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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

N Nakamura1, SA Timmermann1, DA Hart1, Y Kaneda2, NG Shrive1, K Shino3, T Ochi3 and CB Frank1 1McCaig Centre for Joint Injury and 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 , , into ligament fluorescence-labelled phosphorothioate oligodeoxynuleo- 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 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 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) . 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; healing

Introduction this inferior biomechanical quality of scar material gives rise to clinical concern. Skeletal are discrete bands of dense connective For the purpose of designing new therapies, it is which connect 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 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 ;10 elevated adhesion molecule con- ment healing still remain unclear. Previous research has tent such as fibronectin and ;11 decreased type I shown that ligaments heal with scar-like tissue, which is 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 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 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 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 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 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 . Fluor- vivo transfection efficiency in scars is definitely ‘delivery escence areas were mostly ovoid shapes with a longer system dependent’. The delivery of ODN into ligament radius up to 250 ␮m from the center, where the fluor- scar tissue was much more efficient by direct injection escence intensity was strongest (Figure 2a). Needle holes than by intra-arterial injection from the feeding artery. were seen within the most fluorescent areas by viewing While HVJ-liposomes are known for their excellent dif- serial sections (not shown), suggesting that the most flu- fusion capacity compared with other cationic liposomes orescent area corresponds to the position of the needle or viral vectors,27 and despite the fact that early wound tip at the time of injection. Using a 10-␮l syringe dis- vasculature is highly permeable,28 the HVJ-liposome penser and a square mesh grid system, we also systemati- complex did not appear to penetrate well into scar matrix cally injected every 0.5 mm within the ligament scar from from the vasculature. This strongly suggests that vessels the surface. With this method, fluorescence was diffuse in the ligament scar matrix are resistant to the diffusion throughout the scar (Figure 2b). In contrast, by intra- of a HVJ-liposome suspension. Poor diffusion of HVJ- arterial injection delivery, fluorescence was localized only liposomes in the scar matrix is further suggested by the around blood vessels. Spread of fluorescence from vascu- restricted fluorescent area by free-hand direct injection. lar areas was barely detectable, demonstrating that this Cellular transfection occurred within only a small radius method is unlikely to be effective in delivering any lipo- of 250 ␮m around the needle site. We observed that the some-mediated products to ligament scars (Figure 2c). fluorescence was even more restricted to a smaller radius Quantitative comparisons showed that the percentage of when 3-week ligament scars were injected, suggesting fluorescence-labeled cells in MCL scar at 1 day after injec- that as the ligament scar matures, it becomes even more Antisense therapy in ligament healing N Nakamura et al 1457

Figure 1 Fluorescence for DAPI and 6-FAM-labelled ODN in MCL scar. (a) Fluorescence microscopy of DAPI, nuclear stain dye in the MCL scar tissue 1 day after transfection. (b) The corresponding field showing the fluorescence of 6-FAM-labelled ODN in scar cells (×200).

Figure 2 Fluorescence of 6-FAM-labelled ODN in MCL scar on day 1 after injection. Fluorescence microscopy of 6-FAM-labelled ODN in the MCL scar tissue 1 day after transfection. (a) Free-hand direct injection; (b) systematic direct injection using a dispenser and a grid system; (c) Intra-arterial injection (×100). Antisense therapy in ligament healing N Nakamura et al 1458

Figure 3 Transfection efficiency on day 1 after injection. Transfection efficiency (% total cells labelled ± s.d.) in MCL scars at 1 day after HVJ- liposomes containing 6-FAM-labelled scramble ODN by three different delivery methods (n = 4 each): free-hand direct injection, systematic direct injection using a dispenser and intra-arterial injection.

resistant to the diffusion of HVJ-liposome complexes (data not shown). Such poor diffusion of HVJ-liposome complexes in scar matrix demonstrates that systematic injection using a syringe dispenser and a square mesh grid system can improve transfection efficiency by up to six-fold, compared with that by free-hand injection. This allowed a transfection efficiency which was far higher than previous gene transfection studies in ligaments and .19,27,29 We did not investigate the cell types of transfected cells in this study. We previously charac- terized the expression of a ␤-galactosidase gene intro- Figure 4 Effect of antisense treatment on decorin mRNA expression. duced into cells in early ligament scar by direct injection Semiquantiative RT-PCR analysis of total RNA from scar tissues for of the HVJ-liposome vector,19 similar to the method per- GAPDH (293 bp) and decorin (419 bp) mRNA levels at 2 days (a) and formed in this study. With labelling for marker antigens, 3 weeks (b) after injection of HVJ-liposomes containing sense or antisense decorin ODN. The numbers (1–6) refer to individual . For each we identified that approximately one-third of the trans- rabbit the left MCL was injected with sense ODN and the right leg the fected cells were and monocyte lineage cells antisense ODN. Lane designations: S, sense-treated scar, AS, = antisense- and two-thirds were fibroblastic cells. Considering that treated scar. The results were analysed using image analysis and the effect we used the same vector and injection technique (directly of in vivo antisense therapy is presented as the mean percentage of sense control ± s.d. on mRNA levels in MCL scars at 2 days and 3 weeks after into scar tissue) in the present study, we presume that = = the population of transfected (ODN-introduced) cells injection of HVJ-liposomes containing sense (n 3) or antisense (n 3) decorin ODN (c). Differences between groups were assessed using a paired resulting from the direct systematic injection method is t test. similar to that in our former study. Finally, we succeeded, using the systematic direct injection method, in suppressing one specific matrix mol- ecule for over 3 weeks at both the mRNA and protein levels. Notably, inhibition of decorin mRNA was compa- rable or even stronger at 3 weeks after injection, com- pared with that at 2 days. However, our 6-FAM-ODN Materials and methods introduction study showed that the percentage of fluor- escent (transfected) cells in scar decreased by 50% in a Synthesis of oligonucleotides and selection of sequence week. Such discrepancy between the duration of the anti- targets sense effect and that of fluorescence survival in scar sug- The sequences of ODN against rabbit decorin were: anti- gests that even ‘nonfluorescent’ scar cells may contain sense, 5′-GGA-TGA-GAG-TTG-CCG-TCA-TG-3′, sense, sufficient ODN to exert an antisense action, and that the 5′-CAT-GAC-GGC-AAC-TCT-CAT-CC-3′ (−1to+19 of actual percentage of transfected (antisense-influenced) rabbit sequence). This antisense ODN specifically inhibits cells may be higher than that estimated from our decorin mRNA in primary cultured rabbit MCL scar cells fluorescence uptake study. (unpublished data). The sequences for 6-carboxyfluo- Collectively, these results demonstrate that this system rescein (FAM) labelled ODN was 5′-CTT-CGT-CGG- may have promise as a model for future testing of anti- TAC-CGT-CTT-C-3′, with scramble sequence (19 bp). sense therapy in ligament healing and may provide a 6-FAM was labelled on the 3′ and 5′ ends of the ODN. new therapeutic approach for manipulating scar proper- All the ODNs were synthesized and purified by the ties and healing. University of Calgary regional DNA synthesis laboratory. Antisense therapy in ligament healing N Nakamura et al 1459 just before use), to allow formation of HVJ-liposomes. The mixture was incubated at 4°C for 10 min and, after adjusting total volume to 4 ml, incubated for 60 min with gentle shaking at 37°C. Free HVJ was removed from the HVJ-liposomes by sucrose density gradient centrifug- ation.

In vivo gene transfer of fluorescence ODN Twelve, 12-month-old female New Zealand White rabbits had the central 4-mm portion of their MCL removed sur- gically. After 2 weeks of healing, HVJ-liposome complex containing 50 ␮g of 6-FAM-labelled ODN was injected via one of three procedures (four MCLs for each): (1) direct free-hand injection into the ligament scar using a conventional syringe with a 30-gauge needle (n = 4); (2) direct scar injection using a repeating 10 ␮l dispenser with a 30-gauge needle (Hamilton, Reno, NV, USA) and a square mesh grid system which allowed us to inject systematically every 0.5 mm within the ligament gap (n = 4); (3) single shot injection into the feeding (femoral) artery with clamping of the femoral vein and pooling of the HVJ-liposome solution in the MCL scar area for 10 min (n = 4).

Evaluation of transfection efficiency All 12 MCL scars were then harvested 1 day after trans- fection and fixed with 4% formalin. Three sections (from superficial, mid and deep portions) were examined from each specimen by fluorescence microscopy, after nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma, St Louis, MO, USA), and then the ratio of FAM-6-fluor- escent area to DAPI-stained area were calculated using an image analysis system (Kontron Elektronik, Munich, Germany). Efficiency of transfection in scar tissue was determined using 12 sections in each injection group. Data were analyzed with an unpaired Student’s t test. With the most efficient procedure, the ODN was further introduced into another four 2-week MCL scars. Figure 5 Effect of antisense treatment on decorin protein expression. Equal volume (20 ␮l) of scar tissue extracts (each from 50 mg of sample) On day 7 after transfection, the scars were harvested and as analysed by SDS-PAGE with Coomassie blue staining (a) and by the transfection efficiency was measured. immunoblotting using a monoclonal antibody for decorin which recognizes the C-terminal half of the core protein (b) at 4 weeks after injection of Transfer of antisense decorin ODN HVJ-liposomes containing sense or antisense decorin ODN. The numbers Ten, 12-month-old female New Zealand White rabbits (1–4) refer to individual animals. For each rabbit the left MCL was had bilateral MCL gaps as described. After 2 weeks, HVJ- injected with sense ODN and the right leg the antisense ODN. Lane ␮ designations: S, sense-treated scar; AS, antisense-treated scar. The results liposome complex containing 20 g of antisense rabbit were analysed using image analysis and the effect of in vivo antisense decorin ODN was injected into MCL scars. Into the 10 therapy is presented as the mean percentage of sense control ± s.d. for contralateral MCL scars, HVJ-liposomes containing 20 ␮g decorin levels in MCL scars at 4 weeks after injection of HVJ-liposomes of sense ODN were introduced as a control. containing sense (n = 4) or antisense (n = 4) decorin ODN (c). Differences between groups were assessed using a paired t test. Reverse transcription (RT)-PCR Two days and 3 weeks after injection, semiquantitative RT-PCR was performed on RNA extracted from scar tissues (n = 3 for each period) as previously reported30 Preparation of HVJ-liposomes using primer sets specific for rabbit decorin and a constit- HVJ-liposomes were prepared as described pre- uitively expressed housekeeping gene glyceraldehyde-3- viously.16,19 Briefly, phosphatidylcholine, phosphatidyl- phosphate-dehydrogenase (GAPDH). The rabbit decorin and cholesterol were mixed in a weight ratio of 5′ primer (nucleotide 780–800) was 5′-TGT-GGA-CAA- 1:4.8:2. Dried lipid was hydrated in balanced salt solution TGG-TTC-TCT-GG-3′; the 3Ј primer (nucleotides 1239– (BSS; 140 mM NaCl, 5.4 mM KCl, 10 mM Tris-HCl, 1250) was 5′-CCA-CAT-TGC-AGT-TAG-GTT-CC-3′. The pH 7.5) containing sense or antisense ODN. The mixture rabbit GAPDH 5′ primer (nucleotides 211–230 of rabbit was agitated and sonicated for preparation of unilamellar sequence) was 5′-TCA-CCA-TCT-TCC-AGG-AGC-GA-3′; liposomes. The liposome suspension (0.5 ml, containing the 3′ primer (nucleotides 488–507) was 5′-CAC-AAT- 10 mg of lipids) as then incubated with HVJ (10 000 GCC-GAA-GTG-GTC-GT-3′. The rabbit-specific decorin haemagglutinating units), which had been inactivated primers were synthesized and purified by the University with ultra-violet irradiation (100 erg/mm2 per s for 3 min of Calgary regional DNA synthesis laboratory. Cloning Antisense therapy in ligament healing N Nakamura et al 1460 and sequencing of the amplified cDNA fragment con- DAH is the Grace Glaum Calgary Foundation Professor firmed the identity of the product. Reaction mixtures in Arthritis Research. CBF is the McCaig Professor in were assessed by 2% agarose gel electrophoresis, ethid- Joint Injury. ium bromide staining and then quantified using a Scana- lytics Image Analysis system (Billerica, MA, USA). The ratio of decorin to GAPDH in each treatment group was References calculated from the three specimens and compared with 1 Viidik A. Tendons and Ligaments. In: Comper WD (ed). Extra- each other using a paired t test. cellular Matrix, Vol. 1. Harwood Academic: Amsterdam, 1996, pp 303–327. Proteoglycan extraction and immunoblotting 2 Miyasaka KC, Daniel DM, Stone ML. The incidence of knee liga- Proteoglycan extraction from tissue was performed using ment injuries in the general population. 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