Transduction of the central nervous system after intracerebroventricular injection of adeno-associated viral vectors in neonatal and juvenile mice

Abstract

Several neurodevelopmental and neurodegenerative disorders affecting the central nervous system are potentially treatable via viral vector-mediated gene transfer. Adeno-associated viral (AAV) vectors have been used in clinical trials because of their desirable properties including a high degree of safety, efficacy, and stability. Major factors affecting tropism, expression level, and cell type specificity of AAV-mediated transgenes include encapsidation of different AAV serotypes, promoter selection, and the timing of vector administration. In this study, we evaluated the ability of single-stranded AAV2 vectors pseudotyped with viral capsids from serotype 9 (AAV2/9) to transduce the brain and target gene expression to specific cell types after intracerebroventricular injection into mice. Titer-matched AAV2/9 vectors encoding the enhanced green fluorescent protein (eGFP) reporter, driven by the cytomegalovirus (CMV) promoter, or the neuron-specific synapsin-1 promoter, were injected bilaterally into the lateral ventricles of C57/BL6 mice on postnatal day 5 (neonatal) or 21 (juvenile). Brain sections were analyzed 25 days after injection, using immunocytochemistry and confocal microscopy. eGFP immunohistochemistry after neonatal and juvenile administration of viral vectors revealed transduction throughout the brain including the striatum, hippocampus, cerebral cortex, and cerebellum, but with different patterns of cell-specific gene expression. eGFP expression was seen in astrocytes after treatment on postnatal day 5 with vectors carrying the CMV promoter, expanding the usefulness of AAVs for modeling and treating diseases involving glial cell pathology. In contrast, injection of AAV2/9-CMV-eGFP on postnatal day 21 resulted in preferential transduction of neurons. Administration of AAV2/9-eGFP with the synapsin-1 promoter on either postnatal day 5 or 21 resulted in widespread neuronal transduction. These results outline efficient methods and tools for gene delivery to the nervous system by direct, early postnatal administration of AAV vectors. Our findings highlight the importance of promoter selection and age of administration on the intensity, distribution, and cell type specificity of AAV transduction in the brain.

FIG. 1.

(A) Schematic representation of the three different AAV vector constructs studied. The vectors were single-stranded, contained ITR elements from AAV serotype 2, and were packaged in either serotype 5 or serotype 9 capsids. A WPRE was inserted into the AAV2/9 vectors downstream of the eGFP cDNA to enhance transcription. (B) Schematic diagram depicting bilateral intracerebroventricular injections on postnatal day 5 and postnatal day 21. Twenty-five days after AAV administrations (i.e., on postnatal day 30 or 46), animals were killed and brains were analyzed for GFP immunoreactivity. AAV, adeno-associated viral; CBA, chicken β-actin; CMV, cytomegalovirus; i.c.v., intracerebroventricular; ITR, inverted terminal repeat; WPRE, woodchuck hepatitis posttranscriptional regulatory element; eGFP, enhanced green fluorescent protein; hSynapsin, human synapsin-1.

FIG. 2.

eGFP expression in the mouse striatum. AAV2/9 vectors were injected on PND 5 (A–C, G–I) or on PND 21 (D–F, J–L). Different patterns of cell-specific gene expression were observed; AAV2/9-CMV-eGFP transduced predominantly protoplasmic astrocytes in PND 5 injections, as assessed by double labeling with anti-GFP and with the protoplasmic astrocyte-specific anti-S100β antibody (A–C). In contrast, AAV2/9-CMV-eGFP vectors injected on PND 21 and AAV2/9-SYN-eGFP injected on either PND 5 or PND 21 resulted in primarily neuronal transduction (D–L), as confirmed by co-staining with NeuN. Insets: Cells of interest at higher magnification. Scale bars: 50 μm. Color images available online at www.liebertpub.com/hgtb

FIG. 3.

eGFP expression in the retrosplenial cortex. In mice injected with AAV2/9-CMV-eGFP on PND 5, the majority of eGFP-positive cells in the retrosplenial cortex were confirmed to be protoplasmic astrocytes by double labeling with anti-S100β (A–C). In contrast, AAV2/9-CMV-eGFP injected on PND 21 and AAV2/9-SYN-eGFP injected on either PND 5 or PND 21 resulted in transduction of almost exclusively neurons (D–L), as confirmed by co-staining with anti-NeuN. Insets: Cells of interest at higher magnification. Scale bars: 50 μm. Color images available online at www.liebertpub.com/hgtb

FIG. 4.

Transgene expression in the CA1 region of the hippocampus after administration of AAV2/9-CMV-eGFP (A–F) or AAV2/9-SYN-eGFP (G–L) vector on PND 5 or PND 21. Brain sections from the hippocampus of AAV-injected mice were double labeled with anti-GFP and anti-NeuN. Insets: Cells of interest shown at higher magnification. Scale bars: 50 μm. Color images available online at www.liebertpub.com/hgtb

FIG. 5.

Transgene expression in the cerebellum. eGFP expression was seen in the cerebellum after injection of mice with AAV2/9-CMV-eGFP or AAV2/9-SYN-eGFP on PND 5 (A–L) but not in mice injected on PND 21 (data not shown). AAV2/9-CMV-eGFP resulted in widely distributed transduction of Purkinje neuron cell bodies and dendrites in the molecular layer of the cerebellum (A–C, D–F). With AAV2/9-SYN-eGFP, GFP transgene expression was seen in both Purkinje neurons and in some granule neurons (G–I, J–L). Co-staining with anti-calbindin was done to confirm the presence of GFP-positive cells in Purkinje neurons. M, molecular layer; G, granular layer; P, Purkinje cell layer. Insets: Selected Purkinje neurons shown at higher magnification. Scale bars: (A–C, G–I) 200 μm; (D–F, J–L) 50 μm. Color images available online at www.liebertpub.com/hgtb