Title of Invention	"TREATMENT OF EYE DISORDERS CHARACTERIZED BY AN ELEVATED INTRA-OCULAR PRESSURE BY SIRNAS"
Abstract	Sequences and protocols for treatment of eye conditions by use of RNA interference are disclosed. Target genes are selected from those responsible for aqueous flow or aqueous outflow, while particularly preferred conditions to be treated include glaucoma and uveitis.

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FIELD OF THE INVENTION The present invention relates to methods and compositions for the treatment of eye disorders; in particular but not exclusively to the treatment of glaucoma. In preferred embodiments, the invention relates to the use of RNAi technology to downregulate the expression of aqueous formation genes and aqueous outflow genes. Methods and compositions for the treatment of eye disorders are also provided; BACKGROUND OF THE INVENTION RNAi as a tool to downregulate gene expression Gene targeting by homologous recombination is commonly used to determine gene function in mammals, but this is a costly and time-consuming process. Alternatively/ the functions of many genes can be determined after mRNA inhibition with ribozyme or antisense technologies. Although successful in some situations these technologies have been difficult to apply universally. The advent of siRNA-directed "knockdown" has sparked a revolution in somatic cell genetics, allowing the inexpensive and rapid analysis of gene function in mammals. Establishing a convenient and reliable method to knock-out gene expression at the mRNA level has been a recurrent theme in molecular biology over the last 15 years. In efforts to generate loss-of function cells or organisms, various molecules that included, for example, antisense sequences/ ribozymes, and chimeric oligonucleotides have been tested, but the design of such molecules was based on trial and error, depending on the properties of the target gene. Moreover, the desired effects were difficult to predict, and often only weak suppression achieved (Braasch & Corey, 2002). After the discovery of the phenomenon in plants in the early 1990s, i n 1998 Andy Rre and Craig Mello for ttie first time demonstrated with the worm Caenorhabditis elegans that dsRNA (double-stranded RNA) may specifically and selectively inhibit gene expression in an extremely efficient manner (Rre et at., 1998). In their experiment, the sequence of the first strand (the so-called sense RNA) coincides with that of the corresponding region of the target messenger RNA (mRNA). The second strand (antisense RNA) is complementary to this mRNA. The resulting dsRNA turned out to be far more (several orders of magnitude) efficient than the corresponding single-str§rtaedRNA molecules (in particular, antisense RNA). Rre et a/., 1998 named the phenomenon RNAi for RNA interference. This powerftif gene silencing mechanism has been shown to operate in several species among most pbyfogeneHc phyla. RNA! begins when an enzyme named DICER encounters dsRNA and chops it into pieces called small-interfering RNAs or siRNAs. This protein belongs to the RNase El nuclease family. A complex of proteins gathers up these RNA remains and uses their code as a guide to search out and destroy any RNAs in the cell with a matching sequence, such as target mRNA (for review see Bosher & Labouesse, 2000). The RNAi phenomenon (Akashi eta/., 2001) might be summarized as follows: • Step 1: dsRAIA recognition and scanning process. • Step 2: dsRNA cleavage through RNase III activity and production of siRNAs. • Step 3: association of the siRNAs and associated factors in RISC complexes. • Step 4: recognition of the complementary target mRNA. (Figure Removed) • Step 5: cleavage of the target mRNA in the centre of the region complementary to the siRNA. • Step 6: degradation of the target mRNA and recycling of the RISC compl ex. In trying to apply the RNAi phenomenon as a technology for gene knockdown, it was soon realized that mammalian cells have developed various protective phenomena against viral infections that could impede the use of this approach. Indeed, the presence of extremely low levels of viral dsRNA triggers an interferon response, resulting in a global non-specific suppression of translation, which in turn triggers apoptosis (Williams, 1997, Gil & Esteban, 2000). In 2000, a first attempt with dsRNA resulted in the specific Inhibition of 3 genes (MmGFP under the control of the Elongation Factor la, E-cadherin, and c-mos) in the mouse oocyte and early embryo. Translational arrest, and thus a PKR response, "was'TjorTobserved aTtife"embTyicdTitinued"to""develop"(Wian"riy"8r Zernicka-Goetz, 2000). One year later, research at Ribopharma AG (Kulmbach, Germany) first demonstrated the functionality of RNAi in mammalian cells. Using short (20-24 base pairs) dsRNAs - which are called SIRPLEX™- they specifically switched off genes even in human cells without initiating the acutephase response. Similar experiments carried out later by other research groups (Elbashir et aL, 2001; Caplen et ai, 2001) further confirmed these results. A year later, Paddison et aL (Paddison et al, 2002) tried to use small RNAs folded in hairpin structures to inhibit the function of specific genes. This work was inspired by previous studies showing that some genes in Caenorhabditis eiegans naturally regulate other genes through RNAi by coding for hairpinstructured RNAs. Tested in a variety of normal and cancer human and mouse cell lines, short hairpin RNAs (shRNAs) are able to silence genes as efficiently as their siRNA counterparts. Moreover, shRNAs exhibit better reassociatlon kinetics in vivo than equivalent duplexes. Even more important, these authors generated transgenic cell lines engineered to synthesize shRNAs that exhibit a long-lasting suppressing effect throughout cell divisions (Eurogentec). Recently, another group of small RNAs (also comprised in the range of 21-25 nt) was shown to mediate downregulation of gene expression. Th ese RNAs, known as small temporally regulated RNAs (stRNAs), have been described in Caenorhabditis elegans were they regulate timing of gene expression during development It should be noted that stRNAs and siRNAs/ despite obvious similarities, proceed through different modes of action (for review see Banerjee & Slack, 2002. In contrast with siRNAs, 22 nt long stRNAs downregulate expression of target mRNA after translational initiation without affecting mRNA integrity. Recent studies indicate that the two stRNAs first described in nematodes are the members of a huge family with hundreds of additional micro-RNAs (miRNAs) existing in metazoans (Grosshans & Slack, 2002). Scientists have initially used RNAi in several systems, including Csenorhabditis e7eiDrosophila"trvpanosornes, and"vanotB other im/e?!BDTatBsrM6reove7;" using this approach, several groups have recently presented the specific suppression of protein biosynthesis in different mammalian cell lines - specifically in HeLa cells - showing that RNAi is a broadly applicable method for gene silencing in vitro. Based on these results, RNAi has rapidly become a well recognized tool for validating (identifying and assigning) gene functions. RNA interference employing short dsRNA oligonudeotides will, moreover, permit to decipher the function of genes being only partially sequenced. RNAi will therefore become inevitable in studies such as: « Inhibition of gene expression at the post-transcriptional level in eukaryotic cells. In this context, RNAi is a straight-forward tool to rapidly assess gene function and reveal null phenotypes. • Development of the RNAi technology for use in post-implantation embryos. • The predominant economic significance of RNA interference is established by its application as a therapeutic principle. As so, RNAI may yield RNA-based drugs to treat human diseases. Glaucoma Glaucoma is one of the leading causes of blindness. Approximately 15% of cases of blindness world-wide result from glaucoma. The most common type, primary open-angle glaucoma, has a prevalence of 1/200 in the general population over 40 years of age. Glaucoma has been simply defined as the process of ocular tissue destruction caused by a sustained elevation of the Intra Ocular Pressure normal physiological limits. It Is becoming increasingly clear that many forms of glaucoma have a genetic component, and much current research is focused on identifying chrom osomal regions and genes that contribute to glaucoma. It is likely that the aetiology of OAG is multifactorial, resulting from a combination of mutations in more than one gene and as yetlodenWed environmental factors. With?e"p7clT:o"jQv(2fnie" and adult-onset OAG, several loci have been identified. However, only one gene is known, namely the myodfin / TIGR (trabecular meshwork inducibte glucocorticold response) gene at the GLC1A locus on chromosome Iq21-q31. More than thirty mutations of this gene have been identified in ethnically diverse populations worldwide. Sudies have shown that it is responsible for only about 5% of OAG overall (See reviews in Wirtz & Samples, 20O3, and Khaw et al, 2004a). Pathogenesis Most glaucomas are characterised by an elevated IOP, although the level of elevation may vary. In those glaucomas in which the elevation is initially low (i.e., open angle glaucoma, melanocytic glaucoma) and some secondary glaucoma, retinal ganglion cell and optic nerve damage are slow to progress. In angle-closure glaucoma the sudden high rise in IOP often renders the eye blind, undoubtedly primarily due to a cessation of axoplasmic flow at the level of the lamina cribrosa. In human studies, it has been widely accepted that tissue ischaemia has a part to play in the initiation or progression of the optic disc damage that occurs in glaucoma. Retinal ganglion cell degeneration may be necrosis, but the possibility that it is apoptosis triggered by the rise in IOP is plausible and the respective roles of nitric oxide and glutamate are thought to be relevant during progression of the disease (For a recent review on the subject see Osborne et al, 2003). Treatment Although several aetiologies are involved in the glaucoma complex, the absolute determinant in therapy selection is ttie amount of primary and/or induced change in pressure within the iridocorneal angle. aT lowering this" pressure, although the pathophysiojogical mechanisms by which elevated IOP leads to neuronal damage in glaucoma are unknown. Medical suppression of an elevated IOP can be attempted using four types of drugs: the aqueous formation suppressors (among them, carbonic a nhydrase inhibitor beta-adrenergic blocking agents, or alphaZ-adrenoreceptor agonists) miotlcs (i.e. parasympathomimetics - chollnergics-, or antfchoiinesterase inhibitors); uveoscleral outflow enhancers; and the hyperosmofjc agents (that produce an osmotic pressure gradient across the blood/aqueous barri er within the ciliary epithelium). All four are used in the treatment of glaucoma,, the first three commonly as emergency treatment and in long term control uvhile the hyperosmotic agents are invaluable as emergency and preoperative treatment A fifth category of drugs, the neuroprotection agents, is beginning to emerge as an important possible addition to medical therapy. Indeed, observation that the NOS and glutamate levels are elevated in glaucoma and that they are involved in retina! ganglion cell necrosis or apoptosis has raised the possibility of neuroprotective therapies and even neuroregeneration. Thus NOS inhibitors, exciting amino acid antagonists, glutamate receptor antagonists, apoptosis inhibitors and calcium channel blockers are all potential candidates in the ievelopment of future glaucoma therapies. The calcium channel blockers may •educe the effect of impaired microcirculation to the optic nerve head whilst Dotentially increasing outflow facility at the level of the trabecular cells. Reviews of various eye disorders and their treatments are given in the references, in particular in Bunce (2005), Costagliola (1995, 2000), Cullinane (2002), Sakaguchi (2002), Shah (2000), and Wang (2005). Currently our existing therapies must fall short of the mark and the practical difficulties associated with the assessment of outflow facility, the accurate monitoring of therapy and the complexity of surgical techniques all combine to ronfounStfie prognosis; ThlToveTnaing"fa degeneration of the retinal ganglion cell, thus neuroprotection through effective ocular hypotension is the essential requirement of any therapy we utilise (for a recent review on the subject, see Khaw et al 2004b). BRIEF SUMMARY OF THE INVENTION In the present invention we describe a method for the treatment of eye conditions characterised by an altered IOP in animals, including humans. In particular, the eye conditions may include glaucoma, uveitis, and inflammation. The method is based on the downregulation of expression of genes involved in aqueous formation or aqueous outflow in the eye. Downregulation may be effected by the use of double stranded nucleic acid moieties, named siNA or small interfering IMA that are directed at interfering with the mRNA expression of various candidate genes. The siNA are preferably siRNA, although modified nucleic acids or similar chemically synthesised entities are also included within the scope of the invention. Preferred embodiments of the invention relate to topical application of siNA. Embodiments of the invention also provide pharmaceutical compositions for use in the treatment of eye conditions. The invention may be used within the fields of local eye treatments, of target genes involved in glaucoma pathogenesis, as well as the use of chemically synthesized entities to treat animal (including humans) diseases. In addition to the treatment of glaucoma, the present method is also suitable for the treatment of other diseases of the anterior chamber of the eye. In particular, the method may be applied to the treatment of diseases characterised by altered aqueous formation or outflow in the eye. Examples of conditions which may be treated include local conditions such as infections or inflammations, and general conditions such as uveitis or expression of systemic diseases. Further, certain embodiments of the invention provide treatment for DETAILED DESCRIPTION OF THE INVENTION Target genes In the present invention, we define a list of target genes, whose expression levels may alter IOP. These genes can fall within the groups of genes involved in aqueous formation or the group of genes involved in aqueous outflow. Here is a list of our target genes: • Carbonic Anhydrases II, IV and XII • Adrenergic Receptors: betal and 2 and alpha 1A, IB and ID • Acetylcholinesterase • Cyclooxygenases 1 and 2 • ATPases: alphal, alpha2, alphas, betal, beta2 • Endothelial Leukocyte Adhesion Molecule I (ELAM-1) • Angiotensln System: Angiotensin II, Angiotensin II Converting Enzymes (ACE I and ACE II), Angiotensin II Receptors (ATR1 and ATR2) and Renin • Cochtin Design of siNA Although the mechanisms for RNAi remain unknown, the steps required to generate the specific dsRNA oligonucleotides are clear. It has been shown that dsRNA duplex strands that are 21-26 nucleotides in length work most effectively in producing RNA interference. Selecting the right homologous region within the gene is also important Factors such as the distance from start codon, the G/C content and the location of adenosine dimers are important when considering the generation of dsRNA for RNAi. One consequence of this, however, is that one may need to test several different sequences for the most efficient RNAi and this may become costly., In 1999", Tuscfil eTaraeaplieTeTiTe" that" their efficiency is a function of the length of the duplex, the length of the 3'-end overhangs, and the sequence in these overhangs. Based on this founder work, Eurogentec recommends that the target mRNA region, and hence the sequence of the siRNA duplex, should be chosen using the following guidelines: Since RNAi relies on the establishment of complex protein interactions, it is obvious that the mRNA target should be devoided of unrelated bound factors. In this context, both the 5' and 3' untranslated regions (UTRs) and regions dose to the start codon should be avoided as they may be richer in regulatory protein binding sites. The sequence of the siRNA is therefore selected as follows: • In the mRNA sequence, a region located 50 to 100 nt downstream of the AUG start codon or upstream of stop codon is selected. • In this region, the following sequences are searched for: AA(N19), CA(N 19). • The G/C percentage for each identified sequence is calculated. Ideally, the G/C content is 50 % but it must less than 70 % and greater than 30 %. • Preferably, sequences containing following repetitions are avoided: AAA, An accessibility prediction according to the secondary structure of the rnRNA is carried out as well. A BLAST is also performed (i.e. NCBJ ESTs database) with the nucleotide sequence fitting best the previous criteria to ensure that only one gene will be inactivated. order to maximize the result's interpretation, the following precautions should be taken when using siRNAs: Always test the sense and antisense single strands in separate experiments. Try a scramble siRNA duplex. This should have the same nucleotide composition as your siRNA but lack significant sequence homology to any other gene (iniaiiaing yours) " • If possible, knock-down the same gene with two Independent siRNA duplexes to control the specificity of the silencing process. Practically, each of the selected genes is introduced as a nudeotide sequence in a prediction program that takes into account all the variables described above for the design of optimal oiigonucleotides. This program scans any mRNA nucleotide sequence for regions susceptible to be targeted by siRNAs. The output of this analysis Is a score of possible siRNA oligonucleotides. The highest scores are used to design double stranded RNA oligonudeotides (typically 21 bp long, although other lengths are also possible) that are typically made by chem ical synthesis. In addition to siRNA, modified nucleotides may also be used. We plan to test several chemical modifications that are well known in the art These modifications are aimed at increasing stability or availability of the siNA. Examples of suitable modifications are described in the publications referenced below, each of which is incorporated herein by reference. Studies show that replacing the 3'-terminal nucleotide overhanging segments of a 21-mer siRNA duplex having two -nucleotide 3'-overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to four nucleotides on each end of the siRNA with deoxyribonucleotides has been reported to be well tolerated, whereas complete substitution with deoxyribonucleotides results in no RNAi activity (Elbashir 2001). In addition, Elbashir et al. also report that substitution of siRNA with 2'- 0- methyl nucleotides completely abolishes RNAi activity. Affinity modified nudeosides as described in WO2005/044976 may be used. This publication describes oligonudeotides comprising nudeosides modified so the target mRNA and/or in the complementary siNA strand. GB2406568 describes alternative modified oftgonudedides chemically modified to provide improved resistance to degradation or improved uptake. Examples of such modifications indude phosphorotfiioate intemudeotide linkages, 2'-Omethyl rlbonucleob'des, 21-deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base" nucleotides, 5-Omethyl nucleotides, and inverted deoxyabasic residue incorporation. WO2004/029212 describes oligonucleotides modified to enhance the stability of the siRNA or to increase targeting efficiency. Modifications include chemical cross linking between the two complementary strands of an siRNA and chemical modification of a 3' terminus of a strand of an siRNA. Preferred modifications are internal modifications, for example, sugar modifications, nucleobase modifications and/or backbone modifications. 2'-fiuoro modified ribonucleotides and 2-deoxy ribonucleotides are described. WO2005/040537 further recites modified oligonucleotides which may be used in the invention. As well as making use of dsNA and modified dsNA, the present invention may use short hairpin NA (shNA); the two strands of the siNA molecule may be connected by a linker region, which may be a nucleotide linker or a nonnucleotide linker. In addition to siNA which is perfectly complementary to the target region, degenerate siNA sequences may be used to target homologous regions. W 02005/045037 describes the design of siNA molecules to target such homologous sequences, for example by incorporating non-canonical base pairs, for example mismatches and/or wobble base pairs, that can provide additional "targefuences. In iristarxlvheleTnlsTHattt TOrRanonkar base pairs (for example, mismatches and/or wobble bases) can be used to generate siNA motecutes that target more than one gene sequence. In a noniimitjng example, non-canonical base pairs such as UU and CC base pairs are used to generate siNA molecules that are capable of targeting sequences for differing targets that share sequence homotogy. As such, one advantage of using siNAs of the invention is that a single siNA can be designed to include nucleic acid sequence that is complementary to the nucleotide sequence that is conserved between homologous genes. In this approach, a single siNA can be used to inhibit expression of more than one gene instead of using more than one siNA molecule to target different genes. Preferred siNA molecules of the invention are double stranded. A siNA molecule of the invention may comprise blunt ends, that is, ends that do not include any overhanging nucieotides. In one embodiment, an siNA molecule of the invention can comprise one or more blunt ends. In preferred embodiments, the siNA molecules have 3' overhangs. siNA molecules of the invention may comprise duplex nucleic acid molecules with 3' overhangs of n nucieotides (5>n>l). Elbashir (2001) shows that 21-nucleotide siRNA duplexes are most active when containing 3'-terminaI dinucleotide overhangs. Candidate oligonucleotides are further filtered for interspecies sequence conservation in order to facilitate the transition from animal to human clinical studies. In preferred embodiments of the invention conserved oligonucleotides are used; this allows a single oligonucleotide sequence to be used in both animal models and human clinical trials. GenBank Accession Numbers corresponding to our selected human target genes are shown in Rgure 1. In some of these genes, alternative splicing produces a family of transcripts that differ in exon content. The present invention allows individual targeting of each transcript form. Selected oKgonucleotide sequences against which RNAi is directed are shown in Rgure 2. Displayed sequences are the DNA sequences targeted by the sINA. Therefore, the Invention would make use of MA duplexes with sequences complementary to the indicated DNA sequences. The sequences shown in Rgure 2 are not limiting. As a matter of fact, target DNA need not necessarily be preceded by AA or CA. Further, target DNA could be constituted by sequences included in Rgure 2 flanked by any contiguous sequence. In vitro and animal studies. Obtaining siRNA duplexes RNAs are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Substitution of one or both strands of a siRNA duplex by 2-deoxy or 2-0- methyl oligoribonucleotides abolished silencing in fly extract (Elbashir et al. 2001). In mammalian cells, however, it seems possible to substitute the sense siRNA by a 2'-0-methy! oligoribonucleotide (Ge eta/. 2003). Most conveniently, siRNAs are obtained from commercial RNA oligo synthesis suppliers, which sell RNA-synthesis products of different quality and costs. In general, 21-nt RNAs are not too difficult to synthesize and are readily provided in a quality suitable for RNAi. Suppliers of RNA synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK), Qiagen (Germany), Ambion (USA) and Invitrogen (Scotland). The previous custom RNA synthesis companies are entitled to provide siRNAs with a license for target validation. In particular, our siRNA suppliers are Ambion, Dharmacon and service fbr siRNA, and supply the siRNA wfth HPLC purification and delivered in dry form along with RNasefree water. A central web-based resource for RNAi and siRNA methodologies, along with links to additional siRNA related products and services, can be found on the website of above-mentioned suppliers. An annealing step is necessary when working with single-stranded RNA molecules. K is critical that all handling steps be conducted under sterile, Rnase free conditions. To anneal the RNAs, the oligos must first be quantified by UV absorption at 260 nanometres (nm). The following protocol based on Elbashir et al. (2001) is then used for annealing; • Separately aliquot and dilute each RNA oligo to a concentration of 50 uM. • Combine 30 ul of each RNA oligo solution and 15 ul of 5X annealing buffer. Final buffer concentration is: 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4,2 mM magnesium acetate. Final volume is 75 pi. • Incubate the solution for 1 minute at 90 °C, centrifuge the tube for 15 seconds, let sit for 1 hour at 37 °C, then use at ambient temperature. The solution can be stored frozen at -20 °C and freeze-thawed up to 5 times. The final concentration of siRNA duplex is usually 20 uM. Alternatively, already annealed dsRNAs may be purchased from the modified nucleic acids may also be used. For example, an overview of the types of modification which may be used is given in W003/070744, the contents of which are incorporated herein by reference. Particular attention is drawn to pages 11 to 21 of this publication. Other possible modifications are as described above. The skilled person will be aware of other types of chemical modification which may be incorporated into RNA molecules. In vitro system To check the specificity of the siRNA interference different cell cultures that express the target genes were employed. The-cells used for these experiments were: "raBblT nongmentecl" aliafy" epitfieiruTfr epithelium cells OMDC, and human embryonic kidney cells HBQ93. The cells are Incubated with the corresponding siRNA duplexes, and analysis of the downregulat'on of expression of the target gene is carried out For linking siRNA knockdown to specific phenotypes in cultured cells, it is necessary to demonstrate the reduction of targeted protein or at least demonstrate the reduction of the targeted mRNA. mRNA levels of the target gene can be quantitated by real-time quantitative PCR (RT-PCR). Further, the protein levels can be determined in a variety of ways well known in the art, such as Western blot analysis with specific antibodies to the different target allow direct monitoring of the reduction of targeted protein. Transfection of siRNA duplexes Several examples of techniques well known in the art are as follows: We can perform a single transfection of siRNA duplex using a cationic lipid, such as RNAiFect Transfection Reagent (Qiagen) and Lipofectamine 2000 Reagent (Invitrogen) and assay for silencing 24,48 and 72 hours after transfection. A typical transfection protocol can be performed as follows: For one well of a 6- well plate, we transfect using lOOnM as final concentration of siRNA. Following RNAiFect protocol, we seed, the day before transfection, 2-4 x 10s cells per well In 3ml of an appropriate growth medium, containing DMEM, 10% serum, antibiotics and glutamine, and incubate cells under normal growth conditions (37°C and 5% COa). On the day of transfection, cells have to be at 30-50% confluence. We dilute 15ul of 20uM siRNA duplex (corresponding to 100 nM final concentration) in 85ul of Buffer EC-R, to give a final volume of lOOul, and mix by vortexing. For complex formation, we add 19 ul of RNAiFect Transfection Reagent to the diluted siRNA and mix by pipetting or vortexing. After incubating the samples for 10-15 minutes at room temperature to allow formation of "transfection complexes, we add the"complexes drop::wtseinti6aie oells"with2.9 ml of fresh growth medium tow in antibiotics. After swirling the plates to ensure uniform distribution of tile transfection complexes, we incubate the cells under their normal growth conditions. The day after, the complexes are removed and fresh and comptefce growth medium is added. To monitor gene silencing, cells are collected at 24, 48 and 72 hours post-transfection. The LJpofectamine 2000 Reagent protocol is quite similar. The day before transfection, we seed 2-4 x 10s cells per well in 3ml of an appropriate growth medium, containing DMEM, 10% serum, antibiotics and glutamine, and incubate cells under normal growth conditions (37°C and 5% C02). On the day of transfection, cells have to be at 30-50% confluence. We dilute 12.5ul of 20uM siRNA duplex (corresponding to 100 nM final concentration) in 250ul of DMEM, to give a final volume of 262.5ul, and mix. Also, 6ul of LJpofectamine 2000 is diluted in 250ul of DMEM and mixed. After a 5 minutes incubation at room temperature, the diluted oligomer and the diluted Lipofectamine are combined to allow complex formation during a 20 minutes incubation at room temperature, Afterwards, we add the complexes drop-wise onto the cells with 2 ml of fresh growth medium low in antibiotics and mix gently by rocking the plate back and forth, to ensure uniform distribution of the transfection complexes. We incubate the cells under their normal growth conditions and the day after, the complexes are removed and fresh and complete growth medium is added. To monitor gene silencing, cells are collected at 24, 48 and 72 hours post-transfection. The efficiency of transfection may depend on the cell type, but also on the passage number and the confluency of the cells. The time and the manner of formation of siRNA-liposome complexes (e.g. inversion versus vortexing) are also critical. Low transfection efficiencies are the most frequent cause of unsuccessful silencing. Good transfection is a non-trivial issue and needs to be carefully examined for each new cell line to be used. Transfection efficiency may be tested transfecting reporter genes, for example a CMV-driven EGFPexpression plasmid (e.g. from Qontech) or a B-Gal expression plasmid, and 'TheJTaisesseBbyphase contrastahl/6Ffluol:iscercm{croscopyti1ene%ay. Testing of siRNA duplexes Depending on the abundance and the life tame (or turnover) of the targeted protein, a knock-down phenotype may become apparent after 1 to 3 days, or even later. In cases where no phenotype is observed, depletion of the protein may be observed by immunofluorescence or Western blotting. After transfections, total RNA fractions extracted from cells were pre-treated with DNase I and used for reverse transcribed using a random primer. PCR is amplified with a specific primer pair covering at least one exon-exon junction In order to control for amplification of pre-mRNAs. RT/PCR of a non-targeted mRNA is also needed as control. Effective depletion of the mRNA yet undetectable reduction of target protein may indicate that a large reservoir of stable protein may exist in the cell. Alternatively, RealTime PCR amplification can be used to test in a more precise way the mRNA decrease or disappearance. Real-time reverse-transcriptase (RT) PCR quantitates the initial amount of the template most specifically, sensitively and reproducibly. Realtime PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production during each PCR cycle. This signal increases in direct proportion to the amount of PCR product in a reaction. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template. To verify the interference pattern of the differentially expressing genes identified in the cell cultures, qRT-PCR was performed according to the manufacturer protocol.. For quantitative RT-PCR (qRT-PCR), approximately 250 ng of total RNA were used for reverse transcription followed by PCR amplification with specific primers for each gene in reaction mixture containing Master SYBR Green I. Basic PCR conditions comprised an initial step of Quantification of b-actin mRNA was used as a control for data normalization. Relative gene expression comparisons work best when the gene expression of the chosen endogenous/Jntemal control is more abundant and remains constant, in proportion to total RNA, among the samples. By using an invariant endogenous control as an active reference, quantitation of an mRNA target can be normalised for differences in the amount of total RNA added to each reaction. Pharmaceutical formulations The present invention may comprise the administration of one or more species of siNA molecule simultaneously. These species may be selected to target one or more target genes. The siNA molecules of the invention and formulations or compositions thereof may be administered directly or topically (e. g., locally) to the eye as is generally known in the art. For example, a siNA molecule can comprise a delivery vehicle, including liposomes, for administration to a subject. Carriers and diluents and their salts can be present in pharmaceutically acceptable formulations. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins poly (lacticco- g!ycolic) acid (PLGA) and PLCA microspheres, biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors. In another embodiment, the nucleic acid molecules of the invention can also be formulated or complexed. with polyethyleneimine and derivatives thereof, such as polyethyleneimine- polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine- polyethyleneglycol-tri-N-acetylgalactosamine (PH-PEGtriGAL) derivatives. A sINA molecule of the invention may be complexed with membrane disruptive agents and/or a cationic lipid or helper Dpid molecule. Delivery systems which may be used with the invention include, for example, aqueous and non aqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and non aqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e. g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e. g., polycarbophil and polyvinylpyrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer. A pharmaceutical formulation of the invention is in a form suitable for administration, e.g., systemic or local administration, into a cell or subject, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect The present invention also includes' compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art For example, preservatives, stabilizers, dyes and flavouring agents can be provided. These include sodium benzoate, sorblc acid and esters of phydroxybenzoic acid. In addition, antioxidants and suspending agents can be used. A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms)bT1T9isease stateTThe the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between O.lmg/kg and 100 mg/kg body weight/day of active ingredients is administered. The formulations of the invention can be administered in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. Formulations can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavouring agents, colouring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations, Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, com starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and a bsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a" time delay material such "as glyceryi monostearate or glyceryi distearate can be employed. Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed witfri water or an oil medium, for example peanut oil, liquid paraffin or olive oil. Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl- methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooieate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin. Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyi alcohol. Sweetening agents arid flavouring agents can be added toptwJde"palal3He"oral pfaraBonsT These compositions can be preserved by the addition of an antJ-oxidant such as ascorbic add. Dispersibte powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents, can also be present. Pharmaceutical compositions of the invention can also be in the form of oll-inwater emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooieate, and condensation products of the said partial esters with ethylene oxide, for example poiyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavouring age, ,fcs. Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavouring and colouring agents. The pharmaceutical compositions can be in the form of a sterile injeotable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. A sterile"InjertaBlT pVeparatf suspension in a non-toxic parentally acceptable diiuent or solvent, for example as a solution in 1,3- butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonk: sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synttietic mono-or diglycerides. In addition, fatty adds such as oteic add find use in the preparation of injectables. The nucleic acid molecules of the invention can also be administered in the form of suppositories, e. g. , for rectal administration of the drug. These compositions can be prepared by mixing the dmg with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols. Nucleic acid molecules of the invention can be administered parenterally In a sterile medium. The drug, depending on the vehicle and concentration used., can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anaesthetics, preservatives and buffering agents ca n be dissolved in the vehicle. It is understood that the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy. For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formu late the animal feed and drinking water compositions so"tfii£fherainirnar takes" in"a therapeutfcafly appropriate quantity of the composition along with its diet, ft can ateo be convenient to present the composition as a premix for addition to the feed or drinking water. The nudek: add molecules of the present invention can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects. Alternatively, certain siNA molecules of the invention can be expressed within cells from eukaryotic promoters. Recombinant vectors capable of expressing the siNA molecules can be delivered and persist in target cells. Alternatively, vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of siNA molecule expressing vectors can be systemic, such as by intravenous or intra-muscuiar administration, by administration to target cells ex-planted from a subject followed by reintroduction into the su bject, or by any other means that would allow for introduction into the desired target cell. Animal studies The New Zealand rabbit is the gold standard in experimental platforms designed to study IOP. It is easy to handle and it has a big eye, similar in size to the human organ. In addition, present equipment to measure IOP is not suited to use in animals with small eyes such as mice or rats. Finally, rabbits have an IOP (around 23 mm Hg) that can be brought down to up to 40% its value using local commercial hypotensive medication. Thus, although it is possible to generate rabbit glaucoma models (for example, surgically blocking episclerotic veins or artificially occluding the trabecular meshwork), we have used normotensive rabbits since, in our hands, the pharmacological decrease in Deeiy and reprbucl61yrrneasured7 Experimental protocol f*>rmotEnsive New Zealand White rabbits (mates, 2-3 kg) were used. The animals were kept in individual cages with free access tD food and water. They were submitted to artificial 12 hours light/darkness cydes, to avoid uncontrolled arcadian oscillations of IOP Animal handfing and treatment were carried out in accordance with the European Communities Council Directive (86/609/EEC) and the statement of the Association for Research in Vision and Ophthalmology on the Use of Animals in Ophthalmic and Vision Research. The drugs were typically administered by instilling a small volume (typically 40 uL) on the corneal surface. Contralateral eyes were treated with the vehicle alone and could be used as controls in each experiment lest there is a sympathy phenomenon with the other eye. Multiple experiments in the same animal should be abolished. IOP measurements were done using a contact tonometer (TONOPEN XL, Mentor, Norwell, Massachusetts). The TonoPen tonometer is very convenient due to its reliability and small size. Measurements with this instrument were HEK293. The effect of siRNAs over the target gene was analyzed by real time PCR and semi-quantitative PCR according to standard protocols. The gene target transcript levels were normalized using actin as housekeeping gene. Table I below shows representative results of real time PCR experiments for some of the target genes described previously. The values represent the mean of the percentage of siRNA interference over each gene expression once normalized with the control cells and their standard deviations. Compared to the control cells, the level of the different transcripts at both 24 and 48h time points was significantly reduced after the siRNA treatment In the Table are included some of the different siRNAs that were tested and their different efficacies in the interference of the target gene. The siRNAs used in the Table correspond toBiis"RurnansiRhlA targets given in" Rgure 2as "follows. AC2: sjRNAl: rabbit sequence homologous to human SEQ. ID. 73 siRNAZ: rabbit sequence identical to human SEQ. ID. 54 siRNA3: rabbit sequence identical to human SEQ. ID. 65 PTGS1 siRNAl: rabbit sequence homologous to human SEQ. ID. 353 siRNAZ: rabbit sequence homologous to human SEQ. ID. 369 PTGS2 siRNAl: rabbit sequence identical to human SEQ. ED. 426 siRNA2: rabbit sequence homologous to human SEQ. ID. 421 siRNAS: rabbit sequence homologous to human SEQ. ID. 477 gene depends on the efficiency In siRNA silencing. For each target, the most effective siRNA obtained by in vitro studies was administered to .the animal model. RNA was prepared from cells treated with different siRNAs. The samples were analyzed by semi-quantitative PCR using specific primers. The figure shows a representative semi-quantitative gel for Beta Adrenergic Receptor 2 expression (A) and other for Acetylcholinesterase expression (B). M: MW Marker; C: Control cells; TC: Transfection Control; 1: siRNAl; 2: siRNA2; 3: siRNAS; NC: Negative Control. The expression levels for each target depends on the efficiency in siRNA silencing. The siRNAs used in the Figure correspond to the human targets given in Figure 2 as follows: Panel A (Beta Adrenergic Receptor 2) 1: rabbit sequence homologous to human SEQ, ID. 122 2: rabbit sequence identical to human SEQ. ID. 125 3: rabbit sequence homologous to human SEQ. ID. 139 Panel B (Acetylcholinesterase) 1: rabbit sequence homologous to human SEQ. ID. 162 2: rabbit sequence homologous to human SEQ. ID. 177 Example 2 In vivo assays. Previously to the siRNA therapeutical application, the in vivo assays were validated to determine the proper siRNA delivery. Those siRNAs selected by the in vitro assays were applied to the animal model, following the protocol previously described. To avoid the effect of IOP fluctuations due to circadian cycles, all the applications were performed at the same time. To determine the siRNA effect, intraocular pressures (lOPs) were measured as previously mentioned. Since glaucoma pathology presents an Increase in the intraocular pressure, the aim was to obtain a decrease in its levels following siRNA application. Most of the results for the different targets showed a significant decrease in IOP levels comparing with controls and also with commercial drugs (Latanoprost and Dorzolamide) and the animals treated with vehicle alone (negative control),didn't present any significant change in their IOP baseline. The data are summarized in table II where values represent the mean of the maximum percentage of IOP reduction after siRNA treatment once normalized and their standard deviations. The decrease in IOP was statistically significant for all the treated targets. These results indicate that siRNAs and commercial drugs act in a similar way, reducing IOP levels around 20%, although siRNAs present a more maintained effect. No secondary effects were observed in the animals along the experimental protocols. The siRNAs used in these experiments correspond to the human targets given in Rgure 2 as follows: AC2: rabbit sequence homologous to human SEQ. ID. 73 AC4: rabbit sequence identical to human SEQ. ID. 5 AC12: rabbit sequence identical to human SEQ. ID. 522 ADRB1: rabbit sequence identical to human SEQ. ID. 105 ADRB2: rabbit sequence homologous to human SEQ. ID. 139 ADRA1A: rabbit sequence homologous to human SEQ. ID. 546 ADRA1B: rabbit sequence homologous to human SEQ. ID. 619 ACHE: rabbit sequence homologous to human SEQ. ID. 189 PTGS1: rabbit sequence homologous to human SEQ. ID. 322 PTGS2: rabbit sequence identical to human SEQ. ID. 426 SELE: rabbit sequence homologous to human SEQ. ID. 262 ACE1: rabbit sequence homologous to human SEQ. ID. 866 AGTR1: rabbit sequence homologous to human SEQ. ID. 705 AGTR2: rabbit sequence identical to human SEQ. ID. 774 ATP1A1: rabbit sequence Identical to"human SEQ7KO399 ATP1B2: rabbit sequence identical to human SEQ. ID. 1820 (Figure Removed) WE CLAIMS 1. An isolated siNA molecule for use in treatment of an eye condition characterized by an altered intra-ocular pressure (1OP) in the patient, the siNA being complementary to a nucleotide sequence selected from SEQ ID 1 to SEQ ID 1829. 2. An isolated siNA molecule as claimed in claim l^wherein siNA is siRNA. 3. An isolated siNA molecule as claimed in claim l,wherein siRNA is dsRNA. 4. An isolated siNA molecule as claimed in claim 1 wherein cisRNA is shRN A. 5. An isolated siNA molecule as claimed in claim 1, wherein siNA comprises a modified oligonucleotide. 6. A pharmaceutical composition comprising siNA having a sequence which is complementary to a nucleotide sequence selected from SEQ ID 1 to SEQ ID 1829 and pharmaceutically acceptable excipients. 7. A kit useful in down regulating intraocular pressure, said kit comprising a pharmaceutical composition of claim 6, a delivery system such as herein described and an instruction manual. 8. A kit as claimed in claim 7 wherein delivery system is selected from a group comprising a aqueous and non aqueous gels, creams, multiple emulsions, micro emulsions, liposome's, ointments, aqueous and non aqueous solutions, lotion, aerosols, hydrocarbons bases and powders, wherein said delivery system optionally comprises solubilizers, permeation enhancers and hydrophilic polymers. 9. A kit as claimed in claim 8 wherein the pharmaceutical composition is delivered through liposome's, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydro gels, cyclodextrins poly(lactic- co-glycolic) acid (PLGA) and PLCA microspheres, biodegradable nanocapsules, and bioadhesive microsphere, or by proteinaceous vectors. 10. Use of an isolated siNA molecule having a sequence which is complementary to a nucleotide sequence selected from SEQ ID 1 to SEQ ID 1829 in the preparation of a medicament for the treatment of an eye condition. 11. Use of siNA molecule having a sequence which is complementary to a nucleotide sequence selected from SEQ ID 1 to SEQ ID 1829 in the preparation of a medicament for use in a method of treatment of an eye condition, comprising down regulating expression of a target gene in a patient in need of treatment, the gene being selected from the group comprising aqueous formation genes and aqueous outflow genes. 12. A use as claimed in claim 11, wherein the eye condition is characterized by an altered intraocular pressure (IOP) in the patient. 13. A use as claimed in claim 11 or 12, wherein the eye condition is selected from the group comprising" glaucoma, infection; inflammation, uveitis, and" expression of systemic diseases. 14. A use as claimed in claim 10-13, wherein the eye condition is glaucoma. 15. A use as claimed in claim 10-13, wherein the eye condition is diabetic retinopathy. 16. A use as claimed in claim 10-13, wherein the target gene expression is down regulated in the eye of the patient. 17. A use as claimed in claim 10-13, wherein the target gene is selected from the group comprising: Carbonic Anhydrases II, IV and XII; Adrenergic Receptors: betal and 2 and alpha I A, IBand ID; Acetylcholinesterase; Cyclooxygenases 1 and 2; ATPases: alphal, alpha2, alpha3, betal, beta2; Endothelial Leukocyte Adhesion Molecule I (ELAM-I); Angiotensin System: Angiotensin II, Angiotensin II Converting Enzymes (ACE I and ACE II), Angiotensin II Receptors (ATR1 and ATR2) and Renin; Cochlin. 18. A use as claimed in claim 10-13, wherein the siNA is siRNA. 19. A use as claimed in claim 18 wherein the siRNA is dsRNA. 20. A use as claimed in claim 18 wherein the siRNA is shRNA, 21. A use as claimed in claim 10-13 wherein the siNA comprises a modified oligonucleotide. 22. A use as claimed in claim 10-13 wherein the siNA is administered topically to the eye of a patient 23. A use of claim 22, wherein the siNA is administered to the" cornea of a patient. 24. A use as claimed in claim 10-13 wherein a plurality of species of siNA are used. 25. A use of claim 24 wherein said plurality of species are targeted to the same mRNA species. 26. A use of claim 24 wherein said plurality of species are targeted to different mRNA species. 27. An isolated siNA molecule, a pharmaceutical composition, a kit and use of kit in down regulating intraocular pressure substantially as herein described with reference to the foregoing examples and figures.

Documents:

2018-delnp-2007-abstract.pdf

2018-delnp-2007-Claims-(03-04-2014).pdf

2018-delnp-2007-Claims-(08-01-2013).pdf

2018-delnp-2007-claims.pdf

2018-delnp-2007-Correspondence Others-(03-04-2014).pdf

2018-delnp-2007-Correspondence Others-(08-01-2013).pdf

2018-DELNP-2007-Correspondence Others-(24-08-2007).pdf

2018-delnp-2007-correspondence-others.pdf

2018-delnp-2007-description (complete).pdf

2018-delnp-2007-form-1.pdf

2018-delnp-2007-Form-13-(15-03-2007).pdf

2018-delnp-2007-form-13.pdf

2018-delnp-2007-Form-2-(08-01-2013).pdf

2018-delnp-2007-form-2.pdf

2018-delnp-2007-Form-3-(08-01-2013).pdf

2018-delnp-2007-form-3.pdf

2018-delnp-2007-Form-5-(08-01-2013).pdf

2018-delnp-2007-form-5.pdf

2018-delnp-2007-GPA-(08-01-2013).pdf

2018-delnp-2007-pct-101.pdf

2018-delnp-2007-pct-210.pdf

2018-delnp-2007-pct-304.pdf

2018-delnp-2007-Petition-137-(08-01-2013).pdf