Recombinant Adeno-Associated Virus Serotype 6 Efficiently Transduces Primary Human Melanocytes

PLOS ONE, Dec 2019

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.

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Recombinant Adeno-Associated Virus Serotype 6 Efficiently Transduces Primary Human Melanocytes

et al. (2013) Recombinant Adeno-Associated Virus Serotype 6 Efficiently Transduces Primary Human Melanocytes. PLoS ONE 8(4): e62753. doi:10.1371/journal.pone.0062753 Recombinant Adeno-Associated Virus Serotype 6 Efficiently Transduces Primary Human Melanocytes Rod Dunbar 0 Hilary M. Sheppard 0 James E. Ussher 0 Daniel Verdon 0 Jennifer Chen 0 John A. Taylor 0 P. 0 Yiqun G. Shellman, University of Colorado, United States of America 0 1 School of Biological Sciences, University of Auckland , Auckland , New Zealand , 2 Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland , Auckland , New Zealand 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 nonintegrating vector for the transduction of primary human melanocytes. - Funding: This research was conducted during the tenure of a Clinical Research Training Fellowship from the Health Research Council of New Zealand (J.U.). The authors also acknowledge funding from the Maurice Wilkins Centre for Molecular Biodiscovery, NZ (http://www.mauricewilkinscentre.org/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Melanocytes are found in the skin, iris, and uvea and are responsible for the pigmentation which protects against the mutagenesis induced by ultraviolet radiation. Melanocyte dysfunction is important in the pathology of several heritable diseases, including albinism, as well as numerous disorders of pigmentation with more complex causes, such as vitiligo. Studies of melanocyte function are also highly relevant to the study of their malignant transformation, especially now that the first molecularly-targeted drugs with clinical efficacy against malignant melanoma are becoming available [1]. Vectors that efficiently transduce human melanocytes are therefore relevant to both fundamental studies of melanocyte biology and translational research. While transduction of primary human melanocytes by gamma-retroviral vectors has been reported [2,3,4], geneticin selection or co-culture with infected feeder cells, keratinocytes, or producer cell lines have been required to achieve high levels of transduction. Furthermore, retroviruses are unable to transduce non-dividing cells [5] and integration of the vector genome may lead to malignant transformation [6]. Lentiviral vectors have shown more promise, efficiently transducing primary human melanocytes with transduction rates higher than 90% and with transgene expression maintained for at least 4 weeks [7]. Lentiviral vectors have been widely used in in vitro studies of melanocyte biology [8,9,10], however their clinical use may be limited by the risk of insertional mutagenesis, although this risk may be lower than with gamma-retroviral vectors [11,12]. While primary melanocytes can be transduced by adenoviral vectors [13,14] their utility is limited by a lack of persistence and the immunogenicity of the vector [15,16]. Non-viral methods of gene transfer to melanocytes have also been reported. While the efficiency of transduction with lipidbased transfection reagents is low (,5%) [17,18], high rates of transduction of primary human melanocytes have been achieved with the NucleofectorTM (44%), although viability was significantly affected [18]. Adeno-associated virus (AAV) is a helper-dependent parvovirus that is not associated with any known pathology. Recombinant AAV (rAAV) expresses no viral proteins, is able to transduce both dividing and non-dividing cells, and is associated with persistent transgene expression [19,20]. It has been extensively evaluated as a gene therapy vector with hundreds of patients having received rAAV in clinical trials without any significant vector-a (...truncated)


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Hilary M. Sheppard, James E. Ussher, Daniel Verdon, Jennifer Chen, John A. Taylor, P. Rod Dunbar. Recombinant Adeno-Associated Virus Serotype 6 Efficiently Transduces Primary Human Melanocytes, PLOS ONE, 2013, Volume 8, Issue 4, DOI: 10.1371/journal.pone.0062753