Boosting AAV vector development with HiFi sequencing
One of the most important technologies responsible for the acceleration of gene therapy has been the emergence of vectors based on Adeno Associated Viruses (AAVs). In recent years AAVs have become the leading platform for gene therapy delivery due to their compatibility with CRISPR technology, as well as strong safety and efficacy profiles, demonstrated in preclinical studies and clinical trials worldwide.
Since recombinant AAV (rAAV) vectors are currently produced via cell-based methodologies, there is a great deal of opportunity for error and batch inconsistencies. This can result in fluctuations in transgene construct, packaging components, and encapsulated material. As such, vector characterization and quality control (QC) is essential.
While traditional methods for rAAV vector characterization, such as qPCR, can clarify the size distribution of vectors, they fail to identify their prevalence, heterogeneity, or their components. They can also fail to identify fragmentation/truncation events that can impact rAAV vector production and efficacy.
In this blog, we discuss how PacBio’s HiFi sequencing can provide value at various stages of AAV vector development.
Sequencing in AAV vector development
Various sequencing platforms and approaches have been used to gain a deeper understanding of vector populations, and their encapsulated genetic material. Next-generation sequencing (NGS) techniques such as Illumina’s sequencing-by-synthesis can be used, but these are limited by short read lengths, and yield only partial and fragmented genomic data. Meanwhile, longer-read sequencing technologies like Sanger sequencing are labor intensive, limited in their accuracy over ~700 bases, especially over GC-rich regions, and the need for priming means it can fail to distinguish the functionally of distinct AAV species.
Third generation, long-read sequencing approaches may be the answer.
HiFi Sequencing, also known as Single Molecule Real Tine (SMRT) sequencing, is an alternative sequencing technique from PacBio, that provides full genome read lengths at the high accuracy levels associated with short reads. These highly accurate long reads are known as HiFi reads, and can bring great value to a variety of genomic applications.
HiFi sequencing also opens up novel applications, such as providing genome population data for AAV vector characterization and QC .
At Eremid, we recognize the potential of HiFi sequencing for AAV vector development, and have integrated PacBio SMRT sequencing apparatus into our genomics services labs.
Phases of HiFi sequencing in AAV vector development 
What exactly is HiFi Sequencing?
HiFi reads are generated using PacBio SMRT sequencing systems, and can provide base-level resolution with an average of 99.9% single-molecule read accuracy.
In short, the technique involves the creation of a SMRTbell library by ligating adapters to double stranded DNA, creating a circular template and providing a binding site for the polymerase.
In the SMRT sequencing cell, DNA molecules are immobilized into millions of microwells. During sequencing, light of known wavelengths is emitted as the polymerase incorporates labelled nucleotides into the sequence, providing real time sequencing data. Each molecule is sequenced in loop multiple times resulting in subreads that are aligned to produce a circular consensus sequencing (CCS) with high read accuracy.
AAV discovery / vector design
Sequencing data is not only useful for validation and QC, but can also be a powerful tool in the early phases of AAV discovery and vector design. Thanks to its single molecule resolution, HiFi sequencing can be exploited to investigate human tissues for the discovery of novel AAVs, which could be of therapeutic value.
Recently, a novel AAV capsid called AAVv66 was discovered via PacBio SMRT sequencing. The capsid has high sequence similarity to the popular AAV2 capsid, but exhibited enhanced production yields, vector stability, and CNS transduction .
This capsid was discovered via HiFi sequencing with primers targeting conserved regions across AAV serotypes. The ability to sequence through the full-length target region with high accuracy enabled the research group to collate and quantify the AAV species in the sample, leading to the identification of a dominant novel capsid, AAVv66, that made up 45% of the population. AAV discovery in this way may hold the key to developing a library of AAVs with enhanced tissue specificity for specialized gene therapy applications.
When it comes to AAV vector design, fully characterizing the vectors to assess design integrity is an important step. HiFi sequencing can be of great value here, especially in the identification of packaging issues, which wouldn’t be possible with standard NGS approaches.
SMRT sequencing can resolve the full AAV genome in one read, and rapidly provide genomic data for a large rAAV vector population. This enables researchers to identify undesirable gene truncation events, and gain an accurate measure of the number of vector species in a population that have encapsulated the full-length transgene required for therapy .
AAV vector quality control
When producing clinical-grade rAAV vectors for therapeutic applications, ensuring they maintain attributes consistent with safe and durable expression is essential. There is currently no standardization of QC assays for vectors, beyond titration and purity assessments, but it can be advantageous to use NGS to enhance characterization.
Various NGS approaches can be used to analyze the rAAV vector genome to assess the extent of error in genome encapsulation during production, and retrieve measures of identity and purity. The GC-rich nature of AAVs presents a challenge for traditional NGS approaches, however. Traditional NGS also struggles to sequence the flanking inverted terminal repeat (ITR) regions that are essential for genome packaging into the capsid.
SMRT sequencing can overcome the limitations of traditional NGS, and identifies genomic errors that would be difficult to detect with other approaches while providing direct detection of base modifications with high accuracy. The SMRT HiFi sequencing approach can provide a robust assessment of vector population homogeneity and visualization of encapsulated plasmid sequences. This enables researchers to isolate areas of improvement and optimize production.
AAV gene therapy validation
Once an AAV-based gene therapy has been administered, it is key to ensure adequate expression of the therapeutic gene at levels that do not prompt immunogenicity. HiFi reads can provide full length transcript sequencing (Iso-Seq), which provides researchers with full-length isoform RNA reads to give an insight into the effectiveness of a gene therapy treatment.
Data from isoform RNA reads can be used to investigate host integration and transgene expression levels for therapeutic optimization. Furthermore, once a therapeutic has been introduced into the host, SMRT sequencing can determine whether the level of expression will provide the intended treatment, as well as acting as a key safety measure.
A valuable tool throughout the AAV vector R&D journey
With its ability to perform full length reads with high accuracy, HiFi sequencing can bring myriad benefits to AAV discovery, development, optimization, and validation. It can empower R&D programs to identify AAV vectors best suited to their research/therapeutic needs.
At Eremid, we understand the value that HiFi sequencing can bring to AAV R&D. That’s why we have integrated PacBio SMRT sequencing apparatus into our genomics services lab. We’re fully equipped to support you in all aspects of your genomics research, from study design and sample preparation to sequencing and bioinformatics no matter how big or small your project. Why not discover how we could help you with you AAV vector development?
- Ngoc Tan Tran, et al., AAV-Genome Population Sequencing of Vectors Packaging CRISPR Components Reveals Design-Influenced Heterogeneity. Methods & clinical development, 2020. 18, 639–651.
- HiFi sequencing for AAV gene therapy: from discovery and design to production. PacBio: https://www.pacb.com/blog/hifi-sequencing-for-aav-gene-therapy/
- Hung-Lun Hsu, et al., Structural characterization of a novel human adeno-associated virus capsid with neurotropic properties. Nature communications, 2020. 11(1), 3279.
- Philip W M Tai, et al., Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras. Molecular therapy. Methods & clinical development, 218. 9, 130–141.