Adeno-associated virus (AAV) vectors are showing promise in gene therapy trials and have proven to be extremely efficient biological tools in basic neuroscience research. One major limitation to their widespread use in the neuroscience laboratory is the cost, labor, skill, and time intense purification process of AAV. We have recently shown that AAV can associate with exosomes (exo-AAV) when vector is isolated from conditioned media of producer cells, and the exo-AAV is more resistant to neutralizing anti-AAV antibodies compared to standard AAV. Here we demonstrate that simple pelleting of exo-AAV from media via ultracentrifugation, results in high-titer vector preparations capable of efficient transduction of central nervous system (CNS) cells after systemic injection in mice. We observed that exo-AAV is more efficient at gene delivery to the brain at low vector doses relative to conventional AAV, even when derived from a serotype that does not normally efficiently cross the blood brain barrier. Similar cell types were transduced by exo-AAV and conventionally purified vector. Importantly, no cellular toxicity was noted in exo-AAV transduced cells. We demonstrated the utility and robustness of exo-AAV-mediated gene delivery by detecting direct GFP fluorescence after systemic injection, allowing 3-dimensional reconstruction of transduced Purkinje cells in the cerebellum using ex-vivo serial 2-photon tomography. The ease of isolation combined with the high efficiency of transgene expression in the CNS, may enable widespread use of exo-AAV as a neuroscience research tool. Furthermore, the ability of exo-AAV to evade neutralizing antibodies while still transducing CNS after peripheral delivery is clinically relevant.
Neuroinflammation is a key contributor to the pathology of Alzheimer’s Disease (ad). CD33 (Siglec-3) is a transmembrane sialic acid-binding receptor on the surface of microglial cells. CD33 is upregulated on microglial cells from post-mortem ad patient brains and high levels of CD33 inhibit uptake and clearance of amyloid beta (Aβ) in microglial cell cultures. Furthermore, knock-out of CD33 reduces amyloid plaque burden in mouse models of ad. Here, we tested whether a gene therapy strategy to reduce CD33 on microglia in ad could decrease Aβ plaque load. Intracerebroventricular injection of an adeno-associated virus (AAV) vector-based system encoding an artificial microRNA targeting CD33 (miRCD33) into APP/PS1 mice reduced CD33 mRNA and TBS-soluble Aβ40 and Aβ42 levels in brain extracts. Treatment of APP/PS1 mice with miRCD33 vector at an early age (2 months) was more effective at reducing Aβ plaque burden than intervening at later times (8 months). Furthermore, early intervention downregulated several microglial receptor transcripts (e.g. CD11c, CD47 and CD36) and pro-inflammatory activation genes (e.g. Tlr4 and Il1b). Marked reductions in the chemokine Ccl2 and the pro-inflammatory cytokine Tnfα were observed at the protein level in the brain from APP/PS1 mice treated with miRCD33 vector. Overall, our data indicate that CD33 is a viable target for AAV-based knock-down strategies to reduce ad pathology.
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