Introduction

Anti-scarring strategies have been increasingly utilized in surgical practice. This is especially the case in the surgical treatment of glaucoma – a disease that is the major cause of irreversible blindness in both the developed and developing world, accounting for 15% of all blindness and over 500 000 new cases each year.^1,2,3 The treatment of this disease is directed towards the reduction of intraocular pressure (IOP) – the main identifiable risk factor in glaucoma⁴ and includes topical medication, laser and surgical modalities. Of all available therapies, surgery has been shown to be the most effective,^5,6 achieving lower IOPs and preventing progressive vision loss.⁷ However, it is not always successful due to the occurrence of excessive post-operative scarring.^8,9 The introduction of wound-healing modulators has greatly improved glaucoma surgery results, but unfortunately, their use is limited by severe and potentially blinding complications.^10,11,12

Transforming growth factor- (TGF-) is known to be the most potent growth factor involved in wound healing throughout the body.^13,14,15,16 In the eye, all three human isoforms (TGF-1, TGF-2 and TGF-3) have been found although TGF-2 appears predominant.^17,18 TGF- has been implicated in the pathogenesis of several ocular scarring diseases such as corneal scarring,¹⁹ proliferative vitreoretinopathy,^20,21,22 cataract and posterior capsular scarring.^23,24,25 It is also involved in the wound-healing response following ocular surgery. In glaucoma surgery, the conjunctival scarring response is the major determinant of success. We have previously shown TGF- to be an important component of the conjunctival scarring response,^26,27,28 and recently demonstrated that neutralization of TGF-2 activity with repeated application of an antibody during the first week after glaucoma surgery, significantly improves surgical results both in vivo,²⁶ and at 1 year in patients undergoing primary trabeculectomy.²⁹

Antisense oligonucleotides (OGN) are a promising therapeutic strategy that has been successfully applied in oncology, inflammatory and viral infective disease.^30,31,32,33 The possible mechanisms by which antisense molecules result in decreased protein expression include the modulation of protein translation by disrupting ribosome assembly, RNase H mediated cleavage of targeted mRNA, and pre-translational modification of splicing.^30,34 Antisense molecules were described as long ago as 1978,³⁵ but adjustment of their structures has led to improvements in pharmacokinetics and pharmacodynamics.

Here we report the first study of novel antisense oligonucleotides to TGF- which contain both a phosphorothioate backbone and a 2'-methoxyethyl sugar modification which increase nuclease stability and antisense potency.^36,37 We have shown that these antisense molecules effectively inhibit TGF--mediated ocular scarring activity in vitro and in vivo. In two different animal models of ocular scarring, closely related to the surgical procedure performed in glaucoma patients to reduce IOP, we have demonstrated a significant reduction in post-operative scarring following single-dose administration only at the time of surgery.

Results

Rabbit TGF- oligonucleotide sequence identification

To evaluate the effect of TGF- antisense OGN in vivo, we used two different animal models – mouse and rabbit. Although mouse TGF- has been well characterized, that of rabbit is not. For the purposes of this study, therefore, we identified the nucleotide sequences of the cDNA coding regions for rabbit TGF-1 (1179 bp), TGF-2 (1245 p) and TGF- Type II receptor (TR-II – 1704 bp) (Figure 1a-c). We found the primary protein structures of rabbit TGF-1, TGF-2 and TR-II to consist of 392, 414 and 567 amino acids, respectively (Figure 1d–f).

Figure 1.

Rabbit TGF- oligonucleotide sequence identification. (a) Nucleotide sequence representing the coding region and the amino acid sequence of rabbit genes for TGF-1 (a), TGF-2 (b), and TGF- Type II receptor (c). Nucleotide residues are numbered on the left side relative to the first nucleotide of start codon, and amino acid sequences are shown below the DNA sequence and numbered on the right side. Stop codons are indicated by the asterisks. The mature peptide starts from the Arg280 under the arrow to Ser392. Nine conserved cysteines are underlined in TGF-1 and -2. (d,e) Alignment of amino acid sequences for TGF-1(d) and TGF-2 (e) precursors translated from human, rat and mouse mRNAs. Box I indicates the signal sequences and Box II indicates the mature protein sequences. The nine cysteine residues are underlined and show conserved alignment. (f) The optimal alignment of TGF- Type II receptor mRNAs between different species with stop codons indicated by an asterisk.

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Design and selection of antisense oligonucleotides

In order to select the most active antisense oligonucleotide, 20 2'-O-methoxyethyl-modified phosphorothioate oligonucleotides (OGN), designed to hybridize to multiple sites on each of the TGF- RNA targets, were screened in the appropriate cell lines. Thus, for example, the effect of different OGN on expression of TGF-1 mRNA in mouse bENDS cells was investigated to establish an OGN to target mouse TGF-1, and the most active was identified as ISIS 105204 (Figure 2a and b).

Figure 2.

Design and selection of TGF- antisense oligonucleotides. (a,b) RNA blot (a) and graphical display (b) demonstrating the reduction of TGF-1 mRNA expression by different TGF-1 methoxyethyl-modified phosphorothioate oligonucleotides on mouse bEND cells, at concentrations up to 300 nM and 10 g/ml of Lipofectin. The most active oligonucleotide to TGF-1 was found to be ISIS 105204. (c) Sequences of the selected oligonucleotides targeting mouse TGF-1 and rabbit TGF-1, -2 and TR-II. The reporter oligonucleotide ISIS 110410 sequence is an 8bp mismatch to 105204 and was designed to demonstrate the sequence specificity of the oligonucleotide for the target, TGF-1. The universal control oligonucleotide ISIS 29848 is a random mixture of 20-mer nucleotide sequences with the same chemical modifications as the other modified TGF- oligonucleotides. (d) Primer sequences used for cloning TGF-1, TGF-2 and TR-II genes.

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Figure 2c shows the sequences of the selected OGN targeting mouse TGF-1 and rabbit TGF-1, -2 and TR-II. The reporter oligonucleotide ISIS 110410 sequence is an 8 bp mismatch to ISIS 105204 and was designed to demonstrate the sequence specificity of the oligonucleotide for the target, TGF-1. The universal missense control oligonucleotide ISIS 29848 is a random mixture of 20-mer nucleotide sequences with the same chemical modifications as the other modified TGF- OGN.

Mouse model of conjunctival scarring

Localization of antisense oligonucleotide

Assessment of the localization of the TGF- antisense OGN in vivo was made possible by using a reporter oligonucleotide specifically designed to demonstrate the sequence specificity of the mouse TGF-1 antisense OGN. Histological analysis demonstrated the reporter OGN to be present in the subconjunctival space up to 7 days after administration (Figure 3a), and mainly confined to the area of the subconjunctival space around the original injection site. Microscopically, the reporter OGN showed a cellular pattern of staining in the conjunctival fibroblast and inflammatory cells.³⁸

Figure 3.

Mouse model of conjunctival scarring. (a) Demonstration that the reporter oligonucleotide was present in the subconjunctival space up to 7 days after administration. Localization of the reporter confirmed it to be mainly confined to the area of the subconjunctival space around the original injection site, although a trace was found in the adjacent cornea and sclera (magnification 40). The conjunctival epithelium and corneal epithelium were unstained, and microscopically, the reporter oligonucleotide showed an intracellular pattern of staining in the conjunctival fibroblast and inflammatory cells,³⁸ as also an extracellular presence. TGF-1 antisense treated eyes (b, d and f all magnification25) compared to control (c, e and g) were associated with a reduction in total cellularity in the subconjunctival space even at 7 days after surgery, as seen by H&E histological stains (b and c). The presence of oxytalan fibres indicating early elastogenesis showed peak activity at day 7 in controls (e) but with a reduction in the antisense-treated eyes (d). Scar formation and architecture was demonstrated by picrosirius red staining and significant differences were found between treatment groups, with the TGF-1 antisense OGN significantly reducing the amount of subconjunctival picrosirius staining on day 14 (f) compared to control (g).

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Effects on conjunctival scarring

Using a mouse model of conjunctival scarring we have previously characterized,^27,38 that localizes the scarring response to the level of the subconjunctival tissues, we evaluated the effects of a TGF-1 antisense OGN. Histological evaluation confirmed the development of a scarring response similar in the control eyes to that we have previously reported, following injection of phosphate buffered saline (PBS) ^27,38 or the missense Universal control OGN.

TGF-1 antisense treatment significantly inhibited this scarring response. Compared to control, the TGF-1 antisense treated eyes were associated with a delayed peak in the appearance of inflammatory cells (Figures 3b, c and 4a) and a significant reduction in the numbers present on days 2 and 7 (P<0.05). Antisense treatment also reduced conjunctival fibroblast activity, with a significant reduction in the number (P<0.05) seen in antisense-treated eyes compared to controls, on days 7 and 14 (Figures 3b, c and 4b).

Fibroblast activity was closely associated with the deposition of newly laid extracellular matrix (Figure 3d–g). Aldehyde fuchsin stains demonstrated elastic fibres with the oxidized version also showing oxytalan fibres, which are representative of the earliest identifiable stage of elastogenesis indicating early local extracellular matrix production and deposition. The presence of oxytalan fibres showed peak activity at day 7 (dark fibrillar stain, Figure 3d and e). Maximal total elastic fibre demonstration was observed at day 14 (Figure 4d and e). Scar formation and architecture was demonstrated by picrosirius red staining and significant differences were found between treatment groups, with the TGF-1 antisense OGN significantly reducing the amount of subconjunctival picrosirius staining on days 7 and 14 (Figures 3f, g and 4c). Statistical analysis using ANOVA showed significant differences between the extracellular components on days 2, 7 and 14 (P<0.05).

Figure 4.

Grading of scarring response in mouse model. Compared to control, the TGF-1 antisense treated eyes were associated with a delayed peak in the appearance of inflammatory cells (a) and a significant reduction in the numbers present on days 2 and 7 (P<0.05). Antisense treatment also reduced conjunctival fibroblast activity, with a significant reduction in the number (P<0.05) seen in antisense-treated eyes compared to controls, on days 7 and 14 (b). Scar formation and architecture was demonstrated by picrosirius red staining and significant differences were found between treatment groups, with the TGF-1 antisense OGN significantly reducing the amount of subconjunctival picrosirius staining on days 7 and 14 (c). Aldehyde fuchsin stains demonstrated elastic fibres (d) with the oxidized version also showing oxytalan fibres (e), which are representatve of the earliest identifiable stage of elastogenesis indicating early local extracellular matrix production and deposition. The presence of oxytalan fibres showed peak activity at day 7 with maximal total elastic fibre demonstrated observed at day 14 (d and e). Error bars=95% CI. * Activity of mouse TGF-1 antisense OGN significantly different from control (P<0.05).

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Effects of antisense on TGF- expression in mouse model

In addition to examining the histological effects of the TGF-1 antisense OGN on the scarring response in the mouse model, we also investigated its effects on TGF- expression. TGF-1, -2 and -3 protein expression was assessed using immunostaining. TGF-2 was found to be the predominant isoform demonstrated in both untreated and treated control eyes, being present in the conjunctival epithelium and stroma and distributed in varying degrees in the sclera and corneal stroma, as previously described.^18,39 Compared to the control (Figure 5a and c) TGF-1 antisense treatment (Figure 5b and d) appeared to decrease the expression of TGF-1 and -2, especially the degree of TGF-2 expression in conjunctival fibroblasts.

Figure 5.

Effects of antisense on TGF- expression in mouse model. Immunohistochemical assessment of TGF-1 and TGF-2 expression at day 7, with TGF-1 (a and b) and TGF-2 (c and d) protein demonstrated by DAB (brown) in the subconjunctival space. Compared to control (a and c), TGF- antisense treatment (b and d) appeared to decrease the expression of TGF-1 and -2, (Magnification 40) (e) Analysis of TGF- mRNA expression using a ribonuclease protection assay system (RPA) showed that eyes treated with TGF-1 antisense OGN appeared to decrease the expression of both TGF-1 and TGF-2 mRNA compared to PBS controls 7 days after subconjunctival injections.

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Analysis of TGF-1 and TGF-2 mRNA expression showed that control eyes had greater levels of TGF-1 and TGF-2 than those treated with the TGF-1 antisense OGN. This was particularly apparent at day 7 (Figure 5e).

Rabbit model of glaucoma filtration surgery

Local tolerance of subconjunctival rabbit TGF- antisense oligonucleotide

To assess the tolerability of the TGF- antisense OGN in vivo, we investigated the effects of single subconjunctival injections of rabbit TGF-1 antisense OGN at doses of 100 l of 100, 50 and 25 g. We found that all injections were well-tolerated in New Zealand rabbits with no evidence of intraocular inflammation being seen in any rabbit at any time in the study and at any concentration.

Effects on filtration surgery

The rabbit model used in this study is an aggressive model of scarring following surgery and is closely related to the procedure performed in glaucoma patients. However, unlike in standard clinical glaucoma filtration surgery, in this model a patent sclerostomy is maintained with a 22 gauge plastic cannula to negate the effect of scleral healing and isolate the wound-healing response to the level of the subconjunctiva.^26,40 Glaucoma filtration surgery produces a raised area of conjunctiva, called a 'bleb', which is elevated by aqueous fluid draining from the anterior chamber to the subconjunctival space in the eye via a surgically created channel. Any scarring at the surgical wound site impedes the flow of aqueous, leading to 'bleb' failure. Our results in this study showed that TGF-2 antisense OGN treatment significantly prolonged 'bleb' survival compared to control. Figure 6 shows the typical appearances of successful and failed filtration blebs. TGF-2 antisense OGN treatment was associated with an elevated, diffuse, fleshy-looking bleb at day 21 of the study (Figure 6a) compared to the flat, scarred bleb in the PBS-control group (Figure 6b).

Figure 6.

Effects of antisense on rabbit model of glaucoma filtration surgery. Rabbit TGF-2 antisense OGN was found to significantly improve glaucoma filtration surgery outcome in the rabbit, and appeared safe and non-toxic. Glaucoma filtration surgery produces a raised area of conjunctiva, called a 'bleb', which is elevated by aqueous fluid draining from the anterior chamber to the subconjunctival space in the eye via a surgically created channel. Any scarring at the surgical wound site impedes the flow of aqueous, leading to 'bleb' failure. Rabbits undergoing filtration surgery with TGF-2 antisense OGN treatment characteristically had an elevated, diffuse, fleshy-looking 'bleb' at 21 days after treatment (a), compared to the flat, scarred blebs treated with PBS-control (b). (Black arrows demarcate edges of the 'bleb') (c) Rabbit TGF-2 antisense OGN significantly prolonged bleb survival following filtration surgery, compared to treatment with TGF-1 OGN (log rank P=0.0009), Universal missense control (P=0.0072) OGN and PBS control (P=0.0035) treatment groups, as shown in this Kaplan–Meier survival curve. (d) Survival was prolonged in TGF-2 and TR-II antisense OGN groups (mean survival 19.4, 16.5 days, respectively) compared to the TGF-1 and control groups. Compared to PBS control, TGF-2 antisense OGN increased bleb survival by 5.68 days, and TR-II antisense OGN by 2.78 days.

Full figure and legend (514K)

Kaplan–Meier analysis demonstrated prolonged bleb survival in TGF-2 and TR-II antisense OGN groups (mean survival 19.4, 16.5 days, respectively) compared to the TGF-1 and control groups (Figure 6c and d). Treatment with the TGF-2 antisense OGN significantly prolonged bleb survival compared to TGF-1 (log rank P=0.0009), Missense (P=0.0072) OGN and PBS Control (P=0.0035) treatment groups. Compared to PBS Control, TGF-2 antisense OGN increased bleb survival by 5.68 days, and TGF-IIR antisense OGN by 2.78 days.

Analysis of the mean intraocular pressure (IOP) and IOP survival in the operated eyes showed no significant difference between treatment groups and at any time point. Comparison of treatment groups for vascularity at each quadrant showed no significant difference throughout the study period.

Discussion

Although the introduction of anti-proliferative anti-scarring agents has greatly improved results of surgery in the eye, their microscopic, destructive effects and associated blinding complications make them potentially hazardous treatments.^10,11,12 This study describes for the first time a more physiological method of inhibiting post-surgical ocular scarring with modified second-generation phosphorothioate antisense oligonucleotides against TGF-. We suggest that TGF- antisense OGN offer a safer and more controlled strategy than those currently available for modulating scarring not only in the eye, but throughout the body.

In this study, we have used two different in vivo models of post-operative ocular scarring and both have demonstrated a reduction in conjunctival scarring with a single TGF- OGN antisense application at the time of surgery.

From our previous studies, we have demonstrated TGF-2 to be the most important isoform involved in conjunctival scarring.^27,41 We have shown that TGF-2 is produced predominantly by conjunctival fibroblasts during the wound-healing response.³⁹ Furthermore, we have previously reported that repeated application of a human neutralizing antibody to TGF-2 (CAT-152) significantly reduced conjunctival scarring in the same rabbit model of filtration surgery used in this study.²⁶ As inhibiting TGF-2 appears to be so important in reducing the scarring response, it is perhaps not surprising that the antisense OGN to TGF-2, compared to that to TGF-1, was most effective in successfully increasing bleb survival in the rabbit model.

Using the mouse model, we have shown that an antisense OGN specifically designed against the TGF-1 isoform suppresses not only TGF-1 but also TGF-2. This effect may be explained by the fact that TGF- is known to be autoinductive.⁴² Hence by suppressing TGF-1 production released predominantly from platelets at the wound site, application of the TGF-1 antisense OGN also reduced TGF-2 and -3 production from local fibroblasts and inflammatory cells.^17,18,38,43

Unlike the mouse model, the rabbit model used in this study involves a conjunctival scarring response that is complicated by the presence of a fluid called aqueous humour and dynamically changing conditions.²⁸ The TGF- antisense OGN would be expected to have maximal effects on local cellular production of TGF- at the filtration wound site and not in aqueous which is known to contain high concentrations of TGF-2 protein in association with glaucoma and intraocular fibrosis.^20,44,45,46 Aqueous TGF-2 is produced by cells within the eye, ie in the iris, ciliary body⁴⁷ and trabecular meshwork.⁴⁸ As application of the TGF-2 antisense OGN subconjunctivally in this rabbit study is external to the eye, it cannot directly suppress intraocular production of TGF-2, so we can only postulate that it may reduce aqueous TGF-2 by interfering with the TGF-2 autoinduction pathway from the cells within the eye.⁴²

Most cell types simultaneously express the three main TGF- receptors, Type I, II and III (TR-I, TR-II and TR-III, respectively)⁴⁹ which have been shown to be present in the conjunctiva.^50,51 TR-I and TR-II are now believed to initiate signal transduction by involving Smad2 and Smad3.^49,52,53,54 Although less well understood, all three TGF- isoforms are believed to bind to TR-II, although TGF-1 has the greatest affinity. A recent finding has been that TR-IIB, an alternatively spliced variant of the TR-II receptor, is a TGF-2 binding receptor, which mediates signalling via the Smad pathway in the absence of any TR-III.⁵⁵ Interestingly, the expression of TR-IIB is restricted to cells originating from mesenchymal tissues such as bone where the isoform TGF-2 has a predominant role. It would be interesting to investigate an antisense OGN designed against this spliced variant, as it may be more specific in inhibiting TGF-2-mediated activity.

The most encouraging finding in this study has been the demonstration of efficacy of TGF-2 antisense OGN after only a single administration at the time of surgery. This is to be compared to our previous study using a neutralizing antibody against TGF-2 in the same rabbit model, where the regimen consisted of repeated subconjunctival injections (five applications in 7 days from the time of surgery).²⁶ The presence of the reporter OGN even at 7 days after surgery in the mouse conjunctival scarring model confirms the TGF- OGN to have prolonged activity. It also provides an explanation for the long-lasting effects of the TGF-2 OGN seen in the rabbit model of filtration surgery.

Mitomycin-C and 5-fluorouracil are antimetabolites currently used to reduce conjunctival scarring following glaucoma filtration surgery, with application within the first week after surgery having been shown to improve surgical outcome months to years later.^56,57,58,59 However, both treatments result in the production of thin avascular blebs which can leak^60,61 and lead to the development of blinding infections¹¹ and hypotony.^10,62 The complications associated with their use can be partly attributed to the non-selective manner in which these agents work causing widespread cellular destruction.⁶³ By selectively targeting TGF-, the TGF- antisense OGN allows much more controlled and focal treatment with specific inhibition of TGF- production.

We have shown that TGF-2 antisense OGN treatment is effective in reducing the conjunctival scarring response following glaucoma filtration surgery in a model of aggressive scarring. In comparison with TR-II and TGF-1, TGF-2 antisense OGN are more potent in inhibiting the conjunctival wound-healing response. In addition, TGF- antisense treatment appears to be well tolerated in vivo, with no evidence of adverse reactions. These results are very encouraging as they suggest that TGF-2 antisense OGN treatment may be an effective and safe anti-scarring therapeutic agent. This is the first time such an agent has been used in filtration surgery, and represents an exciting new prospect for inhibiting post-surgical scarring in the eye, without the debilitating side effects seen with the existing agents. Furthermore, TGF- antisense OGNs may potentially have widespread applications anywhere in the body where modulation of the wound-healing response is important.

Materials and methods

Sequencing of Rabbit TGF-1, -2 and TR-II

Total RNA was isolated from adult New Zealand White rabbit liver using a standard chloroform/isopropanol extraction technique, following which reverse transcription (RT) and then PCR was performed in 50 l reactions using primers specifically designed using Primer Premier software (Premier Biosoft International, Palo Alto, CA, USA) to TGF-1, TGF-2 and TR-II human and mouse genes (Figure 2d). PCR conditions used were as follows: 94°C for 5 min, followed by 35 cycles of 94°C for 45 s, 59°C for 1.5 min, 72°C for 2 min, and 72°C final extension for 10 min. Purified fresh PCR TGF-1, TGF-2 and TR-II DNA fragments were extracted and then ligated into the pCR2.1 vector (Invitrogen, Invitrogen Corporation, Carlsbad, CA, USA) and the ligated product was transformed into competent E.coli DH5 cell (Gibco/BRL, Life Technologies, Gaithersburg, MD, USA). Plasmid DNA was purified using Wizard Plus Miniprep DNA Purification System (Promega, Promega Corporation, Madison, WI, USA) and digested with 12U EcoR I. Plasmid DNA with the proper insertion was adjusted to 0.5 g/l and sequenced.

Design and selection of antisense oligonucleotides

At least 20 phosphorothioate oligonucleotides which were methoxyethyl modified on nucleotides 1–5 and 16–20; DNA 6–15 were designed to hybridize to multiple sites on each of the TGF- RNA targets shown in Figure 2c.³⁷

For selection of the mouse TGF- 1 antisense oligonucleotide, bEND mouse endothelial cells, grown to 70-80% confluency, were treated with 20 different TGF-1 OGN at concentrations up to 300 nM and 10 g/ml of Lipofectin. Total RNA from these cells was extracted using the RNeasy Mini Kit (Qiagen), and RNA blots were then probed for TGF-1, using a radiolabelled {³²P}100 bp sequence from the TGF-1 gene Accession Number AJ009862. The most active oligonucleotide in terms of inhibiting TGF-1 expression was then selected and scaled up for experimental treatments (Figure 2b).

A similar method was used to select the mouse sequences for TGF-2 and TR-II. To obtain rabbit OGN, the lead mouse OGN were blasted against the rabbit sequences previously identified (Figure 1). TGF-1 and TR-II OGNs were 100% homologous. TGF-2 OGN, ISIS 105009, was 90% homologous, and so two nucleotides were changed to create the rabbit-specific oligonucleotide, ISIS 123285.

Localization of antisense oligonucleotide

Administration of a 'reporter' OGN, constructed as described before, was performed. Subconjunctival injections of 25 g of the reporter OGN were given to mouse eyes using the method previously described.²⁷ Paraffin sections from enucleated eyes were stained for the reporter OGN using a two-step horseradish peroxide immunohistochemistry technique, using DAKO Blocking solution (Carpenteria, CA, USA), and DAKO Proteinase K solution for pretreatment followed by incubation with the primary antibody 2E1-B5 (Berkeley Antibody Company, Berkeley, CA, USA) which is an IgG1 antibody that specifically recognizes a CG or TCG motif in phosphorothiate OGN. Secondary antibody incubation was with Zymed Anti-IgG1 isospecific HRP conjugated secondary (San Francisco, CA, USA), with DAB finally added as the chromogen and revealing agent (DAKO Carpenteria, CA, USA). Sections were counterstained with Gill's haematoxylin and then photographed to document cellular localization of reporter OGN expression.

Mouse model of conjunctival scarring

All experiments were performed on 6-week-old BALB/c mice, and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Mice eyes were randomly allocated to one of the five treatments (4 eyes/treatment/time point): subconjunctival injections of 5 l of either 12.5 or 25 g mouse TGF-1 OGN or 12.5 (test treatments) or 25 g mouse scrambled missense OGN or PBS-carrier (controls).

Surgery was performed using the method previously described,²⁷ whereby all animals under general anaesthesia had a subconjunctival injection of 25 l of each test substance with a 27 gauge needle. Mice were assessed clinically and killed by cervical dislocation at 2, 7, 14 and 30 days after surgery. Development of scar tissue was studied in sequential 5 m thick paraffin sections from enucleated eyes using the following special stains: haematoxylin and eosin (H&E) to assess cellularity, picrocirius red to demonstrate collagen deposition, aldehyde fuchsin for elastic and elaunin fibres. Sequential sections were assessed for cellularity profile and extracellular matrix deposition by two independent and masked-observers (MFC, RAA) using a grading system previously described.^26,40 Parameters assessed included: fibroblast and inflammatory cell profile and collagen and elastic fibre deposition. For each treatment group a mean grade per parameter at each time point (with 95% confidence intervals) was calculated. Analysis was performed using computer software (SPSS for Windows; SPSS Inc.) at individual time points using a one-way analysis of variance (ANOVA). All treatments were compared to control (PBS-carrier). The observed significance levels from multiple comparisons were adjusted using the Bonferroni test with P<0.05 indicating significance.

No statistical differences were found in any of the parameters measured between the two concentration subgroups (12.5 and 25 g) used in the TGF-1 OGN test treatments. These two test treatment subgroups were therefore grouped together as a single TGF- OGN treatment group in the analysis for ease of comparison (group n=8/timepoint). In addition, no statistical differences were found in any of the parameters measured between the two concentration subgroups used in the missense OGN control groups (12.5 and 25 g) or between missense OGN and PBS control groups. For ease of comparison, the two missense OGN concentration subgroups and the PBS control group were grouped together as a single control group in the final analysis (group n=12/timepoint).

Assessment of TGF- expression in mouse model

TGF- protein expression in treated mouse eyes was assessed using immunohistochemistry using a 2-step HRP technique. Antigen retrieval was performed with a citrate buffer (BioGenex, San Ramon, CA, USA) and a rice steamer method (Black & Decker rice steamer from DAKO). The primary antibodies used were rabbit anti-TGF-1 antibody, rabbit anti-TGF-2 antibody and rabbit anti-TGF-3 antibody all at 1:50 dilution (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA). An HRP-conjugated donkey anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA, USA) was used as the secondary antibody and DAB was finally added as the chromogen and revealing agent (DAKO Carpinteria, CA, USA). Sections were then analysed and photographed.

For the analysis of TGF- RNA expression, a ribonuclease protection assay system (RPA) was used. Surgical specimens consisting of a rectangular block of conjunctiva, Tenon's capsule, and sclera at the original site of the subconjunctival injection measuring 2 mm² were trephined from each of 10 enucleated eyes used at 2 and 7 days after surgery. Equal amounts of purified RNA (2 g) from each specimen were then hybridized with mCK3b labelled template (Pharmingen-In Vitro Transcription/RPA Kits). RNase-protected probes were resolved on a denaturing polyacrylamide gel and quantified by phosphoimaging (Molecular Dynamics). Each transcript was normalized to the housekeeping gene G3.

Tolerance of TGF-2 antisense oligonucleotide in rabbit

To determine whether at selected doses subconjunctival TGF- antisense OGN were tolerated and safe to administer in normal rabbits, a randomized, prospective, masked-observer trial was performed.

Nine New Zealand rabbits, aged 12–14 weeks and weighing 2–2.4 kg, were randomly assigned to treatment. Subconjunctival injections of 100 l (100 l is the maximum deliverable volume by the subconjunctival route) of test substances were administered (100 l of 100 g, 50 and 25 g TGF-1 OGN dissolved in PBS giving concentrations of 1, 0.5 and 0.25 mg/ml, respectively). Two subconjunctival injections of the antisense oligonucleotide were administered to the left eye of each rabbit (60 min apart), under topical anaesthesia (Amethocaine 1% eye drops), by a method we have previously described.²⁶ The right unoperated eye acted as control. Animals were killed 30 days after the first dose, using a lethal injection of pentobarbitone intravenously under a general anaesthetic.

Rabbit model of glaucoma filtration surgery

Twenty-five female New Zealand White rabbits, aged 12–14 weeks and weighing 2–2.4 kg, were randomly assigned to treatment which consisted of rabbit TGF-1, TGF-2, TGF-RII OGN test treatments (all at 1 mg/ml), and missense (1 mg/ml) and PBS control groups. All rabbits underwent glaucoma drainage surgery with one of these five treatments (5 eyes/treatment group). The experiment was performed as a randomized, controlled study with masked observers.

Subconjunctival injections of 100 l of test substances were given as described above immediately pre- and postoperatively (ie on day 0 only) to the operated eye of each rabbit. Filtration surgery was performed in the left eye of all rabbits, using the technique previously described.⁴⁰

Clinical evaluation of all animals was made at baseline and every day for the first 3 days after surgery and thereafter every 3rd or 4th day until 30 days after surgery. This included bleb size, bleb vascularity, anterior chamber depth and activity and intraocular pressure, as used in previous studies.^26,40 In addition, a general description was recorded of the injected area in terms of complications such as lid oedema, chemosis, haemorrhage and corneal toxicity.

The effects of TGF- antisense treatments in rabbit filtration surgery was assessed as previously described,²⁶ with all groups being compared to each other and the PBS control. The primary efficacy endpoint was taken as bleb survival, where bleb failure was defined as the appearance of a flat, vascularized, scarred bleb in association with a deep anterior chamber. Kaplan–Meier and log rank statistics were used to compare bleb survival rates across all treatment groups. Bleb area and height, and anterior chamber depth and activity, and conjunctival vascularity were all analysed using the repeated measures procedure and the generalised linear model (SPSS). Avascularity was assessed using the Pearson's -squared test. Finally, intraocular pressures were analysed using the multivariate analysis of variance (ANOVA), with Bonferroni's modification to compare differences between treatments and the effects of time and treatment. IOP was also assessed with a Kaplan–Meier and log rank statistics, where IOP failure was defined as the return of the IOP in the operated eye to its baseline level.

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