IE20090793A1 - A delivery system - Google Patents

Abstract

This invention relates to delivery of siRNAs. In one aspect, the invention relates to method for delivering an siRNA or engineered RNA precursor to a target by bringing a multiplicity of RNA-containing minicapsules into contact with the target cell.

Description

INTRODUCTION A number of studies have implicated the occurrence of hypoxia in mucosal inflammatory diseases sueh ns IBD (inflammatory bowel disease) and surgical specimens from patients with IBD have revealed activation of the so-called HIF (hypoxia-tndueible factor) family of proteins associated with increased vascular density in diseased areas as a defensive response to the hypoxia caused by the disease. This and other evidence (see C.T. Taylor et al. J Mol Med (2()07( 85:1295-1300 the entirety of which is incorporated herein by reference) support a protective role for HIF in mucosal disease.

IE 0 9 07 93 Under normal cellular function, HIF is hydroxyIated (inactivated) by prolyl hydroxylase (PHD) implying that a PHD inhibitor should provide benefit to diseases of this type. In mammalian cells, three PHD isoforms have been identified (PHD 1-3), and shown to hydroxy late HIF-α in vitro (see P. Jaakkola et al. (2001) Science 292:468-472 and WC Hon et al Nature 417:975-978). These enzymes have an absolute requirement for oxygen as the substrate. Reactions conducted in a limited oxygen environment have revealed that the activity ofthe purified enzyme is strikingly sensitive to diminished levels of oxygen in vitro. Ihe three enzymes have different tissue distributions and, at least under conditions of overexpression, have distinct patterns of subcellular localization. PHD I mRNA is expressed in many tissues, with especially high expression in the testis. Likewise, PHD2 mRNA is widely expressed, with particularly abundant expression in adipose tissue. PHD3 mRNA is also expressed in many tissues but is most abundant in the heart and placenta. Expression of all three isol'oinis of PHDs has been found in mouse intestinal mucosal tissue with a distribution of PHD1 Ihe discovery of HIF-selective PHDs as central regulators of HIF expression has now provided the basis for potential development of PHD-based molecular tools and therapies. For example, pharmacological inactivation of the PHDs by 2-OG analogues (eg DMOG or ldimethyloxallyl glycine) is sufficient to stabilize HIF-α but this action is non-specific with respect to individual PHD iso forms. In vitro studies suggest significant differences in substrate specificity. For example, according to Huang et al. (1998 Proc. Nail. Acad Sci. 95: 7987-7992), regulation of HIF o-subunit stability is mediated through a region referred to as the oxygen-dependent degradation domain (ODD) and comparison of enzyme activity in vitro has shown that this sequence is hydroxylated most efficiently by PHD2. These observations have generated Interest in identifying enzyme-modifying therapeutics.

A number of PHD inhibitors have been described, including direct inhibitors of the PHDs. analogs of naturally occurring cyclic hydroxamates, as well as antagonists of a-keloglutarate.

Studies in mice (see C.T. Taylor el al, .1 Mol Med (2007) 85:1295-1300) show that PHD inhibition provides an overall beneficial influence on clinical symptoms (weight loss, colon length, tissue tumor necrosis factor-a/interferon-γ). It is believed that these effects are due to activation of HI F-l-dependent pathways leading to barrier-protective function and enhancement of wound healing at the site of inflammation. 1609 07 93 Related to these observations, Cummins et al. (2006) PNAC (the entirety of which is incorporated herein by reference) observe that mimicking hypoxia by treatment of cells with si RNA against PHD-1 or PHD-2 or the pan-prolyl hydroxylase inhibitor DMOG results in NFkB activation. More specifically, they report that although sensitive to knockdown of both PI ID-1 and PHD-2. NF_B activation appears to be more sensitive to silencing of the PHD-I isoform. suggesting somewhat differential regulation to HIF-I, which is predominantly regulated by PHD-2.

Berra et al. (2003) JEMBO (the entirety of which is incorporated herein by reference) state that silencing (using siRNAs) of PHD1 and PHD3 has no effect on the stability of HlF-la either in normoxia or upon re-oxygenation of cells briefly exposed to hypoxia. They therefore conclude that, in vivo, PHDs have distinct assigned functions, PHD2 being the critical oxygen sensor selling the low steady-state levels of HIF-la in normoxia. They point out that PHD2 is upregulated by hypoxia, providing an HIF-1-dependent auto -regulatory mechanism driven by the oxygen tension.

To achieve their results, Cummins et al. and Berra et al used a transfection technique in which siRNA duplexes of the relevant ribonucleic acid sequences in cationic liposomes were used to transfect (or co-transfect along with reporter plasmid combinations) mammalian including HeLa cells. This technique is described by Elbashir et al (2001) Nature vol 411 p494 lhe entirety of which is incorporated herein by reference.

In tuber therapeutic areas also, RNA interference (RNAi) is emerging as an indispensable strategy for target-specific knockdown of gene expression. However, despite secondgeneration technologies representing advances in RNAi design, efficiency, and efficacy, daunting obstacles remain, such as how to deliver RNAi compounds to the right targets in the right amounts.

Recent advances in RNAi delivery include utilizing tiny delivery vehicles called nanopartieles and applying epigenetics for changing DNA expression without altering the gene sequence. ΙΕ ύ 9 Ο7 93 Successful delivery of RNAi faces a number of challenges including exposure. For example, if the RNAi compound is to be injected, a critical question is whether the tissue targeted will be exposed to the RNAi. If the RNA is injected and delivered intraperitonea I ly or intravenously it enters the systemic circulation (bloodstream), where it is taken up by the kidneys and removed. As a result, standard injections are believed not to provide a likely avenue of RNA delivery.

Exposure issues also depend on the type of tissue targeted. For example, exposure can be enhanced if it is possible to achieve local delivery, such as injecting RNAi into the eye for treating age-related macular degeneration. The physiological structure of the eye permits retention of the injection fluid locally. Local delivery has also been attempted by injection directly into the central nervous system or via inhalation or topically.

A second hurdle is cytoplasmic uptake. Overcoming exposure problems doesn’t mean the RNA can penetrate the cytoplasm of cells. A third problem is the stability of the RNAi compound itself. It is believed that RNA needs to be carefully designed and chemically stabilized.

Nanoiransporters (see for example www.rxipharma.com) are chemicals of defined size that are mixed with an RNAi compound to form minute particles for delivery into target tissues. The nanotransporter has a core to which layers are added by chemical synthesis. The final layer has a positive charge allowing it to attract and bind negatively charged RNAi compounds. This approach has been successfully utilized for delivery into mouse liver as a treatment for amyotrophic lateral sclerosis (ALS) leading to knockdown of the gene for superoxide dismutasel (SOD1) which normally neutralizes oxygen molecules that damage cells. Although ALS can be associated with more than 100 different mutations in SOD1, it was found that RNAi-mediated knockdown of SOD I had a benefit in an animal model of ALS.

Il is possible to combine nanoparticles for both efficient transfection as well as imaging studies. Magnetic nanoparticles with a lipid core have been used in MRI studies in which tags, such as fluorescence are combined with active RNAi (see http://www.genovis.com/RNAi). This approach uses tiny superparamagnetic nanoparticles that feature iron oxide cores coated with a specific cationic lipid formulation. This facilitates IE 0 9 07 93 particle solubility and cellular uptake and enables experiments to be conducted to establish and track the system (such as in a whole cell, endosome, or liposome) in which RNAi is working. However these reagents have so far only been adapted to cell culture (suspension cells, mesenchymal stem cells and adherent standard cell lines). Even in this context, and more so in therapeutic settings, it is important in using iron oxide to keep toxicity to a minimum.

The mammalian RNAi pathway contains an enzyme known as dicer, a natural initiation point for the RNAi cascade. It is possible to create longer-than-natural RNA precursors adapted to be substrates of dicer and in some settings this leads to a more potent and longer-lasting variant of RNA interference (see http://www.dicerna.com/science-technoloiiy.html). The current challenge for this dicer-based approach, in common with other approaches of the prior art, is to optimize the delivery modalities for the highest efficiency.

Delivering RNA for gene knockdown is not equivalent to DNA delivery with DNA delivery techniques having been largely unsuccessful. Some groups are attempting to design synthetic delivery systems, including polymeric systems for efficient RNA delivery and gene knockdown. For example, Egen (see http://www.egencorp.com/index.htm) has the ThcraSilence™ technology platform for delivery of therapeutic siRNA or shRNA. Candidates are being tested for proof of concept in animal models of diseases It has been suggested that any RNAi delivery system must efficiently function to protect the therapeutic cargo from degradation, to promote uptake by target cells, and to facilitate intracellular trafficking and that multiple approaches may be needed depending on a variety of factors, such as the disease being treated, the tissue, mode of administration, dose, and the specific siRNA or short hairpin RNA (shRNA).

RNAi may also have application in epigenetics which refers to stable changes in gene expression that do not involve altering the actual DNA sequence. Epigenetic mechanisms can control or alter gene activity in several ways, such as RNAi, DNA methylation, and modification of histones that encase DNA. It may not be necessary to ensure the permanent presence of a therapeutic compound for a permanent effect. So, in some cases a transient transfection of a gene can produce a permanent change. This approach has been used in the transient transfection of mesenchymal stem cells.

IE 0 9 0 7 9 3 It has been suggested that the nature of epigenetic mechanisms may assist with the utilization of RNAi for therapeutics in that RNAi knocks down the expression of the target by destroying its mRNA. If the target is a regulatory protein, knocking it down would alter the expression of other genes at the transcriptional level and some of these transcriptional effects may in certain circumstances be locked in by epigenetic changes, such as DNA methylation, thereby allowing the RNAi effect to persist after the RNAi-inducing molecule is gone.

None of the foregoing technological approaches relates to RNAi delivery to the cells of the intestinal epithelium which is the focus of the present invention. fhe primary functions of the gastrointestinal tract are the processing and absorption of ingested nutrients, waste removal, fluid homeostasis, and the development of oral tolerance to nonpathogenic luminal antigens. The last of these functions involves the intestinal mucosa being unique among tissues as it is in a constant state of controlled inflammation. This occurs as the mucosal immune system is constantly exposed to new food-borne material in the lumen, which is processed to avoid inappropriate inflammatory reactions to harmless ingested antigens.

A critical cell type in the maintenance of intestinal homeostasis is the epithelial cell of the gastro-intcslinal tract (GIT). The intestinal epithelium is a monolayer of cells that covers an area of approximately 250-300 m2 in an adult human and forms a critical barrier between the external (luminal) and internal (vascular) compartments. This dynamic barrier is maintained primarily by the existence of regulated intercellular tight junctions. As well as being a critical barrier, the epithelium is responsible for the absorption of approximately nine litres of fluid from consumed liquids and secreted digestive fluids per day. This fluid transport function is carried out through coordinated ion transport events and the subsequent regulation of salt and water transport between the lumen of the gut and the bloodstream. Importantly, both the barrier and absorptive functions of the intestinal epithelium can be physiologically regulated by oxygen.

Inflammatory bowel disease (IBD) is an umbrella term for a range of disorders including ulcerative colitis and Crohn’s disease, which are characterized by a breakdown in the intestinal epithelial barrier with subsequent unregulated exposure of the mucosal immune ΙΕ π 9 07 9 3 system lo luminal antigenic material leading to inflammation and further barrier breakdown. Thus, a self-perpetuating cycle of inflammation is initiated leading lo severe pathology. Because ot the limited number of current therapeutic options available, treatment often ultimately resorts to surgical resection of significant amounts of chronically inflamed intestinal tissue.

SUMMARY: STATEMENTS OF INVENTION Ihe present invention is based, in part, upon the discovery of delivery methods for siRNA or an engineered RNA precursor using minieapsules or minispheres.

Ihe expression siRNA or an engineered RNA precursor1* and RNA** are used interchangeably unless the context requires otherwise and includes small interfering RNAs (siRNAs). mieroRNAs (miRNAs). small hairpin RNAs (shRNAs). and others, including conjugates, such as described below particularly in the detailed description, ll is intended that any such mention of RNA includes molecules with or without backbone modi flea lion or conjugated variants thereof (where the conjugate is can be another nucleic acid or a molecule of a different type such as a lipid or peptide).

Ihe terms minieapsules and minispheres and minibeads are used interchangeably unless the context requires otherwise.

In one aspect, the present invention features a method for delivering an siRNA or engineered RNA precursor to a cell, particularly an epithelial cell of the GIT (gastrointestinal tract), by bringing a multiplicity of RNA-containing minieapsules into contact with the target cell, for example by oral administration of a pharmaceutical formulation comprising such minieapsules.

In one embodiment, the target cell is an intestinal epithelial cell.

In another aspect of the invention, the RNA comprised in the minispheres is adapted to interfere, knockdown or inhibit the expression of specific genes or gene products or messenger products (eg mRNA) and/or expression of enzymes, especially those affecting the control of hypoxia in the cells of the GI tract. Of particular interest in the present invention IE 0 9 07 93 are RNAs which affect (in particular knockdown, inhibit or interfere with) enzymes which normally cause IIIF io be upregulaled or retained at beneficial levels.

One embodiment of the invention is the knock-down of a target gene or gene product (including messenger) for a transient period.

Il is particularly preferred that the RNA be adapted lo knockdown, silence or inhibit the expression ol one or more PHDs. including PHD I. 2 and 3. Such RNAs arc referred to in the Examples herein as $iRN/\s for PHD I, 2 and 3 respectively.

In another aspect, the present invention features a method for delivering an siRNA to a cell, preferably a (j| cell, by obtaining, identifying or targeting a cell (or system of cells or tissue), forming a minisphcrc comprising an siRNA and contacting the cell (or system of cells or tissue) with the minisphere ora plurality thereof In another aspect, the invention provides an siRNA or engineered RNA precursor conjugated to a delivery peptide, the conjugate being encapsulated in a minisphere.

In one aspect, the inveniion features biconjugatcs of targeting peptides susceptible of enhancing uptake of siRNA and thus promote gene silencing in vivo.

Also included in (he invention is a pharmaceutical composition comprising mini-beads of solid matrix material wherein the mini-beads comprise one or more si RNAs or engineered RNA precursors dispersed in said solid matrix.

In another aspect, the invention provides an siRNA or engineered RNA precursor associated with a polymer eg a cationic polymer such as ehitosan. to form an aggregate or a complex, the aggregate or complex optionally being encapsulated in a minisphere eg a minibead with or w ithout the presence of liposomal materials.

In one aspect, the invention features such aggregates susceptible of enhancing uptake of siRNA and thus promote gene silencing in vivo. An example is a chitosan-siRNA aggregate which may Ik* lurther associated with liposomal materials and then optionally encapsulated to form a minieapsule or mini-bead.

IE 09 07 93 The invention includes a medicament for delivering active agent selected from an siRNA and engineered RNA precursors to a target cell in the gastrointestinal tract, the malic ament comprising a multiplicity of RNA-containing minicapsutes and being adapted for the active agent lo be released and contact the target cell after administration of the medicament. Also included is a medicament for delivering active agent selected from an siRNA and engineered RNA precursors to a predetermined region of the gastrointestinal tract, the medicament comprising a multiplicity of RNA-containing minicapsules and being adapted lor the active agent to he released in said region: in one embodiment, the medicament is adapted to release the active agent in the colon.

The invention also provides an oral composition comprising minicapsules wherein the minicapsules comprise one or more siRNAs or engineered RNA precursors in a core susceptible of maintaining such RNA in a stable, active form.

While the core may be liquid, semi-solid, or solid core, a preferred embodiment ot' the invention is a liquid core.

In another aspect. the minicapsules have release profiles to release the siRNA or engineered RNA precursor in an active form at one or more sites along the gastrointestinal tract, for example where absorption (for local or systemic benefit) is maximized or therapeutic efficacy is maximized.

Preferably, according to the invention, the siRNA or engineered RNA precursor, regardless of its inherent physicochemical property, when released from the minicapsule is in a soluble form or is readily soluble in the aqueous GIT environment.

According to the invention, the minicapsule may have one layer e.g. a minibead and may be essentially solid throughout or be a solid comprising inclusions selected from liquid inclusions, semi-solid inclusions or combinations thereof. Some minibeads thus have a solid phase with semi-solid interior portions. Included, therefore, are single layer minicapsules which are solid throughout. Alternatively, the minicapsule may have two layers comprising a solid outer shell layer encapsulating a liquid, semi-solid or solid core. For example, the ίο ΙΕ Π 9 0 7 9 3 minicapsule may have three layers comprising a solid outer shell layer; a solid, semi-solid or liquid middle buffer layer; and a liquid, semi-solid or liquid core.

According lo the invention, the minieapsules may be modified to enable modified release of the siRNA or engineered RNA precursors). For example, a modified release coating may be applied to the outer shell layer of the minicapsule. Alternatively, an outer shell layer of the miiiicapsulc may be modified to achieve modified release. In other formats, the minicapsule core or entirety may control the rate of active compound release. For example a buffer layer of the minicapsule may be modified to achieve modified release. Alternatively, the liquid, semi-liquid or solid core of the minicapsule may be modified to achieve modified release. For example, polymeric materials may be used achieve modified release such as polymeric materials that are sensitive to one or more of pH, time, thickness, erosion, and bacterial breakdown.

The minicapsule may comprise of one layer containing one or more active pharmaceutical agents as well as delivery enhancing excipients in addition to the siRNA or engineered RNA precursor and that layer may control the release of the siRNA or engineered RNA preeursor(s).

The invention includes as such mintbeads, minicapsules and minispheres as described herein, e.g. a single minibead, minicapsule or minisphere as described herein. Λ medicament lor delivering an active agent selected from siRNAs and engineered RNA precursors to a target cell in the gastrointestinal tract, the medicament comprising a multiplicity of RNA-containing minieapsules and being adapted for the active agent to be released and contact the target cell after administration of the medicament.

The invention includes product selected from a minicapsule, a minibead or a minisphere and comprising a solid phase and an active agent selected from siRNAs and engineered RNA precursors (e.g. a combination of at least two such agents or a single such agent). The product may have one layer being a solid phase comprising inclusions selected from liquid inclusions, semi-solid inclusions or combinations thereof or have at least two layers comprising a solid phase outer shell layer encapsulating a liquid, semi-solid or solid core. In embodiments, the product has one layer and the inclusions comprise the active agent; in other embodiments the II IE 0 9 07 9 3 product has nt least two layers and the eorc comprises the active agent. In some embodiments the active agent is dispersed in the solid phase, whether additionally to or alternatively lo inclusion of the same or different active agent in a core or in incusions.

The siRNA or engineered RNA preciirsor(s) may be released along the gastrointestinal tract in a form that maximises systemic absorption and/or local absorption. For example, the siRNA or engineered RNA precursor (s) may be released along the gastrointestinal tract in a form that maximises lymphatic absorption. Such delivery is desirable for RNAi molecules targeting inicro-melastatic or metastatic tumour cells that pass through the lymphatic system or for RNAi molecules designed to modulate immune cell function and response.

According to another aspect of the invention, the siRNA or engineered RNA precursor (s) may be released along the gastrointestinal tract in a form that maximises blood brain barrier absorption. Alternatively, the siRNA or engineered RNA precursor (s) may be released along the gastrointestinal tract in a form that maximises pre-systemic and/or local absorption, in particular by the epithelial cells lining the GIT. Alternatively, the siRNA or engineered RNA precursors) may be released along the gastrointestinal tract in a form that maximises local gastrointestinal activity. Alternatively, the siRNA or engineered RNA precursor(s) may be released along the gastrointestinal tract in a form that maximises gastrointestinal lumen activity. Alternatively, the siRNA or engineered RNA precursor(s) may be released along the gastrointestinal tract in a form that maximises chronotherapy. In all cases, the RNA formulation or component(s) thereof is (are) released in such that it (they) is (are) in soluble when released or is (are) readily soluble in the local GIT environment. Another aspect of the invention is the release of RNAi molecules, formulations thereof or components of such formulations to act within the lumen to inhibit bacterial or viral entities.

In relation lo the types of RNA which may be incorporated into the minicapsules of the present invention, they can be selected from sequences appropriate to a target gene ie. a gene whose expression is to be selectively inhibited, knocked down or silenced by such RNA. This silencing is achieved by cleaving the mRNA of the target gene by an siRNA. e. g.. an isolated siRNA or one that is created from an engineered RNA precursor. One portion or segment ol a duplex stem of the siRNA, RNA precursor, or one strand of the siRNA. is an anti-sense strand that is complementary', e. g. fully complementary, to a section, c. g., about 16 to about 40 or more nucleotides, of the mRNA of the target gene.

IE 0 9 0 7 93 An ‘'isolated nucleic acid molecule or sequence is a nucleic acid molecule or sequence that is not iinmediateK contiguous with both ofthe coding sequences with which it is immediately contiguous (one on (he 5' end and one on the 3’ end) in the naturally occurring genome ofthe organism from which it is derived. I he term therefore includes, for example, a recombinant I IN.A of PNA (hat is incorporated into a vector; into an autonomously replica line plasmid or virus: or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (c. g. a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatmenl) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

I he term engineered as in an engineered RNA precursor, or an engineered nucleic acid molecule, indicates that the precursor or molecule is nol found in nature, in that all or a portion of the nucleic acid sequence of the precursor or molecule is created or selected by man. Once created or selected, the sequence ean be replicated, translated, transcribed. or oilierw ise processed by mechanisms within a cell. Thus, an RNA precursor produced within a cell from an engineered nucleic acid molecule, e. g. a transgene, is an engineered RNA precursor. Engineered RNA precursors are artificial constructs that are similar io naturally occurring precursors of small temporal RNAs (stRNAs) that are processed In the body lo form siRNAs. Ihe engineered RNA precursors can be synthesized by standard methods known in the art. e. g. by use of an automated DNA synthesizer (such as arc commercially available Irom Biosearch. Applied Biosystcms. etc.) or encoded by nucleic acid molecules.

I'nl css otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of eonllict. the present specification, including definitions, will control.

In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. π ΙΕ η 9 0 7 9 3 Other features and advantages of the invention will he apparent from the following detailed description. and from the claims.

BRI El· DESCRIRI ION OE II IE FIGURES Figure I shows western blots showing knock-down of p65 NFkB by siRNA in a liposomal formulation.

DETAIL!d) DESCRIPTION I he present invention provides compositions and methods for delivering siRNAs. or siRNA precursors, into cells, e. g. eukaryotic celts such as mammalian cells (for example, human cells). I hese methods are useful both in vivo and in vitro.

Sequence-selective, post-transcriptional inactivation of expression of a target gene can he achieved in a wide variety of eukaryotes by introducing double-stranded RNA corresponding to the target gene, a phenomenon termed RNA interference (RNAi).

‘This approach lakes advantage of the discovery that siRNA can trigger the degradation of mRNA corresponding to the siRNA sequence. To be effective, the siRNA must nol only enter the cell, but must also enter the cell in sufficient quantities to have a significant effect. RNAi methodology has been extended to cultured mammalian cells, but its application in vivo has been limited due to a lack of efficient delivery systems with little or no loxieity. fhe present application provides sueh a system.

Al present most commonly used techniques (sueh as microinjection. transfection using cat ionic liposomes, viral transfection or electroporation of oligonucleotide conjugates) induce in the cells and'or host stress and other limitations and drawbacks.

For example, nucleic acid delivery mediated by cationic liposomes such as EII>(>FEC'FAMINEIM. LIPOFECTIN™. CYTOFECFIN™ as well as transfection mediated hy polymeric DNA-binding cations sueh as poly-E-lysine or polyethylene imine are extensively used 1 runs feet ion techniques. These methods can be associated with cytotoxicity and sensitivity to serum, antibiotics and certain cell culture media. In addition, these methods are limited hy low overall transfection efficiency and time-dependency. Other methods such IE 0 9 0 7 9 3 as micro'injection or electroporation arc simply not suitable for large-scale delivery of nucleic acids in io living tissues. The relevance of these approaches does not appear to have been assessed for oral delivery of siRNA.

RNAi is a remarkably efficient process whereby double-stranded RNA (dsRNA) induces the sequence-specific degradation of homologous mRNA in animals* and plant cells (I luuagner and /anion? (2002). Curr. Opin. Genet. Dev. . 12.225-232 ; Sharp (2001). Genes Dev. , .485-490). In mammalian cells. RNAi can be triggered by 21- nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chiu et al. (2002). Mol. Cell. . 10, 549-561 : Elbasliir et a!. (2001). Nature. 41 1,494-498). or by micro-RNAs (miRNA). functional small-hairpin RNA (sliRNA). or oilier dsRNAs that are expressed in vivo using engineered RNA precursors such as DNA templates, e. g.. with RNA polymerase III promoters (Zeng et al. (2002). Mol. Cell. 9.1327-1333 : Paddison et al. (2002), Genes Dev. . 16. 948-958 ; Lee et al. (2002). Nature Biotechnol. . 20.500-505 : Paul ct al. (2002). Nature Bioteclinol. , 20.505-508 : Tuschl. T. (2002). Nature Biotechnol.. 20,440-448 ; Yu et al, (2002). Proc. Natl. Acad. Sci. USA. 99 (9). 6047- 6052: McManus et al. (2002). RNA. 8.842-850 ; Sui et al. (2002), Proc. Natl. Acad. Sci. USA. 90 (6), 5515-5520.) siRNA Molecules flic nucleic acid molecules or constructs used in (lie invention include dsRNA molecules comprising 16-30. e. g. 16, 17. 18, 19. 20. 21. 22. 23. 24, 25. 26, 27, 28, 29 or 30 nucleotides in each strand, wherein one of the strands is substantially complementary to. c. g. at least 80% identical (or more. e. g. . 85%. 90%, 95%. or 100%) (for example, having 3. 2. I. or 0 mismatched nucleotide (s) ). to a target region, such as a target region that differs by at least one base pair between the wild type and mutant allele of a nucleic acid sequence. 1'or example, the target region can comprise a gain-ol- (unction mutation, and the oilier strand is identical or substantially identical to the first strand. The dsRNA molecules of the invention can be chemically synthesized, or can be transcribed in vitro from a DNA template, or in vivo from an engineered RNA precursor, e. g„ sliRNA. Ihe dsRNA molecules can be designed using any met hod known in the art. for instance, by using the following protocol: STEP I. ΙΕ !) 9 ο 7 9 3 Beginning with ihe AUG start codon ot the target DNA. look for AA dinudcotide sequences: each A A and the Vadjacent 16 or more nucleotides are potential siRNA targets. The siRNA should be specific for a target region that differs by at least one base pair between the wild type and mutant allele, c. g. a target region comprising the gain of function mutation, 'fhe first strand should be complementary to this sequence, and the other strand is identical or substantially identical to the first strand. In one embodiment, the nucleic acid molecules are selected from a region of the target allele sequence beginning at least 50 lo 100 ni downstream of the start codon, e. g. of the sequence of SOD I. Further, siRNAs with lower G/C content (35-55%) may be more active than those with G/C content higher than 55%. Ί hus in one embodiment, the invention includes nucleic acid molecules having 35-55% G/C content. In addition, the strands of the siRNA can he paired in such a way as to have a 3' overhang of I to 4, e. g. 2. nucleotides. Thus in another embodiment, the nucleic acid molecules may have a 3'overhang of 2 nucleotides, such as TT, The overhanging nucleotides may be either RNA or DNA. In one embodiment, the overhang nucleotides are deowlhy inidincs or other appropriate nucleotides or nucleotide analogs. Other embodiments are also envisioned where the strands of the siRNA do not have a 3' overhang. As noted above, it is desirable to choose a target region wherein the mutant: wild type mismatch is a purine: purine mismatch.

STEP 2.

Using any method known in the art. compare the potential targets to the appropriate genome database (human, mouse, rat, etc. ) and eliminate from consideration any target sequences with significant homology to other coding sequences. One such method for such sequence homology searches is known as BLAST, which is available at www. nebi. nhn. nih. gov/BLAST.

STEP 3.

Select one oi mote sequences that meet the criteria for evaluation.

Further general information about the design and use of siRNA may be found in The siRNA User Guide available at www. mpibpc. gwdg. de/abteilungen/100/l05/sirna. html.

IE 0 9 0 7 9 3 Alternatively suitable siRNA may be purchased commercially for example from suppliers such as Dharmacon.

Negative control siRNAs should have the same nucleotide composition as lhe selected siRNA. Ixii without significant sequence complementarity to the appropriate genome. Such negative controls may be designed by randomly scrambling the nucleotide sequence of the selected siRNA: a homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control siRNAs ean be designed by introducing one or more base mismatches into the sequence.

The siRNAs used in the invention include both siRNA and crosslinked siRNA derivatives as described in Ik S. Provisional Patent Application 60/413.529. which is incorporated herein by reference in its entirety. Crosslinking can be employed io alter the pharmacokinetics ol the composition, foi example, to increase half-life in the body. Additionally, other chemical backbone modifications such as phosphorothiolation can be employed for example to enhance stability. Equally, covalent attachment of lipid-based (or other) moieties such as chemical attachment of amphiphilic oligomers to specific sites on the RNA to form a socalled conjugate ean be employed as described in more detail below. Such an approach may be employed to enhance stability against enzymic degradation eg in the GI tract in addition to allowing incorporation of the RNA into formulations discussed in more detail below that facilitate absorption across intestinal mucosal barriers. More detail of this technique is provided Ibr example in the paper by J. Gordon Still (2002) in Diabetes/Metabolism Research and Reviews. 1 8 (Suppl I): S29-S37 the entirety of which is incorporated herein by reference as well as elsewhere in this description.

Thus, the invention makes use of siRNA derivatives that include siRNA having two complementary strands of nucleic acid, such that the two strands arc crosslinked. For example, a 3*011 terminus of one ofthe strands can be modified, or the two strands ean be crosslinked and modified at the 3ΌΗ terminus. The siRNA derivative can contain a single crosslink (e. g., a psoralen crosslink). In some embodiments, the siRNA derivates has al its 3’ terminus a biotin molecule (e. g.. a photocleavable biotin), a peptide (e. g. , a l at peptide). a nonoparticle. a pcptidomimettc. organic compounds (e. g. , a dye such as a fluorescent dye), or dendrimer (such as are available from Sigma-Aldrich and also including PAM AM amine IE 0 9 Ο 7 9 3 terminated and/or PAM AM: carboxylic acid tcnninaled (available from Dendritcch Inc): diaminobutane (DAB)-DAB: amine terminated and/or carboxylic terminated). The siRNAs can also be delivered by mixing with such a delivery agent. Modifying siRNA derivatives in this way may improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative as compared to the corresponding siRNA, are useful for tracing (he siRNA derivative in the cell, or improve the stability of the siRNA derivative compared to the corresponding siRNA.

Ihc nucleic acid molecules used in the present invention can also be labeled using any method known in the art; for instance, the nucleic acid compositions can be labeled with a lluorophme, e. g. . Cy3, fluorescein, or rhodamine. The labeling can be carried out using a kit, e. g. . the SILENCER siRNA labeling kit (Ambion), Additionally, the siRNA can Ik radiolabeled, e. g., using 3H, 32p, or other appropriate isotope.

Nucleic acid molecules described or recited herein are intended to comprise nucleotide sequences with or without 3’ overhangs, e. g., with or without 3’- dcoxythymidincs. Other embodiments are also envisioned in which the 3' overhangs comprise other nucleotides, e. g.. UU or the like.

SiRNA Conjugates I’he siRNA formulations of the present invention, as well as comprising an engineered RNA precursor or engineered nucleic acid molecules that encode the precursors, can comprise such a molecule conjugated to delivery peptides or other compounds to enhance the efficiency of transport of the siRNA into living cells compared to the efficiency of delivery to unmodified siRNA. Such conjugates are described in more detail in US 2004/0204377 AI Ihe entirety of which is incorporated herein by reference.

In particular, the siRNAs used in the present invention, as well as an engineered RNA precursor or engineered nucleic acid molecules that encode the precursors, can be conjugated to delivery peptides or other compounds to enhance the efficiency of transport of the siRNA into living cells compared to the efficiency of delivery to unmodified siRNA. These delivery' peptides can include peptides known in the art to have cell-penetrating properties. For instance, the delivery peptide can be, but is not limited to: TAT derived short peptide from If 0 9 ο 7 9 3 human immunodeficiency virus (HIV-1), such as TAT 47-57 and Cys (amino acid sequence: CYGRKK.RRQRRR), and TAT 49-60 and (Arg)9 (Tat) (amino acid sequence RKKRRQRRRPPQC), and substantially similar variants thereof, e.g., a variant that is at least 65% identical thereto. Of course, the percent identity can be higher, e.g., 65%, 67%, 69%, 70%, 73%. 75%, 77%, 83%, 85%, 87%, 90%, 93%, 95%, 97%; 100% identity (for example, peptides with substitutions at 1, 2, 3, 4 or more residues) (e.g,, amino acid sequence: CYQRKKRRQRRR). In general, the substitutions are conservative substitutions. The methods of making such peptides are routine in the art.

The above mentioned delivery peptides can also have modified backbones, e.g.oligocarbamate or oligourea backbones; see, e.g., Wang et al., J. Am. Chem. Soc., Volume 119, pp. 6444-6445, (1997); Tamilarasu et al., J. Am. Chem. Soc., Volume 121, pp. 1597-1598, (1999), Tamilarasu et al., Bioorg. Of Med. Chem. Lett., Volume 11, pp. 505-507. (2001). The conjugation can be accomplished by methods known in the art, e.g., using the methods of Lambert et al. (2001), Drug Deliv. Rev., 47(1), 99-112 (describes nucleic acids loaded to polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al. (1998), J. Control Release, 53(1-3), 137-43 (describes nucleic acids bound to nanoparticles); Schwab et al. (1994), Ann. Oncol., 5 Suppl. 4, 55-8 (describes nucleic acids linked to intercalating agents, hydrophobic groups, polycations or PACA nanoparticles); and Godard et al. (1995), Eur. J. Biochem., 232(2), 404-10 (describes nucleic acids linked to nanoparticles). The contents of the papers referred to in this paragraph (Wang et al ; Tamilarasu et al (1999 and 2001); Lambert el al, Fattal et al; Schwab et al and Godard et al) are, in their entirety, incorporated herein by reference.

As defined herein, a therapeutically effective amount of an siRNA-peptide conjugate or siRNA delivery agent mixture, e.g., an siRNA-dendrimer mixture (i.e., an effective dosage) depends on the nucleic acid selected. For instance, if a plasmid encoding shRNA is selected, single dose amounts in the range of approximately I /ug to 1000 mg may be administered; in some embodiments, 10, 30, 100 or 1000 pg cna be administered. In some embodiments, 1-5 g of the compositions can be administered. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other IE 0 9 ο 7 9 3 diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a scries of treatments.

The nucleic acid molecules employed according to the invention can also include small hairpin RNAs (shRNAs), and expression constructs engineered to express shRNAs. Transcription of shRNAs is initiated at a polymerase III (pol III) promoter, and is thought to be terminated at position 2 of a 4-5- thymine transcription termination site. Upon expression, shRNAs are thought to fold into a stem-loop structure with 3' UU-overhangs; subsequently, the ends of these shRNAs are processed, converting the shRNAs into siRNA-like molecules of about 21 nucleotides. Brummelkamp et al. (2002), Science, 296, 550-553; Lee et al, (2002). supra; Miyagishi and Taira (2002), Nature Biotechnol, 20, 497- 500; Paddison et al. (2002), supra; Paul (2002), supra; Sui (2002) supra; Yu et al. (2002), supra. More information about shRNA design and use may be found the following web sites: katahdin.cshl.org;933l/RNAi/docs/BseRl-Bam- HI_Strategy.pdf and at katahdin.cshl.org:9331/RNAi/docs/ Web_version_of_PCR_strategyl.pdf, Such stRNAs can then be modified as described herein, e.g. by addition of a peptide, or can be mixed with a dendrimer for delivery, e.g., PAM AM, as described herein.

The expression constructs may be any construct suitable for use in the appropriate expression system and include, but are not limited to retroviral vectors, linear expression cassettes, plasmids and viral or viral ly-derived vectors, as known in the art. Such expression constructs may include one or more inducible promoters, RNA Pol III promoter systems such as U6 snRNA promoters or HI RNA polymerase III promoters, or other promoters known in the art. The constructs can include one or both strands of the siRNA. Expression constructs expressing both strands can also include loop structures linking both strands, or each strand can be separately transcribed from separate promoters within the same construct. Each strand can also be transcribed from a separate expression construct. (Tuschl (2002), supra). Linear constructs may be delivered either by conjugation with a delivery peptide or by mixing with PA MAM; non-linear constructs may be delivered by mixing with PAM AM. siRNA Complexes IE ' 9 0 7 9 3 I lie siRNA formulations of ihe present invention can comprise one or more siRNA molecules (the same or different) associated with polymers of the kind described elsewhere in this specification eg to enhance the efficiency of transport of the siRNA into living cells compared io the efficiency of delivery to uneomplexed siRNA. Complexes of particular interest are those with cationic polymers such as chitosan. Such complexes or aggregates (also referred to as nanoparticles) are described in more detail in Andersen (201)8) Volume 20. Issue 4. ffiomatcrials. Pages 506-512 and Liu et al (2007) Volume 28. Issue 6. Pages 1280-1288 the entirety of both of which is incorporated herein by reference.

Such complexes are made as described in the above cited references and involve simple mixing of chitosan with siRNA solutions in the desired ratio (see examples herein).

Such siRNA-chitosan (or other protein) complexes may be further processed before encapsulation into minisphercs. minicapsules or minibcads. eg by association with liposomes. Thus, in one embodiment, RNA complexed with a protein may be included in or associated with liposomes or liposomal material. The protein is preferably cationic eg chitosan.

SiRNA specific to PHDs The present invention is particularly directed towards siRNAs able to knock down or silence or otherwise inhibit, in whole or in part, the propylhydroxylase (PHD) group of enzymes in mammals particularly man.

All organisms possess mechanisms to maintain oxygen homeostasis, which are essential for survival. The hypoxia-inducible factor-1 (HIF-1), conserved during evolution from worms to fiies to vertebrates, is central to adaptation to low oxygen availability. HIF-1 in turn regulates transcription of many genes involved in cellular and systemic responses to hypoxia, including breathing, vasodilation, anaerobic metabolism, erythropoiesis and angiogenesis. Therefore, hif represents a master’ gene in oxygen homeostasis during embryonic development and postnatal life in both physiological and pathophysiological processes such as tumour growth and metastasis (for a review, see Semenza, 1998).

IE ^90793 HIF-1 is a heierodimer consisting of one of three a-subunits (HIF-la, HIF-2a or HIF-3a) and the b-subunit (HIF-lb, also called aryl hydrocarbon nuclear translocator, or ARNT) (Wang et al., 1995: Ema et ah, 1997; Tian et al., 1997; Gu et ah, 1998). HIF-Ib is a constitutive nuclear protein, which also participates in the cellular response to environmental toxins such as aryl hydrocarbons, whereas HIF-a is specific to the response to hypoxia (Hoffman et ah, 1991). Although oxygen availability regulates multiple steps on HIF-1 transcriptional activation, the dominant control mechanism occurs through oxygen-dependent proteolysis of HIF-a (Huang et al.. 1996). The most extensively studied iso form of the a-subunits is the ubiquitous HIF-la.

In normoxia, HIF-la is constitutively synthesized and sent to destruction by the ubiquitin±proteasoine pathway (half-life <5 min) (Salceda and Caro, 1997; Huang et al, 1998; Kallio et al., 1999). This process is mediated by the specific binding of pVHL, the product of the von Hippel-t Lindau tumour suppressor gene, which is mutated in most sporadic clear cell carcinomas and in VHL disease (Kaelin and Maher, 1998; Maxwell et al., 1999; Cockman et al., 2000; Kamura et al., 2000; Ohh et al., 2000). pVHL is part of a multiprotein complex that includes elongin B, elongin C, Rbxl and Cul2 (Kamura et ah, 1999; Lisztwan el ah, 1999; Stebbins et ah, 1999), This complex functions as an E3 ubiquitin ligase which, only in the presence of oxygen, binds directly to and targets HIF-la for polyubiquitylation and proteasome-dependent degradation. Decreased oxygen levels result in the stabilization of HlF-la and the activation of the transcriptional complex leading to the expression of target genes such as vegf, epo and glut-1 (Semenza, 1998).

Recent major advances have shown that prolyl hydroxylation and acetylation, by controlling HIF-la±pV11L physical interaction, are critical in the regulation of HIF-la steady-state levels (Ivan et al., 2001; Jaakkola et ah, 2001; Jeong et at, 2002). The proline residues subjected to hydroxylation reside in the HIF-la oxygendependent degradation domain (ODDD) within an LXXLAP sequence motif, which is strongly conserved between the HIF-a isoforms. In the same degradation domain, Lys532, when acetylated by ARD I, cooperates with the hydroxyl group in the recruitment of pVHL and subsequent HIF-la degradation (Jeong et ah, 2002).

In mammalian cells, three iso forms, PHD I, PHD2 and PHD3, have been identified and shown to hydroxylate in vitro the key proline residues (Pro402 and Pro564) of IIIF-la (Epstein et ah, 2001). These three orthologues of the Caenorhabditis elegans Egi-9 have also been called EGLN2, EGLN1, EGLN3 and HPH3, HPH2, and HPH1, respectively (Bruick and McKnight, 2001; Ivan et al., 2002). Hereafter, they will be referred to using the PHD nomenclature. PHDs are dioxygenases that utilize oxygen as co-substrate providing the molecular basis for the oxygen-sensing function of these enzymes. Indeed, the activity of the purified PHDs has been reported to be strikingly sensitive to graded levels of hypoxia in vitro, mirroring the progressive increases in HIF-la protein and DNA binding activity that are observed when cells are exposed to gradual hypoxia in culture (Epstein et al., 2001). ln addition, the prolyl hydroxylation reaction requires 2-oxoglutarate and iron as cofactors, thereby accounting for the well known hypoxia-mimic' effects of iron chelators (such as desferrioxamine) and transition metals (such as Co2+, Mn2+ and Ni2+) on HIF-la induction.

Each PHD iso form differs in the relative abundance of their mRNA, but all three show a ubiquitous pattern of expression (Lieb et al., 2002; Cioffi et al., 2003).

To evaluate the role of the three mammalian Egl-9 orthologues with respect to HIF-la hydroxylation and expression in vivo, Berra et al (2003) specifically ablated each isoform using the small interfering RNA (siRNA) approach, developed by Tuschl and co-workers (Elbashir et al., 2001). They showed that specific silencing of PHD2 is sufficient to: (i) stabilize HIF-la steady-state levels in normoxia in all the human cells they analysed ; (ii) fully protect HIF-la degradation upon re-oxygenation of hypoxia-stressed cells; and (iii) trigger HIF-la nuclear accumulation and HIF-dependent transcriptional activation in normoxia. They also reported evidence that PHD2 is upregulated by hypoxia, supporting the auloregulatory mechanism they had previously proposed for oxygen-driven HIF-la regulation (Berra et al., 2001).

More specifically, to evaluate the role of the three mammalian PHDs in the stability of HIFla in vivo, Berra et al, ablated each isoform by transfecting HeLa cells with siRNAs. Two independent sets of 21 bp siRNA duplexes were chosen. They first targeted the sequence coding for the iron-binding site within PHD1, PHD2 and PHD3. This region is 100% conserved at the amino acid level between the three isoforms. However, degeneration of the codons within this region allowed design of three siRNAs differing by five nucleotides; this variation has been described to be more than sufficient to target specifically the different isoforms (Elbashir et al, 2001). HeLa cells transiently transfected with the siRNA duplex corresponding to PHDI (referred to in the Examples herein as the siRNA ‘for PHD I”) displayed a remarkably specific and a virtually complete loss of the PHDI mRNA, whereas the same siRNA had no effect on PHD2, PHD3 or control 36B4 mRNA levels. Equivalent results were obtained following transfection with the siRNAs targeting PHD2 or PHD3 (referred to in the Examples herein as the siRNAs ‘for PHD2” or “for PHD3”). As a control, they used an irrelevant siRNA (D-HIF) which, as expected, had no effect on any mRNA. The same results were also obtained with transfection of a second set of siRNA duplexes targeting an independent and non-conserved region within PHDI, PHD2 and PHD3. They also examined the siRNA action at the protein level. They showed the remarkable efficiency of PHDs siRNAs, used under the same conditions, in lowering protein levels of the corresponding recombinant expressed PHDs. In fact, presumably due to differences in protein turnover, PHDI and PHD3 were much more reduced than PHD2. Therefore, as previously published (Elbashir et al., 2001), their results confirmed the efficiency and specificity of the siRNA silencing strategy.

Berra el al (2003) subsequently described that the silencing of PHD2 upregulates HIF-la steady-state levels in normoxia. To show this, they evaluated the impact of the specific silencing of each PHD isoform on the steady-state levels of the HIF-1 a subunit, monitored by immunofluorescence microscopy or western blotting. Under the same conditions, they transiently transfected HeLa cells with siRNAs corresponding to PHDI, PHD2 or PHD3. As a control, HeLa cells transfected with the irrelevant D-HIF siRNA were incubated either in normoxia. in the presence of Co2+ (200 mM) or in hypoxia for 4 h. Immunofluorescence revealed that extinction of either PHDI or PHD3 had no impact on HIF-la expression in normoxia. However, silencing of PHD2 upregulated HIF-la similarly to hypoxia or Co2+. In addition, as expected, transfection of a siRNA targeting pVHL mimicked PHD2 silencing.

Moreover, HIF-la steady-state upregulation was dependent upon the amount of PHD2 siRNA transfected. Interestingly, they detected HIF-la induction at a concentration as low as 0.5 nM siRNA; at 2 nM, the level of HIF-la surpassed that achieved after 4 h of hypoxic stress, and slightly increased up to 200 nM. In addition, transfection of an siRNA targeting the human HIF-la isoform completely abolished the signal they detected by immunoblotting using the group's anti-HIF-Ia antibody in normoxia as well as in hypoxia. This result also validates that the two immunoreactive species shown in the SDS±gel correspond to the human HIF-la isoform. In addition, it isimportant to note that none of the transfected siRNA had an impact on the expression of p42MAPK.

IE 09 ο7 93 Berra ct al (2003) subsequently noted that PHD2 silencing upregulates HIF-la in all the human cells they investigated. They analysed whether specific silencing of PHD2-induced HIF-la upregulation was a common mechanism. Thus, they investigated a battery of human cells of different origin. First they tested immortalized cell lines such as CAL27 (derived Irom a squamous cell carcinoma of the tongue), CALS 1 (derived from a breast cancer), HaCAT (keratinocyte cell line), HT29 (derived from a colon carcinoma), RCC4/pVHL (derived from a clear cell renal carcinoma in which we have re-introduced wild-type pVHL) and WM9 (melanoma cell line) (Gioanni et al., 1990). Secondly, they assessed nonimmortalizcd cultures of fibroblasts (FHN), keratinocytes and vascular endothelial cells from the umbilical vein (HUVECs), As for HeLa cells, only PHD2 siRNA was able to upregulatc IIIF-Ia in all the cells so far evaluated. Although the efficiency of transfection varied greatly among the cell types, neither PHD I nor PHD3 siRNAs had, under these conditions, an impact on HIF-la. They therefore suggested that PHD2 controls steady-state levels of HIF-la in alt cell types examined.

Berra et al (2003) also noted that HIF-la induced in normoxia by PHD2 silencing is functionally active. In this respect, it has been reported that oxygen deprivation affects the subccllular localization, DNA-binding capacity and transcriptionalactivation function of HIFla in addition to regulating its stability (Kallio et al., 1999). However, Berra et al's results demonstrated that silencing of PHD2 also triggers HIF-la nuclear accumulation. To characterize the functionality of HIF-la induced in normoxia by PHD2 silencing, they performed luciferase assays by using a hypoxia-sensitive reporter gene vector (pRE-DtkLUC) coding lor the LUC gene under the control of a minimal promoter containing three copies of the hypoxia response element (HRE) from the erythropoietin gene. Thus, HeLa cells were transfected with the reporter construct in the presence of either the irrelevant DH1F siRNA, as a control, or siRNAs targeting each of the PHD isoforms, and an expression vector coding for b-galactosidase for normalization. Luciferase activity was measured after 48 h of transfection. As a positive control, cells transfected with D-HIF siRNA were incubated for 16 h either in hypoxia or in the presence of Co2+ (200 mM). LUC reporter gene expression was induced in both conditions by -20-fold over the basal luciferase activity detected in extracts from non-stimulated cells. In agreement with the above reported findings, PHD1 or PI1D3 silencing did no affect the luciferase activity of the reporter vector. However, the specific silencing of PHD2 markedly activated the HREs (14-fold stimulation), showing that upreguiated HIF-la is functionally active in normoxia. Moreover, in parallel |Ε η 9 Ο7 9 3 experiments, we showed that lueiferase activity correlated with the levels of HIF-la achieved by transfection of PHD2 siRNA at different concentrations.

Berra et a I (2003) also reported that PHD2 is essential for HIF-la degradation. Here the finding that PHD1 and PHD3 fail to regulate HIF-la levels, whereas both isoforms share with PIID2 the ability to hydroxylate in vitro the critical Pro564 within the HIF-la ODDD, they found intriguing. So far, all experiments were performed in normoxia. They therefore wanted to explore whether low oxygen availability might not be required to 'activate* PH DI and/or PHD3 in vivo. To investigate this possibility, they stressed cells for 4 h in hypoxia followed by re-oxygenation. This group next measured the HIF-la half-life in total cell extracts from cells transfected with the siRNA control or the specific siRNAs invalidating each PHD isoform. As previously reported, re-oxygenation triggers immediate and dramatic destruction of HIF-la in control cells (Figure 5). Interestingly, cells silenced for either PHD1 or PHD3 displayed identical kinetics for HIF-la degradation compared with control cells. Once again, only the silencing of PHD2 had a significant effect on oxygen-dependent HIF-la degradation. As expected, cells transfected with PHD2 siRNA showed upregulated levels of HIF-la in normoxia. This level was increased further with 4 h of hypoxic stress. Reoxygcnalion for 10 min did not modify HIF-la expression, whereas the protein level progressively decreased to reach that found in normoxia after 60 min. These results clearly excluded the contribution of PH DI and PHD3 lo HIF-la regulation even under these stress conditions. They therefore concluded that PHD2 is the critical oxygen sensor regulating oxygen-dependent HIF-la degradation either in normoxia or following a short exposure to hypoxia.

Finally, in their 2003 paper, Berra et al. reported that PHD2 is a hypoxia-inducible gene product, They had shown previously that HIF-la is subjected to an autoregulatory mechanism, and they had postulated that HIF-la is targeted for degradation by a factor they called HIF-la proteasome targeting factor (HPTF) (Berra et al., 2001). This factor accumulates in an inactive form during long periods of hypoxia, whereas, upon reoxygenation, its reactivation triggers HIF-la degradation at a rate that is inversely proportional to the length of hypoxia. They therefore proposed that HPTF is PHD2. As had been previously reported (Epstein et al., 2001), Berra et al. demonstrated that PHD2 mRNA is upregulated by hypoxia. To evaluate this hypothesis further, they raised antibodies against the last 16 amino acids of the human form of PHD2. The antibodies recognized faint bands at ~50 kDa, which all disappeared following transfection of the siRNA targeting PHD2. In ΙΕϋθ contrast, a siRNA control or those silencing PHD I and PHD3 had no effect on PI ID2 protein levels, showing the specificity of the antibody they generated. HeLa cells were subjected to different periods of hypoxia and total cellular extracts analysed by western blotting. PHD2 protein expression increased progressively with the duration of the hypoxic stress. Upregulation detected after 8 h of hypoxia reached a maximal level after 48 h of incubation at low pO2. Identical results were obtained using the CAL51 tumour cell line. Interestingly, this hypoxic induction of PHD2 is mediated via HIP-1 transcriptional activity since depletion of HIP-1 a with the appropriate siRNA abolishes hypoxia-dependent PHD2 induction.

One embodiment of the invention is a composition of siRNA susceptible of knocking-down the expression of one or more of PHD I, PHD2 and PHD3.

SiRNA specific to NF-kB The nuclear factor-κΒ (NF-κΒ) family is composed of hornodimers and heterodimers ofthe Rcl family proteins, including p65 (RelA), c-Rel, RelB, p52 and p50. For a review, see Ghosh S. Karin M (2002) “Missing pieces in the NF-kappaB puzzle’* in Cell 109: SuppISB 1 96 the entirety of which is incorporated herein by reference.

The most abundant form of NF-κΒ is a heterodimer with two subunits: one p50 and one p65. NF-κΒ is hound to inhibitory' IkB proteins in the cytoplasm. After stimulation by a variety of stimuli. NF-κΒ is released and translocates to the nucleus where it binds to its coaclivators. mainly CBP (CRFB-Binding Protein), and activates expression of pro-inflammatory genes, including the mast cell growth factor stem cell factor (SCF).

NF-κΒ is activated by phosphorylation, which plays a key role in the regulation of its transcriptional activity, and is associated with nuclear translocation, CBP recruitment and DNA-binding activity. For a review, see Chen LF, Greene WC (2004) “Shaping the nuclear action of NF-kappaB” in Nat Rev Mol Cell Biol 5: 392-401 the entirety of which is incorporated herein by reference.

Phosphorylation of p65 occurs on several serine residues. For instance, upon treatment with TNFu, Ser529 is phosphorylated by casein kinase 11, Ser536 by the IkB kinase (IKK) IE 0 θ Ο7 9 3 λ Q ft 7 9 3 complex. Ser31 I by protein kinase C (ΡΚΟζ. and Ser276 by both PKA and mitogen- and stress-activated protein kinase I (MSK1), Suppression of NF-κΒ eg by knock-down using a composition of the invention, especially transient suppression or knock-down, may increase the barrier function of the intestine and/or protect the epithelial barrier and/or decrease epithelial apoptosis and may therefore have medical utility in one or more intestinal diseases as described more fully below.

One embodiment of the invention is a composition comprising siRNA susceptible of knocking-down the expression of NF-κΒ and/or the 65KDa sub-unit of NF-κΒ. A related embodiment is a composition which comprises a siRNA which knocks down p65.

Another embodiment is the transient knock-down of NF-κΒ eg in the intestine.

Preparation of suitable siRNA is as described elsewhere herein or as known in the art - see for example Tao et al (2006) Mol Cell Biol 26(3) 1038-1050 or may be purchased commercially for example from suppliers such as Dharmacon.

RNA-coiuaining Minispheres and Pharmaceutical Compositions The release, including controlled release, of siRNAs. or siRNA precursors at specific sites for cellular absorption is only truly useful if the RNA is released and available in an active form or in a form which becomes active on absorption by the target cell. The invention is a drug delivery format that enables the release therefrom of siRNAs or siRNA precursors in soluble or readily-soluble form and adapted to be absorbed by the target cells eg intestinal epithelial cells.

As the invention permits the release of the siRNAs. or siRNA precursors in soluble or readily-soluble form and maintains them in an appropriate solvent protected from exogenous influences until release, it addresses the question of limited stability and/or a short half-life. the invention also provides an oral drug delivery technology that permits the colon-specific release of pre- or readily-solubilised siRNAs, or siRNA precursors in tandem with a controlled release formulation that permits release of the RNA and absorption of the RNA by ο 9 o7 93 cells in the small intestine, ileum and/or colon. Medicaments of the invention may therefore be adapted Ibr the colon-specific release of active agents as described herein, e.g. siRNAs.

Colon delivery is particularly advantageous as an effective drug delivery mechanism for 5 siRNAs. or siRNA precursors addressing diseases of the colon (ulcerative colitis, Crohn's disease. Gastro-Intestinal Graft-Versus-Host-Disease (GI-GVHD), Irritable Bowel Syndrome, constipation, diarrhoea, carcinomas and other infections) whereby high local concentration can be achieved while minimizing side effects that occur because of release of drugs in the upper GIT or unnecessary systemic absorption.

The colon is rich in lymphoid tissue and uptake of siRNAs. or siRNA precursors, into the mast cells of the colonic mucosa is intended to modulate the cells to enhance or decrease sensitivity. Delivery of this type of RNA according to the invention is therefore relevant to conditions arising from ingestion of oral allergens or dysregulated mast cells in clinical IS conditions such as mastocytosis. Mastocytosis is a group of disorders characterized by proliferation of mast cells and infiltration of organs such as the gastrointestinal tract. RNAi molecules that inhibit the release of mediators from sensitized mast cells represent therefore a preferred embodiment of the invention and may be preferentially used to treat or prevent mastocytosis.

The colon is a site where a drug molecule, particularly but not exclusively a hydrophilic molecule, such as certain siRNAs. or siRNA precursors, that has limited intestinal absorption may have an improved bioavailability or local effect. The colon is recognized as having a somewhat less hostile environment with less diversity and intensity of activity than the stomach and small intestine. Additionally, the colon has a longer retention time and appears highly responsive to agents that enhance the absorption of poorly absorbed drugs such as siRNAs. or siRNA precursors. Apart from retarding or targeting dosage forms, a reliable colonic drug delivery system is also important for the colonic absorption of perorally administered, undigested, unchanged and fully active molecules such as siRNAs. or siRNA precursors.

By-passing the gastric and small intestinal regions, and releasing siRNAs, or siRNA precursors intact and in soluble as well as in a form able to permeate directly into the colon enhances absorption of the drug from the epithelial and other cells lining the colon.

IE»’ In embodiments of the invention, siRNAs, or siRNA precursors are protected from release in the upper gastrointestinal tract (GIT) but are able to be abruptly and/or released in a sustained manner, starting at the ileum or proximal colon and throughout the length of the colon. Such colon targeting is particularly of value for the treatment of diseases of colon such as Crohn's diseases, ulcerative colitis, graft-versus-host-disease (GVHD), colorectal cancer, amebiasis and mastocytosis. The delivery of RNA according to the invention is also applicable to other intestinal conditions, including carcinomas, gastritis, pancreatitis etc, as well as viral infections, including rotavirus, and bacterial infections, including Clostridium difficile.

As compositions of the invention are comprised of a multitude of separate minicapsules or minispheres, either containing liquid, semi-solid or solid RNA formulations, the invention enables the development of novel combination therapies in a single dosage form, each component of the combination (or each population of different tninicapsules/minispheres) IS containing the same or different RNAs or non-RNA active principles and having distinct release profiles, the release being inherent to the core formulation, the shell or the entirety of the minicapsule or some additional polymer coating thereon eg as a membrane.

In embodiments comprising a membrane-controlled dosage form, the polymeric material may comprise methacrylic acid co-polymers, ammonio methacrylate co-polymers, or mixtures thereof. Methacrylic acid co-polymers such as EUDRAGIT™ S and EUDRAGIT™ L (Evonik) are suitable for use in the controlled release formulations of the present invention. These polymers arc gastroresistant and enterosoluble polymers. Their polymer films are insoluble in pure water and diluted acids. They dissolve at higher pHs, depending on their content of carboxylic acid. EUDRAGIT™ S and EUDRAGIT™ L can be used as single components in the polymer coating or in combination in any ratio. By using a combination of the polymers, the polymeric material can exhibit solubility at a pH between the pHs at which EUDRAGIT™ L and EUDRAGIT™ S are separately soluble.

The membrane coating can comprise a polymeric material comprising a major proportion (i.e.. greater than 50% of the total polymeric content) of at least one pharmaceutically acceptable water-soluble polymers, and optionally a minor proportion (i.e., less than 50% of the total polymeric content) of at least one pharmaceutically acceptable water insoluble polymers. Alternatively, the membrane coating can comprise a polymeric material comprising a major proportion (i.e., greater than 50% of the total polymeric content) of at least one pharmaceutically acceptable water insoluble polymers, and optionally a minor proportion (i.e.. less than 50% of the total polymeric content) of at least one pharmaceutically acceptable water-soluble polymer.

Alternatively, or in addition, the membrane may comprise an amylose, especially a “glassy” amylosc as described in US Patent 6534549 and/or 6743445. Other so-called “pore-formers” are also contemplated by the present invention.

Amirtonio methacrylate co-polymers such as EUDRAGIT™ RS and EUDRAGIT™ RL (Evonik) arc suitable for use in the modified release formulations of the present invention. These polymers are insoluble in pure water, dilute acids, buffer solutions, or digestive fluids over the entire physiological pH range. The polymers swell in water and digestive fluids independently of pH. In the swollen state, they are then permeable to water and dissolved active agents. The permeability of the polymers depends on the ratio of ethylacrylate (EA), methyl methacrylate (MMA), and trimethylammonioethyl methacrylate chloride (TAMO) groups in the polymer. Those polymers having EA:MMA:TAMCI ratios of 1:2:0.2 (EUDRAGI'I™ RL) are more permeable than those with ratios of 1:2:0.1 (EUDRAGIT™ RS). Polymers of EUDRAGIT™ RL are insoluble polymers of high permeability'. Polymers of EUDRAGIT™ RS are insoluble films of low permeability.

The amino methacrylate co-polymers can be combined in any desired ratio, and the ratio can be modified to modify the rate of drug release. For example, a ratio of EUDRAGIT™ RS: EUDRAGIT™ RL of 90:10 can be used. Alternatively, the ratio of EUDRAGIT™ RS: EUDRAGIT™ RL can be about 100:0 to about 80:20, or about 100:0 to about 90:10, or any ratio in between. In such formulations, the less permeable polymer EUDRAGIT™ RS would generally comprise the majority of the polymeric material with the more soluble RL, when it dissolves, permitting creating gaps through which solutes can enter the core and dissolved pharmaceutical actives escape in a controlled manner. lhe amino methacrylate co-polymers can be combined with the methacrylic acid copolymers within the polymeric material in order to achieve the desired delay in the release of the drug. Ratios of ammonio methacrylate co-polymer (e.g., EUDRAGIT™ RS) to methacrylic aeid co-polymer in the range of about 99:1 to about 20:80 can be used. The two 31 IE 0 9 0 7 9 3 types of polymers can also be combined into the same polymeric material, or provided as separate coats that are applied to the core.

Eudragil™ FS 30 D is an anionic aqueous-based acrylic polymeric dispersion consisting of methacrylic acid, methyl acrylate, and methyl methacrylate and is pH sensitive. This polymer contains fewer carboxyl groups and thus dissolves at a higher pH (> 6.5). The advantage of such a sysiem is that it can be easily manufactured on a large scale in a reasonable processing time using conventional powder layering and fluidized bed coating techniques. In a study by Gupta ct al (bit,/ Pharm, 213: 83-91, 2001) Eudragit FS 30 D demonstrated its potential for colonic delivery by resisting drug release up to pH 6.5 and the combination of Eudragit™ RL and RS proved successful for the sustained delivery of 5-AS A at the pH of the colon, Thus, Eudragit™ FS 30 D alone or with other controlled release polymers holds great potential to enable delivery of minieapsule formulations specifically to the colon.

In addition to the EUDRAGIT™ polymers described above, a number of other such copolymers can be used to control drug release. These include methacrylate ester copolymers such as the EUDRAGIT™ NE and EUDRAGIT™ NM ranges. Further information on the EUDRAGIT™ polymers can be found in Chemistry and Application Properties of Polymethacrylate Coating Systems, in Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms, cd. James McGinity, Marcel Dekker Inc., New York, pg 109-114.

Several derivatives of hydroxypropyl methylcellulose (HPMC) also exhibit pH dependent solubility and may also be used to enhance permeability, Shin-Etsu Chemical Co.. Ltd. esterified HPMC with phthalic anhydride to produce hydroxypropyl methylcellulose phthalate (HPMCP), which rapidly dissolves in the upper intestinal tract. Due to the limited compatibility of HPMCP with several types of plasticizers, hydroxypropyl methylcellulose acetate succinate (HPMCAS) was developed. The presence of ionizable carboxyl groups in the HPMCAS structure cause the polymer to solubilize at high pH (> 5.5 for the LF grade and >6,8 for the HF grade). This polymer exhibits good compatibility with a variety of plasticizing agents and is commercially available from Shin-Etsu Chemical Co. Ltd, under the proprietary name AQOAT® in a powdered form to be redispersed in water. HPMCP may alternatively or additionally be incorporated into the film-forming solution (eg gelatine) and thus, in the case of single layer minispheres such as beads, be a component of the solidified matrix.

IE 09 θ7 93 It is particularly preferred according to the invention to use a polmeric coating substance which is pll-independent in its dissolution profile and/or in its ability to release active principles incorporated in the mini-beads of the invention. Examples have already been given (e.g.. Eudragit RS and RL). Another example of a pH-independent polymeric coating substance is ethylcellulose, in particular a dispersion of ethylcellulose in a sub-micron to micron particle size range, e.g. from about 0.1 to 10 microns in size, homogeneously suspended in water with the aid of an emulsification agent, e.g. ammonium oleate. The ethylcellulose dispersion may optionally and preferably contain a plasticizer, for example dibutyl scbacate or medium chain triglycerides. Such ethylcellulose dispersions may, for example, be manufactured according to U.S. Rat. No. 4,502,888, which is incorporated herein by reference. One such ethylcellulose dispersion suitable for use in the present invention and available commercially is marketed under the trademark Surelease®, by Colorcon of West Point, Pa. USA. In this marketed product, the ethylcellulose particles are, e g., blended with oleic acid and a plasticizer, then optionally extruded and melted. The molten plasticized ethylcellulose is then directly emulsified, for example in ammoniated water optionally in a high shear mixing device, e.g. under pressure. Ammonium oleate can be formed in situ, for instance tc stabilize and form the dispersion of plasticized ethylcellulose particles. Additional purified water can then be added to achieve the final solids content. See also U.S. Pat. No. 4,123,403. which is incorporated herein by reference.

The trademark Surelease®” is used hereinafter to refer to ethylcellulose coating materials, for example a dispersion of ethylcellulose in a sub-micron to micron particle size range, e.g. from about 0.1 to 10 microns in size, homogeneously suspended in water with the aid of an emulsification agent, e.g. ammonium oleate. In particular, the trademark “Surelease®” is used herein to refer to the product marketed by Colorcon under the Surelease® trademark.

Surelease® dispersion is a unique combination of film-forming polymer, plasticizer and stabilizers. Designed for sustained release and taste masking applications, Surelease is an easy-to-use, totally aqueous coating system using ethylcellulose as the release rate controlling polymer. The dispersion provides the flexibility to adjust drug release rates with reproducible profiles that are relatively insensitive to pH. The principal means of drug release is by diffusion through the Surelease dispersion membrane and is directly controlled by film thickness. Increasing or decreasing the quantity of Surelease® applied can easily modify the IE 0'9 βί $5 rate of release. With Surelease dispersion, reproducible drug release profiles arc consistent right through from development to scale-up and production processes. Pore formers, for example those described in US Patent 6534549 and/or 6743445 may alternatively or additionally be included, In addition to the EUDRAGIT™ and Surelease® polymers discussed above, other enteric, or pH-dependent, polymers can be used. Such polymers can include phthalate, butyrate, succinate, and/or meditate groups. Such polymers include, but are not limited to, cellulose acetate phthalate, cellulose acetate succinate, cellulose hydrogen phthalate, cellulose acetate trimellitate, hydroxypropyl-methylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, starch acetate phthalate, amylose acetate phthalate, polyvinyl acetate phthalate, and polyvinyl butyrate phthalate. Additionally, where compatible, any combination of polymer may be blended lo provide additional controlled- or targeted-release profiles.

The coating membrane can further comprise at least one soluble excipient to increase the permeability of the polymeric material. Suitably, the at least one soluble excipient is selected from among a soluble polymer, a surfactant, an alkali metal salt, an organic acid, a sugar, and a sugar alcohol. Such soluble excipients include, but are not limited to, polyvinyl pyrrolidone, polyethylene glycol, sodium chloride, surfactants such as sodium lauryl sulfate and polysorbates, organic acids such as acetic acid, adipic acid, citric acid, fumaric acid, glutaric acid, malic acid, succinic acid, and tartaric acid, sugars such as dextrose, fructose, glucose, lactose, and sucrose, sugar alcohols such as lactitol, maltitol, mannitol, sorbitol, and xylitol, xanthan gum. dextrins, and maltodextrins. In some embodiments, polyvinyl pyrrolidone. mannitol, and/or polyethylene glycol can be used as soluble excipients. The at least one soluble excipient can be used in an amount ranging from about 1% to about 10% by weight, based on the total dry weight of the polymer. The coating process can be carried out by any suitable means, for example, by using a perforated pan system such as the GLATT. ACCELACOTA. Vector, Diosna, O'Hara, HICOATER or other such coating process equipment. Seamless minicapsules may be manufactured using the method described in US5,882.680 (Ereund), the entire contents of which are incorporated herein by reference.

Solubilisation and Suspension of RNA Various liquid vehicles and/or transfection agents/reagents may be used to suspend or dissolve RNA for encapsulation such that the core of minicapsules according to the invention is a fluid, eg a solution or an emulsion with or without appropriate surfactant. For example, the RNA may be encapsulated in a water-in-oil emulsion either as the fluid core of a true minisphere with outer capsule or as a bead of semi-solid or solid RNA-containing emulsion optionally within a matrix forming the body of the bead.

Thus, in one embodiment, RNA or siRNA-protein complexes, as described in more detail above, may be included in, or associated with, liposomes, particularly cationic or other liposomes suitable for transfection. Examples include Lipofectamine or Lipofectamine 2000 which are established transfection reagents, produced and sold by Invitrogen for the introduction (transfection) of siRNA or plasmid DNA into cells by lipofection. Lipofectamine treatment alters the cellular plasma membrane, allowing nucleic acids to cross into the cytoplasm and other such membrane-altering reagents are contemplated by the present invention. Variant cationic liposomes for nucleic acid delivery according to the invention include those generated by using cationic lipids such as l.ipofectin™ and Cyiolectin,M. Lipofectin is a mixture of N-[ 1-(2, 3-dioleyIoyx) propylJ-N-N-N-trimethyl ammonia chloride (DOTMA) and DOPE (phosphatidylethanolamine). Another possible cationic lipid is DOTAP (dioleoyl triniethylammonium propane.). Other possible transfection agents are those known in the DNA field eg derivatives of phosphatidyl choline including 1)( )PC (dioleoyl- phosphatidylcholine) and I.-DOPC (diolcoyl-sn-glycerol-j-clhylphusphocholinc oi 0-cih>ldiolcoOphospiuitidylcholinc). Alternatively neutral phospholipids such as dipalmitoyl phosphatidyl choline (DPPC) may be used or negatively charged phospholipids such as dipalm itoyl-phosphatidylglycerol (DPPG) may be used in either case with or without phosphatidylethanolamine (DOPE). The liposome suspension may be stabilized for example by addition of a stabilizing agent such as sorbitol and the thus stabilized suspension may optionally be lyophilised for further formulation as described elsewhere herein. The liposomes, eg after rehydration of a lyophilisate, may be converted into beads in the manner described in more detail elsewhere in this description.

The liposome to siRNA quantity ratio may be expressed as molar charge ratio ie the molar ratio of positive charge from the (phospho) lipids to the negative charge of the siRNA component. The molar charge ratio (positive : negative) may range from 1:1 to 55 :1, for IE 0θ Ο7 θ3 example from 1:1 to 25 :1 or from 1:1 to 12 :1 depending on choice of the (phospho) lipid(s). in one embodiment, it is preferable for the charge rato to be from 2:1 to 5 : L It is believed that admixture of liposome stock solution made eg from phosphate buffered saline and one or more of the above lipids or phospholipids to siRNA (associated or not with a polymer) may lead to a loss of unilamellar liposomal structure and the adoption of a more complex molecular arrangement of siRNA and liposomal material. These molecular arrangements are nevertheless generally aqueously soluble and/or hydrophilic owing to constituent (phospho) lipids orientated as in the outside layer of standard liposomes (outwardfacing hydrophilic head). Such liposomal-si RNA structures (with or without protein associated with the siRNA) are stable in aqueous media and may therefore be handled and further processed according to the invention as if they were standard liposomes. For example, the aqueous media which contains such structures may be dispersed in an oil phase (such as described elsewhere herein) to create a water-in-oil emulsion which itself may be dispersed in an aqueous solution of a polymer matrix such as a gelling agent (described elsewhere herein) to form a water-in-oil-in-water (w/o/w) emulsion. This w/o/w emulsion may be extruded to form minispheres, minicapsules or minibeads as described elsewhere herein.

The term “liposome” as used herein, unless the context demands otherwise, therefore includes unilamellar and multilamellar liposomes and/or liposomal components, having aqueous RNA phases enclosed in the aqueous cores (concentric or non-concentric) as well as more complex liposomal structures eg of liposomal components such as flat lamellar structures (eg where RNA is “sandwiched” between lipid bi layers), hexagonal, cylindrical, rod and columnar structures possibly with hexagonal cross-sectional aspect. So called “inverted” liposomal structures behaving as lipophilic entities are also contemplated.

Alternatively to the approaches described above, mini-beads may be generated directly (in the manner described below) without prior formation of liposomes by generating a (water-inoil) emulsion or microemulsion and dispersing droplets of such emulsion or microemulsion (as if it were an oil phase) in an aqueous matrix such as gelatin or other such material described in more detail in the section herein describing the structure of the minispheres/minicapsules.

IE o9»7 93 The emulsion containing liposomes or not, particularly a water-in-oil emulsion, may favourably contain 2-(2-ethoxyethoxy) ethanol which is available under the tradename Transculo! or Cellusolve. Preferably the emulsion may contain a non ionic surfactant, especially of the kind from polyethoxylated sorbitan and oleic acid. An example is the surfactant and emulsifier available under the tradename Tween. Tween 80 is particularly preferred.

I he oil component of the emulsion or microemulsion in this aspect and in other aspects of the invention may be any kind of pharmaceutically appropriate oil for oral administration including for example oleoyl and linoleoyl macrogolglycerides (and other polyoxylglycerides) as commercialised by Gattefosse under the name Labrafil™. A preferred example is Labrafil M 1944 CS. Another example is product number M2125CS also by Gattefosse. Alternative or additional oils are capryloeaproy! macrogolglycerides such as Labrasol by Gattefosse.

Alternative or additional oils which may be included in the oil phase according to the invention are medium chain tryglycerides such as for example Labrafac™ Lipophilc manufactured by Gattefosse in particular product number WL1349.

Other oils which may alternatively or additionally be included as the oil phase of the emulsion include poly-unsaturated fatty acids such as omega-3 oils such as eicosapentanoic acid (EPA). docosohexaenoic acid (DHA), alpha-linoleic acid (ALA). Combinations of such components are also contemplated eg a mixture of EPA and DHA in a ratio of 1.5 available commercially under the trade name Epax 6000.

Further oils which may alternatively or additionally be used as or included in the oil phase are natural triglyceride-based oils which include olive oil, sesame oil, coconut oil, palm kernel oil. Oils which arc particularly preferred include saturated coconut and palm kernel oilderived caprylic and capric fatty acids and glycerin eg as supplied under the trade name Miglyol™ a range of which are available and from which one or more components of the oil phase of the invention may be selected including Miglyol™ 810, 812 (caprylic/capric triglyceride); Miglyol™ 818: (caprylic/capric/linoleic triglyceride); Miglyol™ 829: (caprylic/capric/succinic triglyceride; Miglyol™ 840; (propylene glycol dicaprylate/dica prate). Note that Miglyol™ 810/812 differ only in Cs/C|0-ratio and because ΙΕ ο9 °7 9 3 of its low C10-content, the viscosity and cloud point of Miglyol™ 810 are lower. The Miglyol™ range is available commercially front Sasol Industries. (lie vehicle and/or transfection agent may include cationic surfactants including the Montanide f-unilv of reagents available from Scppie, France.

Other Iransl'cclioit agents/lechniques include use of polymeric DNA-binding catkins such as I to I) -F-lysine or polyethyleneimine. According to the invention, the fin id core of ininicapsulcs or the tluid or semi-solid components of a bead including transfection agents ma> include modified cyclodextrins with cationic moieties.

A lurther vehicle which ntay be utilised in the present invention are nanolipid vesicles or nano lipids illicit are very small spherical bodies arising from the long chain fatly acids which may be used in the formulation of the preparation according to the invention. Such encapsulating vesicles may be used to penetrate and/or are useful to facilitate and/or enhance siRNA penetration of the cell membrane and to migrate to cell organelles.

Another vehicle appropriate for use according to the invention are self-emulsifying drug delivery systems (SEDDS) and other (eg micro-emulsion) systems which enhance the permeability of cells to macromolecules.

Fhe size of the emulsion droplets constituting the above minispheres or minicapsules can be varied according to the invention and microemulsions are preferred for example in the targeting of intestinal epithelial cells as well as the lymphatic system which, according to the invention, represents a route of RNA delivery from the GI tract to other parts of the body. Where lymphatic delivery is desired, emulsifying with bile salts, may be preferable.

For water-soluble RNAs, it is preferred to adopt the liposome or water-in-oil microemulsion approach. For hydrophobic or water-insoluble RNAs and RNA derivatives such as lipid conjugates, it is preferred either to disperse the RNA in an appropriate medium before proceeding to further formulation in accordance with the invention or to select an appropriate solvent cg an oil or lipid, as described elsewhere herein, in which to dissolve the RNA before |Ε Π9 07 93 proceeding to further formulation as described herein such as formation of a waier-in-oil emulsion or microemulsion.

Controlling Release Rate Ί he modifications in the rates of release, such as to create a delay or extension in release, can be achieved in any number of ways. Mechanisms can be dependent or independent of local pi I in the intestine, and can also rely on local enzymatic activity to achieve the desired effect. Examples of modified-release formulations are known in the art and are described, for example, in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5.059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,566.

A number of modified dosage forms suitable for use are described below. A more detailed discussion of such forms can also be found in, for example The Handbook of Pharmaceutical Controlled Release Technology, D. L. Wise (ed.), Marcel Decker, Inc., New York (2000); and also in Treatise on Controlled Drug Delivery: Fundamentals, Optimization, and Applications, A. Kydonieus (ed.), Marcel Decker, Inc., New York, (1992), the relevant contents of each of which are hereby incorporated by reference for this purpose. Examples of modified-release formulations include but are not limited to, membrane-modified, matrix, osmotic, and ion-exchange systems. All of these can be in the form of single-unit or multiunit dosage forms, as alluded to above.

Another approach according to the invention is to have the core itself controlling release of RNA or RNA-containing beads, microemulsions or liposomes.

With membrane-modified extended-release dosage forms, a semi-permeable membrane can surround the formulation containing the active substance of interest. Semi-permeable membranes include those that are permeable to a greater or lesser extent to both water and solute. This membrane can include water-insoluble and/or water-soluble polymers, and can exhibit pH-dependent and/or pH-independent solubility characteristics. Polymers of these types are described in detail below. Generally, the characteristics of the polymeric membrane, which may be determined by, e.g., the composition of the membrane, will determine the nature of release from the dosage form.

In particular, the present invention provides for formulations of minieapsules or minispheres wherein the modified release is dependent upon, where appropriate, any one of the core formulation constituents, the shell composition or the shell coating.

Generation of Minispheres and Minieapsules The minieapsules or minispheres may be produced through the utilisation of surface tension of one or more different solutions which when ejected through an orifice or nozzle with a certain diameter and subject to specific frequencies and gravitational flow, forms into a spherical form and falls into a cooling air flow or into a cooling or hardening solution and the outer shell solution where it is gelled or solidified. Minispheres, minispheres or minibeads of diameter between 500 and 5000 microns are preferred with a range from 1mm to 2mm being particularly preferred.

The core solution of RNA may be a hydrophobic solution or suspension of RNA as described above depending on the physicochemical characteristics of the RNA being formulated, eg whether it is a conjugate eg lipid conjugate or a hydrophilic derivative. Alternatively the core solution can be pre-mixed with a shell solution eg emulsified such that the shell solution acts as a matrix’' within which the core solution is already dispersed (a shell/core mixed suspension) which may be extruded to form single layer minieapsules (beads) without further processing. The outer shell or bead matrix solution can be any gel forming agent but is preferably gelatine- or alginate-based although pectin, carrageenan and others may be used. It is advantageous to include sorbitol with the gelatin in manufacturing the beads, minispheres or minieapsules of the invention. The outer shell (or shell solution) may also include polymers or other materials that enable controlled release. RNA solutions can also be hydrophilic and one of the advantages of the present invention is that hydrophilic solutions can also be encapsulated in the manner described although preferably with the existence of an intermediate solution, which can avoid the direct contact of the hydrophilic core solution (which contains the RNA) with the outer shell. The temperature of the gelatin solution during manufacture depends on the gelatin type and the amount of softener but is typically in the range of60°C to 70°C eg around 65°C.

IE Ο 9 07 93 With the nozzle having a single orifice, a minicapsule or a bead of shell/core mixed suspension can be processed and may further be processed using a melt-extrusion-like process with solidification of the matrix occurring shortly after extrusion e.g. by change in temperature or exposure to cross-linking agents to form mini-beads. The mini-beads may have one layer being a solid phase comprising inclusions selected from liquid inclusions, semi-solid inclusions and combinations thereof With the nozzle having two orifices {centre and outer), a hydrophobic solution can be encapsulated. in one embodiment the minicapsule e.g. mini-bead has one layer and the inclusions comprise the active agent. In another embodiment the minicapsule has at least two layers and wherein the core comprises the active agent In one embodiment the mini-beads are of solidified matrix material and the mini-beads comprise one or more siRNAs or engineered RNA precursors dispersed in said solidified matrix. In an embodiment at least one of the siRNAs or engineered RNA precursors may form an aqueous phase dispersed in an oil phase. The oil phase may be dispersed in the solidified matrix. I'he solidified matrix may be, for example, an aqueous phase.

Where appropriate, it may be possible that both the core and t or shell may be comprised of a material or material composites that have been processed by a wet- or dry-granulation mechanism, melt or otherwise fluidized prior to mixing or granulation.

Ideally, to enable RNA content and release consistency, it is preferred that all processes result in fairly uniform morphologies with a relatively smooth surface to facilitate quite even coaling layers to be added in a uniform manner. With the nozzle having one or more orifices seamless minicapsules for various applications can be processed using minicapsule processing equipment enabled by, but not limited to, Freund Spherex, ITAS/Lambo Globex or lnotech processing equipment. As outlined above the coating process can be carried out by any suitable means, for example, by using a perforated pan or fluidized-baed system such as the GLATT, Vector, ACCELACOTA, Diosna, O’llara and/or HICOATER processing equipment.

IE 0 9 The result is modified release compositions that in operation deliver siRNAs. or siRNA precursors optionally with one or more additional active ingredients in a unique (unimodal), bimodal or multimodal manner.

The present invention further relates to solid oral dosage forms, sachets or suppositories containing such multiple minicapsule or minisphere controlled release compositions of siRNA or RNA precursors as well as methods for delivering one or more active ingredients to a patient in a unimodal, bimodal or multimodal manner.

Furthermore, the invention permits targeted release of orally delivered formulations to specific regions of the gastrointestinal tract to maximize absorption, confer protection on the payload, to optimize treatment of diseased intestinal tissue or enhance oral bioavailability.

Additionally, the invention enables one or more RNAs to be administered sequentially or concomitantly lo improve disease treatment and management and to benefit from the body’s natural circadian rhythms. The invention also permits the release of siRNAs. or siRNA precursors along with, optionally, other pharmaceutical actives into the ileum and colon for the enhanced treatment of local intestinal diseases or to facilitate the absorption of active pharmaceutical agents. The other pharmaceutical actives may be small molecules or biopharmaceuticals such as peptides or proteins. Examples of drugs that have demonstrated limited colonic absorption that could be combined wth the siRNAs, or siRNA precursors in a combination formulation include Tacrolimus, Cyclosporine, ASA etc, Budcsonide and Celecoxib.

The invention enables a siRNAs, or siRNA precursors, optionally co-released alongside a small molecule or macromolecule, to be clinically effective, to reach its intended target cell, cell system or tissue in an active form.

The use of enteric polymer coatings protects the contents of minicapsules from gastric acid degradation while other colon-specific coatings permit release of minicapsule contents only in the colon where the proteolytic enzyme content is significantly less than in the small intestine. Thus, by controlling the minicapsule coatings the invention provides formulations that ensure that the siRNAs, or siRNA precursors and other optional active contents are released intact at sites where absorption or therapeutic activity is optimal.

II Ο7 93 The invention includes RNA delivery in the colon which has been largely overlooked from a drug delivery perspective. Mainly having evolved to regulate electrolyte balance and to further breakdown complex carbohydrate structures, the colon is the site of significant flow of water from the colonic lumen into the body. In addition, the colon is home to a natural bacterial flora lo degrade complex carbohydrates to ensure effective excretion, provide much needed fibre and some nutrient absorption. With a much lower concentration of proteolytic and other enzymes populated in the colon, it is a much more benign environment for nucleic acids, including RNAs and DNAs, proteins and peptides as well as other biological entities such as carbohydrates. From a drug delivery perspective, the colon presents a number of interesting possibilities; the bacteria can be harnessed to break down controlled release coatings that are resistant to acidic breakdown as well as pH differentials; the benign environment ensure than active pharmaceuticals, including biopharmaceuticals, are less likely to be degraded if released locally into the colon; the almost continuous flow of fluids from the colonic lumen to the bloodstream may be harnessed to carry hydrophilic entities from the intestine lo the lumen. Finally, the long transit time in the colon, ranging form 10-20 hours provides greater residence and potential for interaction with the colonic mucus and epithelial cells leading to enhanced absorption.

Technologically, this invention is based on various modifications of basic one- or multilayered minieapsules or minispheres, modulating the core, the shell or the coating to permit enhanced solubility and permeability of the siRNAs. or siRNA precursors or other active or non-active entity as well as conferring protection on siRNAs, or siRNA precursors or entities that are susceptible to various forms of intestinal, mucosal or systemic degradation and targeted release of the therapeutically-active or -inactive entities to predetermined regions of the gastrointestinal tract. The minieapsules or minispheres may be solid or encapsulate solid, semi-solid or liquid drug-containing formulations.

In addition to the above minicapsule modifications, the present invention provides the coating of minieapsules or minispheres with a muco- or bio-adhesive entity which will ensure that they first adhere to the mucosa prior to releasing the fragile payload. The advantages thus enabled include further protection of the active entities but also release of the actives proximal to the site of absorption. As absorption is, in part, related to the surface area exposed lo the active as well as the concentration gradient from intestinal luminal side to the II A 9 ο 7 9 5 intestinal basal side, the higher local yet dispersed concentration has greater potential to ensure enhanced absorption, not only of siRNAs, or siRNA precursors but also of other hydrophilic, lipophilic or hydrophobic drugs.

A barrier to effective colonic delivery of hydrophobic and lipophilic drugs is that the colon did not evolve to solubilize foodstuffs and other entities but rather to ensure electrolyte balance and maximize fibre breakdown and fermentation. The colon remains very porous to hydrophilic entities. By delivering hydrophobic or lipophilic drugs to the colon in a presolubilised or readily soluble format and releasing such in the colon, the potential for absorption is enhanced significantly. The present invention permits the encapsulation of presolubilized or readily soluble drugs in liquid or hydrolysable semi-solids or solids into the minicapsulc core (especially RNA) and then modulation of the shell to include intestinal- or colon-con trolled release polymers or coating the shell with same. The result is release of optimized formulations at specific sites along the intestinal tract for maximal therapeutic efficacy or systemic absorption..

Likewise, delivery of formulations that are readily broken down in an aqueous environment or a bacteria rich environment has the potential, when coated with colon-specific controlled release polymers or include entities that are degraded by bacteria have the potential to protect susceptible entities from the gastric or intestinal environment yet ensure that they are released intact in the colon where, once liberated, will be readily absorbed. Redox-sensitive, pectin-, alginate-, chitosan- or other bacterially susceptible polymer-based matrices, coatings or other sustained release formulations, liquid, semi-solid or solid, can be encapsulated into or coated onto one- or multi-layered minicapsules. Thus, in one embodiment of the invention, chitosan can be directly associated with siRNA to form an aggregate or a complex which may be further formulated eg with liposomal materials before incorporation or encapsulation in a single layer minicapsule, such as a bead, or a multi-layered minicapsule.

The formulations of the present invention can exist as multi-unit or single-unit formulations.

The term multi-unit as used herein means a plurality of discrete or aggregated minicapsules, minispheres, particles, beads, pellets, granules, tablets, or mixtures thereof, for example, without regard to their size, shape, or morphology. Single-unit formulations include, for example, tablets, hard gelatin capsules, caplets, and pills. ΙΕ η θ ο7 9 3 The methods and formulations of the present invention are intended to encompass all possible combinations of components that exhibit modified-release and immediate-release properties. For example, a formulation and/or method of the invention can contain components that exhibit extended-release and immediate-release properties, or both delayed-release and immediate-release properties, or both extended-release and delayed-release properties, or a combination of all three properties. For example, a multi-minicapsule or multi-minisphere formulation including both immediate-release and extended-release components can be combined in a capsule, which is then coated with an enteric coat to provide a delayed-release effect. Or, for example, a delayed- and extended-release caplet may comprise a plurality of discrete extended-release particles held together with a binder in the caplet, which is coated with an enteric coating to create a delay in dissolution.

As used herein, the term modified-release formulation or dosage form includes pharmaceutical preparations that achieve a desired release of the siRNAs. or siRNA precursors from the formulation. A modified-release formulation can be designed to modify the manner in which the siRNAs, or siRNA precursors is exposed to the desired target, preferably intestinal epithelial cells. For example, a modified-release formulation ean be designed to focus the delivery of the siRNAs, or siRNA precursors entirely in the distal large intestine, beginning at the cecum, and continuing through the ascending, transverse, and descending colon, and ending in the sigmoid colon. Alternatively, for example, a modifiedrelease composition can be designed to focus the delivery of the siRNAs. or siRNA precursors in the proximal small intestine, beginning at the duodenum and ending at the ileum. In still other examples, the modified-release formulations can be designed to begin releasing active agent in the jejunum and end their release in the transverse colon, l he possibilities and combinations are numerous, and are clearly not limited to these examples. lhe term modified-release encompasses extended-release and delayed-release formulations, as well as formulations having both extended-release and delayed-release characteristics. An extended-release formulation can extend the period over which drug is released or targeted to the desired site. A delayed-release formulation can be designed to delay the release of the siRNAs, or siRNA precursors for a specified period. Such formulations are referred to herein as delayed-release or delayed-onset formulations or dosage forms. Modified-release formulations of the present invention include those that exhibit both a delayed- and extended-release, for example, formulations that only begin IE releasing after a fixed period of time or after a physicochemical change has occurred, for example, then continue releasing over an extended period.

As used herein, the term immediate-release formulation,” is meant to describe those formulations in which more than about 50% of active ingredient is released from the dosage form in less than about 2 hours. Such formulations are also referred to herein as ’’conventional formulations.

As used herein, the phrase drug-release profile that is independent of surrounding pH means effectively a drug composition comprising a polymeric system that is non-entcric or whose permeability and solubility properties do not change with environmental, i.e., external, pH. Meaning, a drug composition having release characteristics such as dissolution is substantially unaffected by pH or regardless of pH-changes in the environment. This is in comparison lo a release profile that is pH-dependent where the release characteristics vary according to the pH of the environment. ll is known that certain medium and long-chain fatty acids exert an intestinal epithelial effect which leads to an increased permeability of intestinal membranes to entities that may otherwise be impermeable or exhibit limited permeability. The medium chain triglycerides, including but not limited to sodium caprate, enhance absorption to a greater extent in the small intestine than in the ileum or colon. In a study to investigate the effects of the longchain polyunsaturated fatty acids, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on insulin absorption from rat intestinal loops in situ, Suzuki et al demonstrated that both EPA and DHA strongly enhanced insulin absorption and induced hypoglycaemia after rectal and colonic dosing. DHA did not induce gross morphological changes in the structure of the intestinal mucosa (Suzuki et al, Journal of Pharmaceutical Sciences, Vol 87. 10: Pgs. 1196-1202); 1998). Thus, it is apparent that medium chain triglycerides enhance intestinal |>enneability while DHA is a possible means of facilitating the intestinal absorption of insulin and possibly other macromolecules, nucleic acids, peptides and proteins included, without inducing any serious damage to epithelial cells. Combining poorly permeable entities with medium- or long-chain fatty acids and targeted delivery to local regions of the intestine or colon has the potential to enhance absorption of otherwise poorly permeable entities. The current invenlion seeks to enable such delivery through the encapsulation of entities formulated with medium or long chain polyunsaturated fatty acids using a gelling agent, ΙΕ η 9 ο 7 9 3 including, but not limited to one or a mixture of gelatine, pectin, alginate or chitosan, with or without an additional colon-specific coating. In one embodiment of the invention, this mixture of gelling agents may be gelatine and chitosan. For example, the chitosan may be first associated with siRNA to form an aggregate, complex or a nanoparticle which may then be further formulated eg with liposomal materials. The resulting aqueous phase may then be dispersed in a medium or long chain triglyceride oil phase (water-in-oil emulsion) of the kind described above. This w/o emulsion may then itself be dispersed (emulsified) in an aqueous solution of gelatine as the second gelling agent (although alternative second gelling agents such as alginate are possible as described elsewhere herein) to form a w/o/w emulsion. This w/o/w emulsion may be converted eg into minibeads, minispheres or minicapsules (dependent on extrusion nozzle geometry described elsewhere herein) by extrusion followed by solidification by temperature reduction.

Intestinal Diseases Gastrointestinal conditions pose a significant worldwide health problem. Inflammatory bowel diseases, which genus encompass a range of diseases including Crohn's disease and ulcerative colitis, affect nearly 1 million people in the United States each year. The two most common inflammatory conditions of the intestine, ulcerative colitis (UC) and Crohn's disease (CD), are collectively known as inflammatory bowel disease (IBD). These conditions are diseases of the distal gut (lower small intestine, large intestine, and rectum) rather than the proximal gut (stomach and upper small intestine). Between the two, ulcerative colitis primarily affects the colon, whereas Crohn's disease affects the distal small intestine as well.

Inflammatory Bowel Disease (IBP) Although they are distinct IBD conditions, the same drugs are commonly used to treat both UC and CD. Drugs commonly used in their treatment include steroids (e g., budesonide and other corticosteroids, and adrenal steroids such as prednisone and hydrocortisone); cytokines such as interleukin-10; antibiotics; immunomodulating agents such as azathioprine, 6mcrcaptopurine, methotrexate, cyclosporine, and anti-tumor necrosis factor (TNF) agents such as soluble TNF receptor and antibodies raised lo TNF; and also anttnfla minatory agents such as zinc. The most commonly prescribed agents for IBD include sulfasalazine (salicylazo-sulfapyridinc, or SASP) and related 5-aminosalicylic acid (5-ASA) products, including mesalazine. ΙΕ Π 9 0 Ί 9 3 Inflammation of the ileum (the farthest segment of the small intestine) due to Crohn's disease is known as ilclis. When both the small intestine and the large intestine are involved, the condition is called Crohn’s enterocolitis (or ileocolitis). Other descriptive terms may be used as well. Diagnosis is commonly made by x-ray or colonoscopy. Treatment includes medications that arc anti-inflammatories, immune suppressors, or antibiotics. Surgery can be necessary in severe cases. Crohn’s disease is an area of active research around the world and new treatment approaches are being investigated which have promise to improve the lives of affected patients.

Gastrointestinal Graft-Versus-Host-Disease (GI-GVHD) GI GVHD is a life-threatening condition and one of the most common causes for bone marrow and stem cell transplant failure. These procedures are being increasingly used to treat patients with leukemia and other cancers to eliminate residual disease and reduce the likelihood of relapse. Unlike solid organ transplants where the patient’s body may reject the organ, in GVI ID it is the donor cells that begin to attack the patient’s body - most frequently the gut, liver and skin. Patients with mild-to-moderate GI GVHD typically develop symptoms of anorexia, nausea, vomiting and diarrhea. If left untreated, GI GVHD can progress to ulcerations in the lining of the GI tract, and in its most severe form, can be fatal. Systemic immunosuppressive agents such as prednisone, which are the current standard treatments for GI GVHD, are associated with high mortality rates due to infection and debility. Further, these drugs have not been approved for treating GI GVHD in the U.S. or European Union, but rather are used off-label as investigational therapies for this indication.

Minicapsule-enabled colon-targeted immunosuppressant therapy delivering agents such as cyclosporine A to the colon is a novel oral, locally acting active therapy which will reduce the need for systemic immunosuppressive drugs such as prednisone, which is currently used to prevent and control GI GVHD. Drugs such as prednisone have the unwanted and potentially dangerous side effects of weakening the patient's immune system leaving them susceptible to opportunistic infections as well as substantially inhibiting the intended anticancer effect of bone marrow and stem cell transplants. Minicapsule-enabled colon-targeted immunosuppressant therapy is designed to reduce the need for systemic immunosuppressive drugs and thereby improve the outcome of bone marrow and stem cell transplantation. Therefore, it is possible that delivery of intact peptides or proteins to the colon may be achieved.

IE 0 9 0 7 9 3 l'he invention is directed to, among other things, a pharmaceutical composition for administration to a subject in need thereof comprising a dose of RNA, and at least one pharmaceutical ly acceptable excipient, wherein the composition exhibits localized release and exhibits: For Ulcerative Colitis and Crohn’s Disease - a dissolution profile, when tested in a U.S.P. Type II apparatus (paddles) at 37.degree.C. and 50 rpm, in pH 6.8 buffer for the test: Up to 4 hours: less than or equal to about 20% drug released; 6 hours: less than or equal to about 35% drug released; 8 hours: less than or equal to about 50% drug released; 12 hours: less than or equal to about 60% drug released; 18 hours: less than or equal to about 75% drug released; and 24 hours: from about 25% to about 100% drug released.

For GI-GV I-ID - a dissolution profile, when tested in a U.S.P. Type 11 apparatus (paddles) at 37.degree.C. and 50 rpm, in pH 6.8 buffer for the lest: 1 hour: less than or equal to about 20% drug released: 4 hours: less than or equal to about 35% drug released; 6 hours: less than or equal to about 50% drug released; 12 hours: less than or equal to about 60% drug released; 16 hours: less than or equal to about 75% drug released; and 24 hours: from about 25% to about 100% drug released.

This invention relates to formulations and methods for treating inflammatory bowel disease. The term inflammatory bowel disease includes, but is not limited to, ulcerative colitis, Crohn's disease and GI-GVHD. Other diseases contemplated for treatment or prevention by the present invention include non-u Icerative colitis, and carcinomas, polyps, and/or cysts of the colon and/or rectum. All of these diseases fall within the scope of the term inflammatory bowel disease as used in this specification, yet the invention does not require the inclusion of each recited member. Thus, for example, the invention may be directed to the treatment of Crohn's disease, to the exclusion of all the other members; or to ulcerative colitis, io the exclusion of all the other members; or to any single disease or condition, or combination of diseases or conditions, to the exclusion of any other single disease or condition, or combination of diseases or conditions.

Tight Junction Modulators ΙΕ Λ 9 0 7 9 3 A number of small molecule and peptides to regulate the functional state of tight junctions (TJ) and paracellular permeability being molecules that transiently and reversibly open the Us of epithelial and endothelial tissues such as the intestinal mucosa, blood brain barrier and pulmonary epithelia. As increased paracellular permeability is implicated as a causal factor in many disease states, modulation of permeability by TJ regulatory pathways represents a very important therapeutic opportunity. Potential applications range from the treatment of diseases involving light junction dysfunction and autoimmunity to vaccine and drug delivery. Certain TJ modulators such, but not limited to, parozotide acetate, have potential in the treatment of gastrointestinal disorders, including Celiac Disease and Inflammatory Bowel Disease. The current invention permits the local delivery of tight junction modulators simultaneously with local delivery of siRNAs. or siRNA precursors thus further enhancing absorption by target tissue.

RNA Handling In relation to the following examples, the following information is provided regarding RNA handling. Synthesis of RNA is as described above in the detailed disclosure of the invention. Annealing of siRNAs is generally performed as previously described by Tuschl and coworkers (Elbashir et al., 2001 the entirety of which is incorporated herein by reference). A particular annealing buffer is: 100 mM NaCI in 20 mM sodium phosphate buffer, pH 6.8.

Procedures for preparing RNA for formulation include one or more of the following steps: 1. Combine equimolar amounts of sense and antisense strands and adjust the siRNA concentration to 20 μΜ (double stranded) in a final volume of 100 to 500 μί annealing buffer. 2. Heat a glass beaker containing 500 ml water to 90°C on a magnetic stirrer with adjustable temperature. Place tightly capped 1.5-ml reaction tubes containing the RNA in a flotation device and transfer to the water bath. After 3 min switch the heating element off. 3. Allow the water bath to slowly cool to room temperature (-*3 hr) to permit the siRNA to anneal. 4. Slure the siRNA stock solutions at -80°C (long term) or at 20 °C for up to 6 months.

. Do not keep solutions of highly diluted siRNA for more than an hour, as the doublestranded molecules tend to dissociate IE090793 6. Ihe stability of siRNA at room temperature depends on its sequence and the composition of the solvent (e, g. serum-free medium) 7. The pH and salt composition of the solvent may accelerate RNA decay in solution.

LXAMITI'S Ihe following materials, methods, and examples are illustrative only and not intended to be limiting.

Example I - Preparation of siRNA-containing liposomes The appropriate siRNA (see following examples) is incorporated into liposomes as follows. An appropriate amount of lipid (such as DPPC, DPPG with or without addition of DOPE or positively charged agents) is dissolved in chloroform or chloroform-methanol and the solution is placed in a 50-ml round-bottomed flask. The chloroform is evaporated by gently heating so that a thin film of the lipid is formed on the walls of the flask. To the resulting film siRNA (dissolved in a suitable volume of hydrophilic solvent like RNase-free sterile water) solution is added, glass beads were put, which is mixed at room temperature for blending and hydration, 'I'he mixture is well shaken for 10-15 min to produce an almost homogeneous liquid of nmhilamellar liposomes (MLV).

I he sorbitol is added to the suspension as the membrane stabilizing agent in an amount of up 1% wt/v of the suspension. The lyophilisation of the liposomal suspension is preferably carried out by cooling the suspension to a temperature of about -25C. The freeze dried composition of the present invention, upon rehydratatton, forms a suspension of MLVs which substantially maintains its native size distibution, siRNA/lipid ratio and the morphology of the vesicles. To form beads, an appropriate volume of liposomes were mixed with gelatin (90% gelatin, 10% sorbitol), pectin 2-4% or alginate 2-4% solution using Spherex Labo (single nozzle operation) to form beads.

Bead Composition siRNA 10-15 Gelati n/Pectin/Alginate 90-85 Example 2 IE 0 9 0 7 9 3 The 2l-nucleotide siRNA targeting PHDI corresponding to the coding region 538±558 and 835±855 relative to the start codon is chemically synthesized by methods described above in the body ofthe description (annealing of siRNAs is also performed as described above) and is incorporated into liposomes in accordance with example I.

Example 3 The 21-nucleotide siRNA targeting PHD2 corresponding corresponding to regions 885±905 and 1250± 1270 relative to the start codon is chemically synthesized by methods described above in the body of the description (annealing of siRNAs is also performed as described above) and is incorporated into liposomes in accordance with example 1.

Example 4 The 2I-nucleotide siRNA targeting PHD3 corresponding to the coding regions 351±371 and 389±409 relative to the start codon is chemically synthesized by methods described above in the body of the description (annealing of siRNAs is is also performed as described above) and is incorporated into liposomes in accordance with example I.

Example 5 - siRNA Bead Formation The appropriate RNA (see following examples) is prepared in a suitable siRNA solubility system as follows: siRNA is dissolved in water in the proper amount, then siRNA solution is mixed with a pre-blended mixture ofTranscutol HP, Tween 80 and Labrafil M 1944 CS to form a w/o (water-in-oil) microemulsion (w/o ME) (Table 1); Preparation of Gelatin Solution: appropriate amounts of gelatine and D-sorbitol are dissolved in water at up to 70 degrees C until in solution; Preparation of Beads: appropriate quantities of w/o ME and Gelatin Solution are mixed at up to 70 degrees C (using a siRNA derivative stable at this temperature) to form a stable mixture, which is then processed using a Spherex Labo to produce a single layer bead. fable 1, Composition of w/o ME Ingredients % w/w siRNA 1.23 Water 10.10 ΙΕ η 9 Ο 7 9 3 Transcutol HP 26.58 Tween 80 26.82 Labrafil M 1944 CS 35.26 Table 2. Composition of Gelatin Solution Ingredients % w/w Gelatin 18.50 D-Sorbitol 2.50 Water 79.00 Table 3. Composition of siRNA Beads Ingredients % w/w siRNA 5.38 Gelatin 50.49 lranscutol HP 11.17 Tween 80 1L27 Labrafil M 1944 CS 14.81 D-Sorbitol 6.88 Example 6 A derivative of siRNA for PHDI (as described in Example 2) stable to at most 70°C is synthesised as described above in the body of the description. Single layer beads of this siRNA derivative ate produced in accordance with Example 5.

Example 7 A derivative of siRNA for PHD2 (as described in Example 3) stable to at most 70°C is synthesised as described above in the body of the description. Single layer beads of this siRNA derivative are produced in accordance with Example 5.

IB Ο 9 Ο 7 9 3 Example 8 A derivative of siRNA for PHD3 (as described in Example 4) stable to at most 70°C is synthesised as described above in the body of the description. Single layer beads of this siRNA derivalive are produced in accordance with Example 5.

Example 9 flie appropriate siRNA (see following examples) is prepared in a suitable solubility system as follows: an appropriate quantity of siRNA is dissolved in water, then D-sorbilcI and gelatine are added and dissolved at up to 70 degrees C.

Preparation of Self-MicroEmulsifying Drug Delivery System: Labrafil M 1944 CS, Tween 80 10 and Tiansculol IIP are mixed at room temperature until a clear solution is formed. This is referred as Self MicroEmulsifying Drug Delivery System (SMEDDS).

Preparation of Beads: appropriate amounts of the previously prepared siRNA solubility system and SMEDDS are mixed to form a stable mixture, which is then processed through Spherex Labo to produce single layer beads Table 4. Composition of siRNA solubility System Ingredients % w/w siRNA 1.50 Water 77.50 Gelatin 18.50 D-Sorbitol 2.50 Table 5. Composition of SMEDDS Ingredients % w/w Transeutol HP 30.00 Tween 80 30.00 labrafil M I944CS 40.00 Table 6. Composition of siRNA Reads IE 09 Q7 93 Ingredients % w/w siRNA 4.29 Gelatin 52.86 Transcutol HP 10,71 Tween 80 10.71 Eabrafil M 1944 CS 14.29 D-Sorbitol 7.14 Example 10 A hydrophilic or hydrophobic derivative of siRNA for PHD1 (as described in Example 2) stable to at most 70°C is synthesised as described above in the body of the description. The derivative is then used in accordance with Example 9 to generate beads.

Example 11 A hydrophilic or hydrophobic derivative of siRNA for PHD2 (as described in Example 3) stable to at least 70°C is synthesised as described above in the body of the description. Single layer beads of this siRNA derivative are produced in accordance with Example 5.

Example 12 A hydrophilic or hydrophobic derivative of siRNA for PHD3 (as described in Example 4) stable to at least 70°C is synthesised as described above in the body of the description. Single layer beads of this siRNA derivative are produced in accordance with Example 5.

Example 13 - siRNA minieapsules This example is suitable for siRNAs or siRNA derivatives which are dispersible or soluble in the exemplified core materials. An appropriate amount of 80°- stable siRNA (or appropriate derivative) is dispersed in a solid gelling agent as follows to prepare solid minieapsules (ininispheres): Appropriate quantities of siRNA gelatine and sorbitol are added to water and heated lo 80°C, continually stirring until in a homogeneous solution. The solution is then processed into solid minispheres at an appropriate flow rate and vibrational frequency. The resulting minispheres are cooled in oil. The cooled minispheres are harvested and centrifuged to remove residual oil and dried overnight.

Ingredients % w/w Core Composition siRNA 2.5 Gelatin 87.5 Sorbitol 9.7 Tabic 7: Single-Layer siRNA Minicapsules (Minispheres) To enable the development of a once-daily or an ileum- and colon-specific product, the minicapsules are coated with a range of sustained release polymers, namely differing weight gains of Surelcase®, ranging from 0 to 30% weight gain, or variable weight gains of Sureleasc® plus variable concentrations of pectin.

Example 14 Minicapsules prepared as per Example 13 are coated with 10% weight gain Surelcase®.

Example 15 Minicapsules prepared as per Example 13 are, coated with 15% weight gain Surelease®.

Example 16 Minicapsules prepared as per Example 13 arecoated with 20% weight gain Surelcase® Example 17 Minicapsules prepared as per Example 13 are coated with 25% weight gain Surelease®.

Example IS Minicapsules prepared as per Example 13 arc coated with 30% weight gain Surelease®.

Example 19 Minicapsules containing derivatives of siRNA for PHDI, PHD2 and PHD3 are prepared according to Example Band arecoated according to Examples 14to 19.

Example 20 Mammalian HeLa cells are transfected with siRNA duplexes of the ribonucleic acid of Examples 2, 3 and 4 in cationic liposomes following the technique of Elbashir et al (2001). 0 9 07 93 Example 21 Transfection is conducted according to Example 20 but with the addition of MONTAN IDE™ ISA 50 V which is an oily adjuvant composition of mannide oleate and mineral oil.

Example 22 Mammalian HcLa cells are transfected with siRNA duplexes of the ribonucleic acid of Examples 13 using uncoated minicapsules comprising siRNA for PHD1, PHD2 and PHD3 following the technique of Elbashir et al (2001).

Examples 23-28 First a liposome stock solution was prepared as follows. Solutions of lipids in chloroform (eg DOTAP. DOPE, cholesterol etc) were mixed together in the ratios described below. Evaporation of the solvent (chloroform) was achieved by standard methods known in the art. However, gently applying a flow of nitrogen helped in the formation of a thin film of lipid on the walls of the flask. Rehydration of the lipidic film was obtained by introducing buffer (PBS al pH7) previously warmed to 37’C, after which sample was vortexed for 15 minutes for liposome formation. Liposome stock solutions were kept in the freezer till further use. The stock solution concentration was 0.2 mg/ml.

Using the above liposomes, siRNA formulations were prepared as follows (siRNA supplier was Dharmacon). Using a siRNA stock solution kept in PBS buffer, pH 7.0, at -80*C till use. the appropriate amount of sip65 (siRNA for p65 of NFkB) was added to the liposome stock solution (above) to meet the required positive to negative charge molar ratio defined per formulation described below. After an incubation period of 5 minutes, the samples were ready for use.

Where used, the chitosan was from NovaMalrix, named PROTASAN IIP <Ί. 113 which is based on a chitosan where between 75-90 percent of the acetyl groups orc deacety lated. The cationic polymer is a highly purified and well-characterized water-soluble chloride salt. The molecular weight for PROTASAN UP CL 113 is in the 50000-150000 g/mol range. It has ultra low levels of endotoxins and proteins allowing for a wide variety of in vitro and in vivo applications.

IE Ο 9 0 7 9 3 For the formulations containing ehitosan, liposomes and siRNA, the appropriate amount of siRNA was added to the ehitosan solution, to meet the required positive to negative charge molar ratio defined per formulation. After an incubation period of 5 minutes the remaining amount of liposome solution was added to complete the sample volume. After another incubation period of 5 minutes the sample was ready for use.

Example 23 (H in Figure 1) To 247pL of liposome stock solution prepared as above and containing 10 DOTAP:DOPL:cholesterol at the molar ratios of 6:2:2 was added 13pL of siRNA as supplied (2μΜ) at a molar ratio of positive to negative charges of 20:1 to yield a formulation having the following components by weight %: siRNA 0.0033 DOTAP 0.1327 DOPE 0.0459 Choi 0.0239 PBS 99.7942 Example 24 (L2 in Figure 1) To 247gL of liposome stock solution prepared as above and containing DOTAP.DOPC-.cholesterol at the molar ratios of 6:2:2 was added 13pL of siRNA at a molar ratio of positive to negative charges of 20:1 to yield a formulation having the following components by weight %: siRNA 0.0033 DOTAP 0.1327 DOPC 0.0485 “ChoT 0.0239 PBS 99.7916 ΙΕ ο ϊ $ * Example 25 (L3 in Figure 11 Το 247μΕ of liposome stock solution prepared as above and containing DOTAP: cholesterol at the molar ratios of 6:4 was added 13pL of siRNA at a molar ratio of positive to negative charges of 40:1 to yield a formulation having the following components by weight %. siRNA 0.0033 DO'fAP 0.2655 Choi 0.0973 PBS 99.6339 Example 26 (PL1 in Figure 1) io An aggregate of siRNA with chitosan at a molar ratio of chitosan(+)/siRNA(-) of 50:1 was made by simple mixing of IgL of chitosan with 13pL of siRNA. The chitosan-siRNA complex was then added to the liposomes used in Example 23 (containing DOTAP. DOPE.Cholesterol at the molar ratios of 6:2:2) to yield a formulation having the following components by weight %: siRNA 0.0033 Chitosan 0.000077 DOTAP 0.1322 DOPE 0.0458 Choi 0.0238 PBS 99.7949 Example 27 (PL2 in Figure 1) An aggregate of siRNA with chitosan at a molar ratio of chitosan(+)/siRNA(-) of 50:1 was made by simple mixing of IgL of chitosan with !3pL of siRNA to the liposomes used in Example 24 (containing DOTAP:DOPC.cholesterol at the molar ratios of 6:2:2) to yield a formulation having the following components by weight %: 1109 07 93 siRNA 0.0033 Chitosan 0.000077 DOTAP 0.1322 DOPC 0.0483 Choi 0.0238 PBS 99.7923 Example 28 (PL3 in Figure 1) An aggregate of siRNA with chitosan at a molar ratio of chttosan(+)/siRNA(-) of 50:1 was made by simple mixing of lpL of chitosan with 13pL siRNA to the liposomes used in Example 25 (containing DOTAP:cholesterol at the molar ratios of 6:4) to yield a formulation having the following components by weight %: siRNA 0.0033 Chitosan 0.000077 DOTAP 0.2644 Choi 0.0969 PBS 99.6353 Example 29 Experiments on the six formulations of Examples 23-28 were performed to determine gene silencing. The method used was western blot analysis as described below. Samples were prepared according to known techniques - sec for example Tao et al (2006) Mol Cell Biol 26(3) 1038-1050 or following the siRNA manufacturer’s instructions.

Figure I shows scanned western blots of the samples prepared. The bands represent the proteins p65 and beta-actin. The intensity of one band is proportional to the amount of the protein in the sample. Knock down of the target protein (p65) therefore leads to a lighter band (lower intensity) than in the control. The control (panel 1 of Figure 1) shows samples IE 09 π7 93 resulting from transfection with siRNA mixed with lipofectamine. The potential gene knockdown effect of two siRNAs was tested namely a “non-target” (NT) siRNA or siNT which is a siRNA lor which there is no target protein in the cells. The second siRNA is for the p65 portion ot NlkB referred to as sip65. Two proteins were included in the western blot namely p65 and beta-actin. Beta-actin is a protein known not be affected by siRNA. Its inclusion in the experiment is to confirm the system is working and that proteins are detectable. It also acts as a negative control for which therefore no change in intensity between samples is expected. Also, use of this negative control demonstrates that the different intensities of p65 in Figure 1 are not due to loading of different amounts of protein. The p65 protein is obviously the protein whose expression it is desired to inhibit by gene knock-down using siRNA. Two samples of sip65 were used in the control (Panel A of Figure I) namely a laboratory stock (kept in optimal conditions) and an ex-laboratory stock which had intentionally be exposed to movement and ambient temperature to test stability. Panel A of Figure 1 shows that siRNA from lab stock as well as siRNA from ex-lab stock were able to knock down the protein (lighter bands).

Panel B of Figure 1 shows the results for the formulations of this Example. Two different concentrations of the formulated sip65 namely 2 and 20 nM were tested. Controls used the same formulations without siRNA (with PBS instead) in the same volumes as were used of the formulated siRNA (20 or 200 ul, respectively). Panel B of Figure 1 show's that Ll and PLI show a knock down of the p65 protein especially with 2 nM.

Examples 31-36 In these examples, mini-beads were produced from a oil-in-water emulsion which can be referred to also as a water-in-oil-in-water (w/o/w) emulsion in which the oil phase was prepared by dispersing the siRNA-liposome formulations of Examples 23-25 and the siRNAliposoine-protein complex of Examples 26-28 in an oil phase to create a w/o emulsion. This oil phase (in lacl a w/o phase) and the outer aqueous phase were then mixed in a proportion in the range 1:6-10, preferably approximately 1:7 or 1:8 with gentle continuous stirring of the components using a Magnetic Stirrer (manufactured by Stuart). The outer aqueous phase (gelatin with sorbitol) was prepared by adding the appropriate quantities of sorbitol (optionally with surfactant eg SDS) to water, heating to approximately 60-75° C until in solution and then adding gelatin. The “gelatin solution” comprised 15-25% (preferably 1761 18%) of gelatin: 75%-85% (preferably 77-82%) of water plus from 1-5% (preferably 1.5 to 3%) sorbitol· The gelatin solution was maintained at 60°C-70°C to maintain it in a fluid state. In a slightly variant method, SDS was added to the aqueous phase at the same time the other components are added ie. gelatin and sorbitol al the beginning of the processing session SDS (surfactant) if used was present in an amount between 0.8% and 1% (by weight) of the aqueous phase. The oil phase was made at room temperature with stirring until clear. The w/o/w emulsion was formed by addition of the oil phase (or w/o phase) to the heated aqueous phase with stirring as described above. The resultant emulsion then had the composition of the future solidified mini-beads but with water still present. Once the emulsion was formed, the beading step was begun without delay by using a pipette and dropping the fluid emulsion manually into MCT (cooling fluid) maintained in the range 8-12^0 which effected solidification. Beads were then collected in a mesh basket through which the oil was drained and the beads retained, excess oil removed by centrifugation then washed with ethyl acetate then dried. Drying was with the Freund Drum dryer with warm air at between I5°C and °C. Uncoated mini-beads having the following composition were generated: Mg/g siRNA-liposome (+/- chitosan) 80-120 Transcutol HP 150-190 Cremophor EL 80-120 Migyol810 20-60 SDS 15-50 D-Sorbitol 30-80 Gelatin 450-550 Example 37-42 The beads of these Examples were produced initially as for Examples 31-36 but instead of 20 pipetting the emulsion, the mini-beads were produced through ejection of the fluid w/o/w emulsion through a vibrating 3mm diameter single lumen nozzle applied to the Freund Sphcrcx machine. Operation of the Spherex machine manufactured by Freund was in ΙΕ π 9 07 9 3 accordance with the manufacturer’s instructions. The lines to the ortfice/nozzle were maintained al 65-85°C to maintain the fluidity of the solution. The resulting beads had the following composition: Components Lower limit tmg/e) Upper limit ima/g) stRNA-liposomes {+/- ehitosan) 80 140 Gelatin 490 610 D-Sorbitol 55 75 SDS 20 40 Transcutol P 100 180 Crcmophor EL 50 IIP MCToil* 45 180 Labrafil M 1944 CS 40 150 Lpax 6000** 80 150 *MCT brands used include: Mygliol 810, Labrafac Lipophile 1349 WL, Caplex 355, etc...

♦*Omega-3 oil having a EPA (eicosapentanoic acid)/DHA (docosohexaenoic acid) ratio ~ 1.5 Example 43-54 Uncoatcd beads in this Example were made in accordance with Example 31-42 except that no SDS was used.

The invention also relates to the subject matter of the following clauses; I. Λ method for delivering an siRNA or engineered RNA precursor to a target cell In bringing a multiplicity of RNA coma tiling minicapsules into contact with the target cell fe * 9 π 7 g j * * τ σ ί 2. I hc method of clause I wherein the bringing into contact is accomplished by oral administration of a pharmaceutical formulation comprising said minicapsules. 3. Ihe method of clauses I or 2 wherein the target cell is an intestinal epithelial cell. 4. Ihe method of clauses 1-3 wherein the RNA comprised in the minispheres is adapted to interfere, knockdown or inhibit the expression ol an enzyme.

. Ihe method of clause 4 wherein the enzyme is selected from enzymes affecting the control of hypoxia in the cells of the Gl tract. 6. flic method of clause 5 wherein said enzymes are those which normally cause I III·’ lo be upreguiated or retained at beneficial levels. 7. I he mclhixl of clauses l-ύ wherein the RNA is adapted lo knockdown, silence or inhibit the expression of one or more PHDs. including PHD 1, 2 and 3.

X. Λ method for delivering an siRNA to a cell by obtaining, identifying or targeting a cell (or system of cells or tissue), forming a minisphere comprising an siRNA and contacting the cell (or system of cells or tissue) with the minisphere or a plurality thereof 9. Ihe method of clause 8 wherein said cell is a Ci I cell.

. The mciliod of clause 9 wherein the siRNA or engineered RNA precursor is conjugated to a delivery peptide, the conjugate being encapsulated in a mini sphere. 11. I he method oi clauses 9 or 10 wherein ihe conjugate is a biconjugatc ol' targeting peptides susceptible of enhancing uptake of siRNA. 12. An oral composition comprising minicapsules wherein the minicapsules comprise one or more siRNAs or engineered RNA precursors in a eorc susceptible of maintaining such RNA in a stable, active form.

. I lie co m pi isi ι ton of clause 12 wherein the core is liquid, semi-solid, or solid. 14. I he composition of clause 12 or 13 wherein the minicapsules have release profiles to release the siRNA or engineered RNA precursor in an active form at one or more sites along the gastrointestinal tract.

. The composition of clause 12 to 14 wherein the siRNA or engineered RNA precursor, when released from the minicapsule, is in a soluble form or is readily soluble in the aqueous GIT environment. 16. The composition of clauses 12 to 15 wherein the minicapsule has one layer and is solid throughout. 17. The composition of clauses 12-15 wherein the minicapsule has two layers comprising a solid outer shell layer encapsulating a liquid, semi-solid or solid core. 18. The composition of clauses 12 to 17 wherein the minicapsules are modified to enable modified release of the siRNA or engineered RNA prccursor(s). 19. A pharmaceutical composition comprising mini-beads of solidified matrix material wherein the mini-beads comprise one or more siRNAs or engineered RNA precursors dispersed in said solidified matrix.

. The com posit ion of clause 19 wherein at least one of the siRNAs or engineered RNA precursois is associated with liposomal material.

IE0 Λ σ 9 3 211 he composition of clause 19 or 20 wherein at least one of the siRNAs or engineered RN A precursors is complexed with a protein, 22. The composition of clause 21 wherein the protein is cationic and is preferably chitosan. 23. The composition of clause 19-22 wherein at least one of the siRNAs or engineered RNA precursors forms an aqueous phase dispersed in an oil phase. 24. The composition of clause 23 wherein the oil phase is dispersed in said solidified matrix.

. Ihe composition of clauses 19-24 wherein the solidified matrix is an aqueous phase. 26. Ihe composition of clauses 19-24 wherein the composition is adapted for oral administration.

The invention is not limited to the embodiments hereinbefore described which may be varied 15 in detail.

Claims (25)

CLAIMS IE οθ 0 7 9 3

1. Λ method Ibr delivering an siRNA or engineered RNA precursor to a target cell hy bringing a multiplicity of’ RNA-containing minieapsules into contact 5 w ith the target cell.

2. l he method of claim I wherein lhc bringing into contact is accomplished by oral administration ol a pharmaceutical formulation comprising said minieapsules. 5. l he method of claims 1 or 2 wherein the target cell is an intestinal epithelial cell.

3. 4. ] he method of claims 1-3 wherein the RNA comprised in the minieapsules is 15 adapted to interfere, knockdown or inhibit the expression of an enzyme.

4. 5. lhe method of claim 4 wherein the enzyme is selected from enzymes affecting the control of hypoxia in the cells ofthe Cl tract.

5. 6. lhe method of claim 5 wherein said enzymes are those which normally cause Illi lo be upregulated or retained al beneficial levels.

6. 7. I he method of claims 1-6 wherein the RNA is adapted to knockdown. silence or inhibit (he expression ofonc or more PHDs. including PUD 1.2 and 3. S. A method for delivering an siRN/\ lo a cell by obtaining, identifying, or targeting a cell (or system of cells or tissue), forming a minispherc comprising an siRNA and contacting the cell (or system of cells or tissue) with the minispherc or a plurality thereof. ii ©9 0 7 9 3

7. 9. The method of claim 8 wherein said cell is a GI cell.

8. 10. I he method of claim 9 wherein the siRNA or engineered RNA preeutsor is conjugated to a delivery peptide, the conjugate being encapsulated in a minisphere.

9. 11. Ihe method of claims 9 or 10 wherein the conjugate is a biconjugale of targeting peptides susceptible of enhancing uptake of siRNA.

10. 12. An oral composition comprising minicapsules wherein the minicapsules comprise one or more siRNAs or engineered RNA precursors in a core susceptible of maintaining such RNA in a stable, active form. 15. Ihe composition of claim 12 wherein the core is liquid, semi-so I id, or solid.

11. 14. fhe composition of claim 12 or 13 wherein the minicapstiles have release profiles to release the siRNA or engineered RNA precursor in an active form at one or more sites along the gastrointestinal tract.

12. 15. l'he composition of claim 12 to 14 wherein the siRNA or engineered RNA precursor, when released from the minieapsule, is in a soluble form or is readily soluble in the aqueous GIT environment,

13. 16. The composition of claims 12 to 15 wherein the minieapsule has one layer and is solid throughout.

14. 17. I he composition of claims 12-15 wherein the minieapsule has two layers comprising a solid outer shell layer encapsulating a liquid, semi-solid or solid core. IE η 9 η 7 93

15. 18. The composition of claims 12 to 17 wherein the minicapsules are modified to enable modified release of the siRNA or engineered RNA precursors).

16. 19. A pharmaceutical composition comprising mini-beads of solid matrix material wherein the mini-beads comprise one or more siRNAs or engineered RNA precursors dispersed in said solid matrix.

17. 20. I lie composition of claim 19 wherein al least one of the siRNAs or engineered RNA precursors is associated with liposomal material.

18. 21. I he composition of claim 19 or 20 wherein at least one of the siRNAs or engineered RNA precursors is complexed with a protein,

19. 22. I he composition of claim 21 wherein the protein is cationic and is preferably chitosan. 25. Ihe composition of claim 19-22 wherein at least one of the siRNAs or engineered RNA precursors forms an aqueous phase dispersed in an oil phase.

20. 24. I he composition of claim 23 wherein the oil phase is dispersed in said solid matrix.

21. 25. I he composition of claims 19-24 wherein the solidified matrix is an aqueous phase.

22. 26. I he composition of claims 19-24 wherein the composition is adapted for oral administration.

23. 27. A medicament lor delivering an active agent selected from siRNAs and engineered RNA precursors to a target cell in the gastrointestinal tract, the medicament comprising a multiplicity of RNA-containing minicapsules and being adapted for the active agent lo be released and contact the target cell after administration of the medicament.

24. 28. A product selected from a minicapsule, a minibead or a minisphere and comprising a solid phase and an active agent selected from siRNAs and engineered RNA precursors.

25. 29. I he product of claim 28 having one layer being a solid phase comprising inclusions selected from liquid inclusions, semi-solid inclusions or