Capsid serotype and timing of injection determines AAV transduction in the neonatal mice brain - PubMed (original) (raw)

. 2013 Jun 25;8(6):e67680.

doi: 10.1371/journal.pone.0067680. Print 2013.

Awilda Rosario, Pedro Cruz, Zoe Siemienski, Carolina Ceballos-Diaz, Keith Crosby, Karen Jansen, David R Borchelt, Ji-Yoen Kim, Joanna L Jankowsky, Todd E Golde, Yona Levites

Affiliations

Capsid serotype and timing of injection determines AAV transduction in the neonatal mice brain

Paramita Chakrabarty et al. PLoS One. 2013.

Abstract

Adeno-associated virus (AAV) mediated gene expression is a powerful tool for gene therapy and preclinical studies. A comprehensive analysis of CNS cell type tropism, expression levels and biodistribution of different capsid serotypes has not yet been undertaken in neonatal rodents. Our previous studies show that intracerebroventricular injection with AAV2/1 on neonatal day P0 results in widespread CNS expression but the biodistribution is limited if injected beyond neonatal day P1. To extend these observations we explored the effect of timing of injection on tropism and biodistribution of six commonly used pseudotyped AAVs delivered in the cerebral ventricles of neonatal mice. We demonstrate that AAV2/8 and 2/9 resulted in the most widespread biodistribution in the brain. Most serotypes showed varying biodistribution depending on the day of injection. Injection on neonatal day P0 resulted in mostly neuronal transduction, whereas administration in later periods of development (24-84 hours postnatal) resulted in more non-neuronal transduction. AAV2/5 showed widespread transduction of astrocytes irrespective of the time of injection. None of the serotypes tested showed any microglial transduction. This study demonstrates that both capsid serotype and timing of injection influence the regional and cell-type distribution of AAV in neonatal rodents, and emphasizes the utility of pseudotyped AAV vectors for translational gene therapy paradigms.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Intracerebroventricular injection of AAV2/1-EGFP at different neonatal ages results in distinctive transduction pattern.

Newborn mice were injected with AAV2/1-EGFP into cerebral ventricles at intervals of 6–7 hours till 36 hours postnatal. Representative brain sections from mice aged P21 were stained with anti-EGFP antibody. A representative image of an EGFP expressing neuron (arrowhead) and an astrocyte (arrow) from the cortex is shown (inset, a). Areas of interest, referenced in the manuscript, that show appreciable EGFP expression have been marked as; ctx, cortex; cereb, cerebellum; hpc, hippocampal formation; midbr, midbrain; sn, substantia nigra; str, striatum; olf, olfactory bulb; thal, thalamus; *, lateral ventricle; #, 4th ventricle. (n = 4/group). Scale bar, 500 µm.

Figure 2

Figure 2. Comparative biodistribution of pseudotyped AAVs injected at different neonatal ages in mice detected by Xenogen IVIS Spectrum fluorescence imager.

Wild type mice were injected into the cerebral ventricles with pseudotyped AAV2/n-EGFP on different neonatal days (P0, P2 and P3) and analyzed on day P21. Representative pseudo color images from each group (standardized to non-injected control brain) show differential fluorescent intensities depending on serotype injected and timing of virus injection. n = 3–4/time point/serotype.

Figure 3

Figure 3. Immunohistochemical analysis of EGFP biodistribution following intracerebrovcentricular AAV delivery in neonatal mice.

Representative sections of 3 week old mice injected on neonatal day P0, P2 or P3 show that the biodistribution of EGFP is dependent on serotype used and the time of injection. a,g,m, AAV2/1; b,h,n, AAV2/2; c,i,o, AAV2/5; d, j, p, AAV2/7; e, k, q, AAV2/8; f, l, r, AAV2/9; a-f, P0 injection, g-l, P2 injection, m-r, P3 injection.

Figure 4

Figure 4. AAV2/n-EGFP tropism for neurons following intracerebrovcentricular delivery in neonatal mice.

Representative tricolor merged fluorescent photomicrograph from 3-week-old wild type mice injected on neonatal day P0, P2 or P3 with pseudotyped AAV2/n. Paraffin embedded brain sections were co-labeled with anti EGFP antibody (488 nm-green), anti β-tubulin (568 nm-red) and DAPI counterstain (blue). Images were scanned from the cortex of mice injected at P0 (a-f), P2 (g-l) or P3 (m-r) with AAV2/1 (a,g,m), AAV2/2 (b,h,n), AAV2/5 (c,i,o), AAV2/7 (d,j,p), AAV2/8 (e,k,q) and AAV2/9 (f,l,r). Arrowhead, EGFP expressing neuron; arrow, EGFP expressing astrocyte. n = 3–4/serotype/time of injection. Magnification 400x.

Figure 5

Figure 5. AAV2/n-EGFP tropism for astrocytes following intracerebroventricular delivery in neonatal mice.

Representative tricolor merged fluorescent photomicrograph from 3 week old wild type mice injected on neonatal day P0, P2 or P3 with pseudotyped AAV2/n. Paraffin embedded brain sections were co-labeled with anti EGFP antibody (488 nm-green), anti GFAP-Cy5 (568 nm-red) and DAPI counterstain (blue). Images were scanned from the cortex of mice injected at P0 (a-f), P2 (g-l), P3 (m-r) with AAV2/1 (a,g,m), AAV2/2 (b,h,n), AAV2/5 (c,i,o), AAV2/7 (d,j,p), AAV2/8 (e,k,q) and AAV2/9 (f,l,r). Arrow, EGFP expressing astrocyte; arrowhead, EGFP expressing neuron. n = 3–4/serotype/time of injection. Magnification 400x.

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