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

Abstract

Background: The 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.

Methods: Each 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 + .

Results: We 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.

Conclusions: In 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.

Figure 1

A schematic representation of the self-complimentary AAV (scAAV) genome. We constructed a scAAV genome (double black lines) that included an eGFP reporter gene (in green) driven by the CMV promoter (black arrow) that allowed for efficient and quantitative analysis of transduction. This construct also included a poly A sequence (black line) and inverted terminal repeats (ITRs) in blue)).

Figure 2

scAAV transduction of human primary and immortalized cells. A) Human primary cells and B) human immortalized cell lines were transduced with eGFP scAAV at a multiplicity of infection (MOI) of 100,000 viral genomes (vg)/ cell. The cells were analyzed by flow cytometry at 48 hours post-infection for the percentage that were GFP positive. The number in the box is the actual percentage of GFP positive cells with that serotype. * = Transduction less than 0.01% but greater than 0.0%.

Figure 3

scAAV transduction of murine primary cells and murine, hamster, and monkey immortalized cells. A) Murine primary cells and B) murine, hamster, and monkey immortalized cell lines were transduced with eGFP scAAV at a multiplicity of infection (MOI) of 100,000 vg/ cell. The cells were analyzed by flow cytometry at 48 hours post-infection for the percentage that were GFP positive. The number in the box is the actual percentage of GFP positive cells with that serotype.