Effective gene delivery to the CNS by intravenously administered adeno-associated virus (AAV) vectors requires crossing the blood-brain barrier (BBB). To achieve therapeutic CNS transgene expression, high systemic vector doses are often required, which poses challenges such as scale-up costs and dose-dependent hepatotoxicity. To improve the specificity and efficiency of CNS gene transfer, a better understanding of the structural features that enable AAV transit across the BBB is needed. We generated a combinatorial domain swap library using AAV1, a serotype that does not traverse the vasculature, and AAVrh.10, which crosses the BBB in mice. We then screened individual variants by phylogenetic and structural analyses and subsequently conducted systemic characterization in mice. Using this approach, we identified key clusters of residues on the AAVrh.10 capsid that enabled transport across the brain vasculature and widespread neuronal transduction in mice. Through rational design, we mapped a minimal footprint from AAVrh.10, which, when grafted onto AAV1, confers the aforementioned CNS phenotype while diminishing vascular and hepatic transduction through an unknown mechanism. Functional mapping of this capsid surface footprint provides a roadmap for engineering synthetic AAV capsids for efficient CNS gene transfer with an improved safety profile.

A major hindrance in gene therapy trials with adeno-associated virus (AAV) vectors is the presence of neutralizing antibodies (NAbs) that inhibit AAV transduction. In this study, we used directed evolution techniques in vitro and in mouse muscle to select novel NAb escape AAV chimeric capsid mutants in the presence of individual patient serum. AAV mutants isolated in vitro escaped broad patient-specific NAb activity but had poor transduction ability in vivo. AAV mutants isolated in vivo had enhanced NAb evasion from cognate serum and had high muscle transduction ability. More importantly, structural modeling identified a 100 amino acid motif from AAV6 in variable region (VR) III that confers this enhanced muscle tropism. In addition, a predominantly AAV8 capsid beta barrel template with a specific preference for AAV1/AAV9 in VR VII located at threefold symmetry axis facilitates NAb escape. Our data strongly support that chimeric AAV capsids composed of modular and nonoverlapping domains from various serotypes are capable of evading patient-specific NAbs and have enhanced muscle transduction.

SummaryQuiescent neural stem cells (qNSCs) with radial morphology are the only proven source of new neurons in the adult mammalian brain. Our understanding of the roles of newly generated neurons depends on the ability to target and manipulate adult qNSCs. Although various strategies have been developed to target and manipulate adult hippocampal qNSCs, they often suffer from prolonged breeding, low recombination efficiency, and non-specific labeling. Therefore, developing a readily manufactured viral vector that allows flexible packaging and robust expression of various transgenes in qNSCs is a pressing need. Here, we report a recombinant adeno-associated virus serotype 4 (rAAV4)-based toolkit that preferentially targets hippocampal qNSCs and allows for lineage tracing, functional analyses, and activity manipulation of adult qNSCs. Importantly, targeting qNSCs in a non-Cre-dependent fashion opens the possibility for studying qNSCs in less genetically tractable animal species and may have translational impact in gene therapy by preferentially targeting qNSCs.

Central nervous system (CNS) transduction by systemically administered recombinant adeno-associated viral (AAV) vectors requires crossing the blood-brain barrier (BBB). We recently mapped a structural footprint on the AAVrh.10 capsid, which, when grafted onto the AAV1 capsid (AAV1RX), enables viral transport across the BBB; however, the underlying mechanisms remain unknown. Here, we establish through structural modeling that this footprint overlaps in part the sialic acid (SIA) footprint on AAV1. We hypothesized that altered SIA-capsid interactions may influence the ability of AAV1RX to transduce the CNS. Using AAV1 variants with altered SIA footprints, we map functional attributes of these capsids to their relative SIA dependence. Specifically, capsids with ablated SIA binding can penetrate and transduce the CNS with low to moderate efficiency. In contrast, AAV1 shows strong SIA dependency and does not transduce the CNS after systemic administration and, instead, transduces the vasculature and the liver. The AAV1RX variant, which shows an intermediate SIA binding phenotype, effectively enters the brain parenchyma and transduces neurons at levels comparable to the level of AAVrh.10. In corollary, the reciprocal swap of the AAV1RX footprint onto AAVrh.10 (AAVRX1) attenuated CNS transduction relative to that of AAVrh.10. We conclude that the composition of residues within the capsid variable region 1 (VR1) of AAV1 and AAVrh.10 profoundly influences tropism, with altered SIA interactions playing a partial role in this phenotype. Further, we postulate a Goldilocks model, wherein optimal glycan interactions can influence the CNS transduction profile of AAV capsids. IMPORTANCE Understanding how viruses cross the blood-brain barrier can provide insight into new approaches to block infection by pathogens or the ability to exploit these pathways for designing new recombinant viral vectors for gene therapy. In this regard, modulation of virus-carbohydrate interactions by mutating the virion shell can influence the ability of recombinant viruses to cross the vascular barrier, enter the brain, and enable efficient gene transfer to neurons.

AAV Biologythe overexpression of ERI-1 determined an over 10-fold, selective reduction on ssAAV genome association with H3 and H4, while changes were negligible for scAAV DNA and for control cellular genes. Consistent with chromatin exerting a repressive role on ssAAV transduction, we also noticed that the downregulation of the main replication-dependent histone chaperone CAF-1 induced an over 20-fold increase in transduction. Increase of ssAAV2 transduction by ERI-1 also decreased the association, with the viral DNA, of proteins of the cellular DNA damage response (DDR; e.g. Nbs1 and Mre11), which our previous work had indicated as inhibitory of AAV transduction.Interestingly, DNA damage per se induced downregulation of histone gene expression. In particular, hydroxyurea, a drug markedly increasing AAV transduction, also determined histone mRNA degradation, an effect that required integrity of ERI-1.These results underline the importance of chromatin and its dynamic regulation in determining the fate of productive AAV transduction. These findings can be exploited for the development of more effective AAV-mediated gene delivery strategies.

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2017-08-30T02:06:24