1. Field of invention

The present invention relates to the discovery that a specific micro-RNA (micro-

RNA-17-5p also known as micro-RNA-17) is decreased in the circulation of systemic sclerosis patients and therefore may be used to monitor this as well as other fibrotic conditions and to measure the progress of a treatment. It further relates to the involvement of this micro-RNA in the regulation of fibrosis and therefore the use of it as a drug, as the basis of a drug, or as the target of a drug to treat and prevent fibrotic conditions.

2. General background (State of the Art)

Fibrosis is the presence of excessive collagen in an organ or tissue, in other words the replacement of normal tissue with scar tissue. This process is a part of normal wound healing and is seen in various pathological conditions e.g. primarily affecting the liver (liver cirrhosis), the lungs (lung fibrosis), the skin and various inner organs (scleroderma, systemic sclerosis), the retroperitoneal space (retroperitoneal fibrosis), or wound healing (keloid) and other conditions where the amount and cross-linking of collagen are excessive and destructive and generally not reversible.

Systemic Sclerosis (SSc, scleroderma) is an autoimmune connective tissue multisystem disorder characterized by inflammatory, vascular, and fibrotic changes of the skin (scleroderma) and pathological fibrosis in various internal organs such as the gastrointestinal tract, lungs, heart, and kidneys. The course, severity, and extent of the disease are highly variable. A molecular hallmark of scleroderma is excessive accumulation of collagen characterized by increased levels of pyridinoline collagen cross-links derived from hydroxylated lysine residues in the collagen telopeptide domains (the non-helical N- and C-terminal regions). Lysyl hydroxylase 2 (LH2, see below), an important alternatively spliced enzyme in collagen biosynthesis, acts as the collagen telopeptide hydroxylase responsible for the cross-linking ^{1 3} . Collagen accumulation invariably is seen in the skin and in the internal organs that are affected in SSc.

Collagen consists of a triple helix structure and is assembled both within and outside connective tissue cells. The physical properties (tensile strength and stability) of collagens are very dependent on the degree of cross-linking. Enzymes that convert lysine side chains to hydroxylysines enable cross-linking and are called lysyl hydroxylases (LH-1, -2, -3) or telopeptide lysyl hydroxylases (TLH). LH2, which has been identified as the product of the gene PLOD-2 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2) ⁴ , has been shown to be active in vivo on collagen telopeptide lysines and enables the telomeric bivalent pyridinoline cross-links ⁵ that are important for normal and pathological collagen polymerization. These cross-links are significantly increased in fibrotic tissues including in the accumulated collagen in the skin of scleroderma patients ² . The number of collagen cross-links determines the reversibility of fibrosis ⁶ ' ¹ . LH2 mRNA is alternatively spliced and is responsible for producing the long form of LH2 (LH2(long)) that is increased in scleroderma and other fibrotic conditions l, 2, 4, 8-1 1 _suc ^ ^ _m hypertrophic scars, keloids, and Dupyutren's syndrome ² ' ⁹ .

It has been demonstrated that one factor that regulates the expression of the alternatively spliced long form of LH2 is Fox-2 ³ . Silencing of this regulatory RNA-binding protein results in a reduction of the endogenous levels of LH2(long) mRNA in primary cells from scleroderma skin biopsies ³ . Thus, reduction of the activity of this enzyme may be beneficial in the treatment or prevention of fibrosis.

One newly identified regulator of the expression of the LH2 enzyme is a micro- RNA species called miR-17 (or 17-5p), which is a part of the polycistronic miR-17-92 cluster. Micro-RNAs (miRs) are short (about 22 ribonucleotides) single-stranded post-transcriptional regulators of gene expression that function at the level of protein translation. Through base- pairing miRs bind to complementary sequences on the 3'-UTR regions of messenger RNA molecules whereby translation is inhibited and/or the messenger RNA molecule is degraded. In both cases protein synthesis is halted. Most miRs have multiple target mRNAs. Overall, miRs are estimated to regulate 30-60% of all human genes and more than 1100 different miRs are now known in humans. They are extremely conserved phylogenetically and are present in all types of cells and tissues studied so far suggesting their important roles in the regulation of gene expression in all branches of biology. miRs are promising diagnostic and therapeutic targets in dysregulated pathways involving inflammation, infection, immunity, and cancer ¹² . One example is the overexpression of the oncogenic miR-17-92 cluster in lung cancer tissue and various other human cancers ¹³ ' ^M . It has emerged that miRs are frequently transcribed together as polycistronic primary transcripts that are processed into multiple individual mature miRNAs ¹⁵ . The genomic organization of these miRNA clusters is often conserved, suggesting an important role for coordinated regulation and function. The miR- 17-92 cluster is a prototypical example of a polycistronic miR gene. In the human genome, the miR- 17-92 cluster encodes six miRs (miR- 17, miR- 18a, miR- 19a, miR- 20a, miR-19b-l, and miR-92-1) which are tightly grouped within an 800 base-pair region of chromosome 13. Both the sequences of these mature miRs and their organization are highly conserved in all vertebrates. The human miR- 17-92 cluster is located in the third intron of a 7 kb primary transcript known as C13orf25 frequently amplified in diffuse large B-cell lymphoma ¹⁶ . Despite the extreme conservation of the miR sequences, the exonic sequences of C13orf25 are not measurably constrained between species, suggesting that the sole function of this transcript is to produce these miRs ¹³ .

Upon identification of targets of the miR- 17-92 cluster using cell biological and proteomic methods one of the most markedly regulated novel targets was found to be PLOD-2 which is predicted as a miR-17-specific target by miR-target prediction software ¹⁷ . As mentioned above, the PLOD-2 mRNA has been shown to be significantly increased in fibroblasts derived from fibrotic skin lesions of scleroderma patients ⁴ .

Even though many studies have documented increased expression of miRs e.g. in tumor tissues there are also many examples of miR specificities that are decreased in tissues and cells in cancers and various immunological abnormalities (for a review, see ref. ¹² ). To our knowledge no studies have hitherto addressed the characterization of extracellular circulating miRs in immunological disorders such as autoimmune diseases while some studies have examined cells and tissues from SSc and SLE patients ¹⁸ ' ¹⁹ . A proof-of-concept of the use of miRs to substitute for pathologically decreased levels of miR in cancer tissue was the demonstration of the relative deficiency of miR-26a in a mouse model of hepatocellular carcinoma and the therapeutic tumor suppressive effect of systemic administration of this miR encapsulated in adeno-associated virus particles and injected intravenously ²⁰ . Specific miRs that target collagens were predicted by various biocomputational methods and quantitatively measured in SSc-patient skin biopsies and fibroblasts. Using this approach miR-29 was selected as a candidate miR regulating collagen-expression ¹⁹ . Consistent with this notion miR-29 was found to be strongly downregulated in SSc cells and skin tissue compared to healthy controls and also downregulated in a bleomycin-induced mouse model of skin fibrosis. Further, collagen I and ΠΙ mRNA and protein were increased upon knock-down of miR-29. These studies suggest a role for the miR-29 family in the regulation of collagen synthesis. Analysis of the circulating miR-profiles was not performed. We find no significant decrease in the level of miR-29c in the circulation when compared to disease controls (SLE) and healthy controls. In contrast, we find that the levels of miR-29c circulating in SLE patients are significantly lower than in SSc patients.

The concept of using miRs or miR-analogues in the form of locked nucleic acids (LNAs) mimicking miRs has advanced recently with the most notable example being a miR-122 LNA mimick that is now in phase 2 trials in humans. This medicine is intended for the treatmengt of hepatitis C virus infection. The drug is manufactured and developed by Santaris Pharma and has been shown to be efficacious and with few and neglible side effects.

The use of miRs as diagnostic tools are described in several patent applications, e.g. WO2008042231 (risk of heart diseases), WO2008153692 (diagnosis of various neurological disorders) and WO2009025790 (diagnosis of bladder cancer). In WO2010028274 is described the use of serum or plasma protein marker panels consisting of matrix

metalloproteinases, insulin-like growth factors, and tumor necrosis factor receptor and other molecules to diagnose and evaluate the prognosis of idiopathic pulmonary fibrosis. The use of miRs for diagnosing idiopathic pulmonary fibrosis is covered in WO2010039502 and

WO2010033773. Others describe the use of an inhibitor against miRs (e.g. miR-17) as useful in various diseases e.g. WO2008014008, WO2009137807 (anti-angiogenesis), WO2009004632 (against cancer), WO2009043353 (hepatitis C and hypercholesterolemia-related disorders) and WO2009044899 (cell proliferation). Common for these disclosures is that their basis is the inhibition of miRs for treatment of a disease. Other published work (see above) has demonstrated the feasibility of using miRs to prevent disease, such as liver cancer or hepatitis C, by the systemic administration of a miR specificity that was shown to be decreased in affected tissue.

Diagnosis of SSc today relies on a combination of clinical and laboratory parameters and there are no reliable way to predict the course of the disease and the effect of treatment in the individual patient ²¹ . Other fibrotic conditions and syndromes are largely diagnosed through clinical signs, through imaging methods, and through the histological examination of biopsies from affected organs and tissues. None of these methods are very specific and sensitive especially in the early stages of disease and no specific methods exist for the cure or prevention of fibrosis. Thus, in fibrotic diseases there is an unmet need for reliable diagnostic methods and methods useful for the prevention or cure of such conditions.

3. Summary of the invention

The invention is based on the discovery of reduced amounts of circulating rniR- 17 in plasma samples from SSc-patients compared with normal plasma samples and compared with samples from another chronic immunoinflammatory disease, systemic lupus

erythematosus. It is surmised that the reduced amount of miR-17 circulating in SSc-patients reflects a reduced amount in tissues. The reduced levels lead to hyperactivity of the LH2 enzyme whose expression is normally repressed by miR-17. The hyperactive LH2 enzyme catalyzes the formation of pathologically cross-linked collagen leading to fibrosis. Repressing the LH2 enzyme activity by administration of miR-17 or by inducting its endogeneous expression by promoter activation is a novel approach to treat and prevent fibrosis not only in SSc but in all fibrotic conditions.

4. Detailed disclosure of the invention

The invention discloses the use of miR-17, a precursor, regulator or rnimick or ,analogue hereof for preparation of a pharmaceutical composition for treating, modulating, or preventing any fibrotic disease. The pharmaceutical product can be formulated as miR-17 encapsulated in a virus e.g.

adenovirus or adenovirus-like particles for administration by injection into organs or into the blood stream

Said fibrotic disease can be systemic sclerosis, lung fibrosis, liver cirrhosis, retroperitoneal fibrosis or any other type of fibrotic pathology. A further marker for any fibrotic disease optionally included in the method is miR-29.

The invention further discloses kits for diagnosing, monitoring and assessing fibrotic disease activity. The kit is preferably means for PCR quantification of miR-17 comprising a single or separate containers with nucleotide sequences which are able to prime amplification in a nucleotide sequence amplification reaction of miR-17. Most preferably the kit comprises a micro-RNA gene expression array chip. Definitions

By "fibrotic disease" or "fibrosis" is understood the presence of excessive collagen deposited in an organ or tissue, in other words the replacement of normal tissue with scar tissue.

The term "Micro-RNA (miR)" is understood as short single-stranded 21-23 nucleotide (nt) long non-coding RNA molecules that regulate gene-expression post-transcriptionally by inhibiting mRNA translation. Micro-RNAs are produced as ~70-nt hairpin RNA precursors and processed to mature miRs by RNase HI nucleases Drosha and Dicer. For historical reasons some of the first discovered miRs (in C. elegans) are called "let" (for "lethal") ²² , e.g. miR-let-7a. By "Micro-RNA 17-5p" or "miR-17" is understood the nucleotide sequence: 5'-caaagugcuuacagugcagguag-3' (SEQ ID NO 1)

By "Micro-RNA 17-5p analogues" or "miR-17 analogues" or mimicks is understood any single- stranded RNA sequence, a nucleic acid analogue such as locked nucleic acid (LNA) or any other molecule whose structure is based on the specific base sequence of miR-17 that target and thereby regulate the translational activity of PLOD-2 mRNA.

By "locked nucleic acid (LNA)" is understood an inaccessible RNA and is a modified RNA nucleotide. The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge "locks" the ribose in the V-endo (North) conformation, which is often found in the A-form duplexes.

By "miR-17 precursors" is understood the primary precursor micro-RNA-17 gene transcript (pri-miR-17) that through the action of RNAse Ill-like nucleases Drosha and Dicer is converted into mature micro-RNA through an intermediate 60-80 nucleotide hairpin pre-miR-17 stage.

By "Micro-RNA regulators" is understood any molecular factor that regulates the expression of mature functional miR. These factors include regulatory sequences that are present in miR promoters. Host genes harboring miR sequences in their intronic sites impose their pattern of expression to their intronic miRs. Additionally, many miRs are transcriptionally regulated by transcription factors including oncogenes. Finally, regulation may be accomplished by regulation of miR processing, i.e., at the post-transcriptional level by regulation of the activity and efficiency of miR-degrading enzymes ²³ or at gene level by changes in the expression of miR binding RNAs such as pseudogenes or other miR target sequences.

The term "screening" is used in this invention as analysis of a number of samples for a marker. Thus, a screening assay can be used to determine the state of a marker in samples from a population to identify individuals in the population at risk for or suffering from a disease or the progress of the disease.

The term "miR- 17 regulating elements" is used to mean micro-RNA gene promoters, transcription factors, and the other modulators of miR activity including degrading, protecting, and molecules that sequester the activity of miRs from their bona fide targets by binding miRs.

The present invention relates to laboratory methods and kits for diagnosing and evaluating the activity and prognosis of fibrosis based on patient samples as well as to methods to treat and prevent fibrosis. The methods and kits are based on the micro-RNA-17 molecule that regulates an enzyme important for development of fibrosis.

In one aspect the present invention relates to the use of the levels of miR- 17 or precursors thereof as a means of monitoring disease activity and measuring the effect and proper dosage of therapeutic agents for SSc and any other type of fibrosis using standard quantitative molecular biology methods on samples such as plasma, skin biopsies, other biopsies, or urine from patients suspected of suffering from SSc. The levels can be measured using RT-PCR on total RNA exctracted from the biological material or any other appropiate method.

Another aspect of the invention is to use putative polymorphisms in the miR- 17 regulating elements extracted from nuclear material from human cells to ascertain diagnosis and prognosis of fibrotic disorders.

In yet another aspect, the invention relates to a pharmaceutical composition that as the active principle contains the miR-17 sequence, mimicks, analogues or sequences or structures based thereupon that bind specifically to the LH2-mRNA sequence and thereby inhibits the translation of this mRNA into protein. In an additional embodiment the invention also relates to the use of any gene regulatory mechanisms affecting the level of miR-17 expression to adjust for pathological levels associated with fibrotic diseases. Other features and advantages of the invention will be apparent from the following detailed description and from the claims. The materials, methods, and examples are illustrative only and not intended to be limiting.

For clarity of description, and not by way of limitation, the detailed disclosure of the invention is divided into the following subsections:

(I) Marker for diagnosis and monitoring of fibrosis

(II) Methods of use

(ΙΠ) Methods for treating fibrosis

(I) Marker for diagnosis and monitoring of fibrosis

The present invention provides for a specific human micro-RNA molecule (miR- 17) of the sequence 5'-caaagugcuuacagugcagguag-3' the quantitative measurement of which alone or in any combination with other known and future markers may be used to monitor fibrosis in various body sites with substantial confidence and discriminatory ability to distinguish fibrosis from other diseases and pinpoint fibrotic conditions that likely will be amenable for and benefit by miR-17 based treatment.The preferred, but not limiting, performance of such a method would be diagnostic specificities and disease sensitivities exceeding 95 percent. The use of the method would include early as well as late cases of fibrosis and its use as a means to treat, monitor treatment, disease activity, predict prognosis, choose individualized therapy, and as a screening method in populations at risk. In a screening application a method based on measurement of regulatory factors for miR-17 including gene polymorphisms and promoter polymorphisms may be involved with the purpose of identifying individuals at risk that should receive preventive medicine or other measures. Depending on the anatomical sites and on the extent of fibrosis the samples subjected to quantitative analysis for miR-17 may be blood, plasma, serum, urine, other biofluids, tissues, and tissue biopsies. (II) Methods of use

The present invention provides for a method to monitor fibrotic activity in a subject comprising measuring biofluid or tissue levels of miR-17 alone or in combination with other markers in a sample or a series of samples wherein decreases relative and normalized to internal and external standards indicate a diagnosis of fibrosis. A result indicative of this diagnosis may be followed by further diagnostic steps including but not limited to tissue biopsies, measurement of other markers, or the use of imaging methods. Where histological examination of a biopsy is performed accumulation of collagen is indicative of the fibrotic process The quantitation of miR- 17 in biofluids and tissues may be accomplished by any method known in the prior art to quantitate RNA molecules of known sequences ²⁴ (Methods Mol Biol 2010; 631 : 109-22 is incorporated by reference) including but not limited to quantitative real-time reverse transcription-polymerase chain reaction (PCR) using specific primers and fluorescent probes in PCR plates or on chip platforms and using or not using pre- amplification steps. Levels may be evaluated preferably in combination with other miRs to allow normalization of signals. A decrease in levels is used herein as at least 1 cycle threshold value indicating a decrease corresponding to at least 50% of the level of a normal control sample or the mean of a plurality of normal samples or disease control samples.

Another non-limiting embodiment of the method is its use during intervention to evaluate progression of fibrosis in a subject by measuring the miR-17 level in a sample from a subject wherein a decrease relative to a previous value in the subject in the level of miR-17 indicates progression of fibrosis.

Another non-limiting embodiment would be the use of miR-17 measurements to evaluate survival or disease subgroup prognostics wherein a decrease relative to a previous value in the subject would be indicative of the prognostics of survival and disease subgroup progression in fibrotic conditions and be necessary to monitor the effect of treatment and adjustment of dosage of miR-17 based pharmacological compounds modifying fibrosis.

Another non-limiting embodiment of the method is its use to evaluate the effect of therapeutic or surgical or other intervention in a fibrosis patient by measuring miR-17 one or several times after the intervention and comparing to a previous value in the subject before intervention. Another non-limiting embodiment of the method is to use miR-17 determination and its related prognosis to advise patients and clinicians regarding prognosis and treatment options or regarding the use of further diagnostic procedures.

The present invention also provides for kits comprising means of determining the miR-17 levels in all types of biological materials and for all purposes including but not limited to the examples of uses given above and including the determination of normal values of miR-17 and normalizing miR levels in biological material.

(ΙΠ) Methods for treating fibrosis A set of non-limiting embodiments of the present invention relates to its use for therapy of fibrosis based on the regulation of fibrosis-generating enzymes by the miR-17 molecule. This embodiment relates to the fact that sustained levels of miR- 17 are necessary to depress the gene expression of the LH2 enzyme that otherwise would be generating sites of collagen cross-links that are found in fibrotic tissue. A relative deficiency of miR-17 activity relating to its level as determined by its production, regulation, and elimination and to putative mutations affecting mRNA-binding functions or protein-complexation necessary for translation repression would entail increases in LH2 -enzymatic activity and thus a predilection for fibrosis. The invention embodies the use of exogeneous synthetic miR-17 or miR-17 mimics or analogues delivered to a subject administered by any means to normalize the endogenous levels of miR-17 and thereby repress fibrogenic enzymatic activities. It also includes the use of means to regulate or manipulate promoters and other regulators of endogenous miR-17 expression to increase production of miR-17 in vivo.

It is well established from several animal and human studies that synthetic miRs and miR mimeticks such as locked nucleic acids (LNA) are safe and efficacious agents that can be used to specifically target disease mechanisms, e.g. in liver cancer and hepatitis C virus infection. An LNA miR- 122 analogue is now in phase 2 trials in humans with encoouraging results and other companies are using miR-inhibiting molecules (antagomirs) to treat diseases where increased expression of miRs have been shown to be involved in pathological processes. These examples show that it is feasible and proven that targeting of the miR system is useful in various diseases. miR-17 has been shown in vitro to regulate the expression of the LH-2 enzyme (17). In these experiments otherwise identical cell lines were manufcatured without or with miR-17 and target protein levels were measured by quantitative proteome methods. The most highly regulated protein (depressed by a factor of 2.8 in the cell line containing miR-17) was found to be LH-2, the product of the PLOD-2 gene and the enzyme responsible for collagen crosslinking. These findings show that miR-17 regulates the expression of this enzyme.

Formulation of a pharmaceutical product comprising miR-17 is well known to the skilled person. A preferred method is to encapsulate the miR-17 in a virus e.g. adenovirus or adenovirus-like particles for administration by injection into organs or into the blood stream.This enables uptake of the adminstered miR into cells where it will exert its actions on protein synthesis (20)

Use of miR-17 regulation as a target of treatment by induction of transcription (i.e. by the action of transcription factors) of the miR-17 gene, decreased degradation or turn-over of miR-17, and increased production of miR-17 from precursors by induction of prim-miR and pre-miR processing enzymes may be used to increase the availablity of active miR-17 in cells and thereby increase the inhibition of the translation of LH-2 mRNA.

The invention is further described in the following example, the disclosure of which is hereby incorporated in this section by reference and which do not limit the scope of the invention described in the claims.

Figure legends

FIGURE 1. Micro-RNA gene expression array (Fluidigm) used for samples of RNA extracted from 96 different patient sera (left panel). The miR assays (top panel) were run in triplicate after 16 cycles of preamplification of the extracted RNA using a mix of the miR-assays. The picture is a representation of the fluorescent image after the last PCR-cycle and shows the consistent but variable expression of individual micro-RNAs in these samples. The color scale indicates the signal strength in that the black squares correspond to nano-wells with no signal and a gliding color scale up to yellow squares that represent nano-wells with the maximum signal. FIGURE 2. Micro-RNA expression in RNA isolated from plasma samples from SLE patients relative to the levels in RNA isolated from plasma samples from SSc patients. The fold-change is depicted as a function of miR-name and significance. Twenty-three different miR specificities in addition to two external controls (Cel-miRs) were measured in 70 SLE and 88 SSc samples and the geometric means of the intensities (ACT-values) of the miRs in each group are depicted as the signal in the SLE group divided by the signal in the SSc group (fold-change, red columns, left y-axis). Also shown is the false-discovery rates (FDR, black dots, right y-axis) for each fold-change value, i.e., the frequency with which the fold-change in question will lead to placement of a sample in the SSc-group even though it belongs to the SLE group. This is a measure of the statistical power of the the observed differential expression to discriminate between the two disease groups. The FDR=0.05 level (where one out of twenty samples will be falsely assigned to the SSc-group) is marked with the dashed line and miR-specificities that are above this limit are shown in light red. The miRs are ordered according to the statistical significance with which they are differently expressed in the SLE/SSc groups with the most significant to the left and the least significant to the right. From this analysis it is clear that miR-17-5p (marked with an asterisk) is distinguished by a very significant and unique down-regulation in SSc-patients. This experiment show that miR- 17 is down-regulated in the circulation of the SSc-group of patients when compared to a group of patients suffering from another type of chronic immuno-inflammatory condition (systemic lupus erythematosus, SLE). This down-regulation of miR- 17 was also seen when comparing to normal control individuals.

FIGURE 3. Quantitative determination of miR-levels in plasma from patients and controls. Based on quantitative RT-PCR analyses performed on chips as shown in Fig. 1. Each reaction develops its own fluorescence level curve during the PCR cycles and from this curve the cycle thresholds (CT) are determined. The CT-values are normalized using regression and technical controls and are then expressed as ACT values. The higher the level of miR the higher is the ACT. Levels of A, miR-17-5p; B, miR-106a ; and C, C. elegans miR-238 (added external technical control) in RNA extracted from samples from normal controls, systemic lupus erythematosus (SLE) patients, and systemic sclerosis (SSc) patients are shown as indicated. Abundances are normalized using C. elegans synthetic miRs added to the lysis buffer used for RNA extraction and all data are corrected for chip-to-chip variations using linear regression. The miR-17-5p level in SSc-derived RNA is highly significantly decreased (p<0.0001) compared to the level in SLE patients and also significantly decreased compared to the control group (p=0.012).

FIGURE 4. Independent validation of the circulating miR- 17 depression in SSc samples, Comparison of relative mean miR-abun dances in two independent systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) sample sets run on two different chip assays (plate A and B) for 23 different miRs (+2 technical control miRs). miR-17-5p is consistently and specifically decreased in the SSc sample sets. The values represent the average ACT value for all the samples in each (SLE and SSc) group and are depicted for each analyzable miR. No samples from normal controls are incuded in this figure. Consistently changed values in the two sample sets (SLE and SSc) will be placed in the lower left and the upper right quadrant of this plot, respectively. A number of miRs are consistently down-regulated in SLE as indicated by their placement in the lower left quadrant. The fact that miR-17-5p is the only miR in the upper right quadrant indicates that this miR has been confirmed in two independent analyses of two different SLE/SSc sample sets to be significantly down-regulated in the circulation of SSc patients relative to the level in SLE patients.

5. Examples

Example 1: Discovery of miR-17-5p as a differentially regulated micro-RNA in the circulation of SSc-patients Samples

A total of 70 SLE, 88 SSc (including 2 independently processed duplicates) and 28 control plasma samples were included in the study. Thus, RNA purified from a total of 186 samples was included. The SLE ²⁵ and the SSc ²⁶ patients fulfilled the diagnostic criteria and covered a range from inactive to active disease. For each case and control citrate plasma was available except for 8 controls that were EDTA plasma. Venous blood from SLE patients and controls was collected without the use of a tourniquet into citrate tubes. Immediately after collection, blood cells were removed by centrifugation (1800 g, 10 min, room temperature). This was followed by a second centrifugation step (3000 g, 10 min, room temperature) to obtain platelet poor plasma (PPP). The PPP was aliquoted into 250 μΐ, aliquots and snap-frozen in liquid nitrogen before storage at -80 °C until analysis.

RNA purification

Total RNA purification kit (Norgen Biotek Corp., Ontario, Canada) was used to purify RNA from 100 \iL plasma and samples according to the instructions of the manufacturer with small modifications:

10 mM dithiothreitol and C. elegans synthetic miR-39, -54, and -238 (IDT, Coralville, Iowa), each at 0.13 pM were added to a volume of kit lysis buffer sufficient for all the samples. This volume was then aliquotted out into 4 mL portions and kept at -20 C until used. Also, 1 uL of RNAse inhibitor (ABI, 20 U/μΙ-) was added to every elution tube before elution of RNA. Purified RNA was kept at -80 C before being used for reverse transcription.

Reverse transcription

The RT-primer-mix consisted of equal volumes of each of 32 different 5x RT miR-specific stem-loop primers (Applied Biosystems (ABI), Foster City, CA) (Table 2). Reverse transcription reaction volumes were 10 μΐ _^ using 1 \iL Multiscribe, 3 μΙ_ RT-primer-mix, 1 μΐ, 10 X buffer, 0.2 μΐ, 100 mM dNTPs and 0.15 iL RNAse inhibitor, all reagents from ABI. To this was added 4.65 μΐ, RNA purified from sera or plasma. In the latter case the sample had been pretreated with heparinase I as described above. Reverse transcription was performed using a standard protocol (16 °C, 30 min; 42°C, 30 min; 85°C 5 min; hold at 4°C). Reverse transcribed samples were kept at -20°C until used.

Preamplification

Specific target amplification of the cDNA was accomplished using the TaqMan PreAmp master mix and a mix of the TaqMan MicroRNA Assays (ABI) consisting of equal volumes of the 32 different 20x assays diluted with lx TE buffer to a final concentration of 0.2x. Preamplification mixtures (10 contained cDNA (diluted 1 :4 with H ₂ 0), 2.5 μΐ,, mixed with 5 μΐ, 2x TaqMan PreAmp master mix and 2.5 μΐ- of the 0.2x TaqMan miR-assay mix. After 10 min at 95°C the reactions were subjected to 16 cycles of (95°C, 15 s; 60°C, 4 min) and then held at 4°C.

Samples were diluted 1 :5 with H ₂ 0 before next step (q-PCR). Quantitative PCR

Preamplified samples (diluted 5x with H ₂ 0) and miR TaqMan 20x assays were applied to primed 96.96 dynamic array chips using loading and assay reagents according to the manufacturer (Fluidigm Corp., South San Francisco, CA). The dynamic chip array technology (Fig. 1) allows for the simultaneous analysis of 9216 real-time PCR reactions ²⁷ . The fact that the analysis of all samples take place on one chip that is cycled and where TaqMan signals are image captured at the end of each cycle eliminates a considerable amounts of technical variability. Accordingly the standard deviations of triplicate cycle threshold (CT) measurements for CTs < 1 routinely is below 1 CT. All miR-assays were performed in triplicate, i.e., 32 miR assays x 3 and were mixed with 96 different samples on the chip using a Fluidigm integrated fluid circuit controller to prime and load the chips. The samples of this study were

accommodated on two 96x96 arrays (plate A and B, Fig. 4). SLE and SSc samples were mixed randomly on the two arrays and all control samples except one were included on the first array. Three RNA samples were run in duplicate on the two different chips making up for a total of 192 samples. After loading the reaction chambers using the integrated fluid circuit HX controller from Fluidigm the real-time PCR including image capture after each cycle was performed in a BioMark MX real-time PCR system (Fluidigm) using single probe (FAM-MGB, reference: ROX) settings and the default hot-start protocol with 40 cycles. Data processing took place using the Fluidigm real-time PCR analysis software (v. 2.1.1) and yields CT-values for each miR-assay for each sample. As an overall quality measure data sets that yielded standard deviations of >1 CT value for CTs below 25 in more than 2% of the triplicate measurements led to rejection of the whole chip. This did not apply to any of the chips used in this example. No individual data with SDs for the triplicates above 1 CT value were included.

Data Handling and Analysis

In the array analyses for CT-values global settings of the software were disabled and CTs were determined for each miR using individually set values based on inspection of the amplification curves. These settings were then used for data from all the chip runs. Input data were pruned for failed reactions, reactions with standard deviations of the triplicate analyses above or equal to 1 CT, and reactions with no signal. Each remaining average CT value was then subtracted from the average of the two Cel-miR (Cel-miR-54 and -238) CT-values for that particular sample yielding the ACT values used in the further analyses. Graphs of miR-expression and statistical comparison of the distribution of values in cases and population controls using unpaired t-tests as well as Spearman rank analysis were accomplished by Graphad Prism v. 5.00 (Graphpad Software Inc., San Diego, CA). Class comparisons and multivariate predictor analysis (class predictions) of all miRs were performed with BRB-ArrayTools V. 3.8.0

(http://linus.nci.nih.gov/pilot/index.htm).

RESULTS

The results of the study are exemplified in Fig. 1 where the image capture of the chip after the last PCR-cycle is shown. The degree of release of the fluorescent molecules attached to the probes depend on the amount of hybridizable material after each cycle and is represented by the color scale. The color is black when no signal is obtained and yellow with maximum signal. As is apparent there are large differences in the expression of the miRs tested for in this analysis of RNA derived from plasma samples. Cycle threshold (CT) values are a way of expressing the relative differences in the amount of miRs present and are based on the development of signal during the cycling for each miR-probe set. The CT values were normalized as described and could then be compared between the groups of patients involved in this study. The results of this comparative analysis are shown in Table 1 (and shown in Figure 2). Here, the significance of the differences in miR-expression between the groups of SLE patients (named Class 1 in the table) and the SSc patients (Class 2) are ranked from the lowest (most significant) to the highest (least significant) parametric p-values for each of the measurable miRs tested (unique id, right column). The p-values are corrected for multiple testing using the false discovery rate (FDR) that is given in the second column of the table. The absolute numbers of mean values for each of the listed miRs and the fold-change when comparing the SLE and SSc groups (classes) are also listed. In the accompanying figure (Figure 2) the fold-change are depicted as a function of miR- name and significance. From this analysis it is clear that miR-17-5p (marked with an asterisk) is distinguished by a very significant and unique down-regulation in SSc-patients. Class 1 is SLE, Class 2 is SSc

Table 1 Fold change is the expression level in SLE divided by the expression level in SSC. Thus, figures >1 indicate that the miR expression is decreased in SSc. The parametric p-value is the probability that a given miR difference between SLE and SSc is found by chance assuming a normal distribution of the values of miR expression in the individual samples. The lower the p- value the lower the likelihood that the difference between the groups will be due to chance. The false discovery rate (FDR) is the proportion of results that at this level of the p-value is expected to discrimate between the two classes by chance ²⁸ . This experiment shows that miR-17 is down-regulated in the circulation of the SSc-group of patients when compared to a group of patients suffering from another type of chronic immuno- inflammatory condition (systemic lupus erythematosus, SLE). This down-regulation of miR-17 was also seen when comparing to normal control individuals. Example 2: Quantitation of circulating miR-17-5p in the individual samples show significantly down-regulation in SSc-patients when comparing to SLE-patients and normal controls.

The individual data from the analysis described in Example 1 were plotted as the individual ACT-values for Cel-miR-238 (the external control miR added to the samples to allow for normalization), miR-106a , and miR-17-5p (Fig. 3). For miR-17-5p a highly significant change (decrease) in levels are found in the SSc-group compared to SLE and normal controls. The change compared to the SLE group is significant using a two-tailed non-parametric test (Mann- Whitney) with a p-value O.0001 and compared to the control group with a p-value of 0.012. No other of the miRs showed such differences in similar comparisons

Example 3. Independent sample sets show that down-regulation of circulating miR-17-5p is unique for SSc patients.

The sample sets described above were analyzed independently on two different chips (plate A and plate B). The samples on plate A derive from other patients than the samples on plate B. Still, SLE and SSc patients are represented about equally on each plate. Depicted in Figure 4 is the mean expression value of all the samples in each group of patients for each individual miR with the first set of samples (analyzed on plate A) on the X-axis and the next set of patients (analyzed on plate B) on the Y-axis. Values for miRs consistently down-regulated in SLE patients will be placed in the lower left quadrant while miRs consistently down-regulated in SSc will figure in the upper right quadrant. The technical normalizer miRs (Cel-miRs) appear around zero. Only miR-17-5p appears in the upper right quandrant and this shows that this miR by two independent analyses of two independent sample sets is consistently decreased in the circulation of SSc-patients.

Example 4. Examination of miR- 17 regulatory elements in SSc-patients.

Genomic DNA as well as pre-miRs from peripheral leukocytes of normal controls, SSc-patients, and SLE-patients are extracted and the promoter regions as well as the precursor forms of miR- 17 are sequenced in the search for polymorphisms that may explain the lowered expression of this miR in the circulation of SSc patients.

Example 5. Skin biopsy content of miR- 17 and miR-17 isoforms in SSc-skin and normal skin.

RNA was extracted from patient biopsies and using accepted tissue normalizer genes (U6B, 5S or others ²⁹ ) the contents of the miR- 17-92 cluster is going to be qunatitated using RT-qPCR.

This will show that the affected skin contain decreased expression of miR-17 in parallel with the demonstrated depressed presence of miR-17 in the circulation. mRNA levels for LH2(long) enzyme and antibody staining (anti-PLOD-2 antibody) are also being carried out on the same skin sections from SSc-patients and normal controls

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