Cargando…

Recombinant Adeno-Associated Virus Serotype 6 (rAAV6) Potently and Preferentially Transduces Rat Astrocytes In vitro and In vivo

Recombinant adeno-associated virus vectors are an increasingly popular tool for gene delivery to the CNS because of their non-pathological nature, low immunogenicity, and ability to stably transduce dividing and non-dividing cells. One of the limitations of rAAVs is their preferential tropism for ne...

Descripción completa

Detalles Bibliográficos
Autores principales: Schober, Alexandra L., Gagarkin, Dmitriy A., Chen, Ying, Gao, Guangping, Jacobson, Lauren, Mongin, Alexander A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5104754/
https://www.ncbi.nlm.nih.gov/pubmed/27891076
http://dx.doi.org/10.3389/fncel.2016.00262
_version_ 1782466780862611456
author Schober, Alexandra L.
Gagarkin, Dmitriy A.
Chen, Ying
Gao, Guangping
Jacobson, Lauren
Mongin, Alexander A.
author_facet Schober, Alexandra L.
Gagarkin, Dmitriy A.
Chen, Ying
Gao, Guangping
Jacobson, Lauren
Mongin, Alexander A.
author_sort Schober, Alexandra L.
collection PubMed
description Recombinant adeno-associated virus vectors are an increasingly popular tool for gene delivery to the CNS because of their non-pathological nature, low immunogenicity, and ability to stably transduce dividing and non-dividing cells. One of the limitations of rAAVs is their preferential tropism for neuronal cells. Glial cells, specifically astrocytes, appear to be infected at low rates. To overcome this limitation, previous studies utilized rAAVs with astrocyte-specific promoters or assorted rAAV serotypes and pseudotypes with purported selectivity for astrocytes. Yet, the reported glial infection rates are not consistent from study to study. In the present work, we tested seven commercially available recombinant serotypes– rAAV1, 2, and 5 through 9, for their ability to transduce primary rat astrocytes [visualized via viral expression of green fluorescent protein (GFP)]. In cell cultures, rAAV6 consistently demonstrated the highest infection rates, while rAAV2 showed astrocytic transduction in some, but not all, of the tested viral batches. To verify that all rAAV constructs utilized by us were viable and effective, we confirmed high infectivity rates in retinal pigmented epithelial cells (ARPE-19), which are known to be transduced by numerous rAAV serotypes. Based on the in vitro results, we next tested the cell type tropism of rAAV6 and rAAV2 in vivo, which were both injected in the barrel cortex at approximately equal doses. Three weeks later, the brains were sectioned and immunostained for viral GFP and the neuronal marker NeuN or the astrocytic marker GFAP. We found that rAAV6 strongly and preferentially transduced astrocytes (>90% of cells in the virus-infected areas), but not neurons (∼10% infection rate). On the contrary, rAAV2 preferentially infected neurons (∼65%), but not astrocytes (∼20%). Overall, our results suggest that rAAV6 can be used as a tool for manipulating gene expression (either delivery or knockdown) in rat astrocytes in vivo.
format Online
Article
Text
id pubmed-5104754
institution National Center for Biotechnology Information
language English
publishDate 2016
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-51047542016-11-25 Recombinant Adeno-Associated Virus Serotype 6 (rAAV6) Potently and Preferentially Transduces Rat Astrocytes In vitro and In vivo Schober, Alexandra L. Gagarkin, Dmitriy A. Chen, Ying Gao, Guangping Jacobson, Lauren Mongin, Alexander A. Front Cell Neurosci Neuroscience Recombinant adeno-associated virus vectors are an increasingly popular tool for gene delivery to the CNS because of their non-pathological nature, low immunogenicity, and ability to stably transduce dividing and non-dividing cells. One of the limitations of rAAVs is their preferential tropism for neuronal cells. Glial cells, specifically astrocytes, appear to be infected at low rates. To overcome this limitation, previous studies utilized rAAVs with astrocyte-specific promoters or assorted rAAV serotypes and pseudotypes with purported selectivity for astrocytes. Yet, the reported glial infection rates are not consistent from study to study. In the present work, we tested seven commercially available recombinant serotypes– rAAV1, 2, and 5 through 9, for their ability to transduce primary rat astrocytes [visualized via viral expression of green fluorescent protein (GFP)]. In cell cultures, rAAV6 consistently demonstrated the highest infection rates, while rAAV2 showed astrocytic transduction in some, but not all, of the tested viral batches. To verify that all rAAV constructs utilized by us were viable and effective, we confirmed high infectivity rates in retinal pigmented epithelial cells (ARPE-19), which are known to be transduced by numerous rAAV serotypes. Based on the in vitro results, we next tested the cell type tropism of rAAV6 and rAAV2 in vivo, which were both injected in the barrel cortex at approximately equal doses. Three weeks later, the brains were sectioned and immunostained for viral GFP and the neuronal marker NeuN or the astrocytic marker GFAP. We found that rAAV6 strongly and preferentially transduced astrocytes (>90% of cells in the virus-infected areas), but not neurons (∼10% infection rate). On the contrary, rAAV2 preferentially infected neurons (∼65%), but not astrocytes (∼20%). Overall, our results suggest that rAAV6 can be used as a tool for manipulating gene expression (either delivery or knockdown) in rat astrocytes in vivo. Frontiers Media S.A. 2016-11-11 /pmc/articles/PMC5104754/ /pubmed/27891076 http://dx.doi.org/10.3389/fncel.2016.00262 Text en Copyright © 2016 Schober, Gagarkin, Chen, Gao, Jacobson and Mongin. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Neuroscience
Schober, Alexandra L.
Gagarkin, Dmitriy A.
Chen, Ying
Gao, Guangping
Jacobson, Lauren
Mongin, Alexander A.
Recombinant Adeno-Associated Virus Serotype 6 (rAAV6) Potently and Preferentially Transduces Rat Astrocytes In vitro and In vivo
title Recombinant Adeno-Associated Virus Serotype 6 (rAAV6) Potently and Preferentially Transduces Rat Astrocytes In vitro and In vivo
title_full Recombinant Adeno-Associated Virus Serotype 6 (rAAV6) Potently and Preferentially Transduces Rat Astrocytes In vitro and In vivo
title_fullStr Recombinant Adeno-Associated Virus Serotype 6 (rAAV6) Potently and Preferentially Transduces Rat Astrocytes In vitro and In vivo
title_full_unstemmed Recombinant Adeno-Associated Virus Serotype 6 (rAAV6) Potently and Preferentially Transduces Rat Astrocytes In vitro and In vivo
title_short Recombinant Adeno-Associated Virus Serotype 6 (rAAV6) Potently and Preferentially Transduces Rat Astrocytes In vitro and In vivo
title_sort recombinant adeno-associated virus serotype 6 (raav6) potently and preferentially transduces rat astrocytes in vitro and in vivo
topic Neuroscience
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5104754/
https://www.ncbi.nlm.nih.gov/pubmed/27891076
http://dx.doi.org/10.3389/fncel.2016.00262
work_keys_str_mv AT schoberalexandral recombinantadenoassociatedvirusserotype6raav6potentlyandpreferentiallytransducesratastrocytesinvitroandinvivo
AT gagarkindmitriya recombinantadenoassociatedvirusserotype6raav6potentlyandpreferentiallytransducesratastrocytesinvitroandinvivo
AT chenying recombinantadenoassociatedvirusserotype6raav6potentlyandpreferentiallytransducesratastrocytesinvitroandinvivo
AT gaoguangping recombinantadenoassociatedvirusserotype6raav6potentlyandpreferentiallytransducesratastrocytesinvitroandinvivo
AT jacobsonlauren recombinantadenoassociatedvirusserotype6raav6potentlyandpreferentiallytransducesratastrocytesinvitroandinvivo
AT monginalexandera recombinantadenoassociatedvirusserotype6raav6potentlyandpreferentiallytransducesratastrocytesinvitroandinvivo