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Recombinant adeno-associated virus serotype 6 efficiently transduces primary human melanocytes.

ABSTRACT: The study of melanocyte biology is important to understand their role in health and disease. However, current methods of gene transfer into melanocytes are limited by safety or efficacy. Recombinant adeno-associated virus (rAAV) has been extensively investigated as a gene therapy vector, is safe and is associated with persistent transgene expression without genome integration. There are twelve serotypes and many capsid variants of rAAV. However, a comparative study to determine which rAAV is most efficient at transducing primary human melanocytes has not been conducted. We therefore sought to determine the optimum rAAV variant for use in the in vitro transduction of primary human melanocytes, which could also be informative to future in vivo studies. We have screened eight variants of rAAV for their ability to transduce primary human melanocytes and identified rAAV6 as the optimal serotype, transducing 7-78% of cells. No increase in transduction was seen with rAAV6 tyrosine capsid mutants. The number of cells expressing the transgene peaked at 6-12 days post-infection, and transduced cells were still detectable at day 28. Therefore rAAV6 should be considered as a non-integrating vector for the transduction of primary human melanocytes.

SUBMITTER: Sheppard HM

PROVIDER: S-EPMC3640030 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Recombinant adeno-associated virus serotype 6 efficiently transduces primary human melanocytes.

Sheppard Hilary M HM Ussher James E JE Verdon Daniel D Chen Jennifer J Taylor John A JA Dunbar P Rod PR

PloS one 20130430 4

The study of melanocyte biology is important to understand their role in health and disease. However, current methods of gene transfer into melanocytes are limited by safety or efficacy. Recombinant adeno-associated virus (rAAV) has been extensively investigated as a gene therapy vector, is safe and is associated with persistent transgene expression without genome integration. There are twelve serotypes and many capsid variants of rAAV. However, a comparative study to determine which rAAV is mos ...[more]

PMID: 23646140

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Project 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.

Dataset Information

Recombinant adeno-associated virus serotype 6 efficiently transduces primary human melanocytes.

Publications

Recombinant adeno-associated virus serotype 6 efficiently transduces primary human melanocytes.

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