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A comparison of in vivo gene delivery methods for antisense therapy in ligament healing
N Nakamura1, S A Timmermann, D A Hart
1McCaig Centre for Joint Injury and Arthritis Research, University of Calgary, Alberta, Canada.
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 sca injection using a repeating 10 microliters 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.
Insights
Systematic direct injection is the most effective method for delivering oligonucleotides in vivo for ligament healing. This approach significantly suppresses decorin expression at both mRNA and protein levels, establishing a model for scar quality manipulation.
Area of Science:
- Biomedical Engineering
- Regenerative Medicine
- Molecular Biology
Background:
- Ligament healing involves scar formation, impacting tissue quality.
- Oligonucleotide delivery is crucial for antisense therapy in modulating scar composition.
- Efficient in vivo delivery methods are needed to optimize therapeutic outcomes.
Purpose of the Study:
- To identify the most efficient in vivo delivery method for oligonucleotides in rabbit ligament scars.
- To evaluate the efficacy of systematic direct injection for antisense oligonucleotide delivery.
- To assess the impact of decorin suppression on ligament scar healing.
Main Methods:
- Fluorescence-labeled phosphorothioate oligodeoxynucleotides (ODN) were delivered via haemagglutinating virus of Japan (HVJ)-conjugated liposomes.
- Three delivery methods were compared: free-hand injection, systematic direct injection, and intra-arterial injection.
- Antisense ODN targeting decorin was administered using the most efficient method, and gene/protein expression was analyzed.
Main Results:
- Systematic direct injection achieved significantly higher cellular uptake (58.4%) compared to free-hand (9.7%) and intra-arterial (0.2%) methods on day 1.
- This method resulted in 25.9% scar cell labeling at 7 days.
- Antisense ODN significantly inhibited decorin mRNA (up to 60.5% suppression at 3 weeks) and protein (66.6% suppression at 4 weeks) expression.
Conclusions:
- In vivo transfection efficiency in ligament scars is highly dependent on the delivery system.
- Systematic direct injection represents an effective method for delivering antisense ODN in ligament scars.
- This approach enables significant, sustained suppression of decorin expression, offering a model for manipulating scar quality in ligament healing.