Source: {"pile_set_name": "USPTO Backgrounds"}

RNA interference (RNAi) is a naturally occurring post-transcriptional regulatory mechanism present in most eukaryotic cells that uses small double stranded RNA (dsRNA) molecules to direct homology-dependent gene silencing. Its discovery by Fire and Mello in the worm C. elegans {Fire, 1998} was awarded the Nobel Prize in 2006. Shortly after its first description, RNAi was also shown to occur in mammalian cells, not through long dsRNAs but by means of double-stranded small interfering RNAs (siRNAs) 21 nucleotides long {Elbashir, 2001}.
The process of RNA interference is thought to be an evolutionarily-conserved cellular defence mechanism used to prevent the expression of foreign genes and is commonly shared by diverse phyla and flora, where it is called post-transcriptional gene silencing. Since the discovery of the RNAi mechanism there has been an explosion of research to uncover new compounds that can selectively alter gene expression as a new way to treat human disease by addressing targets that are otherwise “undruggable” with traditional pharmaceutical approaches involving small molecules or proteins.
According to current knowledge, the mechanism of RNAi is initiated when long double stranded RNAs are processed by an RNase III-like protein known as Dicer. The protein Dicer typically contains an N-terminal RNA helicase domain, an RNA-binding so-called Piwi/Argonaute/Zwille (PAZ) domain, two RNase III domains and a double-stranded RNA binding domain (dsRBD) {Collins, 2005} and its activity leads to the processing of the long double stranded RNAs into 21-24 nucleotide double stranded siRNAs with 2 base 3′ overhangs and a 5′ phosphate and 3′ hydroxyl group. The resulting siRNA duplexes are then incorporated into the effector complex known as RNA-induced silencing complex (RISC), where the antisense or guide strand of the siRNA guides RISC to recognize and cleave target mRNA sequences {Elbashir, 2001} upon adenosine-triphosphate (ATP)-dependent unwinding of the double-stranded siRNA molecule through an RNA helicase activity {Nykanen, 2001}. The catalytic activity of RISC, which leads to mRNA degradation, is mediated by the endonuclease Argonaute 2 (AGO2) {Liu, 2004; Song, 2004}. AGO2 belongs to the highly conserved Argonaute family of proteins. Argonaute proteins are ˜100 KDa highly basic proteins that contain two common domains, namely PIWI and PAZ domains {Cerutti, 2000}. The PIWI domain is crucial for the interaction with Dicer and contains the nuclease activity responsible for the cleavage of mRNAs {Song, 2004}. AGO2 uses one strand of the siRNA duplex as a guide to find messenger RNAs containing complementary sequences and cleaves the phosphodiester backbone between bases 10 and 11 relative to the guide strand's 5′ end {Elbashir, 2001}. An important step during the activation of RISC is the cleavage of the sense or passenger strand by AGO2, removing this strand from the complex {Rand, 2005}. Crystallography studies analyzing the interaction between the siRNA guide strand and the PIWI domain reveal that it is only nucleotides 2 to 8 that constitute a “seed sequence” that directs target mRNA recognition by RISC, and that a mismatch of a single nucleotide in this sequence may drastically affect silencing capability of the molecule {Doench 2004; Lewis, 2003}. Once the mRNA has been cleaved, due to the presence of unprotected RNA ends in the fragments the mRNA is further cleaved and degraded by intracellular nucleases and will no longer be translated into proteins {Orban, 2005} while RISC will be recycled for subsequent rounds {Hutvagner, 2002}. This constitutes a catalytic process leading to the selective reduction of specific mRNA molecules and the corresponding proteins. It is possible to exploit this native mechanism for gene silencing with the purpose of regulating any gene(s) of choice by directly delivering siRNA effectors into the cells or tissues, where they will activate RISC and produce a potent and specific silencing of the targeted mRNA. RNAi has been applied in biomedical research such as treatment for HIV, viral hepatitis, cardiovascular and cerebrovascular diseases, metabolic disease, neurodegenerative disorders and cancer {Angaji S A et al 2010}.
Many studies have been published describing the ideal features a siRNA should have to achieve maximum effectiveness, regarding length, structure, chemical composition, and sequence. Initial parameters for siRNA design were set out by Tuschl and co-workers in WO02/44321, although many subsequent studies, algorithms and/or improvements have been published since then. siRNA selection approaches have become more sophisticated as mechanistic details have emerged, in addition further analysis of existing and new data can provide additional insights into further refinement of these approaches {Walton S P et al 2010}. Alternatively, several recent studies reported the design and analysis of novel RNAi-triggering structures distinct from the classical 19+2 siRNA structure and which do not conform to the key features of classical siRNA in terms of overhang, length, or symmetry, discussing the flexibility of the RNAi machinery in mammalian cells {Chang C I et al 2011}.
Also, a lot of effort has been put into enhancing siRNA stability as this is perceived as one of the main obstacles for therapy based on siRNA, given the ubiquitous nature of RNAses in biological fluids. Another inherent problem of siRNA molecules is their immunogenicity, whereby siRNAs have been found to induce unspecific activation of the innate immune system. The knockdown of unintended genes (mRNAs) is a well-known side effect of siRNA-mediated gene silencing. It is caused as a result of partial complementarity between the siRNA and mRNAs other than the intended target and causes off-target effects (OTEs) from genes having sequence complementarity to either siRNA strand. One of the main strategies followed for stability enhancement and OTE reduction has been the use of modified nucleotides such as 2′-O-methyl nucleotides, 2′-amino nucleotides, or nucleotides containing 2′-0 or 4′-C methylene bridges. Also, the modification of the ribonucleotide backbone connecting adjacent nucleotides has been described, mainly by the introduction of phosphorothioate modified nucleotides. It seems that enhanced stability and/or reduction of immunogenicity are often inversely proportional to efficacy {Parrish, 2000}, and only a certain number, positions and/or combinations of modified nucleotides may result in a stable and/or non-immunogenic silencing compound. As this is an important hurdle for siRNA-based treatments, different studies have been published which describe certain modification patterns showing good results, examples of such include EP1527176, WO2008/050329, WO2008/104978 or WO2009/044392, although many more may be found in the literature {Sanghvi Y S. 2011; Deleavey et al 2012}.
Allergic diseases are characterized by an overreaction of the human immune system to a foreign protein substance (“allergen”) that is eaten, breathed into the lungs, injected or touched. Allergies have a genetic component. If only one parent has allergies of any type, chances are 1 in 3 that each child will have an allergy. If both parents have allergies, it is much more likely (7 in 10) that their children will have allergies. There are no cures for allergies; however they can be managed with proper prevention and treatment.
About 30% of people worldwide suffer from allergic symptoms and 40-80% of them have symptoms in the eyes {Key B. 2001}. Allergic diseases affecting the eyes or ocular allergies constitute a heterogenic group of diseases with a very broad spectrum of clinical manifestations. An ocular allergy usually occurs when the conjunctiva (membrane covering the eye and the lining of the eyelid) reacts to an allergen. An ocular allergy can happen independently or in conjunction with other allergy symptoms (such as rhinitis or asthma).
Basic and clinical research has provided a better understanding of the cells, mediators, and immunologic events which occur in ocular allergy. The eye, particularly the conjunctiva, has a relatively large number of mast cells. When allergens are present they can bind to immunoglobulin, IgE, in the FcϵRI receptors on the surface of these mast cells and trigger their activation and release of mediators of allergy (a process known as degranulation). Degranulation releases mast cell components, including histamine, prostaglandins, tryptase and leukotrienes, into the environment outside the mast cell. Through a variety of mechanisms these components produce the signs and symptoms of the ocular allergy. The activation of the mast cells of the allergic inflammation is frequently designated as an acute phase response or early phase of the ocular allergy. The acute phase response can progress to a late phase response characterized by recruitment of inflammatory cells to the site of the allergic inflammation, for example as an influx of eosinophils and neutrophils into the conjunctiva.
Ocular allergy represents one of the most common conditions encountered by allergist and ophthalmologists. Ocular allergy is usually associated with the following symptoms and signs: conjunctivitis, blepharitis, blepharoconjuntivitis or keratoconjunctivitis. The eye becomes red and itchy and there occurs lacrimation and slight discharge. Severe cases may also show eye burning sensation, pain and photophobia.
Allergic diseases affecting the eyes include mild forms such as seasonal allergic conjunctivitis (SAC) and perennial allergic conjunctivitis (PAC); and more severe manifestations such as vernal keratoconjunctivitis (VKC); atopic keratoconjunctivitis (AKC) and giant papillary conjunctivitis (GPC). The latter ones can be associated with complications such as corneal damage and may cause vision loss. SAC and PAC are commonly IgE-mast cell mediated hypersensitivity reaction to external allergens; whereas AKC and VKC are characterized by chronic inflammation involving several immune cell types. SAC and PAC allergens, with the help of antigen presenting cells (APCs),