Preexisting humoral immunity to recombinant adeno-associated virus (AAV) vectors restricts the treatable patient population and efficacy of human gene therapies. Approaches to clear neutralizing antibodies (NAbs), such as plasmapheresis and immunosuppression, are either ineffective or cause undesirable side effects. Here, we describe a clinically relevant strategy to rapidly and transiently degrade NAbs before AAV administration using an IgG-degrading enzyme (IdeZ). We demonstrate that recombinant IdeZ efficiently cleaved IgG in dog, monkey, and human antisera. Prophylactically administered IdeZ cleaved circulating human IgG in mice and prevented AAV neutralization in vivo. In macaques, a single intravenous dose of IdeZ rescued AAV transduction by transiently reversing seropositivity. Importantly, IdeZ efficiently cleaved NAbs and rescued AAV transduction in mice passively immunized with individual human donor sera representing a diverse population. Our antibody clearance approach presents a potentially new paradigm for expanding the prospective patient cohort and improving efficacy of AAV gene therapy.

Adeno-associated viruses (AAV) are composed of non-enveloped, icosahedral protein shells that can be adapted to package and deliver recombinant therapeutic DNA. Approaches to engineer recombinant capsids for gene therapy applications have focused on rational design or library-based approaches that can address one or two desirable attributes; however, there is an unmet need to comprehensively improve AAV vector properties. Such cannot be achieved by utilizing sequence data alone, but requires harnessing the 3D structural properties of AAV capsids. Here, we solve the structures of a natural AAV isolate complexed with antibodies using cryo-electron microscopy and harness this structural information to engineer AAV capsid libraries through saturation mutagenesis of different antigenic footprints. Each surface loop was evolved by infectious cycling in the presence of a helper Adenovirus to yield a new AAV variant that then serves as a template for evolving the next surface loop. This stepwise process yielded a humanized AAV8 capsid (AAVhum.8) displaying non-natural surface loops that displays simultaneously tropism for human hepatocytes, increased gene transfer efficiency and neutralizing antibody evasion. Specifically, AAVhum.8 can better evade neutralizing antisera from multiple species compared to AAV8. Further, AAVhum.8 displays robust transduction in a human liver xenograft mouse model with expanded tropism for both murine and human hepatocytes. This work supports the hypothesis that critical properties such as AAV capsid antibody evasion and tropism can be co-evolved by combining rational design and library-based evolution for clinical gene therapy. Importance Clinical gene therapy with recombinant AAV vectors has largely relied on natural capsid isolates. There is an unmet need to comprehensively improve AAV tissue tropism, transduction efficiency and antibody evasion. Such cannot be achieved by utilizing capsid sequence data alone, but requires harnessing the 3D structural properties of AAV capsids. Here, we combine rational design and library-based evolution to co-evolve multiple, desirable properties onto AAV by harnessing 3D structural information.

Multiple herpesviruses have been recently found to encode viral circular RNAs. Like cellular circular RNAs, these RNAs lack poly-A tails and their 5′ and 3′ ends have been joined, which confers protection from RNA exonucleases. We examined the expression patterns of circular RNAs from Kaposi’s sarcoma herpesvirus (KSHV) in various environments. We performed deep sequencing of circRNA-enriched total RNA from a KSHV-positive patient lymph node for comparison with previous circRNA-Seq results. We found that circvIRF4 is highly expressed in the KSHV-positive patient sample relative to both B cell lines and de novo infected primary vascular and lymphatic endothelial cells (LECs). Overall, this patient sample showed a viral circRNA expression pattern more similar to the pattern from B cell lines, but we also discovered new back-spliced junctions and additional viral circular RNAs in this patient sample. We validated some of these back-spliced junctions as circular RNAs with standard assays. Differential expression patterns of circular RNAs in different cell types led us to investigate what cellular factors might be influencing the ratio of viral linear mRNAs to circular RNAs. We found that repression of certain RNA-binding proteins shifted the balance between viral linear mRNAs and circular RNAs. Taken together, examining viral circular RNA expression patterns may become useful tools for discovering their functions, the regulators of their expression, and determining the stage and cell types of infection in humans.

Highlights d A screen to identify natural products that can bind regulatory non-coding RNAs d Identification of natural pharmacophores that can bind precursor microRNA-21 d Butylcycloheptyl prodiginine inhibits microRNA-21 and reduces growth of cancer cells d Regulatory non-coding RNAs can be potential targets for small-molecule therapeutics