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Adeno-associated viruses (AAVs) are advantageous as gene-transfer vectors due to their favorable biological and safety characteristics, with discovering novel AAV variants being key to improving this treatment platform. To date, researchers have isolated over 200 AAVs from natural sources using PCR-based methods. We compared two modern DNA polymerases and their utility for isolating and amplifying the AAV genome. Compared to the HotStar polymerase, the higher-fidelity Q5 Hot Start High-Fidelity DNA Polymerase provided more precise and accurate amplification of the input AAV sequences. The lower-fidelity HotStar DNA polymerase introduced mutations during the isolation and amplification processes, thus generating multiple mutant capsids with variable bioactivity compared to the input AAV gene. The Q5 polymerase enabled the successful discovery of novel AAV capsid sequences from human and nonhuman primate tissue sources. Novel AAV sequences from these sources showed evidence of positive evolutionary selection. This study highlights the importance of using the highest fidelity DNA polymerases available to accurately isolate and characterize AAV genomes from natural sources to ultimately develop more effective gene therapy vectors.

The isolation of adeno-associated virus (AAV) genomes from biomaterials at the molecular level has traditionally relied on polymerase chain reaction-based and cloning-based techniques. However, when applied to samples containing multiple species, traditional techniques for isolating viral genomes can amplify artificial recombinants and introduce polymerase misincorporation errors. In this study, we describe AAV single-genome amplification (AAV-SGA): a powerful technique to isolate, amplify, and sequence single AAV genomes from mammalian genomic DNA, which can then be used to construct vectors for gene therapy. We used AAV-SGA to precisely isolate 15 novel AAV genomes belonging to AAV clades A, D, and E and the Fringe outgroup. This technique also enables investigations of AAV population dynamics and recombination events to provide insights into virus-host interactions and virus biology. Using AAV-SGA, we identified regional heterogeneity within AAV populations from different lobes of the liver of a rhesus macaque and found evidence of frequent genomic recombination between AAV populations. This study highlights the strengths of AAV-SGA and demonstrates its capability to provide valuable insights into the biology and diversity of AAVs.

Background Apoptosis-inducing factor (AIF), as a mitochondrial flavoprotein, plays a fundamental role in mitochondrial bioenergetics that is critical for cell survival and also mediates caspase-independent cell death once it is released from mitochondria and translocated to the nucleus under ischemic stroke or neurodegenerative diseases. Although alternative splicing regulation of AIF has been implicated, it remains unknown which AIF splicing isoform will be induced under pathological conditions and how it impacts mitochondrial functions and neurodegeneration in adult brain. Methods AIF splicing induction in brain was determined by multiple approaches including 5′ RACE, Sanger sequencing, splicing-specific PCR assay and bottom-up proteomic analysis. The role of AIF splicing in mitochondria and neurodegeneration was determined by its biochemical properties, cell death analysis, morphological and functional alterations and animal behavior. Three animal models, including loss-of-function harlequin model, gain-of-function AIF3 knockin model and conditional inducible AIF splicing model established using either Cre-loxp recombination or CRISPR/Cas9 techniques, were applied to explore underlying mechanisms of AIF splicing-induced neurodegeneration. Results We identified a nature splicing AIF isoform lacking exons 2 and 3 named as AIF3. AIF3 was undetectable under physiological conditions but its expression was increased in mouse and human postmortem brain after stroke. AIF3 splicing in mouse brain caused enlarged ventricles and severe neurodegeneration in the forebrain regions. These AIF3 splicing mice died 2–4 months after birth. AIF3 splicing-triggered neurodegeneration involves both mitochondrial dysfunction and AIF3 nuclear translocation. We showed that AIF3 inhibited NADH oxidase activity, ATP production, oxygen consumption, and mitochondrial biogenesis. In addition, expression of AIF3 significantly increased chromatin condensation and nuclear shrinkage leading to neuronal cell death. However, loss-of-AIF alone in harlequin or gain-of-AIF3 alone in AIF3 knockin mice did not cause robust neurodegeneration as that observed in AIF3 splicing mice. Conclusions We identified AIF3 as a disease-inducible isoform and established AIF3 splicing mouse model. The molecular mechanism underlying AIF3 splicing-induced neurodegeneration involves mitochondrial dysfunction and AIF3 nuclear translocation resulting from the synergistic effect of loss-of-AIF and gain-of-AIF3. Our study provides a valuable tool to understand the role of AIF3 splicing in brain and a potential therapeutic target to prevent/delay the progress of neurodegenerative diseases.

The administration of adeno-associated virus (AAV) vectors to nonhuman primates (NHP) via the blood or cerebrospinal fluid (CSF) can lead to dorsal root ganglion (DRG) pathology. The pathology is minimal to moderate in most cases; clinically silent in affected animals; and characterized by mononuclear cell infiltrates, neuronal degeneration, and secondary axonopathy of central and peripheral axons on histopathological analysis. We aggregated data from 33 nonclinical studies in 256 NHP and performed a meta-analysis of the severity of DRG pathology to compare different routes of administration, dose, time course, study conduct, age of the animals, sex, capsid, promoter, capsid purification method, and transgene. DRG pathology was observed in 83% of NHP that were administered AAV through the CSF, and 32% of NHP that received an intravenous (IV) injection. We show that dose and age at injection significantly affected the severity whereas sex had no impact. DRG pathology was minimal at acute time points (

SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. Here we present an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by one of the many pre-emergent, ACE2-dependent CoVs that are poised for zoonosis., Author summary During the COVID-19 pandemic, variants of SARS-CoV-2 have emerged that are more deadly, more transmissible, and that evade immune response. These variants threaten to reduce the efficacy of first-generation vaccine and monoclonal antibody therapeutics. An additional problem with the existing vaccine strategy is that immune-compromised patients do not respond to active vaccinations. To address these issues, we developed a gene therapy-based strategy to prevent COVID-19 using a viral vector delivered by nasal spray. Unlike vaccines, this passive prophylaxis does not require that the patient mount an immune response. Instead, airway cells are engineered to secrete an antiviral decoy protein that can intercept viral particles before they cause infection. This decoy is based on human ACE2, the natural receptor for SARS-CoV-2. This design imparts resistance to SARS-CoV-2 viral evolution: viral mutations that evade the ACE2 decoy are unlikely to emerge because they would also render the virus less capable of infecting cells and therefore be less fit. Indeed, our ACE2 decoy neutralizes all SARS-CoV-2 variants that we tested and even binds distantly related coronaviruses that have not yet crossed over from animals to humans. This suggests that the ACE2 decoy gene therapy approach could be effectively deployed to vulnerable populations in the COVID-19 pandemic and potentially to broader populations at the outset of future coronavirus pandemics.

Adeno-associated viral vectors can be used as a platform for delivering biological countermeasures against pandemic and biological threats. We show that vector delivery of two antibody components of the ZMapp product is effective in mice against systemic and airway challenge with a mouse-adapted strain of Ebola virus. This platform provides a generic manufacturing solution and overcomes some of the delivery challenges associated with repeated administration of the protective protein.

DNA damage-activated nuclease identified Cells that experience stresses and accumulate excessive damage to DNA undergo cell death mediated by a nuclear enzyme known as PARP-1. During this process, apoptosis-inducing factor (AIF) translocates to the nucleus and activates one or more nucleases to cleave DNA. Wang et al. found that macrophage migration inhibitory factor (MIF) is an AIF-associated endonuclease that contributes to PARP-1-induced DNA fragmentation (see the Perspective by Jonas). In mouse neurons in culture, loss of MIF protected neurons from cell death caused by excessive stimulation. Targeting MIF could thus provide a therapeutic strategy against diseases in which PARP-1 activation is excessive. Science , this issue p. 82 ; see also p. 36