In vitro evaluation of a double-stranded self-complementary adeno-associated virus type2 vector in bone marrow stromal cells for bone healing

Abstract

Background: Both adenoviral and lentiviral vectors have been successfully used to induce bone repair by over-expression of human bone morphogenetic protein 2 (BMP-2) in primary rat bone marrow stromal cells in pre-clinical models of ex vivo regional gene therapy. Despite being a very efficient means of gene delivery, there are potential safety concerns that may limit the adaptation of these viral vectors for clinical use in humans. Recombinant adeno-associated viral (rAAV) vector is a promising viral vector without known pathogenicity in humans and has the potential to be an effective gene delivery vehicle to enhance bone repair. In this study, we investigated gene transfer in rat and human bone marrow stromal cells in order to evaluate the effectiveness of the self-complementary AAV vector (scAAV) system, which has higher efficiency than the single-stranded AAV vector (ssAAV) due to its unique viral genome that bypasses the rate-limiting conversion step necessary in ssAAV.

Methods: Self-complementaryAAV2 encoding GFP and BMP-2 (scAAV2-GFP and scAAV2-BMP-2) were used to transduce human and rat bone marrow stromal cells in vitro, and subsequently the levels of GFP and BMP-2 expression were assessed 48 hours after treatment. In parallel experiments, adenoviral and lentiviral vector mediated over-expression of GFP and BMP-2 were used for comparison.

Results: Our results demonstrate that the scAAV2 is not capable of inducing significant transgene expression in human and rat bone marrow stromal cells, which may be associated with its unique tropism.

Conclusions: In developing ex vivo gene therapy regimens, the ability of a vector to induce the appropriate level of transgene expression needs to be evaluated for each cell type and vector used.

Figure 1

Schematic Drawings of the Viral Vectors A) AAV vector that consists of inverted terminal repeats (ITR) at 3' and 5' ends with BMP-2 or GFP under the control of CMV promoter and SV40 poly(A), B) Lentiviral vector that consists of long terminal repeats (LTR) with RhMLV promoter driving the expression of BMP-2 or GFP and C) Adenoviral vector that consists of ITRs and BMP-2 or GFP under the control of CMV promoter as well as SV40 poly(A). The adenoviral vector carries deletions in E1 and E3 regions rendering it replication deficient except in 293 cell lines (including 293T cells) that are capable of substituting E1 function, hence the toxicity of this vector to 293T cells. ψ: packaging signal, cppt: central polypurine tract, RRE: Rev-responsible element, SV40 poly(A): simian virus poly adenilation signal sequence

Figure 2

GFP Expression in Viral Transduced Cells 1 million rat and human bone marrow stromal cells (BMSCs) and 293T cells were transduced with AAV-GFP, LV-GFP and Ad-GFP and the GFP expression was assessed 48 hours after transduction. Non-transduced cells served as negative control. In comparison with the non-transduced controls, AAV-GFP transduced 293T cells show strong eGFP expression, but rat and human bone marrow stromal cells (BMSCs) did not exhibit expression levels as strong as 293T cells. LV-GFP and Ad-GFP transduced cells showed strong GFP expression in all transduced cell types except for Ad-GFP transduced 293T cells in which the viral replication causes cell death.

Figure 3

BMP-2 Production by Viral Transduced Cells 1 million rat bone marrow stromal cells (BMSCs) and 293T cells were transduced with AAV-BMP-2, LV-BMP-2 and Ad-BMP-2 and the BMP-2 production was quantified 48 hours after transduction. Non-transduced cells served as negative control. A) Amongst the three viral vectors used to over express BMP-2, AAV-BMP-2 induced the highest amount of BMP-2 production in 293T cells. Transducing the 293T cells with Ad-BMP-2 induced cell death after 24 hours in culture. B) AAV-BMP-2 transduced rat bone marrow stromal cells (BMSCs) did not produce any detectable amount of BMP-2 as opposed to LV-BMP-2 and Ad-BMP-2 transduced rat bone marrow stromal cells which made significantly higher amounts of BMP-2.*: P value < 0.05 compared to all other groups, †: P value < 0.05 compared to non-transduced group