A Novel Next-Generation Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations from Viral DNA Extracts

Abstract

Recombinant adeno-associated virus (rAAV) vectors are increasingly popular gene delivery tools in biological systems. They are safe and lead to high-level, long-term transgene expression. rAAV are available in multiple serotypes, natural or engineered, which enable targeting to a wide array of tissues and cell types. In addition, rAAVs are relatively easily produced in a well-equipped lab or obtained from a viral vector core facility. Unfortunately, there is no standardization of quality control assays beyond titering and purity assessments. Next-generation sequencing (NGS) can be used to identify rAAV preparations. Because the rAAV genome is single stranded, previous studies have assumed that rAAV genomes must be converted to double strands before NGS. We demonstrate that rAAV DNA extracts exist primarily as doublestranded species. We hypothesize that these molecules form from the natural base pairing of complementary [+] and [-] strands after DNA extraction and show that rAAV DNA extracts are sufficient templates for downstream NGS without the labor-intensive double-stranding step. Here, we provide a detailed protocol for the simple and rapid NGS of rAAV genomes from DNA extracts. With this protocol, users can quickly confirm the identity of an rAAV preparation and detect the presence of contaminating rAAV DNA. In addition, we share custom Python scripts that allow users to accurately determine the serotype and detect Cre-independent DNA recombination events in rAAV containing Lox sites within minutes. We have used these scripts to analyze more than 100 rAAV preparations. Although we focused on the detection of cross-contaminating rAAV DNA and recombination events, our Python scripts can be customized to detect other sequences or events, such as reverse packaging of plasmid backbone or DNA from the packaging cell line. We find that the NGS of rAAV DNA extracts, termed viral genome sequencing, is a simple and powerful method for rAAV validation.

AAV-Genome Population Sequencing of Vectors Packaging CRISPR Components Reveals Design-Influenced Heterogeneity

“…Excluding reads related to spike-in material, we detected about 2.76% (182 total) and 2.53% (250 total) reads mapping to the hg38 genome among the vector libraries with and without spike-ins, respectively ( Table S1 ; Figure 3 A). As demonstrated by us and others, 10 , 19 , 20 , 21 we also obtained reads that also mapped to the trans plasmid, the pAd helper plasmid, and elements of the vector backbones ( Figure S2 ; Table S2 ). As demonstrated by the alignment display and the varied lengths of reads mapping to the vector backbone, these impurities are highly heterogeneous ( Figure S2 B).…”

“…This presumably circumvents the need to convert the single-stranded genomes into a double-stranded configuration via in vitro second-strand synthesis. We note that certain ssAAV vector genomes, under neutral conditions, have been observed to run as “doublets.” 18 , 19 These species have been suggested to be the annealed plus and minus strands that migrate at the predicted size, and non-annealed single-stranded genomes that migrate faster and as a smear. The non-annealed species, we speculate, may reflect an imbalance in packaged plus- and minus-stranded genomes that is typically resolved when run under alkaline conditions.…”

“…Unfortunately, there are still many unknown aspects of the vector genome structure, especially from novel platforms, that may impact the manufacturing of vector and may influence the effective doses for treatment. In recent years, multiple methods of gauging vector genome integrity have been developed to capture the homogeneity of genomes that are packed into AAV capsids, 10 , 19 , 20 , 30 each with its own advantages and disadvantages. Based on the structure of the sgRNA cassette, which harbors four loop regions, we predicted that the sgRNA sequence may serve as a scaffold for truncation events during AAV replication, similar to what we previously found for siRNA transgene cassettes.…”

“… 16 This phenomenon was also key for a recently developed short-read sequencing platform. 19 By improving this action with heating and slow cooling the extracted vector DNA, we were able to obtain double-stranded genomes that are adapterable by SMRTbell adapters. To demonstrate the reliability of adaptering, we showed heterogeneity of ITR sequences, elements that, aside from being difficult to sequence by conventional means, exhibit flip and flop orientations, display unintended mutations, and can terminate within the ITR structure apart from the conventional TRS at low frequencies.…”

SSV-Seq 2.0, a more accurate PCR-free method for high-throughput sequencing of adeno-associated viral vector genomes

“…In addition to the relative percentage of each DNA species, SSV-Seq can provide information regarding vector genome identity with a computational analysis of the single nucleotide variants (SNV) and the sequencing coverage over the recombinant AAV genome. Since then, and as sign of interest for HTS-based methods applied to rAAV quality control, other protocols have been developed to analyze the identity (Guerin et al, 2020;Maynard et al, 2019) or integrity (Radukic et al, 2019;Tai et al, 2018;Xie et al, 2017) of AAV vector genomes by sequencing.…”