Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9.

Chondrocytes form and maintain the extracellular matrix of cartilage. The cells can be isolated from cartilage for applications such as tissue engineering, but their expansion in monolayer culture causes a progressive loss of chondrogenic phenotype. In this work, we have investigated the isolation of human articular chondrocytes from osteoarthritic (OA) cartilage at joint replacement, their expansion in monolayer culture, and their transduction with adenoviral, retroviral, and lentiviral vectors, using the gene encoding green fluorescent protein as a marker gene. The addition of growth factors (transforming growth factor beta(1), fibroblast growth factor 2, and platelet-derived growth factor BB) during cell culture was found to greatly increase cell proliferation and thereby to selectively enhance the efficiency of transduction with retrovirus. With adenoviral and lentiviral vectors the transduction efficiency achieved was 95 and 85%, respectively. Using growth factor-supplemented medium with a retroviral vector, efficiency in excess of 80% was achieved. The expression was stable for several months with both retrovirus and lentivirus when analyzed by fluorescence-activated cell-sorting flow analysis and immunoblotting. Transduction with SOX9 was investigated as a method to reinitiate cartilage matrix gene expression in passaged human OA chondrocytes. Endogenous collagen II expression (both mRNA and protein) was increased in monolayer culture using both adenoviral and retroviral vectors. Furthermore, collagen II gene expression in chondrocytes retrovirally transduced with SOX9 was stimulated by alginate bead culture, whereas in control chondrocytes it was not. These results demonstrated methods for rapid expansion and highly efficient transduction of human OA chondrocytes and the potential for the recovery of key features of chondrocyte phenotype by transduction with SOX9.

Immunization with a recombinant adenovirus encoding a lymphoma idiotype: induction of tumor-protective immunity and identification of an idiotype-specific T cell epitope.

The Ig Id of a B cell lymphoma is a tumor-specific Ag, although as a self-Ag it is likely to be a weak immunogen. Provision of a foreign gene may enhance the immunogenicity of the idiotype. Viral vectors allow highly efficient transfer of genetic material and are themselves innately immunogenic. We have investigated the ability of recombinant adenoviral vectors, encoding the idiotypic gene with or without fusion to the human Fc region, to produce anti-idiotypic Ab- and T cell-mediated responses in a syngeneic BALB/c A20 murine lymphoma model. The idiotypic V(H) and V(L) sequences were assembled as a single chain variable fragment (scFv) and adenoviral vectors encoding the A20 scFv (Ad.A20) and A20 scFv linked to the Fc fragment of human IgG1 (Ad.A20hFc) were constructed. A single immunization of BALB/c mice with Ad.A20hFc but not Ad.A20 induced a specific anti-idiotypic Ab response. T cell lines generated from mice vaccinated with either vector displayed specific cytotoxicity, proliferation, and IFN-gamma release against a syngeneic dendritic cell line transduced using a retroviral vector to express the A20 scFv idiotype (XS52.A1.A20). Importantly, both T cell lines lysed the A20 lymphoma cells. An immunodominant H-2K(d)-restricted CD8(+) T cell peptide, DYWGQGTEL (A20[106-114]), was identified as a naturally occurring A20 scFv epitope. A single immunization with Ad.A20hFc but not Ad.A20 provided protection in >40% of animals challenged with a lethal dose of the A20 tumor line and was more effective, in this model, than a previously optimized plasmid vaccine.