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.
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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)