Hormone Inhibition

The present invention relates to siRNA's capable of mediating target-specific inhibition of proopiomelancortin.

BACKGROUND

Cushing's disease is a devastating condition caused primarily by a pituitary tumour expressing excess proopiomelancortin (POMC) that is cleaved to adrenocorticotropin (ACTH) that in turn drives the adrenals to synthesise and secrete excess Cortisol (Newell-Price et al (2006) Lancet 367:1605-1617). Modern series show that if left untreated Cushing's disease has a five-fold excess mortality (Lindholm J et al (2001 ) . The Journal of clinical endocrinology and metabolism 86:1 17-123). In rare instances Cushing's disease may result from the ectopic production of ATCH from locations other than the pituitary. Ectopic sites of ATCH production can arise from many different tissues including medullary carcinoma of the thyroid, oat cell carcinoma of the lung, thymoma, islet cell tumors, and pheochromocytoma.

Little is known about the pathogenesis of the pituitary tumours that cause Cushing's disease (Newell-Price et al (1998) Endocr Rev 19:647-672, Dahia and Grossman (1999)

Endocr Rev 20:136-155). Notably the tumours have high proliferative activity and low expression of p27, and mis-expression of Brg1 (Bilodeau S et a/ (2006) Genes Dev. Oct

15;20(20):2871 -86). The synthesis and secretion of ACTH has however been studied in detail. Corticotrope tumours express pro-opiomelanocortin gene (POMC) whose product is cleaved to corticotrophin (ACTH).

Traditionally transsphenoidal surgery has been used as primary treatment for Cushing's disease but this has a long term remission rate of only 55% and half of the patients treated in this way develop hypopituitarism (Newell-Price (2002) Clinical endocrinology 56:19-2). Hypopituitarism itself has excessive mortality. Bilateral adrenalectomy is effective treatment but is complicated by lifelong hypoadrenalism and the development of an expanding, and often invasive, pituitary mass (Nelson's syndrome) in up to 30% of cases (Jenkins et al (1995) The Journal of clinical endocrinology and metabolism 80:165-171 ).

To date there is no proven effective medical treatments for lowering ACTH and hence Cortisol levels.

Patients who are not cured or relapse can receive external beam radiation (in certain cases sterotactic radiosurgery maybe appropriate) and/or medical therapy with steroidogenic inhibitors or neuromodulators of ACTH. Metyrapone, ketoconazole, etomidate and aminoglutethimide inhibit steroid biosynthesis. Valproic acid has also been used in patients with Cushing's and Nelson's syndrome, which may enhance GABA inhibition of hypothalamic CRH release, however the efficacy is unproven in Cushing's disease. Cyproheptadine may suppress ACTH by acting at hypothalamo- pituitary level but further studies are required. Very recent studies are embarking on testing the ability of mifeprostone to block the action of Cortisol in the body.

Recent data has shown that high dose cabergoline maybe partly effective but only in 40% of cases (Shraga-Slutzky I et al (2006) Pituitary 9:151 -154, lllouz F et al (2006) Ann Endocrinol (Paris) 67:353-356, Pivonello R et a/ (2004) J Clin Endocrinol Metab 89:1674- 1683) . Interest has also focused on PPAR gamma agonists such as rosiglitazone in mouse models of Cushing's disease (Heaney et al., (2002) Nat Med 8:1281 -1287). Responses to rosiglitazone in clinical trials of patients with Cushing's disease have been disappointing with responses in only 50 % and escape of control whilst on treatment in half of these subjects (Ambrosi et al (2004) Eur J Endocrinol 151 :173-178, Cannavo et al (2005) Clin Endocrinol (Oxf) 63:1 18-1 19). Recently we have specifically assessed the effectiveness of high dose rosiglitazone given for two months on ACTH levels in a series of patients with Nelson's syndrome, but we have not found this to be effective (Munir et al (2007) J Clin Endocrinol Metab 92:1758-1763).

Spampinato et al used an AtT20 model and treated cells with an oligonucleotide complementary to a region of β-endorphin mRNA. This markedly reduced the levels of ACTH and β-endorphin. In addition they performed an in vivo infusion of the oligonucleotide in rats via an implanted cannula in to the hypothalamic arcuate nucleus and noted a reduction in the ACTH and β-endorphin immunopositive neurons. Grooming behavior was reduced (Spampinato et al (1994) Proc Natl Acad Sci U S A, 91 , 8072-6).

Woloschak et al treated human ACTH-secreting pituitary adenoma cells cultured from two uncommon ACTH-secreting macroadenomas. The tumour tissue was enzymatically dispersed and cultured for four days. They were then treated for 18 hours with antisense

POMC oligomer or control, and one set of tumour cells was treated with dexamethasone. They showed a reduction in POMC mRNA levels and ACTH levels by 76% and 62% in the two tumours and 58% and 48% relative to controls (Woloschak et al (1994) J Endocrinol Invest, 17, 817-9).

Accordingly, there remains a continuing need to develop medical therapy for Cushing's disease aimed at lowering ACTH.

BRIEF SUMMARY OF THE DISCLOSURE

In a first aspect the invention provides a short interfering RNA (siRNA) comprising a sense strand and an antisense strand which each independently comprise from about 19 to about 25 nucleotides, wherein said sense strand is at least 80% identical to an RNA sequence encoding POMC or a portion thereof, and said antisense strand is complementary to said sense strand.

In a preferred embodiment said sense strand is at least 80% identical to an RNA sequence selected from: i) RNA target sequence of siRNA 1 (SEQ ID NO:1 ); ii) RNA target sequence of siRNA 2 (SEQ ID NO:2); iii) RNA target sequence of siRNA 3 (SEQ ID NO:3).

Preferably said sense strand and antisense strand each comprise about 21 nucleotides.

Preferably said siRNA comprises a nucleotide overhang at the 3' end, the 5' end or both the 3' and 5' ends of said siRNA.

Preferably said siRNA comprises at least one nucleotide wherein a methylene bridge connects a 2'- oxygen of a ribose to a 4'carbon.

More preferably said sense strand of said siRNA comprises an RNA sequence selected from: i) an RNA sequence of SEQ ID NO:4; ii) an RNA sequence of SEQ ID NO:5; and iii) an RNA sequence of SEQ ID NO:6.

Preferably, said sense strand comprises an RNA sequence of SEQ ID NO:4 and said antisense strand comprises an RNA sequence of SEQ ID NO:7. Alternatively, said sense strand comprises an RNA sequence of SEQ ID NO:5 and said antisense strand comprises an RNA sequence of SEQ ID NO:8. Alternatively, said sense strand comprises an RNA sequence of SEQ ID NO:6 and said antisense strand comprises an RNA sequence of SEQ ID NO:9.

Still more preferably, said sense strand consists of an RNA sequence of SEQ ID NO:4 and said antisense strand consists of an RNA sequence of SEQ ID NO:7. Alternatively, said sense strand consists of an RNA sequence of SEQ ID NO:5 and said antisense strand consists of an RNA sequence of SEQ ID NO:8. Alternatively, said sense strand consists of an RNA sequence of SEQ ID NO:6 and said antisense strand consists of an RNA sequence of SEQ ID NO:9.

In a further aspect the invention provides a short interfering RNA (siRNA) comprising a sense strand and an antisense strand which each independently comprise from about 19 to about 25 nucleotides, wherein said sense strand is at least 80% identical to a DNA sequence encoding pituitary promoter region of POMC or a portion thereof and said antisense strand is complementary to said sense strand.

Preferably, said sense strand is at least 80% identical to an DNA sequence selected from: i) DNA target sequence of ProPOMCI (SEQ ID NO:10); ii) DNA target sequence of ProPOMC2 (SEQ ID NO:1 1 ); iii) DNA target sequence of ProPOMC3 (SEQ ID NO:12); iv) DNA target sequence of ProPOMC4 (SEQ ID NO:13); v) DNA target sequence of ProPOMCδ (SEQ ID NO:14); vi) DNA target sequence of ProPOMCθ (SEQ ID NO:15).

Preferably said sense strand and antisense strand each comprise about 25 nucleotides.

Preferably said siRNA comprises a nucleotide overhang at the 3' end, the 5' end or both the 3' and 5' ends of said siRNA.

Preferably said siRNA comprises at least one nucleotide wherein a methylene bridge connects a 2'- oxygen of a ribose to a 4'carbon.

Preferably, said sense strand of said siRNA comprises an RNA sequence selected from: i) an RNA sequence of SEQ ID NO:16; ii) an RNA sequence of SEQ ID NO:17; iii) an RNA sequence of SEQ ID NO:18; iv) an RNA sequence of SEQ ID NO:19; v) an RNA sequence of SEQ ID NO:20; and vi) an RNA sequence of SEQ ID NO:21.

Preferably, said sense strand comprises an RNA sequence of SEQ ID NO:16 and said antisense strand comprises an RNA sequence of SEQ ID NO:22. Alternatively, sense strand comprises an RNA sequence of SEQ ID NO:17 and said antisense strand comprises an RNA sequence of SEQ ID NO:23. Alternatively, said sense strand comprises an RNA sequence of SEQ ID NO:18 and said antisense strand comprises an RNA sequence of SEQ ID NO:24. Alternatively, said sense strand comprises an RNA sequence of SEQ ID NO:19 and said antisense strand comprises an RNA sequence of SEQ ID NO:25. Alternatively said sense strand comprises an RNA sequence of SEQ ID NO:20 and said antisense strand comprises an RNA sequence of SEQ ID NO:26. Alternatively, said sense strand comprises an RNA sequence of SEQ ID NO:21 and said antisense strand comprises an RNA sequence of SEQ ID NO:27.

More preferably, said sense strand consists of an RNA sequence of SEQ ID NO:16 and said antisense strand consists of an RNA sequence of SEQ ID NO:22. Alternatively, said sense strand consists of an RNA sequence of SEQ ID NO:17 and said antisense strand consists of an RNA sequence of SEQ ID NO:23. Alternatively, said sense strand consists of an RNA sequence of SEQ ID NO:18 and said antisense strand consists of an RNA sequence of SEQ ID NO:24. Alternatively, said sense strand consists of an RNA sequence of SEQ ID NO:19 and said antisense strand consists of an RNA sequence of SEQ ID NO:25. Alternatively, said sense strand consists of an RNA sequence of SEQ ID NO:20 and said antisense strand consists of an RNA sequence of SEQ ID NO:26.

Alternatively, said sense strand consists of an RNA sequence of SEQ ID NO:21 and said antisense strand consists of an RNA sequence of SEQ ID NO:27.

In a further aspect the invention provides a short interfering RNA (siRNA) comprising a sense strand and an antisense strand which each independently comprise from about 19 to about 25 nucleotides, wherein said sense strand is at least 80% identical to an RNA

sequence encoding Tpit or a portion thereof, and said antisense strand is complementary to said sense strand.

Preferably, said sense strand is at least 80% identical to an RNA sequence selected from: i) RNA target sequence of TpitsiRNA 1 (SEQ ID NO:28); or ii) RNA target sequence of TpitsiRNA 2 (SEQ ID NO:29).

Preferably, said sense strand and antisense strand each comprise about 21 nucleotides.

Preferably, said siRNA comprises a nucleotide overhang at the 3' end, the 5' end or both the 3' and 5' ends of said siRNA.

Preferably, said siRNA comprises at least one nucleotide wherein a methylene bridge connects a 2'- oxygen of a ribose to a 4'carbon

Preferably, said sense strand of said siRNA comprises an RNA sequence selected from: i) an RNA sequence of SEQ ID NO:30; and ii) an RNA sequence of SEQ ID NO:32. Preferably, said sense strand comprises an RNA sequence of SEQ ID NO:30 and said antisense strand comprises an RNA sequence of SEQ ID NO:31. Alternatively, said sense strand comprises an RNA sequence of SEQ ID NO:32 and said antisense strand comprises an RNA sequence of SEQ ID NO:33.

More preferably, said sense strand consists of an RNA sequence of SEQ ID NO:30 and said antisense strand consists of an RNA sequence of SEQ ID NO:31 . Alternatively, said sense strand consists of an RNA sequence of SEQ ID NO:32 and said antisense strand consists of an RNA sequence of SEQ ID NO:33.

In a further aspect of the invention there is provided a therapeutic composition comprising an siRNA according to the invention and a pharmaceutically acceptable carrier.

In a further aspect of the invention, there is provided an siRNA according to the invention for use as a medicament.

In a further aspect of the invention, there is provided an siRNA according to the invention for treating a disorders associated with the expression or activity of a POMC nucleic acid or polypeptide encoded by the POMC nucleic acid. Preferably, said disorder is Cushing's disease.

In a further aspect the invention provides use of an siRNA according to the invention to down-regulate the expression or activity of proopiomelancortin (POMC).

In a further aspect the invention provides methods of treating a subject having a disease or disorder associated with the expression or activity of a POMC nucleic acid or polypeptide encoded by the POMC nucleic acid by administering an siRNA of the invention to the subject. Preferably, there is provided a method of treating a disorder associated with the expression or activity of a POMC nucleic acid or polypeptide encoded by the POMC nucleic acid in a subject in need thereof, comprising administering to the subject an effective amount of an siRNA in accordance with the invention. Preferably, said disorder is Cushing's disease.

In a further aspect there is provided a method of down-regulating the expression or activity of proopiomelancortin (POMC) in a subject in need thereof, comprising administering to the subject an effective amount of an siRNA in accordance with the invention.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described hereinafter, by way of example only, with reference to the accompanying drawings in which:

Figure 1 is a schematic representation of the cellular RNAi response;

Figure 2 is a schematic representation of the polycystronic products of the POMC gene;

Figure 3 is a schematic representation of mouse PO/WC with positions of siRNAs aligned

Figure 4a is a graphical representation of siRNA silencing of pome mRNA with siRNA 1 :. siRNAI silences pome mRNA by 86% at 24 hours; Figure 4b is a graphical representation of siRNA silencing of pome mRNA with siRNA 2:. siRNA2 silences pome mRNA by 89% at 24 hours; Figure 4c is a graphical representation of siRNA silencing of pome mRNA with siRNA 3:. siRNA3 silences pome mRNA by 95% at 24 hours; Figure 4d is a graphical representation of the stability of beta-actin mRNA at 48 hours after treatment with siRNA3: beta-actin expression remains stable in keeping with pomc- specific effect of siRNA3;

Figure 5a is a graphical representation of ACTH levels upon siRNA-3 treatment - effect at 48, 120 and 240 hours respectively; Figure 5b is a graphical representation of siRNA silencing of pome mRNA with siRNA 3, demonstrating significant silencing of pome mRNA over 48, 120 and 240 hours, respectively;

Figure 6a is a graphical representation of the knockdown of pome mRNA following a single transfection with siRNA targeting the pome promoter showing <95% reduction of expression of pome at 96 hours p<0.0001 ; Figure 6b is a graphical representation of the knockdown of pome mRNA following a single transfection with siRNA targeting the pome promoter showing recovery of expression to 50% by 120 hours;

Figure 7a and b provide a graphical representation of methylation analysis of the promoter of pome. Each individual CpG site is represented by a circle: unfilled circles = CpG unmethylated, filled circles = CpG site methylated. The number of rows is the number of clones sequenced, Figure 7a illustrates lack of methylation-induction of pome

after AtT20 cells are treated with 6 siRNAs targeted to the pome promoter region, figure 7b illustrates methylation of the pome promoter in non-expressing tissue (3T3 cells);

Figure 8 is a schematic representation of the regulatory effect of Tpit on the expression of POMC;

Figure 9 illustrates a BLAST alignment of mouse Tpit with human TPIT.The target site for TpitsiRNA 1 is highlighted in dark grey (top of diagram). The target site for TpitsiRNA 2 is highlighted in light grey.(bottom of diagram). The numbers correspond to bases in mouse Tpit and human TPITmRUA;

Figure 10a is a graphical representation of the relative quantities of Tpit mRNA in TpitsiRNA 1 - treated samples 24 hours post-transfection. Figure 10b is a graphical representation of the Relative quantities of Tpit mRNA in TpitsiRNA 2- treated samples 24 hours post-transfection. Neg siRNA = Negative control siRNA;

Figure 1 1 a is a graphical representation of the relative quantities of POMC mRNA in TpitsiRNA 1 - treated samples 24 hours post-transfection. Figure 1 1 b is a graphical representation of the Relative quantities of POMC mRNA in TpitsiRNA 2- treated samples 24 hours post-transfection. Neg siRNA = Negative control siRNA;

Figure 12 is a graphical representation of the relative quantities of beta Actin mRNA in samples 24 hours post-transfection. Neg siRNA = Negative control siRNA;

Figure 13a is a graphical representation of the relative quantities of Tpit mRNA in TpitsiRNA 1 - treated samples 72 hours post-transfection. Figure 13b is a graphical representation of the relative quantities of Tpit mRNA in TpitsiRNA 2- treated samples 72 hours post-transfection. Neg siRNA = Negative control siRNA;

Figure 14a is a graphical representation of the relative quantities of POMC mRNA in TpitsiRNA 1 - treated samples 72 hours post-transfection. Figure 14b is a graphical representation of the relative quantities of POMC mRNA in TpitsiRNA 2- treated samples 72 hours post-transfection. Neg siRNA = Negative control siRNA;

Figure 15a is a Western blot with anti-Tpit antibody, transfected and control protein samples. Expected band size = 48 kDA. Figure 15b is a western blot with anti-alpha Tubulin antibody, transfected and control protein samples. Expected band size = 50 kDa. In each blot Lane 1 = TpitsiRNA 1 , Lane 2 = TpitsiRNA 2, Lane 3 = Negative control siRNA and Lane 4 = No siRNA;

Figure 16a is a graphical representation of the relative levels of ACTH in TpitsiRNA 1 - treated samples 24 hours post-transfection. Figure 16b is a graphical representation of the relative levels of ACTH in TpitsiRNA 2- treated samples 24 hours post-transfection. Neg siRNA = Negative control siRNA;

Figure 17a is a graphical representation of the relative levels of ACTH in TpitsiRNA 1 - treated samples 72 hours post-transfection. Figure 17b is a graphical representation of the relative levels of ACTH in TpitsiRNA 2- treated samples 72 hours post-transfection. Neg siRNA = Negative control siRNA;

Figure 18 is a Light microscope image of live primary human corticotroph tumour cells (x 20);

Figure 19 is a graphical representation of the knock down of ACTH levels with siRNA 2 74%, siRNA 3 27%, TpitsiRNA 2 14 % when compared with Negative siRNA control at 24 hours;

Figure 20 is a graphical representation of the knock down of ACTH levels with siRNA 2 68%, siRNA 23%, TpitsiRNA 2 23 % when compared with Negative siRNA control at 48 hours;

Figure 21 is a graphical representation of ACTH levels 5 days post siRNA 3 treatment in mice with tumours less than or equal to 5mm;

Figure 22 is a graphical representation of ACTH levels 5 days post siRNA 2 treatment in mice with tumours less than or equal to 5mm;

Figure 23 is a graphical representation of tumour growth 5 days post siRNA 3 treatment.

DETAILED DESCRIPTION

The present inventors have used RNA Interference (RNAi) to target POMC coding and promoter sequences, together with a POMC transcription factor, Tpit, and have provided short interfering RNA molecules (siRNA's) that mediate long lasting and reproducible suppression of POMC and ACTH. The siRNA's of the invention may be used to provide effective systemic therapy for the treatment of Cushing's disease.

RNA interference is a process for sequence specific control of gene expression at a transcriptional, post-transcriptional and translational level using short interfering RNA molecules (Fire et al (1998) Nature, 391 , 806-1 1 ). It has been found that dsRNA mixture is at least ten fold more potent as a silencing trigger than sense or anti-sense alone (Guo and Kemphues, (1996) Nature, 382, 455-8).

The presence of dsRNA in a cell triggers a cellular RNAi response, as illustrated in figure 1 . The effector molecule of RNAi is the RNA-induced silencing complex (RISC). RISC is a high molecular weight multienzyme protein complex that endonulceolytically cleaves the target mRNA's before they are translated to protein. This complex was first isolated by Hannon's lab, with a species of small RNA about 25 nuleotides in length (Hammond et al (2000) Nature, 404, 293-6). It was these RNAs that were termed small interfering RNAs (siRNAs) and were shown to be required for degradation of the mRNA.

The introduction of dsRNA into a cell, stimulates the activity of an enzyme responsible for processing the dsRNA to siRNAs (Bernstein et al (2001 ) Nature, 409, 363-6). This enzyme from the Ribonuclease III family of enzymes is referred to as Dicer. The Dicer enzyme possesses dual dsRNA catalytic domains: PAZ and helicase. Dicer is highly conserved with homologues in nearly every species.

siRNAs produced by the enzymatic reaction of Dicer are double stranded, and typically between about 20 to 25, more typically 21 to 23, nucleotides in length with 5'-phosphate and 3'-hydroxyl termini. They comprise about 19 to 22, more typically about 19, central complementary paired bases and have short 3'-nucleotide overhangs, typically 3'- dinucleotide overhangs.

The initiation of silencing occurs upon recognition of dsRNA by Dicer, which converts the dsRNA in to 21 -25nt RNAs. These small interfering RNAs (siRNAs) are incorporated in

to RISC and guided to the mRNA of interest (the guide strand which is anti-sense to the target mRNA) (Hannon GJ (2002) Nature, 418, 244-51 ). Degradation of the mRNA of interest is mediated through endonucleolytic cleavage by Argonaute (AGO) proteins, which are components of RISC.

RISC mediates several different modes of silencing. Sequence specific degradation of mRNA targets, termed slicing, is one method of post-transcriptional gene silencing. The target RNA is cleaved at the position opposite the phosphate between the 10 ^th and 1 1 ^th base from the 5 prime end of the guide. Read through is prevented and mRNA degraded. The alternative method is one of translational repression used by microRNA (endogenous non coding RNAs processed in to stem loops called pre-imRNAs; important in development, oncogenesis and immunity). The third method is transcriptional gene silencing (TGS) initially observed in plants.

The 5 prime nucleotide of the siRNA is unpaired and the base and phosphate interact with residues of the middle domain via base-stacking and ionic interactions. Consistent with the seed locator of nucleotides 2-8 in the guide RNA, Argonaute substrate specificity is determined by the sequence of the bound guide RNA. One strand of the siRNA is incorporated in to RISC this is mediated by Dicer and dependent on thermodynamic differences between the 2 ends of the double stranded siRNA. The passenger strand undergoes destruction requiring an active slicing Argonaute. The loading of the guide RNA strand is also reliant on the presence of a 5 prime phosphate for entry in to the RISC loading complex (RLC), and this enhances binding of the guide to Argonaute. The complex once loaded is capable of multiple rounds of target binding and cleavage (Tolia and Joshua-Tor, (2007) Nat Chem Biol, 3, 36-43).

siRNA's

As used herein, the term "RNA" relates to a polymer comprising at least one ribonucleotide monomer.

As used herein, the term "siRNA" relates to an isolated double stranded RNA molecule, comprising a sense strand and an antisense strand, wherein each strand is from about 19 to 25 nucleotides in length, more particularly from about 19 to 23 nucleotides in length, more particularly 19, 20, 21 , 22, 23, 24 or 25 nucleotides in length. Preferably, at least one strand has a 3' overhang of from about 1 to 5 nucleotides, more preferably a 3'

overhang of 2 nucleotides. Alternatively, at least one strand has a 5' overhang of from about 1 to 5 nucleotides, more preferably a 3' overhang of 2 nucleotides. Alternatively, least one strand has a 3' overhang and a 5' overhang of from about 1 to 5 nucleotides, more preferably a 3' overhang and a 5' overhang of 2 nucleotides.

The sense and antisense strands of an siRNA may comprise two single stranded RNA molecules. Alternatively, the siRNA may comprise a single RNA molecule in which two sections are complimentary and base pair by hydrogen bonding to give a "hair pin" conformation.

As used herein, the term "sense strand" or " sense RNA strand" relates to a nucleic acid sequence which identical to a target mRNA sequence. As used herein, the term "antisense strand" or "antisense RNA strand" relates to a nucleic acid molecule which is complementary to a sense RNA strand, i.e. may anneal to a sense strand by complementary base-pairing.

As used herein the term "complementary" or "complement" refers to the ability of a first nucleic acid molecule, i.e. an RNA, to hydrogen bond to with another nucleic acid, either via conventional nucleotide complementarity, i.e. Watson-Crick base pairing or by an alternative non traditional pairing. The percentage complementarity is a measure of the number of contiguous nucleic acid residues in a first nucleic acid molecule that are capable of hydrogen bonding with a second nucleic acid molecule.

As used herein the term "RNA target", "target mRNA" or "target DNA" refers to the mRNA sequence or DNA sequence to which the sense strand of an siRNA is identical. Preferably the RNA target is a contiguous stretch of between 19 and 25 nucleotides of an mRNA. Alternatively, the RNA target is a contiguous stretch of between 19 and 25 nucleotides of an DNA.

Preferably, the target mRNA, to which the siRNA's of the present application are directed, encodes POMC (Proopiomelanocortin) or a portion thereof. Preferably, the target mRNA comprises a transcribed POMC gene or a portion thereof. The target mRNA may also include a mutant, homologue or alternative splice form of POMC mRNA.

The POMC gene is expressed in the anterior and intermediate lobes of the pituitary gland (in corticotrope cells), the hypothalamus (3000 neurones in the arcuate nucleus, dorsomedial hypothalamus and the brainstem) and the skin (in melanocytes) (Chretien et al., (1979) Can J Biochem, 57, 1 1 1 1 -21 ). The primary product of the POMC gene is a 285 amino acid precursor peptide that can undergo differential processing to yield at least 8 peptides dependent upon the location of stimulus i.e. POMC is polycistronic, as shown in Figure 2.

POMC is processed by the convertases PC1/3 and generates pro-adrenocorticotrophin and β-lipotrophin. Pro-ACTH in turn generates a joining peptide (JP-not shown on the figure), N-terminal pro-opiocortin (NPOC-not shown on the figure) and ACTH (1 -39). This ACTH is cleaved to generate ACTH 1 -17 and corticotrophin like intermediate lobe peptide by PC2. A number of post translational modifications result in ACTH 1 -17 which is cleaved to α-melanocyte stimulating hormone (MSH). N-POC is processed in to v- MSH in humans, but not in rodents. γ-lipotrophin is processed to β-MSH. β-lipotrophin also generates lipotrophin and endorphin which in turn are cleaved to β-MSH , y- endorphin and α-endorphin (Pritchard LE and White A (2007) Endocrinology. 2007 Sep;148(9):4201 -7).

The inventors have surprisingly found that siRNA's directed to the exonic coding sequence of POMC are capable of mediating long duration post-transcriptional silencing of the POMC gene.

Preferably, the POMC gene is a mammalian POMC gene. More preferably, the POMC gene is a human POMC gene (Chang AC et al (1980) Proc Natl Acad Sci U S A, 77,

4890-4, Cochet et al (1982) Nature 297, 335 - 339). Alternatively, the POMC gene is a feline, canine, equine or proboscine POMC gene. As used herein, the term "gene" refers to nucleic acid molecules which include an open reading frame encoding protein, and can further include non-coding regulatory sequences, such as promoter regions and introns.

In humans the POMC gene is assigned to chromosome 2p23.3 and originally isolated in 1980 (Chang et al (1980) Proc Natl Acad Sci U S A, 77, 4890-4). In mouse the POMC gene is located on chromosome 12 and was sequenced in 1982 (Uhler and Herbert, (1983) J Biol Chem, 258, 257-61 ).

In a preferred embodiment, the target mRNA is specific to POMC. More preferably the target mRNA comprises a nucleic acid sequence as illustrated in SEQ ID NO: 1 , 2 or 3 of table 1 below. Preferably, the target mRNA is at least 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, 99 or 100% identical to the target mRNA of SEQ ID NO:1 , 2 or 3.

Table 1

The inventors have surprisingly found that siRNA's directed to the POMC promoter region are capable of mediating transcriptional silencing of the POMC gene.

Alternatively, the target DNA to which the siRNA's of the present application are directed, are located at the POMC promoter region. More preferable, the target mRNA is directed to the transcription binding sites of the factors: Nurr 77, Ptx 1 , Tpit and NeuroDI (NCBI accession (NM_008895), Lamolet B ef a/ (2001 ) Cell. Mar 23;104(6):849-59, Poulin G ef a/ (1997) MoI Cell Biol. Nov;17(1 1 ):6673-82, Philips A et al (1997) MoI Cell Biol. Oct;17(10):5946-51 ,Lamonerie T ef a/ (1996) Genes Dev. May 15;10(10):1284-95).

In a preferred embodiment, the target DNA is specific to a POMC promoter region. More preferably the target mRNA comprises a nucleic acid sequence as illustrated in SEQ ID NO: 10, 1 1 , 12, 13, 14 or 15 of table 2 below. Preferably, the target mRNA is at least 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, 99 or 100% identical to the target mRNA of SEQ ID NO:10, 1 1 , 12, 13, 14 or 15.

Table 2

Alternatively, the target mRNA, to which the siRNA's of the present application are directed, encodes Tpit or a portion thereof. Preferably, the target mRNA comprises a transcribed Tpit gene or a portion thereof. The target mRNA may also include a mutant, homologue or alternative splice form of Tpit mRNA.

The Tpit gene is a pituitary restricted transcription factor, present only in POMC expressing cells and is a partner of Pitxi on the POMC promoter (Lamolet B et al (2001 ) Cell, 104:849-859). Tpit mutations have been identified in patients with ATCH deficiency, suggesting a role for Tpit in POMC regulation (Pulichino AM et al (2003) Genes Dev 17:71 1 -716). Mutations in the human and mouse Tpit genes are known to cause a neonatal onset form of ACTH deficiency (Vallette-Kasic S et al (2005) J Clin Endocrinol Metab 90:1323-1331 ). In human pituitary Tpit expression was restricted to the nucleus of both adenomatous and normal human corticotroph cells and is a marker for POMC-expressing pituitary cells (Vallette-Kasic S et al (2005) J Clin Endocrinol Metab 90:1323-1331 ).

Tpit plays a crucial role in corticotroph development and in the corticotrope-specific expression of POMC, and interacts with other factors to drive POMC expression (Fig 8).

Preferably, the Tpit gene is a mammalian Tpit gene. More preferably, the Tpit gene is a human Tpit gene (NCBI accession no. NG_008244). Alternatively, the Tpit gene is a feline, canine, equine or proboscine Tpit gene.

In a preferred embodiment, the target mRNA is specific to Tpit. More preferably the target mRNA comprises a nucleic acid sequence as illustrated in SEQ ID NO: 28 or 29 of table 5 below. Preferably, the target mRNA is at least 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, 99 or 100% identical to the target mRNA of SEQ ID NO:28 or 29.

Table 5

The sense strand of the siRNA must be sufficiently complementary to the target mRNA to mediate RNAi. Preferably, the sense strand is at least 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, 99 or 100% identical to the target mRNA. Preferably, the percentage identity of the siRNA to the target mRNA is calculated over double stranded portion of the siRNA. Alternatively, the percentage identity of the siRNA to the target mRNA may be calculated including any 3' or 5' overhang. The percentage identity between two sequences may be determined using alignment algorithms known in the art, see for example the Needleman algorithm (Needleman et al. (1970) J. MoI. Biol. 48:444-453) which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com) or algorithm of Meyers (Meyers et al. (1989) CABIOS 4:1 1 -17) which has been incorporated into the ALIGN program (version 2.0) or for example, the Smith-Waterman algorithm as incorporated in the BESTFIT software program using default parameters.

The sense strand of the siRNA may contain insertions, deletions or point mutations when compared to the target mRNA sequence. Preferably, the double stranded portion of the sense strand of the siRNA is identical to the target mRNA.

Preferably, the siRNA sense strand comprises a nucleotide sequence which is at least at least 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, 99 or 100% identical to the nucleotide sequence as represented in SEQ ID NO:4, 5, 6, as illustrated in table 3 below or SEQ ID NO:16, 17, 18, 19, 20 and 21 as illustrated in table 4 below, or SEQ ID NO:30 and 32.

Preferably, the siRNA sense strand has the nucleotide sequence as represented in SEQ ID N0:4, 5, 6, 16, 17, 18, 19, 20, 21 , 30 and 32.

Table 3 POMC exonic siRNA's

siRNA 1 (SEQ ID NO:4 and SEQ ID NO:7): rat and mouse homology siRNA 2 (SEQ ID N0:5 and 8): rat, mouse and man homology siRNA 3 (SEQ ID N0:6 and 9): rat, mouse and man homology

Table 4 POMC promoter siRNA's

POMCPRO1 (SEQ ID NO:16 and 22) rat and mouse homology: Nurr 77 P0MCPR02 (SEQ ID N0:17 and 23) mouse only: Neuro D1 P0MCPR03 (SEQ ID N0:18 and 24) mouse only: Ptx 1 and Tpit P0MCPR04 (SEQ ID N0:19 and 25) mouse only P0MCPR05 (SEQ ID NO:20 and 26) mouse only P0MCPR06 (SEQ ID N0:21 and 27) mouse only: Nurr 77

Table 6 Tpit siRNA's

In one embodiment, the siRNA sense strand is identical to an exonic region of the POMC gene and and comprises SEQ ID N0:4 and the antisense strand comprises SEQ ID N0:7. Alternatively, the sense strand comprises SEQ ID N0:5 and the antisense strand comprises SEQ ID N0:8. Alternatively, the sense strand comprises SEQ ID N0:6 and the antisense strand comprises SEQ ID N0:9.

Alternatively, in a further embodiment, the siRNA sense strand is complementary to the POMC promoter region and comprises SEQ ID N0:16 and the antisense strand comprises SEQ ID NO:22. Alternatively, the sense strand comprises SEQ ID N0:17 and the antisense strand comprises SEQ ID NO:23. Alternatively, the sense strand comprises SEQ ID N0:18 and the antisense strand comprises SEQ ID NO:24. Alternatively, the sense strand comprises SEQ ID N0:19 and the antisense strand comprises SEQ ID NO:25. Alternatively, the sense strand comprises SEQ ID NO:20 and the antisense strand comprises SEQ ID NO:26. Alternatively, the sense strand comprises SEQ ID N0:21 and the antisense strand comprises SEQ ID NO:27. Preferably, the siRNA sense strand is complementary to the Nurr 77 transcription binding site, more preferably the siRNA sense strand comprises SEQ ID N0:21 and the antisense strand comprises SEQ ID NO:27. Alternatively, the siRNA strand is complementary to the Nurr 77 transcription binding site and the siRNA sense strand comprises SEQ ID N0:16 and the antisense strand comprises SEQ ID NO:22.

Alternatively, the siRNA sense strand is complementary to the Neuro D1 transcription binding site, more preferably the siRNA sense strand comprises SEQ ID N0:17 and the antisense strand comprises SEQ ID NO:23. In one embodiment, the siRNA sense strand is complementary to the Ptx 1 transcription binding site, more preferably the siRNA sense strand comprises SEQ ID N0:18 and the antisense strand comprises SEQ ID NO:24.

Alternatively, in a further embodiment, the siRNA sense strand is complementary to an exonic region of the Tpit gene and comprises SEQ ID NO:30 and the antisense strand comprises SEQ ID N0:31 . Alternatively, the sense strand comprises SEQ ID NO:32 and the antisense strand comprises SEQ ID NO:33.

Figure 3 illustrates the mouse POMC gene region with positions of each of the POMC exonic siRNA's and each of the POMC promoter siRNAs of the invention aligned.

Modification of the siRNA's of the invention may be used to improve nuclease resistance and cellular uptake of the siRNA molecules. Modification may comprise modification of the phosphate backbone. For example, the siRNA may include phosphate backbone modifications, such as phosphorothioate and 2'-fluoropyrimidine RNA backbone modifications (Harborth J et al. Antisense Nucleic Acid Drug Dev. 2003;13:83-105), the introduction of 2'-deoxy-2'-fluorouridine (Braasch D. A et al, RNA interference in mammalian cells by chemically-modified RNA. Biochemistry. 2003;42:7967-7975), 2'-O- methylation or 2'-O-allylation (Amarzguioui M et al, Nucleic Acids Res. 2003;31 :589- 595). In addition, LNA (Locked Nucleic Acid) modification, for example modification using an LAN containing a methylene bridge connecting the 2'-oxygen with the 4'-carbon of the ribose ring (Elmen J et al, Nucleic Acids Res. 2005; 33(1 ): 439-447) may be used.

The siRNA's of the invention may also include modifications of the sugar group (Choung S et al (2006) Biochem Biophys Res Commun. Apr 14;342(3):919-27. The siRNA's may comprise combinations of the aforementioned modifications.

Methods for the synthesis of siRNA's are well known in the art (Birmingham, A. et al (2006) Nat Methods, 3, 199-204 and Jackson, A et al, (2006) Rna, 12, 1 197-205). Systems for both transient and permanent expression of siRNA have been developed which may be incorporated into the expression vectors, such as Ad vector

(Brummelkamp, Bernards et al. Science. 2002 Apr 19;296(5567):550-3. Epub 2002 Mar

21 ). In one embodiment the invention provides an expression vector comprising a promoter and a polynucleotide sequence that encodes an siRNA of the invention.

Preferably, the polynucleotide encodes a nucleic acid sequence of any one of SEQ ID NO's: 4 to 9, or SEQ ID NO's: 16 to 26. In one embodiment the vector is a viral vector. Alternatively the vector is a non-viral vector.

Uses

siRNA's of the invention may be used to inhibit, the expression or activity of POMC gene or polypeptide encoded by the POMC gene, i.e. ACTH or β-lipotropin. As used herein, the terms "inhibit", "down-regulate" and "reduce" refer to an alteration of the level of gene expression or the level of RNA molecules encoding POMC, or one or more protein subunits thereof, such that the aforementioned expression, level, or activity of the POMC nucleic acid or polypeptide is than less that observed in the absence of the modulation.

The inhibition can be performed in vitro (e.g., by culturing the cell with the siRNA) or, alternatively, in vivo (e.g, by administering the siRNA to a subject).

In vitro inhibition provides a method of reducing POMC activity or expression in a cell, comprising contact a cell with an siRNA of the invention. The cell is contacted under conditions wherein modulation of siRNA mediated inhibition of POMC may occur. Preferably, said contact comprises introducing the siRNA into the cell.

In vitro inhibition provides methods for treating cells in culture. Preferably the cells are corticotrope cells, neurones in the arcuate nucleus, dorsomedial hypothalamus and the brainstem, or melanocytes. Alternatively the cell is isolated from a cancerous tissue. More preferably the cell is isolated from pituitary adenoma, such as a corticotrope tumour. Alternatively the cell is isolated from an ACTH-producing tumour, such as small cell lung cancer, a thymoma, a pancreatic islet cell tumour, or a medullary carcinoma of the thyroid or an adrenal cancer, such as an adrenocortical carcinoma, or adrenal adenomas, including phaeochromocytomas. Preferably the cells are mammalian cells. More preferably the cells are human.

In vitro inhibition methods, including cell culture, may be carried out in any suitable vessel. Preferably the vessel is selected from the group consisting of: a petri-dish; cell

culture bottle or flask; multiwell plate. "Vessel" is construed as any means suitable to contain a cell culture.

In vivo inhibition provides methods of treating a subject having a disease or disorder, or at risk of having a disease or disorder, associated the expression or activity of a POMC nucleic acid or polypeptide derived there from, for example a disease or disorder associated with the activity of ACTH. Accordingly, the invention provides both prophylactic and therapeutic methods. The siRNA's disclosed herein may be used alone or in combination with one another in prophylactic and therapeutic methods.

Diseases and disorders associated with the activity of ACTH include diseases and disorders associated with the expression of Cortisol.

Accordingly, the invention provides a method of treatment of a subject, preferably a mammal, more preferably a human, comprising administering to said subject an siRNA according to the invention. The invention also provides the use of an siRNA according to the invention for the treatment of diseases or disorders associated with the expression or activity of a POMC nucleic acid or an ACTH polypeptide. The invention also provides siRNA's according to the invention for the treatment of diseases or disorders associated with the expression or activity of a POMC nucleic acid or an ACTH polypeptide.

Diseases or disorders associated with the expression or activity of a POMC nucleic acid or an ACTH polypeptide derived therefrom include, but are not limited to, Cushing's disease, Cushing's syndrome and impaired immune response due to hypersecretion of corticosteroid. In addition, siRNA's which inhibit POMC may be used to exert an effect upon the hypothalamus-pituitary-adrenal axis for initiation of pre-term labour, and for congenital adrenal hyperplasia where untreated there are high levels of pituitary POMC expression and high circulating levels of ACTH. If delivered to the CNS antagonism of POMC will be of benefit for the treatment of anorexic syndromes, including those associated with cancer cachexia and anorexia nervosa.

The siRNA's of the present invention may also be used to treat congenital adrenal hyperplasia, a condition in which an enzyme defect inherited in the steroid synthetic pathway in the adrenal gland results in lower levels of circulating Cortisol and hence causes ACTH levels to rise. The higher levels of ACTH drive the adrenal to make pre- cursor steroids that cause hypertension, premature puberty, hirsutism, and ultimately short stature in the child, and infertility in the adult. Treatment is with glucocorticoids, but

be effective the levels of ACTH have to be inhibited and the side effects of existing treatments resemble Cushing's syndrome. The siRNAs of the present invention could be used to lower ACTH levels to control the androgenic and mineralocorticoid effects associated with high levels of ACTH, which would allow the condition to be treated with lower doses of glucocorticoid. The siRNA's of the present invention could be used alone or in combination with glucocorticoid in the treatment of congenital adrenal hyperplasia, for example the siRNA's may be administered, or prepared for administration, in a combined dosing regimen with with glucocorticoid.

The siRNA's of the present invention may also be used to treat small cell lung cancer (SCLC). In approx 50% of SCLC the POMC gene is activated giving rise to an mRNA product that is similar in length to that seen in the pituitary, and it is likely that Cushing's syndrome is frequently not recognized. The CpG island promoter of the human proopiomelanocortin gene is methylated in nonexpressing normal tissue and tumors and represses expression (Newell-Price J et al. (2001 ) MoI Endocrinol 15(2):338-48). Moreover, the peptide products from POMC cleavage may confer a cellular survival advantage in an autocrine or paracrine fashion. Beta-endorphin and neurotensin stimulate in vitro clonal growth of human SCLC cells (Davis TP et al (1989) Eur J Pharmacol 161 (2-3):283-5. Accordingly administration of any of the siRNA's of the present invention would be of therapeutic benefit.

The siRNA's of the invention can be incorporated into pharmaceutical/ therapeutic compositions, suitable for administration. Such compositions typically comprise the siRNA and a pharmaceutically acceptable carrier. The term "pharmaceutically- acceptable carrier" as used herein, refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.

In the present case, the targeted gene, POMC, has limited tissue expression, thereby reducing the possibility of any off-target inhibition. The restricted expression of the Tpit gene also makes it an attractive target for siRNA. This specificity is advantageous for an siRNA therapy. In addition, the pituitary is outside the blood brain barrier and has a dense blood supply, providing an accessible administration route.

Methods of delivery for nucleic acids are well known in the art (Hawley-Nelson P and

Ciccarone V (2003) Curr Protoc Cell Biol. Chapter 20:Unit20.6.PMID: 18228430,Gulick T (2003) Curr Protoc Cell Biol. Chapter 20:Unit20.4.PMID: 18228428, Kingston RE et al (2003) Chapter 20:Unit20.3.PMID: 18228427). The conjugation of siRNA, with lipophilic derivatives of cholesterol, lithocholic acid, or lauric acid has been shown to enhance cellular uptake. . In addition, cholesterol conjugation with the 3' end of the sense strand by means of a pyrrolidine linker has prolonged in vivo half life to at least 95 minutes(Soutschek et al (2004) Nature, 432, 173-8). Liposomal encapsulation may also be used, for example encapsulation by iontophoresis. The stable nucleic acid lipid particle (SNALP) stabilized the siRNA used to target HBV RNA in a mouse model of HBV replication (Morrissey et al (2005) Nat Biotechnol, 23, 1002-7.). This consists of a lipid bilayer containing a mixture of cationic and fusogenic lipid that enables the cellular uptake and endosomal release of the particle's nucleic acid. It is also coated with a diffusible polyethylene glycol lipid conjugate to neutralize the exterior and stabiles the particle. This also prevents rapid systemic clearance.

An siRNA coding sequence may be linked to the c-terminus of the heavy chain Fab fragment in order to deliver siRNA's to specific cell targets (Song et al (2005) Nat Biotechnol, 23, 709-17). siRNA's may be delivered utilising chemically modified liposomes conjugated to monocloncal antibodies (Kumar et al (2007) Nature, 448, 39- 43). In addition RVG-Arg-siRNA (rabies virus in a glycoprotein envelope fused with a peptide conjugated to the siRNA) may be used as a delivery construct (Cantin and Rossi (2007) Nature, 448, 33-4).

Viral vectors, such as retroviruses, may also be used as a delivery method (Ralph et al (2005) Nat Med, 1 1 , 429-33) as may adenovirus recombinant derivatives.

In addition, the invention provides a method of administering to a subject an siRNA according to the invention comprising contacting the subject with an siRNA, under conditions suitable for administration, i.e. in the presence of a delivery agent such as a lipid, liposome, phospholipid or liposome.

When administered, the pharmaceutical compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.

The compositions of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be topical, oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal, intranasal, intracerebral or epidural and compositions of the invention are prepared accordingly.

The compositions of the invention are administered in effective amounts. An "effective amount" is the amount of a composition that alone, or together with further doses, produces the desired response.

The compositions used in the foregoing methods preferably are sterile and contain an effective amount of the active ingredient for producing the desired response in a unit of weight or volume suitable for administration to a patient. The response can, for example, be measured by measuring the physiological effects of the composition, such as decrease of disease symptoms etc. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response.

siRNA's can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. ScL USA 91 :3054-3057).

Examples

Example 1 : POMC and POMC promoter siRNA's

1.1 Preparation of siRNAs

siRNAs were designed siRNAs to avoid homology to any other areas of the genome, and to regions of exact sequence homology in the mouse, rat and human genomes, as assessed by NCBI BLAST. Thus the sequences were the same for the coding sequence of human POMC and mouse pome. Both POMC exonic and promoter sequences were targeted. Chemically synthesised pre-annealed lyophilised siRNAs (Ambion, Applied Biosystems, Warrington UK, and Dharmacon, Perbio, Northumberland, UK) were reconstituted in filtered de-ionised distilled water prior to transfection. The

Nucleotide RNAs were chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.

The siRNA's used are illustrated in table 3 and table 4.

1.2 Cell Culture and Transfections

AtT20v D16:16 cells were obtained from the European Collection of Cell Cultures. Cells were maintained in DMEM + glutamax, 10% fetal calf serum, and 1 % penicillin and streptomycin, and cultured at 37° in 5% carbon dioxide.

Transfections were optimized using fluorescently labelled siRNAs with a FITC fluorophor (Block-iT™ Fluorescent oligos) and Lipofectamine 2000 (both Invitrogen, Loughborough, Leicestershire, UK). For transfections reagents were prepared in antibiotic free OptiMEM I reduced serum media (Invitrogen, Fisher LTD Loughborough, Leicestershire, UK) as recommended by the manufacturer. Following transfection culture was maintained in antibiotic-free conditions. Fluorescent activated cell sorting (FACS) analysis and confocal microscopy were used to assess transfection efficiency. Trypan blue and cell counting were used to confirm cell viability following transfection, and found to be the same for all treatments. Transfection efficiency was >95% (data not shown).

For the exonic and promoter targets a final concentration of 3OnM of siRNA was used in all transfections.

1.3 RNA Extraction and Synthesis of cDNA by Reverse Transcription

Total RNA was extracted immediately from harvested cells, or cells snap frozen in liquid nitrogen and stored at -80 ⁰ C until processing. The RNeasy kit (Qiagen, Crawley, Uk) was used in accordance with the manufacturers instructions. RNA was treated twice with DNAse 1 (DNA free protocol, Ambion, Warrington, UK) prior to quantification in order to eliminate any contamination with genomic DNA. Quantification of RNA was performed using Ribogreen® chemistry (Invitrogen, Paisly, UK) and plate reader settings at excitation 485 nm and emission 525 nm according to manufacturers instructions. The RETRO-script® kit (Ambion) was used to make cDNA in accordance with manufacturers instructions. 2 μg of input RNA was used to make cDNA using random decamers as first strand primers (RETRO-script® kit Ambion, Applied Biosystems, Warrington UK) in

accordance with manufacturers instructions.

1.4 Analysis of POMC mRNA by quantitative RT-QPCR

RT-qPCR was carried out in MicroAmp® (Alpha Labs, Eastleigh, Hampshire, UK) Optical 96 well plates sealed with optically clear caps. Components of the reaction were made up to a final volume of 25 μl_ using the SYBR Green Core Reagent Kit and The Stratagene M _x 3000 real time thermocycler (Stratagene, Europe, Amsterdam, The Netherlands). Standard curve efficiencies of 95-1 10% with Rsq values of > 0.98 were generated. cDNA for the standard curve. Standards were generated by serially diluting the stock cDNA in ddH ₂ O. The same standards were included on every plate and amplified in triplicate. Total cDNA input amounts for the standards ranged from 100ng to 0.41 ng in five 1 in 3 dilutions. These dilutions were found to empirically to produce optimum standard curves with all samples amplifying within the standards. A negative control PCR reaction with nuclease free water was added in place of the sample cDNA, as was a no RT control from the same RT step.

Primer Sequences

MPOMCmRNA FORWARD For qPCR 5'GAG AGC AAC CTG CTG GCT TGC3', MPOMCmRNA REVERSE For qPCR 5'AGG TCA TGA AGC CAC CGT AAC G3',

MBETA-ACTIN mRNA FORWARD For qPCR 5'CTC GGT CAG GAT CCT CAT GAG G

3',

MBETA-ACTINmRNA REVERSE For qPCR 5'CTC GGT CAG GAT CCT CAT GAG G3'.

1.5 Analysis of ACTH in culture media

Media samples were stored at -80 ⁰ C until analysis and diluted 1 in 500 in sodium phosphate buffer 50 mM and analyzed using an immunometric assay (Immulite 2000, with an assay range of 0.04 to 1250 pmol, intra assay coefficient of variation of 2.4-8.5 %. All samples were run in the same assay in duplicate. All media samples were assessed from the same volume and cell number in treated cells and controls.

1.6 DNA Methylation Analysis: Bisulfite Sequencing Genomic DNA Extraction

DNA was extracted from the snap frozen pelleted cells using the DNeasy kit (Qiagen, Crawley, West Sussex ,UK ) in accordance with the manufacturers instructions and quantified using the Nanodrop ND-1000 spectrophotometer (Fisher Ringmer, East Sussex, UK). Bisulfite treatment was performed as previously described (Newell-Price J, et al (2001 ) MoI Endocrinol 15:338-48). Primers for amplification of converted DNA of POMC promoter were as follows: MBSPOMC FORWARD 5'CGG AAT TCC GTT TTT GTT TAG TTT TAA GTG GAG3' MBSPOMC REVERSE 5'GCT CTA GAC AAA ACT AAA ACA CCC TTA CCT ATC 3'. The PCR product was cloned into the TOPO TA vector (Invitrogen, Loughborough, Leicestershire, UK ) and sequenced with T7 primers, using an ABI Big Dye Terminator kit and resolved on an ABI 3100 platform (both ABI, Epsom, UK).

1.7 Statistical analysis

Each experiment unless stated was performed in triplicate. Data is presented as means with standard errors. GraphPad Prism® was used to generate graphical data. Statistical significance was generated using SPSS 14.0®, using an ANOVA. Cell Quest Pro® software was used for the analysis of FACS data on the MAC and confocal microscopy pictures were taken using Simple PCI software®.

1.8 Results

To determine the effectiveness of RNAi in the knock down of pome mRNA and ACTH in culture media of AtT20 cells, we designed and chemically synthesized siRNAs targeted to the exons 2 and 3 of POMC/pomc, regions of (Figure 2). Sites were selected with homology to mouse, rat and man, but with no homology to other areas of the genomes. Three siRNAs, named siRNA-1 to 3, were used. Transfection efficiency as assessed by FACS analysis was >95%, (data not shown). A final concentration of 3OnM of siRNA was used in all transfections.

Following a single transfection of each siRNA, 1 -3, the effect on expression of pome mRNA and ACTH secreted into the cell culture media was assessed. At 24 hours use of siRNA-1 caused silencing of pome expression by 86 % (p=0.001 , Figure 4a). Similarly, siRNA-2 silenced POMC expression by 89 % (p=0.001 , Figure 4b). Finally, siRNA-3 silenced POMC expression by 95 % (p=0.001 , Figure 4c). At 48 hours Beta-actin mRNA was found to be stable (Figure 4d).

Since siRNA-3 had the most potent effect on pome expression a longer time course was explored. After a single transfection with siRNA-3, pome mRNA was significantly reduced at 48h (p <0.05), 12Oh (p<0.005) and 24Oh (p<0.005) (Figure 5b). Importantly, ACTH concentration in the media at the same time points were also reduced at 240 hours by 85 % (p<0.001 ), relative to controls of cells alone or vehicle alone (Figure 5a). Thus a single application of siRNA-3 resulted in highly effective gene silencing and highly significant reduction in secreted protein expression for at least 10 days

1.9 siRNAs directed to the promoter region of POMC exhibit potent knock down of POMC expression and significantly lower levels of secreted ACTH .

To determine if siRNAs targeting the promoter could induce knockdown of POMC we designed 6 siRNAs to the pituitary promoter region of POMC. We specifically targeted regions including response elements for the following positively-acting transcription factors, nurr 77, neuro D1 , ptx1 and tpit, since DNA methylation may induce interference of transcription factor binding (Newell-Price J et al (2000) Trends Endocrinol Metab 1 1 :142-8). After transfection of all six siRNAs at a final combined concentration of 3OnM, cells and media were harvested at 96 and 120 hours. At 96 hours POMC expression was reduced by 96 % (p<0.001 , Figure 6a). At 120 hours POMC expression was reduced by 50 % (p=0.06 and p<0.0005) (Figure 6b). Thus, siRNAs designed to induce transcriptional gene silencing were initially as potent as targeting the exonic region. The effect on POMC expression appears to start to reverse by 120 hours.

1.10 siRNAs directed to the promoter region of POMC do not induce promoter methylation in AtT20 Cells

Since RNAi directed against promoter regions has been shown to inhibit gene expression and in some genes cause de novo DNA methylation (Morris KV et al (2004) Science 305:1289-92), we used bisulfite sequencing to examine the possibility that siRNAs directed against the POMC promoter had exerting their inhibitory effects on expression by causing de-novo DNA methylation we used bisulfite sequencing assessed to assess this region. Bisulfite sequencing revealed that siRNAs targeted to the POMC pituitary promoter did not induce de-novo DNA methylation of the promoter and all PO/WC-expressing and ACTH-secreting cells remained free of methylation at all

time points (Figure 7a). In sharp contrast 3T3 cells show high levels of methylation, consistent with their POMC non-expressing state (Figure 7b).

These results show that it is possible to cause potent long-duration knockdown of pome expression, and, crucially, reduction in secreted ACTH levels in a cell culture model of

Cushing's disease. Accordingly the identified siRNAs would be particularly useful for treatment of man since in the majority of patients with Cushing's disease control of hormonal hypersecretion is a clinical priority, particularly for those 40% of patients in whom no tumour is visible on MRI (Newell-Price J et al (2006) Lancet 367:1605-17, Newell-Price J et a/ (1998) Endocr Rev 19:647-72).

Despite only one transfection being performed, siRNA-3 (with the greatest activity against exonic sequences) maintained potent knockdown for up to 10 days. It is predicted that repeated dosing would be even more effective.

The results show that targeting the promoter region causes knockdown of a similar magnitude and the effect on ACTH secretion is maintained for at least 5 days, although no evidence of induction of DNA methylation has been found. Whilst plants exhibit DNA methylation of the promoters of genes whose mRNA was degraded by RNAi (Mette MF et al (2000) Embo J 19:5194-201 ), the existence of such a system in mammalian cells has been a controversial. Some groups have reported promoter silencing by siRNAs in the presence of DNA methylation (Morris KV et al (2004) Science 305:1289-92), however more recent observations indicate that dsRNAs may cause promoter silencing in the absence of DNA methylation (Ting AH et al (2005) Nat Genet 37:906-10). The present data is in keeping with these latter data.

The clinical potential of RNA interference is enormous, and has clear applicability to the field of endocrinology where highly-tissue restricted hypersecretion is a common pathology.

2. Tpit siRNA's

2.1 Preparation of TpitsiRNAs

TpitRNAs were prepared as described in section 1 .1 -1.4 above using the following primers:

Table 7:

PCR Primer sequences

The siRNA's used are illustrated in table 6. Target sites for TpitsiRNAs on mouse and human Tpit are illustrated in figure 9.

2.2 Effect of siRNAs on Tpit expression at 24 hours

The two test TpitsiRNAs and a negative control siRNA (siRNA designed to rat growth hormone sequence) were each transfected into AtT20 cells at a concentration of 30 nM in duplicate. Cells were incubated for 24 hours post transfection. A 62.0% knockdown in Tpit mRNA levels was observed in TpitsiRNA 1 samples relative to No siRNA samples (p < 0.001 ) as illustrated in figure 10a. The knockdown in Tpit mRNA levels in TpitsiRNA 2 samples relative to No siRNA samples was 75.4% (p < 0.001 ) as illustrated in figure 10b.

2.3 Effect of siRNAs on POMC expression at 24 hours

Using cDNA from the same stock used for qPCR with Tpit primers, a qPCR was run with POMC primers to determine the effect of TpitsiRNA 1 and TpitsiRNA 2 on POMC mRNA levels in cells 24 hours post-transfection. A 33.7% knockdown in POMC mRNA levels was observed in TpitsiRNA 1 samples relative to No siRNA samples (p = 0.032) as illustrated in figure 1 1 a. The knockdown in Tpit mRNA levels in TpitsiRNA 2 samples relative to No siRNA samples was 54.3% (p = 0.002) as illustrated in figure 1 1 b.

2.4 Effect of siRNAs on beta Actin expression at 24 hours

It was considered that the low levels of Tpit and POMC mRNA in samples transfected with Negative Control siRNA could be due to low levels of total mRNA in these samples. In order to study relative levels of total mRNA in transfected and control samples and to check for non-specific effects as a result of siRNA transfection, expression of the house

keeping gene beta Actin was assessed. No significant differences in beta Actin levels were observed between no siRNA and samples transfected with TpitsiRNA 1 (p = 0.621 ), TpitsiRNA 2 (p = 0.313) or Negative control siRNA (p = 0.720), as illustrated in figure 12.

2.5 Effect of siRNAs on Tpit expression at 72 hours

The two test TpitsiRNAs and a negative control siRNA were each transfected into AtT20 cells at a concentration of 30 nM in duplicate. Cells were incubated for 72 hours post transfection. A 39.8% knockdown in Tpit mRNA levels was observed in TpitsiRNA 1 samples relative to No siRNA samples (p < 0.001 ) as illustrated in figure 13a. The knockdown in Tpit mRNA levels in TpitsiRNA 2 samples relative to No siRNA samples was 62.3 % (p < 0.001 ) as illustrated in figure 13b. No significant difference in Tpit mRNA levels was observed in Negative control siRNA samples relative to No siRNA samples (p = 0.561 ).

2.6 Effect of siRNAs on POMC expression at 72 hours

Using cDNA from the same stock used for qPCR with Tpit primers, a qPCR was run with POMC primers to determine the effect of TpitsiRNA 1 and TpitsiRNA 2 on POMC mRNA levels in cells 72 hours post-transfection. A 55.2% knockdown in POMC mRNA levels was observed in TpitsiRNA 1 samples relative to No siRNA samples (p < 0.001 ) as illustrated in figure 14a. The knockdown in Tpit mRNA levels in TpitsiRNA 2 samples relative to No siRNA samples was 71.67% (p < 0.001 ) as illustrated in figure 14b. No significant difference in POMC mRNA levels was observed in Negative control siRNA samples relative to No siRNA samples (p = 0.076).

2.7 Western blotting with protein from transfected cells

Cells cultured in 6-well plates were transfected with a TpitsiRNA or control solution. Complexes contained Lipofectamine 2000 and either TpitsiRNA 1 , TpitsiRNA 2, a Negative control siRNA or no siRNA. Fresh media was changed 24 hours post- transfection, and cells were cultured for a total of 72 hours post-transfection. Cells were washed, dissociated, pelleted, then resuspended in PBS to wash. Cells were then pelleted again, resuspended in PBS and counted. In the previous Western blots, 5 x 10 ⁶

cells from a T75 cells had been lysed in 125 μl of lysis buffer. The cell counts of cells cultured in 6-well plates in were lower (Around 1 x 10 ⁶ cells per well), so 30 μl of lysis buffer was used. Optimisation of Western blotting indicated that ideally around 80 μg of protein should be loaded into the SDS PAGE gel, but that 40 μg would suffice. After quantification by Bradford assay, it was calculated that after mixing with 4X SDS gel- loading buffer, 55 μg of protein could be loaded per well. The proteins were resolved through the gel then transferred onto a PVDF membrane. The membrane was blotted with an anti-Tpit antibody solution, then with a goat anti-rabbit antibody solution, and visualised using a POD system.

2.8 Effect of siRNAs on Tpit protein

The bands produced in the lanes containing protein from cells transfected with TpitsiRNA 1 or TpitsiRNA 2 were significantly fainter than those in the lanes containing protein from cells transfected with a control solution, as shown in Figure 15a. This knockdown effect was not present in lanes containing protein from cells transfected with a Negative control siRNA.

2.9 Effect of siRNAs on alpha Tubulin protein

To determine whether the reduction in Tpit protein from cells transfected with TpitsiRNA

1 or TpitsiRNA 2 was caused by the siRNAs or due to lower levels of total protein in these samples, the membrane shown in Figure 15a was re-probed with an anti-alpha Tubulin antibody solution. The results, pictured in Figure 15b, show that the bands produced from samples treated with TpitsiRNA 1 or TpitsiRNA 2 were as strong as those from samples treated with a control solution. This indicates that TpitsiRNA 1 and TpitsiRNA 2 caused a reduction in Tpit protein.

It appears that there was a small bubble in the gel, which can be seen in Lane 2 of figures 15a and 15b (sample TpitsiRNA 2). When the blot was probed with the anti- alpha Tubulin antibody, the band in Lane 2 did not migrate as far as the corresponding band produced using the anti-Tpit antibody. This provides evidence that the re-probing procedure worked correctly, as the band produced with the anti-alpha Tubulin antibody (50 kDa) should be larger than that produced with the anti-Tpit antibody (48 kDa) and should therefore not migrate so far through the gel.

2.10 Effect of siRNAs on ACTH levels at 24 hours

The two test TpitsiRNAs and a negative control siRNA were each transfected into AtT20 cells at a concentration of 30 nM in duplicate. Cells were incubated for 24 hours post transfection. Media samples from each well were diluted 1/500 in 50 mM phosphate buffer and ACTH levels in the samples were analysed by immunometric assay. A 14.6% knockdown in ACTH levels was observed TpitsiRNA 1 samples relative to No siRNA samples (p = 0.016) as illustrated in figure 16a. The knockdown in ACTH levels in

TpitsiRNA 2 samples relative to No siRNA samples was 14.4 % (p = 0.009) as illustrated in figure 16b. ACTH levels in Negative control siRNA samples were 8.3 % higher than in

No siRNA samples (p = 0.016).

2.1 1 Effect of siRNAs on ACTH levels at 72 hours

The two test TpitsiRNAs and a negative control siRNA were each transfected into AtT20 cells at a concentration of 30 nM in duplicate. Cells were incubated for 72 hours post transfection. Media samples from each well were diluted 1/500 in 50 mM phosphate buffer and ACTH levels in the samples were analysed by immunometric assay. A 53.4% knockdown in ACTH levels was observed TpitsiRNA 1 samples relative to No siRNA samples (p < 0.001 ) as illustrated in figure 17a. The knockdown in ACTH levels in TpitsiRNA 2 samples relative to No siRNA samples was 62.9 % (p < 0.001 ) as illustrated in figure 17b. No significant knockdown in ACTH was observed in Negative control siRNA samples relative to No siRNA samples (p = 0.063).

2.12 Results

The results of RT-qPCR analysis indicate that at 24 hours post-transfection, TpitsiRNA 1 reduced Tpit mRNA levels by 62.0 % (p < 0.001 ) and POMC mRNA levels by 33.7%. TpitsiRNA 2 reduced Tpit mRNA levels by 75.4% (p < 0.001 ) and POMC mRNA levels by 54.3 %. Expression of the housekeeping gene beta Actin was constant across all treatment groups.

72 hours post-transfection, TpitsiRNA 1 reduced Tpit mRNA levels by 39.8% (p < 0.001 ) and POMC mRNA levels by 55.2% (p < 0.001 ). TpitsiRNA 2 reduced Tpit mRNA levels by 62.3% and POMC mRNA levels by 71.67%. Evidence was provided for the specificity

of the effects, as the Negative control siRNA caused no significant effect on Tpit (p = 0.561 ) or POMC (p = 0.076) mRNA levels.

The results of the ACTH immunometric assay analysis show that at 24 hours post- transfection, ACTH levels were 14.6% lower in media samples from cells transfected with TpitsiRNA 1 (p = 0.016), and 14.4% lower in samples from cells transfected with

TpitsiRNA 2 (p = 0.009), than in samples from cells treated with transfection reagent alone (mock-transfected cell). ACTH levels were 8.3% higher in media samples from cells transfected with the Negative Control siRNA (p = 0.016) than samples from mock- transfected cells.

3. Ex vivo Culture data

3.1 Primary Human Pituitary Culture

Ethical permission was granted from the National Research Ethics Service, South Yorkshire Research Ethics Committee REC reference: 07/H1310/29 on 5 ^th September 2007 to proceed with primary pituitary culture in consenting patients undergoing transsphenoidal hypophysectomy for pituitary disease. Full authorization for the project was granted by Sheffield Teaching Hospitals NHS trust ref: STH14747. Nineteen pituitary tumours have been processed. Additional authorization from Grampian NHS trust was granted and a materials transfer document drawn up and signed.

For initial processing the tumour specimen was placed on a glass petri dish and washed twice with HBSS. Large blood clots were carefully dissected out and tissue chopped finely with a sterile scalpel. The specimen was then placed in complete media and centrifuged for 5 minutes at 2000rpm. Supernatant was removed and washing process repeated twice. This was followed by enzyme digestion with 5 mis of dispase (Gibco)

50U/ml (0.5g/10ml). The specimen was placed in a sterile container with a magnetic stirrer and placed in the 37^ incubator to enzymatically digest the tissue. Depending on the amount of tissue this was for 30-60 minutes. To check for cell dispersal a small amount was viewed on the haemocytometer, if adequate 5mls of complete media was added, passed through a 19 gauge needle and filtered through sterile gauze. The sample was then centrifuged at 2000rpm for 5 minutes. The supernatant was removed and a further wash step with 5 mis media performed. The cell pellet was then vortexed with 1 ml of media and the cells counted. Generally 12 x10 4 cells were plated in a 24

well plate. In general the cells remained in suspension (see figure 18).

Tumour cells were maintained in MEM plus 1% L-glutamine, 1% penicillin, streptomycin and 0.1 % fungizone, and 10 % FCS were added to complete the media. Cells were cultured in a 37 ⁰ C incubator with 5% carbon dioxide.

For transfection and for the duration of the experiments complete media was used.

3.2 Transfection Techniques and Optimisation

The fluorescently labeled siRNAs (siRNA 2, siRNA3 and TpitsiRNA2), scrambled siRNA with no known homology to any gene sequence was used as controls (Block-iT Fluorescent oligos, Invitrogen, UK) and siRNA's of no known homology, with a FITC fluorophor, were individually transfected in to human pituitary cells with Lipofectamine 2000. Reagents were prepared in OptiMEM I reduced serum media (Invitrogen) as recommended by manufacturers. Controls of no transfection reagent and no flurorescent oligo were included. 500 μL of the complete transfection reagent was added to each well and plates gently rocked back and forth to distribute the complexes through the well evenly. The plate was then protected from light using an aluminium foil wrapping (to avoid degradation of the fluorophor) and returned to the incubator for analysis at 24 hours. The encapsulated fluorescent siRNA labelled with Cy3 was viewed live with nuclei counter stained green with Syto 9.

ACTH levels were measured using a chemoluminescent immunometric assay (Immunolite 2000).

3.3 Results

siRNAs 2,3 and TpitsiRNA2 were transfected using lipofectamine and compared to the siRNA controls (scrambled siRNA controls and siRNA's of no known homology) at a final concentration of 30 nM. The data shown here is up to 48 hours. As the cells were predominantly in suspension, plated cells were spun to remove media then re-seeded in fresh media.

Lipofectamine transfection was associated with a lower rate of transfection compared to stable cell line culture (40% micrograph not shown).

At 24 hours there was a significant knock down of ACTH levels (74% reduction) compared with the negative siRNA controls following treatment with siRNA 2 (p=0.03), whilst siRNA 3, and TpitsiRNA2 reduced ACTH levels by 27% and by 14 % respectively (as illustrated in figure 19). At 48 hours ACTH levels remained significantly reduced following siRNA 2 (68% reduction) (p=0.02), whilst siRNA 3 and TpitsiRNA2 both reduced levels by 23 % (as illustrated in figure 20).

These data demonstrate that a single treatment of siRNAs of the present invention are effective in significantly reducing secreted ACTH levels for up to 48 hours from human corticotroph tumour cells grown ex vivo, even though transfection efficiency was lower than that we had obtained in the AtT20 cell line. ACTH levels were lowered to a greater degree by siRNA 2 compared to siRNA 3. These data confirm the potency of these siRNAs, and further demonstrate effectiveness in human tissue.

4. In vivo animal model data

The experiments were designed to study the efficacy of siRNA 2, siRNA3 and TpitsiRNA2, compared to control siRNAs (Block-iT Fluorescent oligos, Invitrogen, UK), on circulating ACTH levels in an established model of Cushing's disease -

BALB/cANnCRL-Foxn1 nu female mice with AtT20 cells inoculated subcutaneously to form ACTH-secreting tumours (Leung C et al (1982) Pathol Anat 396:303-312; Paez- Pereda M et al (2001 ) 108:1 123-1 131 ; Heaney A et al (2002) Nature Med 8:1281 -1287). Mice were maintained at Charles River Laboratories, Kent , UK ender an handled on a defined protocol and MTA.

4.1 Protocol

BALB/cAn NCrI- Foxni nu Female were transferred to the isolator at 5-6w/o for 1 week acclimatisation. At 6-7w.o all animals underwent subcutaneous inoculation of murine

AtT20 cells (2,000,000cells in 20OuI vol per animal) in to the flank. Animals were observed daily for 3 weeks to monitor how the tumour development was progressing.

After 2 weeks 39 out of 50 animals were observed to have tumours between the sizes of

~ 2mm to ~ 9mm.

Animals were randomly selected for the study groups and individually identified. Those animals which had not developed tumours were distributed evenly through out the groups. It was also ensured that there was an even spread of animals with larger and smaller tumours, throughout the groups. The animals were placed into groups as follows:

Grp 1 . Scrambled siRNA (fluorescent) control plus invivofectamine Grp 2. LNA alone (control) Grp 3. Invivofectamine alone (control) Grp 4. siRNA 2 plus invivofectamine Grp 5. siRNA 3 plus invivofectamine Grp 6. TpitsiRNA2 plus invivofectamine

All animals underwent a submandibular bleed (< 100 ul per animal). Bloods were collected into individual EDTA tubes, cold spun at 4 ⁰ C, and then individually identified plasma samples were snap frozen on dry ice. Plasma samples were stored at -7O ⁰ C. After blood collection had completed, one i.v inoculation was given via the tail vein (100 ul volume) to each animal as per the study groups and inoculation type outlined above. In all cases the total dose of siRNA administered was 125 meg.

Six days after inoculation, the animals were euthanized via CO2 euthanasia and terminally bled via the abdominal vena cava. Each animal underwent bio-specimen collection to retrieve tissues as requested. Animals were either processed completely for snap-frozen whole specimens or formalin fixed whole specimens in groups that required specimens processed in both ways (e.g groups 1 & 3). This was randomised within groups.

Initially all ACTH levels were measured on the mouse plasma. ACTH levels were measured using a chemoluminescent immunometric assay (Immunolite 2000), in duplicate. Due to poor sample volume limiting data points to only few mice in the pre- siRNA dose group analysis was only performed on the samples obtained at euthanisation. Statistical analysis was performed on data from the tumours less than 10mm against the scrambled siRNA + in vivo fectamine control group. Graphpad prism version 5 was used for statistical analysis and to generate graphical data.

4.2 Results

4.2.1 ACTH Data on Day 5 Post siRNA Treatment

Six mice in each group had tumours that were 5mm or less at inoculation and had plasma ACTH data that was available for analysis. At 5 days after inoculation with siRNA 3 in these animals there was a significantly lower level of ACTH (39 %) compared to scrambled siRNA control p=0.039, as illustrated in figure 21.

Data for siRNA 2 was analysed as outlined above (n=6 in each group). At 5 days after inoculation with siRNA 2, these animals showed a shows 16% knockdown of ACTH (p=0.13) as illustrated in figure 22.

There was no significant ACTH-lowering effect observed for TpitsiRNA2.

4.2.2 Tumour Size Data

There was no significant difference between the size of the tumours at the time of siRNA inoculation (p=0.5), but after treatment with siRNA 3 there is a significant difference in the size of the tumours in the two groups at day 5 since there was an increase in six of the tumours in the control group, but only three of those treated with siRNA 3 (p=0.03), as illustrated in figure 23.

These data demonstrate a significantly lower level of plasma ACTH in a mouse model of Cusing's disease at five days after a single inoculation with siRNA 3 in vivo siRNA 3. Moreover, with siRNA3 there is an unexpected inhibition of tumour growth, not seen with other siRNA's.

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.