Direct ITR-to-ITR Nanopore Sequencing of AAV Vector Genomes.

Recombinant adeno-associated viruses (rAAVs) are currently the most prominently investigated vector platform for human gene therapy. The rAAV capsid serves as a potent and efficient vehicle for delivering genetic payloads into the host cell, while the vector genome determines the function and effectiveness of these biotherapies. However, current production schemes yield vectors that may consist of heterogeneous populations, compromising their potencies. The development of next-generation sequencing methods within the past few years have helped investigators profile the diversity and relative abundances of heterogenous species in vector preparations. Specifically, long-read sequencing methods, like single molecule real-time (SMRT) sequencing, have been used to uncover truncations, chimeric genomes, and inverted terminal repeat (ITR) mutations in vectors. Unfortunately, these sequencing platforms may be inaccessible to investigators with limited resources, require large amounts of input material, or may require long wait times for sequencing and analyses. Recent advances with nanopore sequencing have helped to bridge the gap for quick and relatively inexpensive long-read sequencing needs. However, their limitations and sample biases are not well-defined for sequencing rAAV. In this study, we explored the capacity for nanopore sequencing to directly interrogate rAAV content to obtain full-length resolution of encapsidated genomes. We found that the nanopore platform can cover the entirety of rAAV genomes from ITR to ITR without the need for pre-fragmentation. However, the accuracy for base calling was low, resulting in a high degree of miscalled bases and false indels. These false indels led to read-length compression; thus, assessing heterogeneity based on read length is not advisable with current nanopore technologies. Nonetheless, nanopore sequencing was able to correctly identify truncation hotspots in single-strand and self-complementary vectors similar to SMRT sequencing. In summary, nanopore sequencing can serve as a rapid and low-cost alternative for proofing AAV vectors.

Novel Combinatorial MicroRNA-Binding Sites in AAV Vectors Synergistically Diminish Antigen Presentation and Transgene Immunity for Efficient and Stable Transduction.

Recombinant adeno-associated virus (rAAV) platforms hold promise for in vivo gene therapy but are undermined by the undesirable transduction of antigen presenting cells (APCs), which in turn can trigger host immunity towards rAAV-expressed transgene products. In light of recent adverse events in patients receiving high systemic AAV vector doses that were speculated to be related to host immune responses, development of strategies to mute innate and adaptive immunity is imperative. The use of miRNA binding sites (miR-BSs) to confer endogenous miRNA-mediated regulation to detarget transgene expression from APCs has shown promise for reducing transgene immunity. Studies have shown that designing miR-142BSs into rAAV1 vectors were able to repress costimulatory signals in dendritic cells (DCs), blunt the cytotoxic T cell response, and attenuate clearance of transduced muscle cells in mice to allow sustained transgene expression in myofibers with negligible anti-transgene IgG production. In this study, we screened individual and combinatorial miR-BS designs against 26 miRNAs that are abundantly expressed in APCs, but not in skeletal muscle. The highly immunogenic ovalbumin (OVA) transgene was used as a proxy for foreign antigens. In vitro screening in myoblasts, mouse DCs, and macrophages revealed that the combination of miR-142BS and miR-652-5pBS strongly mutes transgene expression in APCs but maintains high myoblast and myocyte expression. Importantly, rAAV1 vectors carrying this novel miR-142/652-5pBS cassette achieve higher transgene levels following intramuscular injections in mice than previous detargeting designs. The cassette strongly inhibits cytotoxic CTL activation and suppresses the Th17 response in vivo. Our approach, thus, advances the efficiency of miRNA-mediated detargeting to achieve synergistic reduction of transgene-specific immune responses and the development of safe and efficient delivery vehicles for gene therapy.

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

The gene therapy field has been galvanized by two technologies that have revolutionized treating genetic diseases: vectors based on adeno-associated viruses (AAVs), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene-editing tools. When combined into one platform, these safe and broadly tropic biotherapies can be engineered to target any region in the human genome to correct genetic flaws. Unfortunately, few investigations into the design compatibility of CRISPR components in AAV vectors exist. Using AAV-genome population sequencing (AAV-GPseq), we previously found that self-complementary AAV vector designs with strong DNA secondary structures can cause a high degree of truncation events, impacting production and vector efficacy. We hypothesized that the single-guide RNA (sgRNA) scaffold, which contains several loop regions, may also compromise vector integrity. We have therefore advanced the AAV-GPseq method to also interrogate single-strand AAV vectors to investigate whether vector genomes carrying Cas9-sgRNA cassettes can cause truncation events. We found that on their own, sgRNA sequences do not produce a high degree of truncation events. However, we demonstrate that vector genome designs that carry dual sgRNA expression cassettes in tail-to-tail configurations lead to truncations. In addition, we revealed that heterogeneity in inverted terminal repeat sequences in the form of regional deletions inherent to certain AAV vector plasmids can be interrogated.