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Recombinant adeno-associated virus (AAV) has emerged as one of the primary vectors for gene therapy. This is because it has many desirable properties, including lack of pathogenicity, efficient infection of dividing and non-dividing cells, and sustained maintenance of the viral genome. With an excellent safety profile in numerous preclinical studies and hundreds of clinical trials worldwide, AAV vectors are widely used as an excellent platform for gene editing, gene knockout as well as insertion.

What is AAV?

AAV consists of a protein capsid that protects a small single-stranded DNA genome, approximately 4.7 kb. More than 100 naturally occurring AAVs have been isolated in humans and animals, 32 of which vary in capsid composition and exhibit different cellular tropisms, transduction efficiency, and immunogenicity.

How does AAV work?

Different AAV vectors, either naturally occurring or hybrid/synthetically derived, can be utilized to target different tissues, organs and cells to compensate for defects, with replaced viral DNA, correct dysfunctions and potentially confer novel abilities to fight infections and even cancer. And optimized AAV vectors can help evade any pre-existing immunity to gene therapy vectors, thus expanding therapeutic applications and commercialization.

The advantages of AAV Genome Sequencing using PacBio SMRT sequencing

It was found that PacBio SMRT sequencing technology provides access to the full length of the AAV genome, including inverted terminal repeats (ITRs) and those regions with high GC content (>100bp) that are missed by short-read sequencing approaches. PacBio SMRT sequencing fill the gap of short-read sequencing technology by improving sequencing accuracy to Q30 (99.9%) or higher while taking into account long read length. The researchers analyzed three self-complementary AAV (scAAV, two copies of a gene were put into a vector) genomes using PacBio SMRT sequencing, including the conventional scAAV vector and an shRNA cassette-containing vector similar to scAAV-eGFP. It was found using PacBio SMRT sequencing was able to directly identify and quantify the distribution of encapsulated sense/antisense strands and flip/flop in the rAAV preparations, after comparing to reference sequences. This indicates that AAV Genome Sequencing by PacBio SMRT sequencing technology is extremely advantageous and promising for AAV vector discovery, design and quality control.

How does PacBio SMRT Sequencing facilitate AAV Vector Development?

AAV Vector Design

During the AAV viral vector development phase, several key issues need to be considered, including how to perform AAV capsid optimization, improve target gene expression, and AAV production optimization.

Full-length AAV vector genome data is not only to obtain high-precision data but also to discover new AAV species and identify different features to help further application and development of AAV vectors in later stages. It helps to find AAV viruses that are more suitable for the requirements of specific therapeutic targets. This detailed and accurate insight from Pacbio HiFi sequencing makes it possible for gene therapy developers to create AAV delivery vectors tailored to their therapeutic deliverability.

AAV Vector Quality Control

Recombinant adeno-associated viruses (rAAVs) have emerged as one of the most promising vectors for expressing therapeutic gene products. The specific needs of medical applications for clinical-grade vectors require stringent quality control (QC) testing to assess vector purity and integrity. However, current standard QC processes are limited to titration and quantification of vectors by qPCR; verification of genome size by agarose gel electrophoresis; and characterization of viral purity by silver staining polyacrylamide gels. None of these methods can characterize the composition and structure of the fragmented AAV genome and its proportion of it.

Next-generation sequencing (NGS) can be used to analyze the genome of single-stranded (ss) AAV to assess the extent of "error-prone" genome encapsulation during rAAV production, including measures of identity and purity for AAV viral vectors. However, the ITR region on the AAV vector is a challenge for NGS due to its GC-rich nature, and the short read length of NGS makes it a challenge to explain individual intact molecules of the AAV vector.

In contrast, PacBio SMRT sequencing helps ensure that AAV vectors contain the correct therapeutic gene sequences, captures errors that are difficult to detect with second-generation sequencing, and allows for direct detection of base modifications. This highly accurate technique for a comprehensive assessment of vector genomic heterogeneity and observation of plasmid sequences and the presence of host gene chimerism enables better characterization of the various sequences on the AAV vector and the population distribution of vector molecules, identifying whether structural regions of the AAV vector may need improvement and reducing the difficulty of optimization. The data also shows that sequencing can identify low levels of contaminating plasmid DNA.

PacBio Iso-Seq in AAV validation

The key to successful gene therapy is to ensure adequate gene expression levels of GOI at doses that do not cause unplanned inflammation, otherwise it could lead to reduced or lost efficacy, require re-dosing or cause tissue damage, or even be life-threatening. As a vehicle for gene therapy drugs, it is important to determine their therapeutic effective load. PacBio Iso-Seq technology has the advantage of full-length isoform sequences of RNA obtained to help assess whether a potential therapeutic gene has been introduced into the host and will provide the intended treatment, as well as being an important safety test. Full isoform expression data helps monitor the dynamics of the gene therapy development pipeline for AAV, providing detailed information on codon optimization, and also allows early confirmation of active efficacy and dosing of rAAV, etc.

Applying the advantages of long read length and accuracy of PacBio SMRT sequencing to AAV development, optimization and validation enables more accurate and rapid access to suitable AAV vectors in R&D programs.


  1. Tai P W L, Xie J, Fong K, et al. Adeno-associated virus genome population sequencing achieves full vector genome resolution and reveals human-vector chimeras. Molecular Therapy-Methods & Clinical Development, 2018, 9: 130-141.

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