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 (AAVs) are promising viral vectors for therapeutic gene delivery, and the approval of an AAV1 vector for the treatment of lipoprotein lipase deficiency has heralded a new and exciting era for this system. However, preclinical and clinical studies show that neutralization from pre-existing antibodies is detrimental for medical application and this hurdle must be overcome before full clinical realization can be achieved. Thus the binding sites for capsid antibodies must be identified and eliminated through capsid engineering. Towards this goal and to recapitulate patient polyclonal responses, a panel of eight new mouse monoclonal antibodies (MAbs) has been generated against AAV8 and AAV9 capsids, two vectors in development for therapeutic application. Native (capsid) dot blot assays confirmed the specificity of these antibodies for their parental serotypes, with the exception of one MAb, HL2372, selected to cross-react against both capsids. Furthermore, in vitro assays showed that these MAbs are capable of neutralizing virus infection. These MAbs will be utilized for structural mapping of antigenic footprints on their respective capsids to inform development of the next generation of rAAV vectors capable of evading antibody neutralization while retaining parental tropism.

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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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.