Gene therapy using recombinant adeno-associated viral (AAV) vectors is emerging as a promising approach to treat central nervous system disorders such as Spinal muscular atrophy, Batten, Parkinson and Alzheimer disease amongst others. A critical remaining challenge for central nervous system-targeted gene therapy, silencing or gene editing is to limit potential vector dose-related toxicity in off-target cells and organs. Here, we characterize a lab-derived AAV chimeric (AAV2g9), which displays favorable central nervous system attributes derived from both parental counterparts, AAV2 and AAV9. This synthetic AAV strain displays preferential, robust, and widespread neuronal transduction within the brain and decreased glial tropism. Importantly, we observed minimal systemic leakage, decreased sequestration and gene transfer in off-target organs with AAV2g9, when administered into the cerebrospinal fluid. A single intracranial injection of AAV2g9 vectors encoding guide RNAs targeting the schizophrenia risk gene MIR137 (encoding MIR137) in CRISPR/Cas9 knockin mice resulted in brain-specific gene deletion with no detectable events in the liver. This engineered AAV vector is a promising platform for treating neurological disorders through gene therapy, silencing or editing modalities.
Adeno-associated viruses (AAV) are thought to spread through the central nervous system (CNS) by exploiting cerebrospinal fluid (CSF) flux and hijacking axonal transport pathways. The role of host receptors that mediate these processes is not well understood. In the current study, we utilized AAV serotype 4 (AAV4) as a model to evaluate whether ubiquitously expressed 2,3-linked sialic acid and the developmentally regulated marker 2,8-linked polysialic acid (PSA) regulate viral transport and tropism in the neonatal brain. Modulation of the levels of SA and PSA in cell culture studies using specific neuraminidases revealed possibly opposing roles of the two glycans in AAV4 transduction. Interestingly, upon intracranial injection into lateral ventricles of the neonatal mouse brain, a low-affinity AAV4 mutant (AAV4.18) displayed a striking shift in cellular tropism from 2,3-linked SA ؉ ependymal lining to 2,8-linked PSA ؉ migrating progenitors in the rostral migratory stream and olfactory bulb. In addition, this gain-of-function phenotype correlated with robust CNS spread of AAV4.18 through paravascular transport pathways. Consistent with these observations, altering glycan dynamics within the brain by coadministering SA-and PSA-specific neuraminidases resulted in striking changes to the cellular tropisms and transduction efficiencies of both parental and mutant vectors. We postulate that glycan signatures associated with host development can be exploited to redirect novel AAV vectors to specific cell types in the brain.
IMPORTANCEViruses invade the CNS through various mechanisms. In the current study, we utilized AAV as a model to study the dynamics of virus-carbohydrate interactions in the developing brain and their impact on viral tropism. Our findings suggest that carbohydrate content can be exploited to regulate viral transport and tropism in the brain.
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.
macrophages to the therapeutic effects. Importantly, the degree of therapeutic improvement was similar in the absence or presence of GW2580 as demonstrated by similar changes in BAL turbidity, SP-D, GM-CSF, and M-CSF (n=4-7/group; P>0.05; all comparisons).Conclusions: Results demonstrate that GM-CSF but not M-CSF contributes to the efficacy of PMT therapy that restores surfactant homeostasis in mice with hPAP.
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