WO2009065226A1

WO2009065226A1 - Modulation of tdag51 to inhibit scar formation in skin wound healing and internal organ fibrosis

Info

Publication number: WO2009065226A1
Application number: PCT/CA2008/002054
Authority: WO
Inventors: Richard Austin; Jeffrey Dickhout; Kenneth Maclean
Original assignee: Mcmaster University
Priority date: 2007-11-23
Filing date: 2008-11-21
Publication date: 2009-05-28

Abstract

Methods and compositions for the modulation of wound healing, scar formation and organ fibrosis are provided. The methods and compositions involve modulation of the T cell death associated gene (TDAG51) and/or the related protein. The modulation of TDAG51 expression and/or activity may be effected by genetic or pharmacological means.

Description

MODULATION OF TDAG51 TO INHIBIT SCAR FORMATION IN SKIN WOUND HEALING AND INTERNAL ORGAN FIBROSIS

FIELD OF INVENTION

[0001] The present invention relates to modulation of mechanisms involved in wound healing, scarring and organ fibrosis.

BACKGROUND OF THE INVENTION

[0002] Wound healing in higher vertebrates involves fibrotic scar formation. This process replaces tissue regeneration found in lower vertebrates. Fibrotic scar formation is often modeled in skin. This process involves several stages after initial injury, these are coagulation, local inflammatory response, mesenchymal cell recruitment, proliferation of mesenchymal cells and differentiation and extracellular matrix deposition. Many of these processes are under the control of cytokines. Cytokines are released during coagulation with platelet degranulation and include TGF-beta and PDGF. TGF-beta has been shown to have broad effects on wound healing. Of particular importance is its role in myofibroblast differentiation involving the promotion of the de novo expression of smooth muscle alpha actin and its incorporation into stress fibers.

[0003] T-cell death associated gene 51 (TDAG51) is a gene found to be induced by the process of endoplasmic reticulum (ER) stress. Over expression of TDAG51 in vascular endothelial cells leads to a loss of cell to matrix attachment and programmed cell death, a process known as anoikis. TDAG51 , also referred to as pleckstrin homology-like domain, family A, member 1 (PHLDA1 ) has been shown to be required for the up-regulation of Fas expression in T-cell hybridomas and Fas mediated T-cell apoptosis. However, investigation of the role of TDAG51 in TDAG51^"7" mice revealed that these mice express normal levels of Fas and had normal T-cell apoptosis. Therefore, the role of TDAG51 as a critical mediator of T- cell apoptosis appears to be an in vitro phenomena since loss of this gene in the whole animal had no effect on the process after which it is named.

[0004] Insulin-like growth factor-l (IGF-I) has been shown to up-regulate TDAG51 in NIH-3T3 (NWTb3) cells that overexpress the human IGF-I receptor. IGF-I and ER stress inducing agents (homocysteine, peroxynitrite, thapsigargin) are the only known up-regulators of TDAG51. There is no published material linking TDAG51 to a role in wound healing or fibrosis.

[0005] Regulation of fibrosis by control of the levels of TGF-beta has been attempted; however, it has resulted in contradictory outcomes. This may be due to the many roles of TGF-beta in the wound repair process including effects on leukocyte recruitment and activation, fibroblast recruitment and matrix synthesis and inhibitory effects on immune cells to resolve inflammation. The development of the TGF-beta1 knockout mouse revealed the detrimental effects of complete TGF-beta1 inhibition as these mice succumb early in life, 2-3weeks of age, to excessive inflammation leading to vital organ failure. Thus, there remained a need for methods and agents that reduce scar formation and fibrosis.

SUMMARY OF THE INVENTION

[0006] Loss of TDAG51 prevents myofibroblast differentiation. The present invention involves modulation of expression of the gene TDAG51 by genetic or pharmacological means to prevent the differentiation of fibroblasts to myofibroblast and prevent scar formation or organ fibrosis. The present invention provides methods to both up-regulate and down-regulate TDAG51 by genetic means [up-regulation : TDAG51 expression mammalian plasmid, pBABE retrovirus; down-regulation: TDAG51 knockout, TDAG51 siRNA] are illustrated. It has been demonstrated by genetic means, that disruption of the gene TDAG51 (TDAG51^"A mouse) prevents the differentiation of fibroblasts to myofibroblast and delays scar formation in dermal wound healing. Preventions of scar formation by down-regulation of TDAG51 allows tissue regeneration and prevents the loss of critical organ function associated with fibrosis.

[0007] In one aspect of the invention, a method of modulating scar formation or organ fibrosis in a mammal is provided. The method comprises altering the expression or activity of T cell death associated gene 51 (TDAG51).

[0008] In a preferred embodiment, the expression of TDAG51 is modulated by administering lariat-form RNA, antisense RNA, short temporary RNA (stRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) micro RNA, aberrant RNA containing mismatches, double stranded RNA (dsRNA) or long deoxyrobonucleotide containing RNA (D-RNA).

[0009] In another preferred embodiment, the TDAG51 gene is silenced by an intracellular mechanism selected from the group consisting of post-transcriptional gene silencing (PTGS), RNA interference, antisense or mRNA directed translation inhibition, gene replacement, RNA repairing or homologous recombination.

[0010] In another aspect of the invention, TDAG51 expression is upregulated by a mechanism selected from the group consisting of plasmid transfection, viral infection, growth factor or hormonal upregulation, or use of a pharmacological agent.

[0011] In a further aspect of the invention, a composition for the modulation of TDAG51 expression is provided. The composition may comprise an agent selected from the group consisting of: lariat-form RNA, antisense RNA, short temporary RNA (stRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) micro RNA, aberrant RNA containing mismatches, double stranded RNA (dsRNA) and long deoxyrobonucleotide containing RNA (D-RNA). Alternatively, a composition for the modulation of TDAG51 activity comprising a pharamacological agent, such as a small molecule, a binding peptide, an RNA aptamer or a DNA aptamer or a known drug is provided.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A shows scar formation in normal C57 wildtype versus mice lacking the gene TDAG51 ;

Figure 1 B shows quantification of wound area in wildtype versus TDAG51 lacking mice;

Figure 2 shows characterization of TDAG51 protein expression in fibroblasts;

Figure 3A shows the effect of siRNA to knockdown the expression of TDAG51 protein Figure 3B shows the effect of IGF-1 to upregulate the expression of TDAG51 protein

Figure 4 shows myofibroblast differentiation in wildtype versus TDAG51 knockout fibroblasts;

Figure 5 shows the influence of TDAG51 gene on myofibroblast differentiation marker alpha-smooth muscle actin:Subcellular organization;

Figure 6A shows TGF-β1 signaling through SMAD2- phosphorylation in TDAG51 expressing fibroblasts.

Figure 6B shows the effect of TDAG51 gene knockout on TGF-beta1 signaling through SMAD2- phosphorylation.

Figure 7 shows effect of loss of TDAG51 gene on myofibroblast differentiation: Growth characteristics.

DETAILED DESCRIPTION

[0013] The present invention demonstrates that a deficiency in T cell death- associated gene 51 (TDAG51 ) inhibits scar formation in dermal wound healing. This inhibition delays wound healing in mice lacking the gene TDAG51 (TDAG51- /- mice) as seen by increased wound size in TDAG51-/-. This process appears to be due to an inability of fibroblasts deficient in the TDGA51 protein to enter into a differentiation process that leads to myofibroblast formation. Myofibroblast formation is characterized by the expression of alpha smooth muscle actin which has been shown to be down regulated in fibroblasts lacking TDAG51. Further, the alpha smooth muscle actin present in fibroblasts lacking TDAG51 is not organized into cytoskeletal elements of the cell referred to as stress fibres. The myofibroblast is the cell type responsible for connective tissue deposition that leads to scar formation and end organ fibrosis in the process of internal injury. Down regulation of the gene TDAG51 by genetic means or pharmacological means can be used to prevent scar formation in the dermis and the same process can be used to prevent fibrosis of critical organ systems including lung, kidney, heart and liver. [0014] The present invention provides methods and compositions to modulate wound healing, scar formation and organ fibrosis by altering the expression and/or function of TDAG51.

[0015] In one aspect, the invention provides a method of treating or preventing scar formation or organ fibrosis in a mammal. The method comprises administering an agent that alters the expression or activity of T cell death associated gene 51 (TDAG51 ) or the related protein. Various types of agents can be used to obtain this effect. For example the agent could be a small molecule, nucleic acid, nucleic acid analogue, protein, aptamer, antibody or fragments thereof. Compositions containing these agents are also encompassed.

[0016] Combinations of different agents and combination of a TDAG51 inhibitor with other therapeutic agants can also be employed in the methods of the invention. In preferred embodiments, the expression or activity of TDAG51 gene or protein is downregulated. However, the invention also encompasses methods that increase the level of TDAG51 which may be desirable in certain situations.

[0017] One preferred agent is an RNAi nucleic acid. RNAi refers to any types of interfering RNA. In the methods of the invention the RNAi may be lariat-form RNA, antisense RNA, short temporary RNA (stRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) micro RNA, aberrant RNA containing mismatches, double stranded RNA (dsRNA) or long deoxyrobonucleotide containing RNA (D-RNA). The molecules may be natural or artificial.

[0018] Another method of the invention relates to preventing or reducing scarring or fibrosis in a patient. The risk of scarring or organ fibrosis can be determined based on TDAG51 levels. If the levels indicate it, a clinician directs the patient to be treated with an agent that inhibits TDAG51 expression or activity. This is particularly useful in situations where a patient is scheduled for surgery.

[0019] Referring now to the figures, Figure 1A illustrates scar formation in normal C57 wildtype versus mice lacking the gene TDAG51. Mice lacking the gene TDAG51 (TDAG51-/-) show delayed dorsal skin wound healing versus wildtype mice. [0020] Figure 1 B shows quantification of wound area in wildtype versus TDAG51 lacking mice. Mice lacking the gene TDAG51 (tdko) showed a statistically significant increase in wound area (p<0.05) at the 8 and 11 day time points versus normal C57 wildtype controls (C57). The results shown in Figure 1A and 1 B suggest that TDAG51 plays an important role in preventing scar formation and wound healing.

[0021] Figure 2 illustrates TDAG51 protein expression in fibroblasts. Western blot analysis indicated a complete loss of TDAG51 protein expression in TDAG51 knockout fibroblasts (TDAG51^"'^"). C57 control fibroblasts were positive for TDAG51 protein expression and TDAG51 +/- fibroblasts showed an intermediate level of TDAG51 protein expression (TDGA51 +/-) as shown in Panel A. Panel B demonstrates that stable cell lines generated using the pBabe retroviral system showed increased expression of TDAG51 protein over untreated (U) or pBabe vector (pB) controls in both C57 and TDAG51 -/- fibroblasts when treated with the retrovirus pBabe-TDAG51 construct (pB-TD).

[0022] Figure 3A illustrates the effect of siRNA knockdown on TDAG51 protein expression. To assess the influence of siRNA mediated knockdown of the TDAG51 gene on TDAG51 protein expression, HeLa cells were transfected with a TDAG51-GFP fusion protein (TD-GFP) or with TD-GFP followed 2 hours later with TDAG51 siRNA in the amount of 6 or 18 micro litres. Gel indicates suppression of TD-GFP protein expression via siRNA transfection. The level of beta actin (β- Actin) is used in this case as a protein loading control for the gel measured by Western blotting.

[0023] Figure 3B illustrates the ability of IGF-1 to upregulate TDAG51 protein expression. Insulin like growth fact -1 (IGF-1 ) was used to upregulate the expression of TDAG51 protein in NIH 3T3 fibroblasts. A dose of 5OnM was found to increase the expression level of the TDAG51 protein.

[0024] Figure 4 shows myofibroblast differentiation in wildtype versus TDAG51 knockout fibroblasts. To assess the influence of the loss of TDAG51 gene on myofibroblast differentiation fibroblasts were grown on a type I collagen gel matrix and the myofibroblast marker alpha-smooth muscle actin was examined 7-days post confluence. It was found that fibroblasts lacking the gene TDAG51 (TDAG51-/-) showed a significantly reduced level of alpha-smooth muscle actin (α-SMActin) expression and hence myofibroblast differentiation than normal fibroblasts (TDAG51 +/+). The level of beta actin (β-Actin) is used in this case as a protein loading control for the gel measured by Western blotting.

[0025] Figure 5 demonstrates the influence of the TDAG51 gene on the subcellular organization of the myofibroblast differentiation marker, alpha-smooth muscle actin. In addition to the reduction in expression of myofibroblast differentiation marker alpha-smooth muscle actin seen in Figure 4, loss of the gene TDAG51 in fibroblasts (Figure 5A) resulted in a different subcellular organization pattern for this protein versus wildtype fibroblasts (Figure 4B). Loss of TDAG51 prevented the organization of alpha-smooth muscle actin into stress fibres (arrows) which are readily seen in wildtype fibroblasts (B) undergoing the differentiation process to myofibroblasts.

[0026] Figure 6 shows the effect of loss of TDAG51 gene on TGF-beta1 signaling through SMAD2- phosphorylation. SMAD2 is phosphorylated as part of the TGF- betai mediated myofibroblast differentiation pathway. Examination of SMAD2- phosphorylation in response to TGF-beta1 treatment revealed robust activation in wildtype TDAG51 containing fibroblasts consistent with the myofibroblast differentiation program (Figure 6A). However, in fibroblasts lacking the TDAG51 gene SMAD2-phosphorylation via TGF-beta1 stimulation is dramatically reduced at similar doses (Figure 6B). This data indicates that TDAG51 down regulation mutes TGF-beta1 signaling that leads to myofibroblast differentiation.

[0027] Figure 7 shows the effect of loss of TDAG51 gene on myofibroblast differentiation as determined by growth characteristics. The loss of the gene, TDAG51 , is shown to significantly increase cell division among fibroblast that lack TDAG51 (tdko) versus fibroblast that express it normally (tdwt). The reintroduction of the gene TDAG51 into cells that lacked it normalized the increase in cell division acting as a proof the effect was due to the gene TDAG51. The reduced growth effects seen in the TDAG51 containing fibroblasts is consistent with the effects of myofibroblast differentiation to bring about cell cycle arrest and apoptosis.

[0028] The results illustrated in the figures demonstrate that modulation of TDAG51 has a profound effect on wound healing and scar formation, including organ fibrosis. While the description has focused on downregulation of TDAG51 to prevent scar formation, there may be other situations where it would be desirable to enhance wound healing. The invention therefore also encompasses methods and compositions that upregulate the expression and/or activity of TDAG51. The specific regulation of TGF-beta induced effects on fibrosis through TDAG51 offers great promise in reducing fibrotic disease while leaving critical immunoregulatory effects of TGF-beta1 intact.

The above disclosure generally describes the present invention. It is believed that one of ordinary skill in the art can, using the preceding description, make and use the compositions and practice the methods of the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely to illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Other generic configurations will be apparent to one skilled in the art. All reference documents referred to herein are hereby incorporated by reference

EXAMPLES

[0029] Although specific terms have been used in these examples, such terms are intended in a descriptive sense and not for purposes of limitation. Methods of microbiology, molecular biology and chemistry referred to but not explicitly described in the disclosure and these examples are reported in the scientific literature and are well known to those skilled in the art. Example 1. Animal model of wound healing

[0030] Wild type C57/BL6 mice and C57/BL6 mice with the gene TDAG51 disrupted (TDAG5V^A) were used for in vivo wound healing experiments. An incisional wound model was applied by cutting a 1 cm wound into the dorsal skin after shaving of either type of mice with a surgical scalpel. The wound was then closed with a single suture. Ten C57/BL6 wildtype mice and eight TDAG51^"Λaged 30 to 32-weeks were used for the wounding experiments. The mice were imaged with a dissecting microscope at 2, 8, or 11 -days after the wound to assess the rate of healing (Figure 1A). At each of these time points the wound area on each of the images was quantified using ImageJ software (NIH) (Figure 1 B).

Example 2. Preparation of mouse embryonic fibroblasts

Mouse embryonic fibroblasts (MEFs) were prepared from C57, TDAG51^+/" and TDAG51^"A mice as described by Abbondanzo et al. (1993) . After appropriate pairing to obtain the desired genotype, the copulatory plug was used as a marker of impregnation. Thirteen and a half days later, the pregnant females were sacrificed to collect C57 wildtype (wt), TDAG51^+/" or TDAG51^"'^" embryos. The embryos were separated from their placenta and brain and organs cut away. The embryos were then minced and cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, lmmunobloting was performed to confirm the genotype of the cells (Figure 2A). In brief, total protein lysates were subjected to immunoblot analysis using goat polyclonal antibody (Santa Cruz, Sc-6142) against TDAG51 (1 :250 dilution) or β- actin antibody (Sigma) (1 :2000) to control for protein loading as previously.

Example 3. Reintroduction of TDAG51 into TDAG51 Null MEFs

[0031] C57 control and TDAG51^"7" MEFs were infected with or without retroviral vectors pBabe or pBabe encoding TDAG51 for 48 hr as described previously. Infected MEFs were then selected for with purimycin (1 mg/mL). Following six to seven days of selection, cell lines were established. Total protein lysates were collected and these cell lines were subjected to immunoblot analysis using antibodies against TDAG51 to determine TDAG51 protein expression or β-actin to control for protein loading (Figure 2B).

Example 4. Smooth muscle alpha-actin expression in MEFs

[0032] In order to determine the expression of myofibroblast differentiation marker smooth muscle alpha actin in response to loss of TDAG51 gene both Western blotting and immunofluorescence antibody labeling were performed. For Western blot detection MEFs from wildtype C57/BL6 and TDAG51^"'^" mice were grown post confluence for 7-days on a type I collagen gel matrix (PureCol, Inamed, Fremont, CA). Gels were prepared as described by Arora et al. Briefly, 1 part each of 10X concentrated DMEM medium, 0.26 mol/L NaHCO₃, 100% FBS, 0.05 mol/L NaOH were added to 5 parts of 3 mg/mL PureCol for neutralization. After neutralization, gelation was then allowed to occur at 37⁰C for a period of 1 hr.

[0033] MEFs from either TDAG51^+/+ wild type or TDAG51^"A mice were added at 1x10⁶ cells/mL. The gels were then incubated with 10% FBS DMEM and collagen-coated plates were used to produce gels of high resistance to deformation previously shown to allow maximum alpha smooth muscle actin expression due to TGF-beta signaling . Total protein lysates from cultures of either TDAG51^+/+ or TDAG51^"'^" fibroblasts were dissolved in SDS-PAGE sample buffer and separated on 10% SDS-polyacrylamide gels under reducing conditions and transferred to nitrocellulose membranes (Bio-Rad). The smooth muscle actin specific primary mouse monoclonal antibody clone 1A4 (Sigma, dilution 1 :1000) was then incubated with blots, blots washed and then incubated with anti-mouse horseradish peroxidase-conjugated secondary antibodies (DakoCytomation, Carpinteria, CA). Membranes were developed using the Renaissance Western blot chemiluminescence reagent for antibody detection as in our previous work (Figure 3) . For immunofluorescence, TDAG51^+/+ or TDAG51^"7" fibroblasts were grown on 1 % gelatin (Sigma) coated glass cover slips to confluence in 10% FBS DMEM. Once confluent cells were switch to 0.1 % FBS DMEM for 24hrs to induce quiescence and then fixed in 4% paraformaldhyde. Cells were permeablized in 0.1% Triton-X, incubated with mouse monoclonal antibody clone 1A4 (Sigma,

4& dilution 1 :200), washed, and labeled with secondary antibody Alexa 488 (Molecular Probes, Eugene, OR) (1 :200 dilution) (Figure 4).

Example 5. Cell Proliferation assays

[0034] Since terminal differentiation of MEFs into myofibroblasts is associated with loss of the ability to proliferate, cell growth assays were performed. C57 control, TDAG51^+/" or TDAG51^"'^" MEFs were seeded at 5,000 or 15,000 cells onto fibronectin coated 96 well plates. On the following day, [³H]-Thymidine was added to the medium and allowed to incorporate for 4 hrs. The cells were then placed at -8O⁰C to block further incorporation. The cells were harvested and radiolabelling of DNA was quantified using a beta counter. Cell proliferation was also assessed utilizing a semiautomated system of cell counting with ethidium bromide (ETB) based fluorescent nuclear labelling and quantification on a Typhoon 9410 imager (Amersham) with laser excitation at 532nm and signal collected through a 610nm BP30 filter. C57 wild type, TDAG51^"7' and TDAG51^"'^" MEFs reconstituted with TDAG51 using the pBabe retroviral system were grown on 24-well plates coated overnight with 10 μg/mL fibronectin. After seeding at 1x10⁴ cells/mL, cells were allowed to attach overnight, washed and medium refreshed with 10% FBS DMEM. The cells were fixed at 0 time, 24 or 48 hrs with 0.1 % paraformaldehyde, permeablized with 0.1 % Triton-X, stained for 5 mins with 5μg/ml_ ETB in PBS, washed 3x in PBS, and placed in 65% glycerol in PBS for imaging on the Typhoon imager. Cell number was calculated by background subtracted volume intensity quantification of ETB fluorescence using ImageQuant software (Molecular Dynamics) as previously . Fluorescence intensity was fit to cell number by scaling through the ratio of fluorescence to cell number emitted from a known number of cells (Figure 5).

Example 6. In Vitro Wound-Healing and Migration assays

[0035] Wild type C57 and TDAG51 null MEFs were plated onto glass coverslips coated overnight with 10 μg/mL fibronectin. The cells were allowed to grow to near confluence in 10% FBS DMEM. The cell layer was then wounded with a 1 ml_ plastic pipette tip introducing a disruption into the monolayer. Cells were then

41- allowed to grow to assess the rate at which the MEFs from wild type or TDAG51 null mice could migrate into the denuded area. Cells were fixed in 4% paraformaldehyde, stained for F-actin with FITC labeled phalloidin, and imaged at the site of the wound to determine the rate of filling at 0, 3, 8, 16 and 30 hrs. To assess the effect on MEF migration of TDAG51 protein expression levels, equal numbers (1x10⁵ cells) of C57, TDAG51^+/" and TDAG51^"A MEFs were plated into collagen coated Boyden chambers. Cells were cultured in the upper chamber with 1 % FBS while 10% FBS containing DMEM was placed in the bottom chamber. The numbers of cells migrating through the membrane into the lower side of the chamber were counted by DAPI nuclear staining in a sampling.

Example 7. Assessment of 3r-lnteqrin binding

[0036] Flow cytometry was used to determine the percentage of active βi-integrin binding sites on the cell surface of TDAG51^+/+ versus TDAG51^''^" MEFs. Total βi- integrin was determined by cell permeablization with Triton-x-100 combined with βi-integrin antibody staining. Active βi-integrin was measured in non- permeablized cells with the BD Biosciences integrin βi chain antibody clone 9EG7. Since active βi-integrin binding to the fibronectin matrix is a critical component of fibroblast mobility, the 9EG7 clone was also used as an inhibitory antibody to determine if the increased rate of migration found in the TDAG51 null MEFs accounted for their increased proliferation rate. TDAG51^+/+ and TDAG51^"'^" MEFs were seeded at 1x10⁴ cells/mL in 24-well plates coated with 10 μg/mL fibronectin. Once cells had attached to the matrix the medium was changed to 10% FBS DMEM with or without 1 :100 dilution of integrin βi chain antibody clone 9EG7. Cells were fixed for ETB fluorescent quantification procedure at times 0, 24 or 48 hrs as previously.

Statistical Analysis

[0037] Values are expressed as mean + standard error. Comparison between the means was performed by student's unpaired T-test. ANOVA was used for multiple comparisons among the means. Significance was recognized at the 95%-confidence level.

42 Example 8. Vinculin and F-actin co-localization in MEFs

[0038] To assess the effect of loss of the gene TDAG51 on the focal adhesions of fibroblasts, MEFs were grown on fibronectin-coated (10 μg/mL) coverslips for laser scanning confocal microscopy. Co-localization of vinculin and F-actin was determined in wild type (wt), tdag51 ko (tdko) and tdag51 ko cells reconstituted with tdag51 (tdko_recon). Coverslips were incubated with the primary monoclonal anti-vinculin (Sigma) antibody (1 :100 dilution) and phalloidin conjugated to fluorphore (Sigma) targeting filamentous actin (F-actin) (1 :40 dilution). Secondary antibody to vinculin, Alexa 488 (Molecular Probes, Eugene, OR) (1 :100 dilution). Images were collected on a Zeiss LSM 510 confocal microscope (North York, ON, CA). Images were merged to determine vinculin and F-actin co-localization.

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Claims

WHAT IS CLAIMED IS:

1. A method of treating or preventing scar formation or organ fibrosis in a mammal, said method comprising identifying a subject with or at risk of developing excess scarring or fibrosis and administering to the subject an agent that alters the expression or activity of T cell death associated gene 51 (TDAG51 ) or the protein.

2. The method of claim 1 wherein the agent is selected from the group consisting of small molecule, nucleic acid, nucleic acid analogue, protein, antibody, aptamer or fragment thereof and combinations thereof.

3. A method according to claim 1 wherein the expression or activity of TDAG51 gene or protein is downregulated

4. The method of claim 2 wherein the nucleic acid is an RNAi agent.

5. The method of claim 4 wherein the RNAi agent is selected from the group consisting of lariat-form RNA, antisense RNA, short temporary RNA (stRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) micro RNA, aberrant RNA containing mismatches, double stranded RNA (dsRNA) or long deoxyrobonucleotide containing RNA (D-RNA).

6. The method of claim 1 further comprising administering additional therapeutic agents.

7. A method according to claim 3 wherein downregulation of the I activity of TDAG51 inhibits or retards scar formation and fibrosis.

8. A method according to claim 3 wherein the TDAG51 gene is silenced by an intracellular mechanism selected from the group consisting of post- transcriptional gene silencing (PTGS), RNA interference, antisense or mRNA directed translation inhibition, gene replacement, RNA repairing or homologous recombination.

9. A method according to claim 3 wherein TDAG51 activity is downregulated through the use of a pharmacological agent.

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10. A method according to claim 1 wherein TDAG51 expression or activity is upregulated.

11. A method according to claim 10 wherein expression is upregulated by a mechanism selected from the group consisting of plasmid transfection, viral infection, growth factor or hormonal upregulation, or use of a pharmacological agent.

12. A composition for the modulation of TDAG51 expression, said composition comprising an agent selected from the group consisting of: a pharmaceutical agent, small molecule, nucleic acid, nucleic acid analogue, protein, antibody, aptamer or fragment thereof , lariat-form RNA, antisense RNA, short temporary RNA (stRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) micro RNA, aberrant RNA containing mismatches, double stranded RNA (dsRNA) and long deoxyrobonucleotide containing RNA (D-RNA).

13. A composition according to claim 12, wherein the agent is siRNA.

14. A composition according to claim 12 wherein said agent comprises a pharamacological agent.

15. A composition according to claim 14 wherein the pharmacological agent is a small molecule, a binding peptide, an RNA aptamer or a DNA aptamer.

16. A composition according to claim 12 further comprising a known drug.

17. A method of preventing or reducing scarring or fribrosis in a patient, said method comprising assessing the risk based on TDAG51 levels, wherein a clinician directs the patient to be treated with an agent that inhibits TDAG51 expression or activity if the patient is at risk.

18. A method according to claim 17, wherein the patient is scheduled for surgery.

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