High-Efficiency Transduction of Fibroblasts and Mesenchymal Stem Cells by Tyrosine-Mutant AAV2 Vectors for Their Potential Use in Cellular Therapy

Abstract

Adeno-associated virus 2 (AAV2) vectors transduce fibroblasts and mesenchymal stem cells (MSCs) inefficiently, which limits their potential widespread applicability in combinatorial gene and cell therapy. We have reported that AAV2 vectors fail to traffic efficiently to the nucleus in murine fibroblasts. We have also reported that site-directed mutagenesis of surface-exposed tyrosine residues on viral capsids leads to improved intracellular trafficking of the mutant vectors, and the transduction efficiency of the single tyrosine-mutant vectors is *10-fold higher in human cells. In the current studies, we evaluated the transduction efficiency of single as well as multiple tyrosine-mutant AAV2 vectors in murine fibroblasts. Our results indicate that the Y444F mutant vectors transduce these cells most efficiently among the seven single-mutant vectors, with >30-fold increase in transgene expression compared with the wild-type vectors. When the Y444F mutation is combined with additional mutations (Y500F and Y730F), the transduction efficiency of the triple-mutant vectors is increased by *130-fold and the viral intracellular trafficking is also significant improved. Similarly, the triple-mutant vectors are capable of transducing up to 80-90% of bone marrow-derived primary murine as well as human MSCs. Thus, high-efficiency transduction of fibroblasts with reprogramming genes to generate induced pluripotent stem cells, and the MSCs for delivering therapeutic genes, should now be feasible with the tyrosine-mutant AAV vectors. Overview SummaryFibroblasts and mesenchymal stem cells (MSCs) are promising targets for gene and cell therapy. Recombinant adeno-associated virus 2 (AAV2) vectors are currently in use in several Phase I/II clinical trials for gene therapy. However, the existing data show that AAV2 vector-mediated transduction of fibroblasts and MSCs is inefficient. We observed that AAV2 vectors containing mutations in three surface-exposed tyrosine residues significantly increase transduction efficiency in fibroblasts, as well as in both human and murine primary MSCs. The increased transduction efficiency of these triple-mutant AAV2 vectors correlates well with improved viral intracellular trafficking in fibroblasts. These data provide strong evidence that high-efficiency gene delivery and transgene expression by AAV vectors are indeed feasible, which should facilitate the generation of induced pluripotent stem cells, as well as the use of MSCs in combinatorial gene and cell therapy.

Reprogramming Adipose Tissue-Derived Mesenchymal Stem Cells into Pluripotent Stem Cells by a Mutant Adeno-Associated Viral Vector

“…S1C). Consistent with observations from previous studies (Blin et al, 2010;Li et al, 2010), this finding indicated that tyrosine mutant AAV vectors have an advantage for gene delivery to AT-MSCs. Quantitative analysis by FACS also showed that rAAV2.3m infected AT-MSCs efficiently ( Fig.…”

“…Previous studies, including our research, showed that rAAV-mediated transgene expression could be sustained for several weeks in nondividing cells (Song et al, 1998(Song et al, , 2001a. In addition, triple tyrosine mutant AAV2 vectors (Y444 + 500 + 730F) showed high infection efficiency toward primary murine bone marrow-derived mesenchymal stem cells (BM-MSCs) and fibroblasts (Li et al, 2010). These features make the tyrosine mutant rAAV vector a good candidate to generate safer iPS cells.…”

“…There is no evidence showing that wild-type or recombinant AAV is associated with any human disease. One study reported that AAV2.3m shows relatively high infection efficiency toward murine bone marrow-derived mesenchymal stem cells and fibroblasts (Li et al, 2010), which makes AAV2.3m a unique candidate vector for generating iPS cells. In the present study we successfully generated mouse iPS cells, using a single polycistronicalAAV2.3m vector expressing three transcription factors: mouse Klf4, Oct4, and Sox2.…”

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

“…Indeed, we have used AAV3 vectors containing the AFP promoter to target transgene expression in liver cancer cells, but not in normal hepatocytes 2 , and studies are currently underway to test the efficacy of this approach in a murine xenograft model of liver cancer. In our additional studies, we have observed that the transduction efficiency of various AAV serotype vectors can be significantly augmented by site-directed mutagenesis of surface-exposed tyrosine residues in the viral capsids [6][7][8][9][10][11][12][13][14] . Since 6 of 7 surface-exposed tyrosines are also conserved in AAV3, we have performed site-directed mutagenesis of these residues and observed that the transduction efficiency of tyrosine-mutant AAV3 vectors is significantly enhanced in human liver cancer cells (unpublished data).…”

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

“…They have rapid initial proliferation and no requirement for specialized medium or activation protocols. Fibroblasts can be efficiently transfected using biological, chemical, and physical protocols 4,5 . There is a possibility to store ears for up to 10 days at RT prior to establishing cell cultures.…”