Gene Therapy (1998) 5, 1455–1461 1998 Stockton Press All rights reserved 0969-7128/98 $12.00 http://www.stockton-press.co.uk/gt A comparison of in vivo gene delivery methods for antisense therapy in ligament healing N Nakamura1, SA Timmermann1, DA Hart1, Y Kaneda2, NG Shrive1, K Shino3, T Ochi3 and CB Frank1 1McCaig Centre for Joint Injury and Arthritis Research, University of Calgary, Calgary, Alberta, Canada; 2Institute for Molecular and Cellular Biology, Osaka University; and 3Department of Orthopaedic Surgery, Osaka University Medical School, Osaka, Japan 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 oligodeoxynuleo- scars 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 per- sense control ± s.d.; P Ͻ 0.024) after treatment, compared centage of all cells in each scar using three delivery pro- with 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 signifi- system 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) system- expression 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. Keywords: gene therapy; antisense; Sendai virus; liposome; ligament; wound healing Introduction this inferior biomechanical quality of scar material gives rise to clinical concern. Skeletal ligaments are discrete bands of dense connective For the purpose of designing new therapies, it is tissue which connect bones across a joint, serving important to note that ligament scar tissue is different important roles in stabilizing joints and guiding joint from normal ligament tissue in many aspects, which col- 1 motion. Ligament sprains are one of the most common lectively appear to contribute to the inferior biomechan- 2 injuries to joints. At present, however, despite a variety ical properties; elevated proteoglycan content such as of treatments, the optimal conditions for promoting liga- decorin and biglycan;10 elevated adhesion molecule con- ment healing still remain unclear. Previous research has tent such as fibronectin and tenascin;11 decreased type I shown that ligaments heal with scar-like tissue, which is collagen and elevated type III collagen; different collagen inferior to normal ligament tissue both biologically and organization; and abnormal collagen cross-linking.3–6 3–6 biomechanically. Whereas the tensile strength of Therefore, manipulation of these (or other) scar compo- 7 injured skin recovers by 10 weeks following injury, gap- nents toward those in normal tissue may improve scar healing medial collateral ligament of the knee (MCL) quality. reach only 30–40% of normal material strength of liga- Gene transfer has received attention for its potential 3 ment at 1 year. Since a major mechanical role of liga- biological manipulation of scarring.12 As one possibility, ments is to stabilize joints and since loose ligaments can the antisense method appears to be a feasible method for lead to significant joint instability, which may predispose this purpose which blocks the transcription or translation 8,9 the joint to the development of degenerative arthritis, of specific genes by binding of antisense phosphorothio- ate oligodeoxynucleotides (ODN) to target mRNA.13 To increase the efficiency of cellular uptake of ODN in vivo, we have developed a highly efficient Sendai virus Correspondence: CB Frank, McCaig Centre for Joint Injury and Arthritis Research, University of Calgary, 3330 Hospital Drive NW, Calgary, (haemagglutinating virus of Japan; HVJ)-liposome- 14–17 Alberta, Canada, T2N 4N1 mediated gene transfer method. HVJ promotes fusion Received 17 November 1997; accepted 30 June 1998 of the liposome with target cells and delivers the ODN Antisense therapy in ligament healing N Nakamura et al 1456 into the cells.18 We have previously reported that this tion was 9.7 ± 7.6% (average ± s.d.) by direct free-hand gene delivery method can be applied to healing ligament injection, 58.4 ± 15.9% by systematic direct injection, and by a direct injection of HVJ-liposomes into the ligament 0.2 ± 0.1% by intra-arterial injection (Figure 3). With the wound.19,20 However, the success of gene delivery into systematic injection method, 25.9 ± 13.0% of scar cells the glomerulus of kidney21 and the cardiac muscle22 via were still labelled 7 days after injection. intra-arterial injection made us revisit the idea of using such potentially less invasive intra-arterial delivery of Antisense decorin therapy in vivo HVJ-liposome complexes into hypervascular ligament Results of testing the in vivo introduction of antisense scars.23 ODN for the small proteoglycan, decorin, into MCL scars The purpose of this study was two-fold. First, we using systematic direct injection confirmed a significant wanted to optimize the number of cells within early liga- suppression of decorin mRNA expression in antisense ment scars into which ODN can be transferred. To do ODN-treated scar tissues in vivo both at 2 days this, we wanted to compare the three most promising (42.3 ± 14.7% of sense control ± s.d.; P Ͻ 0.0025) and 3 delivery techniques into MCL scars; specifically compar- weeks (60.5 ± 28.2% of sense control ± s.d.; P Ͻ 0.024) ing cellular uptake of fluorescence (6-carboxyfluorescein; after injection, compared with sense ODN-treated scars 6-FAM)-labeled ODN in vivo. The others involved direct (Figure 4). Additional experiments have revealed that injection into scars and intra-arterial injection into feed- levels of decorin mRNA expression are similar, following ing arteries of the scar. Second, we wanted to use the injection of HVJ-liposomes alone, and injection of sense most efficient of these three methods to introduce anti- ODN in HVJ-liposomes, to uninjected controls (data not sense decorin ODN into MCL scars in vivo, since decorin shown). Furthermore, we compared decorin protein has been implicated in the control of fibrillogenesis of col- expression in antisense and sense-ODN-treated scars at 4 lagen type I in vitro24 and in vivo.25 Therefore, excess deco- weeks following injection. We first analysed equal vol- rin expression potentially inhibits normal collagen fibril umes of tissue extract by SDS-PAGE followed by Coom- assembly in early MCL healing and its transient inhi- assie blue staining to confirm that the amount of the bition could improve scar quality. Specifically in this loaded protein was similar (Figure 5a), and then perfor- study, we wanted to determine whether or not inhibition med immunoblotting using a specific antibody to decorin of decorin mRNA and protein expression could be achi- which recognizes the C-terminal half of the core protein. eved and whether its inhibition would persist in a liga- We observed that decorin levels in the tissues were sig- ment scar for a period of weeks, increasing the likelihood nificantly suppressed in antisense-treated scars compared of generating a functional antisense effect in vivo. with sense ODN-treated scars (33.4 ± 35.7% of sense control ± s.d.; P Ͻ 0.045) (Figure 5b and c). Results Discussion Cellular uptake of 6-FAM-labeled ODN in ligament scar Scar tissue is a difficult target for gene transfer because When scar cells contained 6-carboxyfluorescein (FAM) of rapid proliferation and high turnover of wound cells.6 fluorescence-labeled ODN, their fluorescent areas corre- Therefore, to address the difficulty in obtaining sponded to 4′, 6-diamidino-2-phenylindole (DAPI)- maximum gene introduction into scar cells, an optimiz- stained areas. This suggests that most ODN introduced ation study of in vivo gene transfection was required. into scar cells accumulates within the nucleus (Figure 1). There have been two studies reporting the in vivo intro- duction of marker gene into ligament and ligament Comparison of three ODN delivery methods scar.19,26 These studies were done by a free-hand direct With lower power magnification, a clear difference was injection technique, where the importance of vector selec- observed in the distribution of 6-FAM fluorescence in tion was emphasized. To date, no study has considered MCL scar tissue among the three delivery methods the significance of delivery mode to maximize gene trans- (Figure 2). By freehand direct injection, fluorescence was fer. This study is the first demonstration that optimal in seen in a patchy distribution in the scar matrix.