A COMPARISON OF IN-VIVO GENE DELIVERY METHODS FOR ANTISENSE THERAPY IN LIGAMENT HEALING

To determine the most efficient in vivo delivery method of oligonucleo tides for antisense therapy in ligament healing, fluorescence-labelled phosphorothioate oligodeoxynuleotides were introduced into 12 rabbit ligament scars 2 weeks after injury using haemagglutinating virus of J apan (Sendai virus; HVJ)-conjugated liposomes. We compared the efficie ncy of cellular uptake of fluorescence as a per: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 s car using a repeating 10 mu l dispenser and a square mesh grid system; and (3) injection into the feeding (femoral) artery. Results showed t hat 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 dir ect, 58.4 +/- 15.9% and (3) intra-arterial, 0.2 +/- 0.1%. Systematic d irect injection was most efficient and it resulted in 25.9 +/- 13.0% o f scar cells being labeled at 7 days after transfection. We then intro duced antisense ODN for the rabbit proteoglycan, decorin, into ligamen t scars with this: delivery method and confirmed a significant inhibit ion of decorin mRNA expression in antisense-treated scar tissues in vi vo both at 2 days (42.3 +/- 14.7% of sense control +/- s.d.; P < 0.002 5) and 3 weeks (60.5 +/- 28.2% of I 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 s cars at 4 weeks (66.6+/-35.7% of sense control +/- s.d.; P < 0.045) af ter treatment. These results demonstrate that in vivo, transfection ef ficiency in ligament scars is 'delivery system dependent' and that int roduction of antisense ODN for the small proteoglycan, decorin, with t his delivery method can lead to significant suppression of ifs express ion over 3 weeks both at mRNA and protein levels. Thus, an effective m odel for the potential manipulation of scar composition and quality in ligament healing has been established.