of constructs, all aimed at maximizing the predicted likelihood of manufacturing success.
This is a critical step to reduce the risk of side effects, costly lot failures in manufacturing, or wasting money on products that may not get approved.
Research on the safety and efficacy of these gene therapy products is crucial, which is why experts in the field are invested in designing the best AAV vectors to minimize potential risks while improving the efficiency of novel treatments.
Enabling the Development of Pure AAV Gene Therapy Products Starts with Sequencing Data That is Both Accurate and Complete
PacBio sequencing technology has evolved to a different type of long read, known as highly accurate long reads, or HiFi reads. HiFi reads are produced using circular consensus on PacBio long-read systems such as Sequel IIe or Revio. HiFi reads provide base-level resolution with 99.9% (Q30) single-molecule read accuracy and read lengths of up to 25 kb.
Accurate long-read sequencing information enables optimization to help minimize truncations and impurities that may affect the function of the vector.
PacBio HiFi sequencing can enable researchers to:
- Optimize AAV vector design – sequence AAV preparations to identify truncation hotspots, characterize ITR configurations, and read through challenging secondary structure elements and use this information to optimize the vector sequence.
- Minimize impurities – understand the composition of all the full-length and partial genomes in vector prep by sequencing.
- Characterize the relative distribution of truncated vs full-length genomes, detect reverse-packaged genomes, and identify sequences from helper plasmids, and host-cell genomic sequences.
- Use the sequencing information to optimize AAV vector design to minimize those impurities for the product or to find the best bioreactor settings to minimize them.
Streamlined PacBio AAV Sequencing with Form Bio AI tools for Easy AAV Vector Design Optimization
Many researchers struggle with the time and expertise required to optimize novel methods and implement them in their existing workflows. This is especially true at a time when scarcity of trained personnel and resources has been a burden on the cell and gene therapy field as a whole.
The PacBio AAV sequencing workflow requires less time and resources to implement since we offer an end-to-end workflow with Form Bio. Use the flexible AAV sequencing protocol for either scAAV or ssAAV, run the sequencing reaction with on-instrument AAV mode to generate consensus reads, and analyze your resulting data with Form Bio analysis tools to fast-track your journey from sample to insight.
Using Form Bio tools to analyze your PacBio HiFi data is a perfect match as this will enable you to quickly implement the best sequencing technology for your AAV, and analyze it with specifically designed workflows and a graphical interface.