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Title:
SIRNA AGAINST P22PHOX
Document Type and Number:
WIPO Patent Application WO/2012/038759
Kind Code:
A1
Abstract:
The invention relates to siRNA against p22phox, compositions comprising the siRNA, methods of treating diseases with the siRNA and cell based systems for studying the effect of p22 phox modulation by siRNA or cells.

Inventors:
SINGER, Donald (37 Newbold Terrace East, Leamington Spa Warwickshire CV32 4EY, GB)
Application Number:
GB2011/051802
Publication Date:
March 29, 2012
Filing Date:
September 23, 2011
Export Citation:
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Assignee:
SINGER, Donald (37 Newbold Terrace East, Leamington Spa Warwickshire CV32 4EY, GB)
International Classes:
C12N15/113; A61K31/713; F16D55/40; F16D65/853; F16D55/00; F16D65/12
Domestic Patent References:
WO2005103297A12005-11-03
Other References:
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Attorney, Agent or Firm:
WITHERS & ROGERS LLP (4 More London Riverside, London SE1 2AU, GB)
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Claims:
Claims

1. An siRNA comprising a sense region and an anti-sense region, wherein said sense region and said anti-sense region form a duplex region, said sense region and said anti-sense region are each 18-30 nucleotides in length and said anti-sense region comprises a nucleotide sequence that is at least 80% complementary to a contiguous sequence of at least 18 nucleotides from position 209 to 279 of the nucleotide sequence of p22phox shown in Figure 1.

2. An siRNA according to claim 1 , wherein the anti-sense region and the sense region are each 19-25 nucleotides in length.

3. An siRNA according to claim 1 or claim 2 wherein the anti-sense region comprise a nucleotide sequence which is complementary to a contiguous sequence of at least 18 nucleotides of at least a portion of the nucleotide sequence from positions 239-259.

4. An siRNA according to claim 3, wherein the anti-sense region comprises a nucleotide sequence which is complementary to a contiguous sequence of at least 18 nucleotides of the nucleotide sequence from 239-259.

5. An siRNA according to any preceding claim, wherein the siRNA comprises two separate complementary strands of RNA or comprises a single hairpin loop of RNA.

6. An siRNA according to any preceding claim comprising the following sense and anti- sense sequences:

5'-AAG CAC ATG ACC GCC GTG GTG-3' (sense, 239-259), and 5'-AAC ACC ACG GCG GTC ATG TGC-3 ' (antisense).

7. A composition comprising an siRNA according to any preceding claim and a delivery agent.

8. A method of modulating angiogenesis and/or modulating oxidative stress in a mammal comprising administering an effective amount of an siRNA or a composition according to any preceding claim.

9. A method according to claim 8 to suppress tumour angiogenesis, for atheromatous plaque stabilisation or to reduce abnormal blood vessel formation in diabetes mellitus in retinal, renal and other circulations, or retinosis.

10. A method of modulating one or more of infective and/or inflammatory disorders, neoplastic disease and/or systemic and neuro-degenerative diseases comprising administering an effective amount of an siRNA or a composition according to any one of claims 1 to 7.

11. A method of treating tumours comprising administering an effective amount of an siRNA or a composition according to any one of claims 1 to 7.

12. An siRNA or a composition according to any one of claims 1 to 7 for use in modulating angiogenesis and/or modulating oxidative stress.

13. An siRNA or a composition according to claim 12 for use in suppressing tumour angiogenesis, atheromatous plaque stabilisation, or to reduce abnormal blood vessel formation in diabetes mellitus in retinal, renal, and other circulations, or retinosis.

14. An siRNA or a composition according to any one of claims 1 to 7 for use in one or more of modulating infection and/or inflammatory disorders, neoplastic disease and/or systemic and neuro-degenerative diseases.

15. An siRNA or a composition according to any one of claims 1 to 7 for use to treat tumours.

16. A method of studying an effect of p22phox modulation on a cell comprising contacting the cell with an siRNA or composition according to any one of claims 1 to 7 and detecting one or more physiological and/or chemical changes to the cell.

17. A method according to claim 16, wherein the cell is an endothelial cell.

18. A method according to claim 16 or claim 17, wherein the method is used to identify the effect of p22phox, or another compound, on angiogenesis.

19. A method of studying angiogenesis comprising contacting an endothelial cell with an siRNA specific for p22phox.

20. A method according to any one of claims 16 to 19 additionally comprising the step of contacting a cell with angiotensin II.

21. A method according to any one of claims 16-20, comprising contacting the cell with a further compound to determine the effect of that further compound on the one or more physiological and/or chemical changes to the cell.

22. An isolated nucleotide sequence encoding at least one of the sense region and/or antisense region of an siRNA according to any one of claims 1 to 6, but no more than 30 contiguous nucleotides of the sequence shown in Figure la or lb, or a sequence complementary to the nucleotide sequence shown in Figure la or lb.

Description:
siRNA

The invention provides siRNA against p22phox, compositions comprising the siRNA, methods of treating diseases with the siRNA, and cell based systems for studying the effect of p22phox modulation by the siRNA on cells.

Angiogenesis, the formation of new blood vessels, is critical to many important physiological processes. Abnormal angiogenesis plays a key role in a wide range of serious cardiovascular disorders including hypertension, atherosclerosis and ischemic heart disease 1 "3 . Data from in vivo and in vitro studies in a wide range of non-human and human tissues including myocardium '* , skeletal muscle 3 , conjunctiva, buccal membranes and retinal circulation 6 , suggest impaired angiogenesis as an important feature in hypertension 6 ' 7 . The inventors and others have shown that microvascular rarefaction is a common feature in subjects with borderline hypertension 8 in patients with established essential hypertension 8 and in pregnant women with pre-eclamptic toxaemia 10 . These findings suggest that capillary rarefaction is likely to occur as a primary or a very early structural abnormality in essential hypertension (see review").

Reactive oxygen species (ROS), such as superoxide anion and hydrogen peroxide (H2O2), are involved in signalling pathways in many aspects of growth factor-mediated angiogenesis 3 . High concentrations of ROS cause oxidative stress and apoptosis, important features of common disorders associated with cardiovascular disease, including hypertension, atherosclerosis, diabetes mellitus and renal impairment 3 ' 12 . In contrast, ROS at low concentrations are involved in endothelial cell (EC) proliferation and migration, and are key to angiogenesis in vivo 3 ' 13~16 . The major source of ROS in ECs is generation via the NAD(P)H oxidase complex 17 . NADPH oxidase subunits (Noxl , Nox2 (gp91phox), Nox4, p22phox, p47phox, p67phox and the small G protein Racl) are expressed in ECs 3 ' 18 ' l 9 . However, the levels of expression of different NAD(P)H oxidase subunits vary between different cell types including vascular smooth muscle cells (VSMC), neutrophils and ECs 20 . The p22phox subunit is an essential component of the NAD(P)H oxidase enzymatic complex 21 . In VSMC, Nox2 and p22phox form the electron transfer component of the oxidase; p47phox and p67phox are cytosolic components interacting with Nox 2 and p22phox to modulate enzyme activity 20 . Increasing evidence suggests that p22phox may play a key role in hypertension . For example, angiotensin II (Ang II), a potent stimulator for NADPH oxidase 12, 11 , activates p22phox in VSMCs in hypertensive rats during infusion with Ang II 23 , with a parallel increase of p22phox mRNA in the aorta in these rats 24 . Ang II increases arterial blood pressure and renal p22phox mRNA and protein levels 25 , whereas knocking down p22phox with small interference RNA (siRNA) reduces Ang-II-induced hypertension in rats 25 . The authors of that paper 25 reported RNA silencing in vivo of p22phox in rats. Double-stranded interfering RNAs (siRNAs) targetted to p22phox tested its role in mediating responses to Ang-II and hypertension in rats. Two siRNAs were observed to reduce mRNA in cultured smooth muscle cells. The siRNAs were observed to decrease protein expression for Nox -1 , -2 and -4 in Ang-II infused rats 25 . It was reported that p22phox is required for renal NADPH oxidase activity, expression of Nox proteins and contributes to hypertension during Ang-II slow-repressor response. Two different siRNAs are indicated as having been used. The online supplement for the paper indicates that the two sequences were directed towards 299-320 and 574-595 of the rat p22phox sequence. These correspond to 284-304 (with a 3 nucleotide mismatch) and 574-595 of the human p22phox sequences. As discussed below, the first sequence was not found to be effective by the inventors using siRNA against the human p22phox sequence.

In human studies, the 930G allele of p22phox polymorphism is associated with higher promoter activity, increased NADPH oxidase-mediated oxidative stress and hypertension . Furthermore, the C242T polymorphism of p22phox is associated with vascular ageing in elderly Korean subjects 27 , and is also associated with progression of asymptomatic atherosclerosis in Japanese subjects with type 2 diabetes, and with insulin resistance in non- diabetic subjects ' , or coronary heart disease in the Chinese population .

Although p22phox is abundantly expressed in VSMCs, its expression in ECs is much lower 20 . However, the p22phox subunit may also play an essential role in NAD(P)H oxidase enzymatic complex in ECs 21 . For example, p22phox is required for Ang II and thrombin- stimulated ROS formation in ECs 31 ' 32 . Angll stimulated NADPH oxidase activity in ECs is regulated by serine phosphorylation of p47phox and enhanced binding of p47phox to p22phox . ROS generated by NADPH oxidase activation stimulates diverse redox signalling pathways, such as mitogen-activated protein kinases, Akt and endothelial nitric oxide synthase (eNOS), which are essential for EC migration and proliferation 34 . The discovery of RNA interference (RNAi) has offered great potential for therapeutic application using siRNA in the treatment of diseases (RNAi therapy), and deeper understanding of regulation of gene function through knocking down expression of specific genes by siRNAs. RNAi therapy offers a powerful option to treat diseases difficult to treat with existing drugs 35 . For example, phase 1 studies to investigate RNAi-based drugs for age- related macular degeneration (a leading cause of blindness) and respiratory syncytical virus, (the leading cause of paediatric hospitalisations) have already been completed 35 . RNAi therapy for the treatment of HIV is about to enter clinical trials 36 . Given the critical role of NADPH oxidase in the modulation of vascular function, NADPH oxidase has been proposed to be a therapeutic target in vascular disease 37 . To develop RNAi therapy targeted at NADPH oxidase, the inventors aimed to target against p22phox, as p22phox might be a critical subunit regulating NADPH oxidase in human ECs. For initial testing of proof of concept, they used the human umbilical vein endothelial cell system, which is a well-established model for the study of EC survival and angiogenesis 38 . Thus, the aims of the inventors were: 1) to determine siRNA targeting p22phox (sip22phox) sequences that might effectively knock down p22phox expression; and 2) to determine whether angiogenesis and key parameters relevant to angiogenesis in human umbilical vein endothelial cells were affected by knocking down p22phox with sip22phox.

A first aspect of the invention provides an siRNA comprising a sense region and an anti- sense region, wherein said sense region and said anti-sense region form a duplex region, said sense region and said anti-sense region are each 18-30 nucleotides in length and said anti- sense region comprises a nucleotide sequence that is at least 80% complementary to a contiguous sequence of at least 18 nucleotides from position 209 to 279 of the nucleotide sequence of p22phox.

The nucleotide sequence is preferably that of human p22phox, and is preferably as shown in Figure la.

However, the sequence may be from any mammalian nucleotide sequence encoding p22phox, depending, for example, on whether the siRNA is to be used in human cells or another mammal's cells. Figure lb shows the alignment of rat and human p22phox. Positions 239- 259 of the human sequence have 85.6% homology with the rat sequence. The sequence either side extending to 209 on one side has 93.3% homology and extending to 279 on the other 90.4% homology.

Small interfering RNA (siRNA) are double-stranded RNA molecules that interfere with the expression of a specific gene. In the case of the current invention they restrict expression of the p22phox gene. Preferably the maximum amount of reduction in the expression of the p22phox gene, as determined by mRNA levels, is at least 40%, most preferably at least 50%, at least 60% or at least 70%, in comparison to the absence of the siRNA molecule.

Preferably the length of the duplex region is 19-28 nucleotides in length, more preferably 20- 25 nucleotides in length, most preferably 21 nucleotides in length.

The anti-sense region is preferably at least 85, at least 90, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, preferably 100% complementary to the contiguous sequence.

Preferably the anti-sense region comprises a nucleotide sequence which is complementary to a contiguous sequence of at least 18 nucleotides of at least a portion of the nucleotide sequence from positions 239-259.

Preferably the nucleotide sequence is complementary to a contiguous sequence of at least 18 nucleotides of the nucleotide sequence from 239-259.

Preferably the anti-sense region is not complementary to a region of the sequence which overlaps with positions 284-304 of the p22phox sequence shown in Figure la. siRNA may be in the form of two separate strands of RNA which are complementary to each other and hybridised together to form a double stranded region of RNA. However, it may also be formed from a single strand of RNA, in the form of a so-called hairpin loop with the complementary regions hybridised together. Such hairpin loops typically comprise a linking region between the duplex region of 4-1 1 nucleotides. The siRNA may comprise a short overhang at one end or the other, or indeed both ends of the duplex region. Typically this may be one or two nucleotides. The overhang may comprise one or more deoxythymidine nucleotides.

Preferably the sense and anti-sense sequences used to form the siRNA are:

5'-AAG CAC ATG ACC GCC GTG GTG-3 ' (sense, 239-259), and 5'-AAC ACC ACG GCG GTC ATG TGC-3' (antisense).

In an alternative embodiment to the invention, the siRNA comprising a sense region and an anti-sense region, may comprise an anti-sense region that is at least 90% complementary to a contiguous sequence of at least 18 nucleotides from position 584-604 of the p22 phox sequence shown in Figure la.

The level of complementarity, preferred lengths of the regions and construction of the siRNA may be as defined for the siRNAs of the first aspect of the invention.

Preferably the siRNA sequences are:

5'-AAC CCC ATC CCG GTG ACC GAC-3' (sense, 584-604) and 5'-AAG TCG GTC ACC GGG ATG GGG-3' (antisense)

The siRNAs, according to any aspect of the invention, may be made by methods generally known in the art.

The siRNAs of the invention may utilise one or more uracil residues instead of thymidine residues. The siRNA may also comprise one or more modified nucleotides, such as 2'-0- methyl nucleotide or a 2'-0-(2-methoxyethyl) nucleotide.

The siRNAs, according to the invention, may be provided in a composition comprising the siRNA in combination with a suitable delivery agent for introducing the siRNA into a cell either in vitro or indeed in vivo. Methods of introducing siRNAs into cells are generally known in the art. For example, Modlinger (Ref. 25) utilises a commercially available polymer sold under the trademark "TransIT", which is available from Minis Inc. Alternatively, an active siRNA may be produced from a plasmid or viral vector expressing, for example, a short hairpin RNA (shRNA) that is subsequently processed into the active siRNA. Such vectors, including adenoviral vectors, are generally known in the art.

Methods of modulating angiogenesis and/or oxidative-stress using the siRNA or compositions of the invention are also provided.

Preferably the method is used to suppress tumour angiogenesis, for atheromatous plaque stabilisation or to reduce abnormal blood vessel formation in diabetes mellitus in retinal, renal and other circulations, or retinosis.

The invention also provides a method of modulating infective and/or inflammatory disorders, neoplastic disease and/or systemic and neuro-degenerative diseases comprising administering an effective amount of a siRNA or a composition according to the invention.

A further aspect of the invention provides a method of treating tumours comprising administering an effective amount of a siRNA or a composition according to the invention.

A further aspect of the invention provides an isolated nucleotide sequence encoding at least one of the sense region and/or anti-sense region of a siRNA according to the invention, but no more than 30 contiguous nucleotides of the sequence shown in Figure la or lb or a sequence complementary to the nucleotide sequence thereof. The nucleotide sequence may encode a short hairpin RNA. The nucleotide sequence may be a part of a plasmid or adenoviral vector to allow the siRNA to be produced either in vivo or alternatively in vitro. siRNA molecules, according to the invention, may be delivered intravenously, intradermally, intramuscularly, intrathecally, intranasally, orally or intraperitoneally.

The siRNAs of the invention may be used to study the effect of modulating p22phox on cells. Such a use may be used to identify, for example, the pathways in which p22phox is involved, be used to model, for example, angiogenesis, and may be used to identify new compounds capable of interacting with the angiogenesis pathways, or other pathways, with which p22phox is involved. Accordingly, a further aspect of the invention provides a method of studying an effect of p22phox modulation on a cell comprising contacting the cell with a siRNA or composition according to any aspect of the invention and detecting one or more physiological and/or chemical changes in the cell.

The cell may be, for example, grown in vitro such as in tissue culture. Alternatively, the cell may be within an organism, such as a rodent (e.g. rat) animal model.

Preferably the cell is a tissue culture cell, or other cell grown in vitro. Preferred cells include endothelial cells from human or animals. Preferably the cell is a human endothelial cell, such as a human umbilical vein endothelial cell or a tissue culture cell such as HUV-EC-C.

The inventors have identified that preferred concentrations are between 5 nmol/L and 10 nmol/L siRNA in growth media.

The method may be used to identify an effect of p22phox or another compound on angiogenesis.

The inventors have newly identified the role of p22phox in angiogenesis. Accordingly a still further aspect of the invention provides a method of studying angiogenesis comprising contacting an endothelial cell with an siRNA specific for p22phox and capable of inhibiting mRNA levels of p22phox in a cell as defined above. This may be an siRNA according to the invention or alternatively another siRNA capable of inhibiting the production of p22phox mRNA.

As discussed in the Materials and Methods, it has been observed that the presence of angiotensin II has an effect on the pathways in which p22phox is involved. Accordingly the methods of the invention may additionally comprise a step of contacting a cell with angiotensin II.

The physiological and/or chemical changes to the cell include, for example, the degree of cell adhesion, proliferation, migration and/or vascular tube formation. For example, adhesion may be measured by observing VE-caherin levels. As discussed in the Materials and Methods section, cell migration assays may be undertaken using an invasion chamber. Vascular tube formation may be observed using a Matrigel layer as discussed below. The inventors also observed that the siRNA in cells had an effect on Akt phosphorylation. This is a key molecule involved in endothelial cell migration and proliferation. Moreover, apoptosis may be observed. The inventors have found that by studying the effect on caspase 3-clevage, p22phox has an effect on apoptosis in cells.

A still further aspect of the invention provides a cell comprising an siRNA according to the invention. The cell may be in vitro or in vivo, and may be an isolated cell type or alternatively a part of a whole mammalian organism.

The invention will now be described by way of example only, with reference to the following figures:

Fig. la. p22phox human nucleotide sequence (GenBank access No. M21186) showing positions of siRNAs studied.

Fig. lb. Comparison of rat (Rattus norvegicus) and human p22phox sequences.

Fig. 2. Quantification of mRNA levels of p22phox transfected with siRNAs against three target sites (Fig 2A) or a dose response curve (Fig 2B) in human ECs. Total RNAs were prepared ~24 hrs after transfection with siRNAs. To determine the efficiencies of suppression of p22phox mRNA levels, siRNAs against three target sites (PI, P2, P3, Fig 1A) were used to transfect human ECs (at 10 nmol/L). *** pO.001. Fig IB. p22phox mRNA levels showed a dose responsive reduction following transfection with siRNA (P2) at 1 , 5 and 10 nmol/L. *** p<0.001.

CN: control, lipo: lipofectamine, cells treated with lipofectamine only.

Fig. 3. Quantification of p22phox protein levels and ROS generation. 20 g of protein were loaded on each lane for the analysis of p22phox protein levels with western blotting (Fig. 2A, AU: arbitrary units) in the presence of angiotensin II (1 μΜ) and siRNA at either 5 or 10 nmol/L approximately 24 hrs post transfection. Levels of β-actin were analysed to control for equal loading. *p<0.05, ***p<0.001 (siRNA treated group vs. control group); *x* p<0.001 (siRNA plus Ang 11 treated group vs. Ang II only treated group). Fig. 3B. ROS generation analysed using the DHE staining method. Transfected ECs with 10 nmol/L siR A were cultured in Ham's F-12K medium (10% FCS) to -80% confluence, and replaced with serum free medium and cultured for overnight. The cells were then treated either with or without Ang II at 1 μΜ for 4 hrs before DHE 10 μΜ (diluted by DMSO under nitrogen gas) was added to the medium and incubated for 30 min. Generation of ROS was recorded under a fluorescence microscope (magnification: 20x).

Fig. 4. Analysis of angiogenic parameters in ECs.

Fig 4A. Vascular endothelial cadherin (VE-Cadherin) protein expression was analysed using fluorescence labelled antibody. After overnight incubation with the first antibody at 4°C, cells were washed with PBS and added with the second antibody (goat - anti-rabbit FITC) at 1 :200 dilution and incubated at 37°C for l hr. The cells were washed with PBS (*3) and mounted with DAPI. The positive staining was calculated by Image J software. Magnification: 40 x .

Fig. 4B. Cell proliferation rate analysed using the MTT assay. Cells were seeded onto gelatine-coated 96-well plates at a density of ~ 1-3 x lO 4 cells per well. After an overnight culture the cells were transfected with siRNAs (either 5 or 10 nmol L) and added with either Ang II (1 μΜ) ~4 hrs post transfection. MTT (2 mg/ml in PBS) was added to each well and incubated for a further 4 hrs. The media containing MTT was removed, and formazan crystals were dissolved in 200 μΐ of DMSO per well. The absorbance was read at 570 nm

Fig. 4C. Cell migration assay undertaken using an invasion chamber (BD Bioscience).

Endothelial cells were seeded (in triplicates) on the upper chambers at ~10 4 cells/well on 24- well plates pre-coated with 0.1% matrigel on the lower chamber. After overnight incubation at 37° 5% CO2, non- invading cells were removed from the upper surface of the membrane by scrubbing. The migrated cells attached to the bottom surface were stained with hematoxylin- eosin (H-E).

Fig 5. Vascular tube formation in endothelial cells treated with siRNA or angiotensin II.

Thawed Matrigel (200 μΐ) was added to 24-well plates and then incubated at 37°C for 30 minutes to form a gel layer. Transfected cells (~10 4 cells/well) were seeded to the Matrigel-coated 24-well plates, treated with/without Ang II (1 μΜ) before transfection and incubated in Ham's F- 12K medium containing 10% FBS for 4 hr. Tube formation at 4 hrs or 18 hrs was recorded with phase contrast microscopy. Images were captured using a Scion digital camera (magnification: 10 x) and analyzed using Imaging J software. *p<0.05, ***p<0.001 (treated group vs. control group).

Fig 6. Analysis of Akt phosphorylation and caspase-3 cleavage pathways in endothelial cells with western blotting. ~20 μg of supernatant was resolved on SDS-PAGE gel. Separated proteins were transferred on to nitrocellulose membrane. The membrane was blocked with 5% low-fat milk for 1 hr at room temperature and incubated with specific antibodies against either phosphorylated Akt or cleaved caspase-3 (1 : 1000, Cell-Signalling) at 4°C overnight. An antibody against β-actin (1 : 100) was used as a control for equal loading of protein. The membrane was washed twice and incubated with a second antibody (1 :2000) at room temperature for 1 hr. The immunoreactive protein was detected using enhanced chemiluminescence (ECL) plus (Amersham) and developed with KODAK film in dark room. The blotting film was quantitatively analyzed with Syngene Genetools (Bio Imaging systems) software. ***p<0.001 (treated group vs. control group).

Methods

Synthesis of siRNAs targeting p22phox (sip22phox) and transfection of human endothelial cell line

Three target sequences were identified from human p22phox (Genebank a/c No. M21 186) mRNA sequence for synthesis of sip22phox, based on the inventors' previous experience in siRNA design 39 ' 0 . A number of potential sequences were considered. However, the best with which to proceed were considered to be: 1) 5'-AAT TAC TAT GTT CGG GCC GTC-3 ' (sense, 284-304) and 5'-AAG ACG GCC CGA ACA TAG TAA-3' (antisense); 2) 5'-AAG CAC ATG ACC GCC GTG GTG-3' (sense, 239-259) and 5'-AAC ACC ACG GCG GTC ATG TGC-3 ' (antisense); and 3) 5'-AAC CCC ATC CCG GTG ACC GAC-3 ' (sense, 584- 604) and 5'-AAG TCG GTC ACC GGG ATG GGG-3 ' (antisense). DNA oligos were synthesised commercially (Thermofisher) and sip22phox were synthesized in vitro using a kit according to the manufacturer's instruction (Ambion).

Human endothelial cells (HUV-EC-C, ATCC-CRL- 1730) were cultured in Ham's F- 12K medium (ATCC) containing 10% foetal calf serum (FCS), 100 μg/ml heparin (Sigma) and 75 μg/ml human ECGS (Sigma). Cells (-80% confluence) were replaced with growth medium without antibiotics the day before transfection. To establish a transfection protocol, fluorescence labelled siRNAs (BLOCK-iT) were mixed with Lipofectamine 2000 (Invitrogen) in serum-free medium and incubated at room temperature (RT) for 20 min, before being added to cells and incubated in serum- free medium at 37°C in 5% C0 2 for 4-6 hrs. Then, the medium was replaced with growth medium containing 10% FCS and antibiotics overnight. The transfection efficiencies for fluorescence labelled siRNAs were: -51% at 1 nmol/L, -87% at 5 nmol/L and -96% at 10 nmol/L. Thus, siRNAs at 5nmol/L or 10 nmol/L were used in our studies.

Quantification of p22phox mRNA levels

Total RNAs were prepared from cells -24 hrs post-transfection using a SV total RNA isolation kit (Promega). To synthesise cDNA, total RNA (1 μg in 12.5 μΐ of RNAse free H 2 0) was denatured at 70°C for five minutes and chilled to 4°C, added with 0.5 mM dNTP, 5 μΜ random hexamers, 10 units/μΐ of MMLV reverse transcriptase (Promega, Southampton, UK), 1.0 units/μΐ RNAsin (Promega), in 1 x MMLV reverse transcriptase buffer containing 50mM Tris-HCl (pH 8.3 at 25°C), 75 mM KC1, 3 mM MgCl 2 , 10 mM DTT and DEPC-H 2 0 to a final volume of 20 μΐ. The cDNA reaction was incubated for 1 hr at 42°C and stopped by heating at 95°C for 5 minutes.

Primers and probe for real-time PCR were designed and synthesized by ABI. The PCR primers were: 5'-GTCCGGCCTGATCCTCATC-3' (sense) and 5'- GCCCGCCACAATGGAGTA-3 ' (antisense). The probe sequence was: 5'- CACCCAGTGGTACTTTG-3 ' . The PCR reactions were undertaken in a 96-well plate with a total volume of 25 μΐ/reaction, containing 12.5 μΐ TagMan universal PCR Master Mix, 1.25 μΐ 20 x Assay Mix (Primer & probe) and 1 1.25 μΐ cDNA (either diluted by 1 :5 or 1 : 10 before added to the reactions). 18S rRNA was amplified as positive control. The PCR reaction conditions were: a pre-run of 50°C for 2 min and a single denature step at 95 °C for 10 min, followed by 95 °C for 15 sec and annealing at 60 °C for 1 min for up to 40 cycles. ABI 7500 relative quantification system software was applied for data analysis.

Western blotting

Cells were lysed with l x modified RIPA buffer with proteases (Upstate, 1% Triton X-100, 158 mM NaCl, 5 mM EDTA, 10 mM Tris-HCl, pH 7.2, 100 mM sodium vanadate, 10 μg/ml leupeptin, 10 μg ml aprotinin and lmM PMSF) on ice -24 hrs post siRNA transfection. Cell lysates were sonicated for 15 seconds, boiled at 100°C for 5 min and cooled at room temperature, and was centrifuged at 12000 g for 10 min at 4°C and cell debris was removed. An aliquot of the cell lysate was used for protein assay using a kit (Bio-Rad). 20 μg of supernatant per sample was resolved on SDS-PAGE gel. Separated proteins were transferred on to nitrocellulose membrane. The membrane was blocked with 5% low-fat milk for 1 hr at room temperature and incubated with specific antibodies against either p22-phox (FL- 195,1 :400, Santa-Cruz), phosphorylated Akt or cleaved caspase-3 (1 : 1000, Cell-Signalling) at 4°C overnight. An antibody against β-actin (1 : 100) was used as a control for equal loading of protein. The membrane was washed twice and incubated with a second antibody (1 :2000) at room temperature for 1 hr. The immunoreactive protein was detected using enhanced chemiluminescence (ECL) plus (Amersham) and developed with KODAK film in dark room. The blotting film was quantitatively analyzed with Syngene Genetools (Bio Imaging systems) software.

ROS production measured using DHE staining

The ROS production in ECs was measured using dihydroethidium (DHE, Sigma) which generated rhodamine in response to stimulation of ROS with phorbol 12-myristate 13-acetate. Fluorescence was monitored by excitation at 480 nm and 540 nm wavelengths. ECs transfected with 10 nmol/L siRNA were cultured in Ham's F-12K medium (10% FCS) to ~80% confluence, and replaced with serum free medium and cultured overnight. The cells were then treated either with or without Ang II at 1 μιηοΙ/L for 4 hrs before DHE 10 , umol/L (diluted by DMSO under nitrogen gas) was added to the medium and incubated for 30 min. Generation of ROS was recorded under a fluorescence microscope.

Immunofluorescent staining of endothelial cells adhesive ability

Anti-human VE-cadherin (vascular endothelium cadherin, also known as cadherin-5) antibody (Santa Cruz) was used to analyze the adhesive ability of ECs. Second antibody was goat - anti-rabbit FITC. After overnight incubation with the first antibody at 4°C, cells were washed with PBS, and the second antibody was added at 1:200 dilution and incubated at 37°C for lhr. The cells were washed with PBS (x3) and mounted with DAPI. The cells were observed under fluorescent microscopy. The positive staining was calculated by Image J software.

MTT (Thiazolyl Blue Tetrazolium Bromide) assay

The proliferation rate of ECs was estimated by measuring the ability of live cells to metabolise MTT to formazan 41 . Cells were seeded on to a gelatine-coated 96-well plate at a density of ~ 1-3 χ 10 4 cells per well. After overnight culture the cells were transfected with siRNAs and added with either Ang II ~4 hrs post transfection. MTT (2 mg/ml in PBS) was added to each well and incubated for a further 4 hrs. The media containing MTT was removed, formazan crystals were dissolved in 200 μΐ of DMSO per well, and absorbance was read at OD570 nm. In vitro migration assay

The migration assay was undertaken using an invasion chamber (BD Bioscience). Endothelial cells were seeded (in triplicates) on the upper chambers at ~10 4 cells/well on 24-well plates pre-coated with Matrigel on the lower chamber. After overnight incubation at 37° in 5% C0 2 , non-invading cells were removed from the upper surface of the membrane by scrubbing. The migrated cells attached to the bottom surface were stained with hematoxylin-eosin (H-E).

Matrigel coated 3-D tube formation assay

Matrigel was stored at 4°C. Thawed Matrigel (200 μΐ) was added to 24-well plates and incubated at 37°C for 30 minutes to form a gel layer. Transfected cells (~10 4 cells/well) were seeded on to the Matrigel-coated 24-well plates and cultured in Ham's F- 12K medium containing 10% FCS for 4 hr. Tube formation was recorded with phase contrast microscopy. Images were captured using a Scion digital camera and analyzed using Imaging J software.

Statistical Analysis

Results were presented as mean ± standard deviation. Statistical significance was assessed by ANOVA with post-hoc paired testing of parametric data (n = 3 for all statistical analyses). A value of p < 0.05 was considered to be statistically significant.

Results

Suppression of p22phox gene expression and ROS generation by sip22phox

Among the three siRNAs synthesised for targeting against three difference fragments of the p22phox mRNA sequence, a sip22phox against the first target sequence had little effect in knocking down p22phox mRNA levels (PI, Fig 2A). A sip22phox against the second site (P2, Fig 2A) suppressed p22phox by ~70% (P2, p < 0.001, Fig 2A), and a sip22phox targeting the third sequence suppressed p22phox by -42% (p< 0.001 , Fig 2A). There was -68% reduction in p22phox mRNA levels 24 hrs after transfection with sip22phox (P2) at 5 nmol/L and ~72% reduction at 10 nmol/L concentrations (Fig 2B, p<0.001). In contrast, Lipofectamine had no significant effects on p22phox mRNA levels (Fig 2B). Similarly, the inventors measured mRNA levels of 18S rRNA and did not observe any difference in 18S rRNA 24 hrs post-transfection with 1, 5, and 10 nM sip22phox (data not shown). Thus, the sip22phox targeting the second site (sequences: 5'-AAG CAC ATG ACC GCC GTG GTG-3' (sense) and 5'-AAC ACC ACG GCG GTC ATG TGC-3 ' (anti-sense)) was used for all subsequent studies.

To further establish the potency of sip22phox in knocking down p22phox expression, p22phox protein levels were measured and Ang II was used as a positive control in this study, as Ang II increases vascular p22phox expression 42 . As expected, Ang II (at 1 μπιοΙ/L) increased p22phox protein levels in ECs (by -69%: Fig 3 A, pO.001). Consistent with mRNA data, there were -15% (p = 0.018, Fig 3A) and -58% (pO.001 , Fig 3A) reductions in p22phox protein levels in cells treated with sip22phox at 5 nmol/L and 10 nmol/L respectively. In the presence of Ang II, the sip22phox reduced p22phox protein levels by -20% at 5 nmol/L (p<0.001 , Ang II+5 nmol/L siRNA vs. Ang II, Fig 3A), and by -35% at 10 nmol L (pO.001 , Ang 11+10 nmol/L siRNA vs. Ang II, Fig 3 A), and the increase in p22phox protein levels stimulated by Ang II was almost blocked by treatment with sip22phox at 10 nmol/L (p = 0.10, Ang 11+10 nmol/L siRNA vs. CN). These data showed that sip22phox not only reduced p22phox protein levels, but also antagonised the stimulatory effects of Ang II, despite a high Ang II concentration. ROS production was estimated by assessment with the oxidative fluorescent dye dihydroethidium (DHE). As expected, ROS production was stimulated by Ang II, whereas sip22phox reduced ROS generation either in the presence of Ang 11 or without Ang II (Fig 3B).

Effect of knocking down p22phox on angiogenesis in ECs

The inventors then determined the functional efficacy of sip22phox through studies of angiogenesis and key parameters (adhesion, proliferation, migration and vascular tube formation) relevant to angiogenesis in ECs.

The cell proliferation rate as measured with MTT was reduced by -39% (p < 0.001) and -75% (p < 0.001) with sip22phox at 5 and 10 nmol/L respectively (Fig 4B). Ang II per se stimulated ECs proliferation by -18% (Ang II vs. CN, p < 0.01 ; Fig 4B). The stimulatory effect of Ang II was blocked with 5 nmol/L sip22phox in ECs (Ang II + 5 nmol/L siRNA vs. CN, p = 0.26; Fig 4B), and a reduced cell proliferation rate was observed (by -14%) with sip22phox at 10 nmol/L (Ang II + 10 nmol/L siRNA vs. CN, p < 0.05, Fig 4B). These data established the potency in the reduction of cell proliferation through knocking down p22phox.

VE-cadherin is an endothelial-specific cadherin localized at intercellular junctions 43 , and involved in EC migration 43 , contact-induced growth inhibition 44 and endothelial cell assembly into tubular structures 45 . The sip22phox at 10 nmol L markedly reduced expression of VE-cadherin by -98% (pO.0001, Fig 4A) or by -79% in the presence of Ang II (p<0.001 , Fig 4A). However, Ang II did not affect VE-cadherin expression in EC cells.

The sip22phox reduced EC migration as measured using the Matrigel migration chamber, by -26% (p < 0.001) and -70% respectively (p < 0.001; Fig 4C) at 5 and 10 nmol L. Although Ang II per se had no significant effects on EC migration (p = 0.97, Fig 4C), sip22phox reduced EC migration by -13% and -26% (p < 0.05 and p < 0.01 respectively, Fig 4C) at 5 or 10 nmol/L in the presence of Ang II. Thus, although Ang II per se did not affect VE- cadherin or EC migration, the effects of sip22phox on VE-cadherin and EC migration can be partly antagonised with high dose of Ang II. The inventors then evaluated the effects of sip22phox on angiogenesis in human umbilical vein endothelial cells.

Lipofectamine had no effects on angiogenesis either on its own or in the presence of Ang II at both 4 and 18 hrs (Fig 5b). Ang II increased vascular tube formation by -35% (p < 0.001) at 4 hrs or by -13% at 18 hrs (p < 0.001, Fig 5c, d). The sip22phox at 5 nmol/L reduced vascular tube formation by -23% at 4 hrs and by -57% at 18 hr (Fig 5e, p<0.001). The sip22phox at 10 nmol/L reduced vascular tube formation by -87% at 4 hrs and by -94% (at 10 nmol L, p < 0.001 for all differences, Fig 5f) at 18 hrs. Furthermore, sip22phox at 5 nmol/L blocked enhancement of vascular tube formation by Ang II (Fig 5g) and there was a further reduction in tube formation in cells treated with 10 nmol/L sip22phox in the presence of Ang II at either 4 hrs (by - 45%) or 18 hrs (by -90%, p < 0.001 ; Fig 5h). Thus, the effects of sip22phox on angiogenesis were extremely potent, as almost all angiogenesis was blocked by addition of 10 nmols/L sip22phox, despite the presence of Ang II. p22phox on ECs apoptosis and their key survival signal transduction pathways

To investigate the potential mechanisms underlying reduced vascular tube formation due to knocking down p22phox with siR As, we determined the effects of sip22phox on Akt phosphorylation, a key molecule involved in EC migration and proliferation 3 ' 17 ' 46 .

Ang II stimulated Akt phosphorylation by -44% (p < 0.001 , Fig 6A). The increased phosphorylation of Akt stimulated by Ang II was blocked by sip22phox at 5 nmol/L and Akt phosphorylation was reduced by -58% in ECs treated with sip22phox at 10 nmol/L (p < 0.001, Fig 6A).

To determine whether the apoptosis signalling pathway was affected in reduced vascular tube formation due to knocking down p22phox, caspase-3 cleavage (19KD product, Fig 6B) was measured. Caspase-3 is a key mediator of apoptosis, and cleavage of this enzyme to its active form correlates with the onset of apoptosis 47 . Ang II per se had no significant effect on caspase-3 cleavage in ECs (Fig 6B). However, there was a marked increase in caspase-3 cleavage in ECs treated with siRNA against p22phox despite the presence of Ang II. There was also a further -3 fold increase in caspase-3 cleavage in ECs treated with sip22phox in the absence of Ang II (Fig 6B). Discussion

The inventors have shown that: 1) one of the 3 siR As designed against the human NADPH oxidase subunit p22phox (sip22phox) was effective in knocking expression and functions of p22phox in human ECs, as almost all angiogenesis was blocked by addition of only 10 nmol/L of sip22phox; 2) sip22 phox markedly suppressed basal and Ang II stimulated angiogenesis in human ECs, suggesting a critical role of p22phox signalling in angiogenesis; 3) expression of VE-cadherin was markedly lower in human ECs with p22phox knocked down, suggesting a novel pathway between p22phox and VE-cadherin.

SiRNAs suppress gene function through base pairing with the target mRNA molecules, via an RNA interference mechanism 48 ' 9 . However, although all 3 siRNAs designed in this study form perfect matches with the p22phox mRNA coding sequence, the magnitude of knocking down of p22phox by siRNAs varied depending on the region targeted by a siRNA. In these studies, the PI sequence had little effect whereas the P2 sequence (sip22phox) had a major effect in knocking down p22phox mRNA and protein levels. Similar results have been reported in siRNAs targeting different mRNA transcripts 50 ' 51 . For example, out of 14 siRNAs designed to target the human telomerase reverse transcriptase, 5 siRNAs have almost no effects 51 . Thus, it is very important to confirm effective sequence targeting against a given mRNA transcript.

RNAi-based gene silencing can be highly specific, as evidenced by selective silencing of alleles containing single nucleotide polymorphisms 52 . A commercially available scrambled siRNA has been used as negative control to determine whether the effects of knocking down the target mRNA transcripts are specific to the designed siRNA. The inventors did not use scrambled siRNAs as additional negative controls for two reasons. Firstly one of the specific siRNA sequences (PI) had little effect on p22phox expression. Secondly they have observed in a previous study that scrambled siRNA can generate off-target effects (e.g. induction of adipocyte differentiation 53 ). The off-target effects of siRNAs against specific mRNA have been observed in other studies 54 . The off-target effects of siRNA are generated due to the complementarity between siRNA sequence at position 2-8 heptamer and the "seed" sequence in the 3'UTR of the mRNA transcripts 55 . Off target effects of siRNA can be minimised through chemical modifications of the siRNA backbone . The primary aim this study was to determine functional effects of sip22phox.

ROS at low concentrations can act as signalling molecules and are involved in EC proliferation and migration, which may contribute to angiogenesis 3 ' 13"16 . P22phox, as an integral component of NADPH oxidase, is critically involved in ROS generation in endothelial cells. The data showing that knocking down of p22phox reduces ROS generation in human ECs is consistent with data from studies using VSMC 21 and cultured ECs derived from porcine aorta 57 . This is consistent with the observation in transgenic mice that over- expression of p22phox in smooth muscle cells elevates generation of ROS 58 . In this study the inventors have further demonstrated that knocking down p22phox markedly reduces vascular tube formation in human ECs, with parallel reduction in Akt phosphorylation and activation of caspase-3. This supports previous work suggesting a key role of background ROS generation in signalling pathways for angiogenesis 59 .

Phosphoinositide 3-kinase (P13-K) and its downstream serine/threonine kinase Akt/protein kinase B play a central role in promoting the survival of a wide range of cells including ECs 46 . Three iso forms of Akt (Aktl , Akt2 and Akt3) have been reported. Akt 1 (referred to as Akt in this study) is predominantly expressed in ECs and plays a crucial role in angiogenesis 46 ' 60 . Akt knockout mice demonstrate growth retardation and elevated apoptosis 61 ' 62 . The data showing that knocking down p22phox expression reduced Akt phosphorylation in parallel to decreasing vascular tube formation, suggest that Akt phosphorylation may be involved in p22phox regulated angiogenesis.

Caspase-3 is a key molecule involved in apoptosis 63 , and cleavage of this enzyme to its active form correlates with the onset of apoptosis. The active-site cysteine residue is susceptible to oxidation, resulting in caspase inactivation and thus potentially inhibition of apoptosis, which is illustrated by the findings that ROS such as H2O2 can both induce 64"66 and inhibit apotosis 66"68 . The data showing that knocking down p22phox reduces ROS generation and markedly increases caspase-3 activation, suggest that basal activity of p22phox (hence ROS generation) may be important in suppressing caspase-3 activation.

These data provide direct evidence indicating that basal expression of p22phox is required for vascular tube formation in human ECs. The data obtained in human ECs are consistent with previous studies in mice in which over-expression of p22phox in smooth muscle cells

58

resulted in stimulation angiogenesis . Interestingly, the inventors' data showing that Ang 11 upregulates in human ECs both p22phox protein expression and ROS generation, is consistent with a report showing that stimulation by Ang II of NADPH oxidase activity in human ECs is regulated by enhanced binding of p47phox to p22phox 33 . The findings are also consistent with findings of an in vivo study showing that Angll infusion increases p22phox mRNA and protein levels in the renal cortex in rats 69 , and that the expression of p22phox can be knocked down by siR A against p22phox in rats 69 . In this regard, Ang II can have similar effects to over expression of p22phox. The inventors further show that Ang II induced cell proliferation, AKT phosphorylation and vascular tube formation are antagonised by sip22phox in human ECs, supporting the notion that the effects of Ang II are mediated at least in part, through p22phox 25 . Although Ang II per se does not have stimulatory effects on cell migration, VE cadherin expression and caspase 3 activation, as shown in this study, sip22phox has dose dependent effects on reduction of cell migration, VE-cadherin expression, caspase 3 activation and vascular tube formation. These data suggest that the effects of p22phox can occur independent of Ang II. However, Ang II can also antagonise the effects of sip22phox on cell migration, expression of VE-cadherin and activation of caspase 3, suggesting interactions between Ang II signalling pathways and NADPH oxidase pathways.

VE-cadherin is an endothelial-specific cadherin localized at intercellular junctions 43 , and is an important mediator in angiogenesis. VE-cadherin is critically involved in the maintenance of the integrity of the endothelium 70 , EC migration 43 , contact-induced growth inhibition 44 and most notably, endothelial cell assembly into tubular structures 45 . Tumour necrosis factor (TNF-oc) caused tyrosine phosphorylation of VE-cadherin, which appears to be regulated by intracellular oxidant production through endothelial NAD(P)H oxidase, as antioxidants and expression of a transdominant inhibitor of the NADPH oxidase, p67phox, effectively block the effects of TNF-ct on all 3 parameters of junctional integrity 71 . The novel data showing that suppression of p22phox by siRNA markedly reduces expression of VE-cadherin is consistent with the view that VE-cadherin expression may be mediated by ROS generation. More studies are needed to explore further mechanisms underlying reduced VE-cadherin expression caused by sip22phox. Therefore, the inventors' data have provided convincing evidence showing that sip22phox can effectively knock down p22phox expression and function in human ECs. The studies provide a new siRNA tool for understanding the role of p22phox and NADPH oxidase in pathophysiology and in mechanisms for efficacy. The findings clearly also suggest the need for further work to explore the therapeutic potential of sip22phox. siR As are a potential new class of drugs with the advantage of high specificity and high potency (effective with nanomolar levels), which is at least 100-fold greater than that of anti-sense RNA (which usually requires microM levels).

Potential applications of sip22phox include any pathophysiological setting in which activity of NAD(P)H oxidase adversely affects cell-signalling. Particular examples of possible applications concern angiogenesis and modulation of oxidant stress generation, for example, to suppress tumour angiogenesis 17 , an anti-angiogenic approach to atheromatous plaque stabilisation 72 and reduction in abnormal blood vessel formation in diabetes mellitus in retinal, renal and other circulations. Other therapeutic areas of interest include a wide range of infective and inflammatory disorders, neoplastic disease and both systemic and neurodegenerative disorders.

Anti-tumour therapy with sip22phox is promising as evidenced by the inventors' data showing that sip22phox can block angiogenesis both on its own, and by antagonising the effects of Ang II. Of note, inhibition of angiogenesis with an Angiotensin II Type 1 receptor blocker has been linked with reduced tumour formation in a number of studies 73"76 .

NADPH oxidase is recognized to be a major contributor to the oxidative stress implicated in

12 endothelial injury, intimal hyperplasia, remodelling and stenosis or restenosis in bypass grafts or at sites of primary angioplasty and stenting 77"79 . The inventors anticipate that sip22phox treatment may reduce the occurrence of early ischaemic syndromes after bypass grafting or primary angioplasty and may be clinically useful in helping to prevent or reduce later in-stent restenosis and bypass graft stenosis.

Thus, the inventors are convinced from the consistent data in the present studies that they have identified a potent sip22phox with efficacy in knocking down expression and function of p22phox. They have also shown that constitutive expression of p22phox is required for vascular tube formation in human endothelial cells. References

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