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

Gene intervention in ligament and tendon: current status, challenges, future directions

“…Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed a significant decrease in decorin mRNA levels in the antisense group when compared to the sense group 2 days and 3 weeks following injection. 28 Western blot analysis of the MCL scars showed a corresponding significant decrease in the protein levels of decorin in the antisense group when compared to the sense group 4 weeks after injection. 28 Electron microscopic evaluation of the scars showed both smaller and larger fibril diameters in the antisense group, similar to normal MCLs while the sense group and the HVJliposomes or 'poke' control groups had only smaller fibril diameters as seen in previous studies of MCL scars ( Figure 2).…”

“…28 These authors used adult rabbit MCL scars as the ligament model. The vector used was a hemagglutinating virus of Japan (HVJ)-conjugated liposome construct that delivered a fluorescent marker.…”

“…27,28 The methods include direct injection into the ligament, intra-articular injection or intra-arterial injection of the vectors. Injection of viral vectors, or transduced allografted cells, directly into ligaments was used by Hildebrand et al, 24 and Menetrey et al 25 With the injection into one area of the ACL, or the ruptured ends of the MCL, most of the b-galactosidase activity was found near the injection sites ( Figure 1).…”

“…16,17,28 Using a well-established model of MCL healing in an adult female rabbit knee where a 4 mm gap is created and marked, the scar that forms to fill the gap was injected with HVJ-liposomes containing antisense ODNs to decorin 2 weeks after the first surgery. 16,17,28,33 Decorin is a small leucine-rich proteoglycan that is implicated in collagen fibrillogenesis, where increased levels of decorin are associated with smaller diameter collagen fibrils. 17 Decorin is a component of normal and healing rabbit MCLs.…”

“…16,17,[24][25][26][27][28] The medial collateral ligament (MCL) and anterior cruciate ligament (ACL) have been evaluated. The MCL heals but has inferior properties compared to normal MCLs, while the ACL does not heal in an appreciable manner to recover biomechanical function.…”