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High throughput screening of novel AAV capsids identifies variants for transduction of adult NSCs within the subventricular zone.

ABSTRACT: The adult mammalian brain entails a reservoir of neural stem cells (NSCs) generating glial cells and neurons. However, NSCs become increasingly quiescent with age, which hampers their regenerative capacity. New means are therefore required to genetically modify adult NSCs for re-enabling endogenous brain repair. Recombinant adeno-associated viruses (AAVs) are ideal gene-therapy vectors due to an excellent safety profile and high transduction efficiency. We thus conducted a high-throughput screening of 177 intraventricularly injected barcoded AAV variants profiled by RNA sequencing. Quantification of barcoded AAV mRNAs identified two synthetic capsids, peptide-modified derivative of wild-type AAV9 (AAV9_A2) and peptide-modified derivative of wild-type AAV1 (AAV1_P5), both of which transduce active and quiescent NSCs. Further optimization of AAV1_P5 by judicious selection of the promoter and dose of injected viral genomes enabled labeling of 30%-60% of the NSC compartment, which was validated by fluorescence-activated cell sorting (FACS) analyses and single-cell RNA sequencing. Importantly, transduced NSCs readily produced neurons. The present study identifies AAV variants with a high regional tropism toward the ventricular-subventricular zone (v-SVZ) with high efficiency in targeting adult NSCs, thereby paving the way for preclinical testing of regenerative gene therapy.

SUBMITTER: Kremer LPM

PROVIDER: S-EPMC8427210 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The adult mammalian brain entails a reservoir of neural stem cells (NSCs) generating glial cells and neurons. However, NSCs become increasingly quiescent with age, which hampers their regenerative capacity. New means are therefore required to genetically modify adult NSCs for re-enabling endogenous brain repair. Recombinant adeno-associated viruses (AAVs) are ideal gene therapy vectors due to an excellent safety profile and high transduction efficiency. We thus conducted a high-throughput screening of 157 intraventricularly injected barcoded AAV variants profiled by transcriptome sequencing. Transcriptome analysis identified two synthetic capsids, AAV9_A2 and AAV1_P5 transducing active and quiescent NSCs. Further optimization of AAV1_P5 by judicious selection of promoter and dose of injected viral genomes enabled targeting of 57% of the NSC compartment. Importantly, transduced NSC readily produced neurons. The present study identifies AAV variants with a high specificity and transduction efficiency of adult NSCs thereby paving the way for preclinical testing of regenerative gene therapy. We identified a synthetic Adeno-associated virus (AAV) capsid, AAV1_P5, that transduces active and quiescent neural stem cells (NSCs). In order to fully characterize the identity of AAV1_P5-transduced cells, as well as potential changes arising by the AAV-transduction itself, we profiled these cells and untransduced ones from the same mouse by single cell RNA sequencing. To this end, three months-old eYFP-reporter mice were injected with 109 vg/mouse AAV1_P5 harboring the CMV_Cre construct. Transduction induces expression of eYFP. 37 days post injection, we isolated cells from the v-SVZ and other brain regions. More precisely, we isolated labeled cells of the ventricular-subventricular zone and the striatum, rostral migratory stream (RMS) and olfactory bulb, here referred to as rest of the brain (RoB). To capture the remaining unlabeled cells of the NSC-lineage in the v-SVZ, we also isolated GLAST+ v-SVZ cells. Two samples of two pooled mice each were subjected to single cell RNA sequencing.

Project description:The subventricular zone (SVZ) in lateral ventricles is the largest neurogenic region in adult brain containing high amounts of copper (Cu). This study aims to define the role of Cu in adult neurogenesis by chelating labile Cu ions using a well-established Cu chelator D-Penicillamine (D-Pen). A neurosphere model derived from adult mouse SVZ tissues was established and characterized for its functionality with regards to neural stem/progenitor cells (NSPCs). Applying D-Pen in cultured neurospheres significantly reduced intracellular Cu levels and reversed the Cu-induced suppression of NSPC's differentiation and migration. An in vivo intracerebroventricular (ICV) infusion model was subsequently established to infuse D-Pen directly into the lateral ventricle. Metal analyses revealed a selective reduction of Cu in SVZ by 13.1% (p = 0.19) and 21.4% (p < 0.05) following D-Pen infusions at low (0.075 μg/h) and high (0.75 μg/h) doses for 28 days, respectively, compared to saline-infused controls. Immunohistochemical studies revealed that the 7-day, low-dose D-Pen infusion significantly increased Ki67(+)/Nestin(+) cell counts in SVZ by 28% (p < 0.05). Quantification of BrdU(+)/doublecortin (DCX)(+) newborn neuroblasts in the rostral migration stream (RMS) and olfactory bulb (OB) further revealed that the short-term, low-dose D-Pen infusion, as compared with saline-infused controls, resulted in more newborn neuroblasts in OB, while the high-dose D-Pen infusion showed fewer newborn neuroblasts in OB but with more arrested in the RMS. Long-term (28-day) infusion revealed similar outcomes. The qPCR data from neurosphere experiments revealed altered expressions of mRNAs encoding key proteins known to regulate SVZ adult neurogenesis, including, but not limited to, Shh, Dlx2, and Slit1, in response to the changed Cu level in neurospheres. Further immunohistochemical data indicated that Cu chelation also altered the expression of high-affinity copper uptake protein 1 (CTR1) and metallothionein-3 (MT3) in the SVZ as well as CTR1 in the choroid plexus, a tissue regulating brain Cu homeostasis. Taken together, this study provides first-hand evidence that a high Cu level in SVZ appears likely to maintain the stability of adult neurogenesis in this neurogenic zone.

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High throughput screening of novel AAV capsids identifies variants for transduction of adult NSCs within the subventricular zone.

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