Use of a Chimeric Adenovirus Vector Enhances BMP2 Production and Bone Formation

Abstract

Recombinant adenoviral vectors have potential for the treatment of a variety of musculoskeletal defects and such gene therapy systems have been a recent research focus in orthopedic surgery. In studies reported here, two different adenovirus vectors have been compared for their ability to transduce human bone marrow mesenchymal stem cells (hBM-MSCs) and elicit bone formation in vivo. Vectors consisted either of standard adenovirus type 5 (Ad5) vector or a chimeric adenovirus type 5 vector that contains an adenovirus type 35 fiber (Ad5F35), which has been recently demonstrated to bestow a different cellular tropism, and a complete cDNA encoding human bone morphogenetic 2 (BMP2). Studies were also conducted to compare the transduction efficiency of these vectors using enhanced green fluorescent protein (GFP). hBM-MSCs transduced with Ad5F35 vectors had higher levels of transgene expression than those transduced with Ad5 vectors. The results also demonstrate that hBM-MSCs lack the coxsackie-adenovirus receptor (CAR), which is responsible for cellular adsorption of Ad5. Therefore, the data suggest that Ad5 virus adsorption to hBM-MSCs is inefficient. Ad5BMP2- or Ad5F35BMP2-transduced hBM-MSCs were also compared in an in vivo heterotopic bone formation assay. Mineralized bone was radiologically identified only in muscle that received the Ad5F35BMP2 transduced hBM-MSCs. In summary, Ad5F35BMP2 can efficiently transduce hBM-MSCs leading to enhanced bone formation in vivo.

Progenitors in Peripheral Nerves Launch Heterotopic Ossification

“…Perhaps the reduced number of cells expressing PS in the mouse compared to human nerve arises because of the nature of the mouse model. Previous studies have shown that the BMP2-producing cells are rapidly cleared from the tissues approximately 5 days after their injection [11, 24, 25]. Thus, the less robust response in mice may reflect the low-level short-term expression of BMP2.…”

“…022137) transgenic mouse [17] was purchased from Jackson Laboratories and crossed with the R26R td Tomato red mouse to generate the mouse termed Wnt1 Cre2 :Ai9Tm. Heterotopic bone formation was established in the mice through an intramuscular injection into the quadriceps of C57BL/6 mouse fibroblasts (1 x 10 5 , 5 x 10 5 , 1 x 10 6 , or 5 x 10 6 in 300 μL of phosphate-buffered saline) transduced with an E1–E3 deleted adenovirus expressing BMP2 [11]. …”

Osteoinduction by ex vivo adenovirus-mediated BMP2 delivery is independent of cell type

“…BM-MSCs were derived from discarded human bone marrow from healthy donors as previously described. 11,12 Cells were isolated by gradient density centrifugation on Ficoll-Paquet PLUS (Amersham Pharmacia Biotech, Piscataway, NJ, USA), plated at 5 Â 10 6 cells/cm 2 in the medium above, and enzymatically collected before becoming confluent. 13 …”

“…Recently, we have described, a novel recombinant chimeric adenoviral BMP2 vector, 11,12 which consists of Ad5 containing a fiber gene of adenovirus type 35 (Ad5F35-BMP2). Comparison of Ad5F35-BMP2 with Ad5-BMP2 demonstrated the ability of the former to elicit significantly higher expression of BMP2 in vitro in human bone marrow MSCs (BM-MSCs).…”

“…Using nonobese diabetic severe immunodeficiency (NOD/SCID) mice, BM-MSCs transduced with Ad5F35-BMP2 demonstrated significantly greater potential to induce heterotopic bone formation than the same cells transduced with Ad5-BMP2. 11,12 We have previously demonstrated 12 by FACS analysis with a monoclonal antibody against the Coxsackie-adenovirus receptor (CAR) that human BM-MSCs are deficient in CAR that is a prerequisite for transduction with Ad5-BMP2, whereas Ad5F35-BMP2 transduces cells in a CAR-independent fashion.…”