A comparison of in vivo gene delivery methods for antisense therapy in ligament healing

Abstract

To determine the most efficient in vivo delivery method of oligonucleotides for antisense therapy in ligament healing, fluorescence-labelled phosphorothioate oligodeoxynuleotides (ODN) were introduced into 12 rabbit ligament scars 2 weeks after injury using haemagglutinating virus of Japan (Sendai virus; HVJ)-conjugated liposomes. We compared the efficiency of cellular uptake of fluorescence as a percentage of all cells in each scar using three delivery procedures: (1) direct free-hand injection into the ligament scar using a conventional syringe; (2) systematic direct scar injection using a repeating 10 μl dispenser and a square mesh grid system; and (3) injection into the feeding (femoral) artery. Results showed that there was a significant difference in fluorescence uptake by scar cells on day 1 after injection between the three delivery methods: (1) direct free-hand, 9.7 ± 7.6% (average ± s.d.); (2) systematic direct, 58.4 ± 15.9%; and (3) intra-arterial, 0.2 ± 0.1%. Systematic direct injection was most efficient and it resulted in 25.9 ± 13.0% of scar cells being labeled at 7 days after transfection. We then introduced antisense ODN for the rabbit proteoglycan, decorin, into ligament scars with this delivery method and confirmed a significant inhibition of decorin mRNA expression in antisense-treated scar tissues in vivo both at 2 days (42.3 ± 14.7% of sense control ± s.d.; P < 0.0025) and 3 weeks (60.5 ± 28.2% of sense control ± s.d.; P < 0.024) after treatment, compared with sense ODN-treated scars. Decorin was significantly suppressed also at protein level in antisense-treated scars at 4 weeks (66.6 ± 35.7% of sense control ± s.d.; P < 0.045) after treatment. These results demonstrate that in vivo transfection efficiency in ligament scars is 'delivery system dependent' and that introduction of antisense ODN for the small proteoglycan, decorin, with this delivery method can lead to significant suppression of its expression over 3 weeks both at mRNA and protein levels. Thus, an effective model for the potential manipulation of scar composition and quality in ligament healing has been established.