A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1-9) and one engineered adeno-associated virus serotype.

Jon P Connelly,Matthew H Porteus,Jenny C Barker,Robert J Steininger,Matthew L Hirsch,Brian L Ellis

doi:10.1186/1743-422x-10-74

Abstract

BackgroundThe ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research. Viruses can be a useful tool for this delivery, particularly in difficult to transfect cell types. Adeno-associated virus (AAV) is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without inducing any known pathogenicity. There are now a number of natural for that designed AAV serotypes that each has a differential ability to infect a variety of cell types. Although transduction studies have been completed, the bulk of the studies have been done in vivo, and there has never been a comprehensive study of transduction ex vivo/in vitro.MethodsEach cell type was infected with each serotype at a multiplicity of infection of 100,000 viral genomes/cell and transduction was analyzed by flow cytometry + .ResultsWe found that AAV1 and AAV6 have the greatest ability to transduce a wide range of cell types, however, for particular cell types, there are specific serotypes that provide optimal transduction.ConclusionsIn this work, we describe the transduction efficiency of ten different AAV serotypes in thirty-four different mammalian cell lines and primary cell types. Although these results may not be universal due to numerous factors such as, culture conditions and/ or cell growth rates and cell heterogeneity, these results provide an important and unique resource for investigators who use AAV as an ex vivo gene delivery vector or who work with cells that are difficult to transfect.

Highlights

The ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research
Even though single-strand AAV (ssAAV) has a larger cloning capacity than self-complementary AAV (scAAV), we chose scAAV because of the overall improved transgene expression of its vectors compared to ssAAV vectors as this report was intended to be a straightforward capsid comparison
Because some cells have been reported to be refractory to Adenoassociated virus (AAV) transduction, we wanted to use the most efficient genome technology helping to reduce the possibility that timing and amount of transgene expression would bias the results

Summary

Introduction

The ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research. Adenoassociated virus (AAV) is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without inducing any known pathogenicity. AAV is a generally useful vector for gene transfer in a wide range of cell types. One of the barriers to efficient expression of the transgene is the conversion of the single-strand AAV (ssAAV) genome into a duplexed single DNA molecule [4]. The limitation in transgene expression from ssAAV vectors has been improved by the development of self-complementary AAV (scAAV) vectors in which the single-stranded AAV genome self-hybridizes to form duplex DNA (Figure 1). ScAAV vectors have shown earlier onset of transgene expression and overall higher transduction efficiencies than ssAAV vectors [4,5] The limitation in transgene expression from ssAAV vectors has been improved by the development of self-complementary AAV (scAAV) vectors in which the single-stranded AAV genome self-hybridizes to form duplex DNA (Figure 1). scAAV vectors have shown earlier onset of transgene expression and overall higher transduction efficiencies than ssAAV vectors [4,5]

Methods

Results

Conclusion