GB2420119A - RNA interference molecules targeting human Keap1 - Google Patents

Abstract

A duplex small inhibitory RNA (siRNA) against human Keap1 comprising a sense, and an antisense RNA strand which form a double-stranded molecule where the sense RNA strand comprises a nucleotide sequence substantially identical to a target sequence of contiguous nucleotides in human Keap1 mRNA, or an alternative splice form or variant. Preferably the molecule comprises the sequence of SEQ ID 1, possesses 3' overhangs and is 21 nucleotides long. As Keap1, which comprises a broad complex, tramtrack and bric-a-brac/pox virus and zinc finger domain (BTB/POZ), negatively regulates Nrf2 by ubiquitinating it and facilitating its degradation by the proteasome, the inhibition of Keap1 increases the level of Nrf2. Therefore, the Keap1 siRNA may be a cancer chemopreventive agent and pharmaceuticals comprising it are also claimed. Examples relate to targeting Keap1 in in vitro keratinocyte cell culture resulting in increased Nrf2 protein and ARE (antioxidant response element) driven luciferase reporter gene activity.

Description

Introduction

The invention is concerned with short inhibitory RNA (sIRNA) molecules and their use in various formulations as therapeutic agents.

For convenience, the various prior art documents referred to in this specification are listed under the section headed "References". The whole contents of each of these documents are hereby incorporated into this specification by reference.

Cancer chemoprevention holds the promise of greatly reducing the incidence of neoplastic disease. Many chemopreventive mechanisms have been proposed including induction of detoxication enzymes, stimulation of apoptosis, promotion of cellular differentiation, and inhibition of hormone receptors, angiogenesis and inflammation (1-4). Those chemicals that:*" up-regulate detoxication enzymes and prevent mutagenesis have been designated as chemopreventive blocking agents (5). For many years blocking agents were thought to solely increase the expression of genes encoding drug-metabolising enzymes, such as: NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione transferases, but more recently: . .

it has become clear that they also induce many antioxidant genes including those for glutamate cysteine ligase catalytic (GCLC) and modifier (GCLM) subunits that catalyse the rate-limiting step in GSH biosynthesis (6-8).

All blocking agents are thiol-active (9, 10) and it is thought that their ability to induce gene expression represents cellular adaptation to redox stress (11). Those genes that are transcriptionally activated by blocking agents contain antioxidant response elements (AREs) in their 5(-upstream regions (11, 12). Cells respond to treatment with these agents through accumulation of the cap'n' collar basic-region leucine zipper (bZlP) transcription factor Nrf2 (nuclear factor-erythroid 2 p45-related factor 2) in the nucleus where it is recruited, as a heterodimer with a small Maf bZlP protein, to AREs and stimulates expression of cytoprotective genes (13, 14).

Central to induction of ARE-driven genes is antagonism of Keapl, a protein that negatively regulates Nr12 by facilitating its proteasomal degradation (15, 16). Keapl is a redox-active protein of 624 amino acids that contains a BTB/POZ (Broad complex, Tramtrack, and Bric-a- Brac/Poxvjrus and zinc finger) domain in its N-terminal portion, six Kelch repeats in its C-terminal half, and reactive cysteine residues located between the BTB/POZ and Keich repeat domains (17-20). Humans possess 51 BTB/Kelch proteins, and Keapl is classified by sequence homology as a member of subclass 1 (21). Keapi controls the half-life of Nr12 by acting as a substrate adaptor protein for the ubiquitin E3 ligase cullin 3 (21, 22). Under homeostatic conditions Keapi promotes ubiquitinization and degradation of Nrf2 via the 26S proteasome. Exposure of cells to chemopreventive blocking agents causes inhibition of the interaction between Keapi and Nrf2 that results in accumulation of the transcription factor (15, 16).

Two principal models have been proposed to explain how Nrf2 protein is stabilized during induction of ARE-containing genes. In one of these, Keapi is modified by blocking agents at Cys-273 and Cys-288, so preventing it from capturing Nrf2 and targeting it for degradation (18, 19). In the other, Nrf2 is phosphorylated at Ser-40 by protein kinase C (PKC) in response to inducing agents, thereby enabling the bZIP protein to either evade Keapi or dissociate from Keapi (24-27). It requires to be clarified whether accumulation of Nrf2 is sufficient for gene induction, or whether phosphorylation of the bZlP protein is also necessary. :...:.

A possible disadvantage of using thiol-active agents as a prophylactic cancer therapy is that their redox activity may lead to undesirable side effects. SiRNA has been found to activate RNA interference (28), and the present inventors hypothesized that siRNA against Keapi could be utilized as an alternative relatively non-toxic strategy to activate the ARE- gene battery. The current invention concerns two siRNA species that can knockdown Keapi and up-regulate cytoprotective genes in human cells. *v..

S S...

A first aspect of the present invention is set out in claim 1 attached hereto.

A second aspect of the present invention is set out in claim 11 hereto.

A third aspect of the present invention is set out in claim 12 hereto.

The term "variant thereof" in relation to a polynucleotide sequences means any substitution of, variation of, modification of, replacement of deletion of, or the addition of one or more nucleic acid(s) from or to a polynucleotide sequence providing the resultant protein sequence encoded by the polynucleotide exhibits the same properties as the protein encoded by the basic sequence. The term therefore includes alleleic variants and also includes a polynucleotide which hybridises to the basic polynucleotide sequence. Preferably, such hybridisation occurs at, or between low and high stringency conditions. In general terms, low stringency conditions can be defined as 3 x SSC at about ambient temperature to about 55 C and high stringency condition as 0.1 x SSC at about 65 C. SSC is the name of the buffer of 0.15M NaCI, 0.015M tn-sodium citrate. 3 x SSC is three times as strong as SSC and so on.

Preferably, variants have 85% or more of the nucleotides in common with the polynucleotide sequence of the present invention and, especially preferred are 90%, 95%, 98% or 99% or more identity.

When comparing nucleic acid sequences for the purposes of determining the degree of identity, programs such as BESTFIT and GAP (both from Wisconsin Genetics Computer Group (GCG) software package) can be used. BESTFIT, for example, compares two sequences and produces an optimal alignment of the most similar segments. GAP enables sequences to be aligned along their whole length and finds the optimal alignment by insertir' spaces in either sequence as appropriate. Suitably, in the context of the present invention.

when discussing identity of nucleic acid sequences, the comparison is made by alignment ot 0s**I* the sequences along their whole length. S... *

The inventors have shown that human Keapl is amenable to knockdown using siRNA. Two: siRNA species, Targeti and Target2 have been designed that possess this property. One of' these, Targeti, has been characterized further, and shown to increase Nrf2 protein resulting in up-regulation of ARE-driven genes and an elevation in intracellular GSH.

By contrast with conventional cancer chemopreventive blocking agents, the use of siRNA has the advantage that it should specifically target the Keapl -Nrf2 pathway. Blocking agents can have pleiotropic effects on cells and are likely to modulate other pathways besides Keapl -Nrf2 (1, 2) . Of potential concern is the fact that all blocking agents are thiol- active redox stressors (10) and can presumably modify intracellular proteins other than Keapi. In the case of Sul, it is expected that thiocarbamylation by the isothiocyanate and/or oxidation of cysteine residues in essential proteins will be harmful to the cell. Whereas low doses of Sul induce ARE-driven gene expression, high doses of the isothiocyanate cause cell cycle arrest and apoptosis through decreasing cyclin Bi, cdc25B and cdc25C (42). It seems unlikely that siRNA against Keapi will suffer from the same problems of toxicity as can occur following exposure to isothiocyanates. Using 100 nmol/l Targeti, the inventors have found no evidence from flow cytometry that it causes apoptosis or cell cycle arrest (data not shown).

The ability of siRNA reagents to interfere only with the intended target is clearly important because lack of specificity can give rise to spurious results (43). The belief that Targeti siRNA is highly selective in its action is based on the observation that it can block ectopic expression of human Keapi but is unable to block expression of mouse Keapi.

Comparison of cDNAs for human and mouse Keapl reveal that nucleotides + 186 to +204 contain mismatches at +189 and +196 suggesting that failure of the Targeti siRNA to inhibit expression of the murine protein is due to loss of homology in the mRNA. Blast searching of the human DNA database revealed that the most homologous cDNA sequence to Targeti contained 4 mismatches. It therefore seems unlikely that Targeti 5iRNA elicits off-target effects.

The fact that Targetl sIRNA could induce ARE-containing genes suggested that accumulation of Nrf2 protein is both necessary and sufficient for activation of the gene battery. Indeed, as induction of AKR1C1 by Targeti siRNA was quantitatively similar to that obtained using Sul, it appears unlikely that a regulated limiting step(s) exists downstream of:" Keapi. These results are consistent with the observed upregulation of NQO1, GST, GCLC.

and GCLM in the keapl-/- mouse (34). However, the findings of this study and that S.....

describing the keapl-/- mouse are surprising given the reports that release of Nrf2 from Keapl and/or nuclear translocation of Nrf2 is controlled by PKC (24-27). We conclude that if: ..

PKC activity is required for phosphorylation of Nrf2 at Ser-40, it either occurs constitutively or....

is primarily involved in antagonising the interaction between Nrf2 and Keapi.

The inventor's data provide evidence that down-regulation of Keapi in human cells can alter" expression of ARE-driven genes. This represents a significant advance because it will allow the battery of genes regulated by Nrf2 to be defined in this species. The question of identifying genes regulated by the Keapl-Nrf2 pathway in humans is an important issue because species-specific differences exist in inducible proteins. For example, AKR1C1 is highly inducible in human cell lines (36, 37), yet the orthologous genes in the mouse and rat appear to be unresponsive to xenobiotics. Conversely, glutathione transferases do not appear to be particularly inducible in human cell lines, yet they are highly inducible in rodents (6, 33, 44).

The inventors have also shown that treatment of HaCaT cells with Targeti siRNA resulted in a 1.75-fold increase in GSH levels. Results suggest that this increase is achieved by upregulation of GCL. However, the cystine/glutamate exchange transporter gene in mouse is regulated through an ARE (45), and although the promoter of the human gene has not been characterized it may also contribute to the increase in GSH because it is responsible for the uptake of cystine into the cell (46). In the rat, Nrf2 activity decreases with age resulting in a decline in GSH in old animals (47). Antagonising Keapi expression could potentially be employed as a remedial measure to enhance GSH synthesis in the elderly.

The invention will now be further described with reference to the following figures in which: Fig. 1. Illustrates Knockdown of Keapi with 5iRNA. HaCaT cells were transfected with Keapl siRNA and scrambled siRNA. Levels of Keapl mRNA were quantified by RT-PCR TaqMan( using 1 8S rRNA as an internal standard. Each experiment was performed in triplicate and data are presented as the mean standard error (SE). (A) HaCaT cells were transfected with 100 nM Targeti, Target2 and scrambled 5iRNE 48 h before RNA was isolated and analysed. (B) Different concentrations of Targeti were used to transfect HaCaT cells. RNA was isolated and analysed 48 h post-transfection. (C) Cells were transfected with nM Targeti, and RNA was analysed 24 h, 48 h and 72 h post-transfection.

0SS**0 Fig. 2. Demonstrates Depletion of ectopically-expressed Keapi protein by siRNA. (A) HaCaT: cells were transfected with 0.4 (g of the human pCMV-Tag/Keapl expression construct (29) Keapl-V5 transfected cells respectively.

Fig. 3. Demonstrates that Knockdown of Keapi increases Nrf2 protein and induces ARE- driven gene expression. (A) HaCaT cells were transfected with 100 nM Targeti and were harvested at various times thereafter. Cell lysates (10 (g protein) were analysed by western..:.

blotting using antiserum raised against mouse Nrf2. (B) HaCaT cells were transfected with nM Targetl or scrambled s1RNA. Co-transfections w 48 h later. In addition, untransfected HaCaT cells were treated with 0.01% DMSO or 5 (M SuP (in DMSO to a final conc. of 0.01%) before being harvested 24 h later. (A) Western blotting was performed on cell lysates, 10 nM Targeti or scrambled siRNA. They were harvested 48 h later and GSH levels measured. Data are presented as the mean of duplicate experiments SD.

the numbering of the nucleotides is from the translational initiation site, with the A of the ATG codori being assigned 1 Figure 4 Demonstrates that Keapi 5iRNA causes induction of antioxidant and detoxication proteins. HaCaT cells were transfected with either lipofectamine alone (mock), lOOnM Targeti or 1 OOnM scrambled siRNA and harvested 48hour later. In addition, untransfected HaCat cells were treated with 0.01% DMSO or 5pM SuP (in DMSO to a final conc. of 0.01%) before being harvested 24h later. (A) Wesern blotting was performed on cell lysates, I Opg protein total. (B) TaqMan chemistry was performed on a total RNA to quantify message levels for (B) GCLC, (C) NQO1, and (D) AKR1 Cl.

Figure 5 Demonstrates that Keapi siRNA can elevate intracellular GSH levels. HaCaT cells were either mock transfected or transfected with lOOnM Target 1 or scrambled 5iRNA. They were harvested 48h later and GSH levels measured. Data are presented as the mean of duplicate experiments plus or minus SD. * * * S...

S * S. * S *. S * S S * . * S *5* S

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Materials and methods Preparation of siRNA against Keapi These were designed by Cenix according to the current guidelines for effective knockdown by this method ("http://www.ambion.com"). The two doublestranded siRNAs targeting human Keapl, and the scrambled control, with the same GC content, were as follows: Targeti for Keapi, sense 5'GGCCUUUGGCAUCAUGAACTT-3' and antisense 5' - GUUCAUGAUGCCAAAGGCCTG - 3'; Target2 for Keapi, sense 5' - GGUCMGUACCAGGAUGCATT -3' and antisense 5' GCAUCCUGGUACUUGACCTG - 3'; scrambled 5iRNA, sense 5' GACGAGCGGCACGUGCACATT -3' and epitope tag, pcDNA3.lN5HisCmKeapl (15), the mouse nqol -ARE (nucleotides -454 to -414) cloned into the pGL3-Promoter lucif erase reporter vector, and the pRL-TK Renilla luciferase vector* " (Promega) have been described elsewhere (14).

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Cell culture and transfection. : The immortalised human keratinocyte HaCaT cell line was maintained in DMEM supplemented with 10% FBS and 2mM L-Glutamine (Invitrogen Corp) under sterile mycoplasma-free conditions. The cells were seeded in 6-well dishes at a density of 2.5 x 105- cells 24 h prior to each experiment. They were allowed to grow until they were 30-40% confluent before being transfected, using Lipofectamine 2000 (Invitrogen), with siRNA, expression vector or lucif erase reporter construct. Co-transfection of HaCaT cells with siRNA and either an expression construct or a reporter construct was carried out by applying simultaneously two transfection reaction mixes that had been prepared separately.

Biochemical analyses. ARE-driven luciferase activity was assayed using the Dual Lucif erase Reporter Assay (14).

Total cellular RNA was extracted using Trizol reagent (Invitrogen). Relative levels of mRNA species were determined by TaqMan( Chemistry (30). The sequences of the primers and probes employed to measure human AKR1C1, GCLC, GCLM, Keapl and NQO1 are listed in the sequence listings. Measurement of 1 8S rRNA was used as an internal control.

Protein and GSH levels were measured by the methods of Bradford and of Tietze as described elsewhere (31).

For Western blothng, protein extracts were prepared by harvesting cells in RIPA buffer (50 mM Tris-HCI, pH 7.4, 150 mM NaCl, 1% (v/v) NP-40, 0.5% (w/v) deoxycholic acid, 0.1% SDS) supplemented with Complete EDTA-free protease inhibitor cocktail (Roche).

Immunoblotting was performed using antiserum raised against GCLC, GCLM, AKR1C1, NQO1, Nr12, FLAG (Sigma), V5 (Invitrogen) or GAPDH (Ambion) as described previously (6, 32, 33).

Results Based on data from the keapi -I- mouse (34), it seemed possible that knockdown of Keapl by siRNA might up-regulate ARE-driven genes in human cells. Human HaCaT keratinocytes were chosen to examine this possibility because they appear to be able to mount an ARE- driven response to sulforaphane (35). Initial experiments indicated that transfection of HaCa5b cells with FITC-tagged oligonucleotides resulted in 60%-70% of cells staining positively * under fluorescence microscopy. This level of transfection efficiency was considered adequate to allow gene induction by Keapl siRNA to be tested by transient transfection.

Example 1 Knockdown of Keapi mRNA and protein using siRNA.

Two siRNA sequences, Targeti and Target2, were designed against human Keapl. They each contained a IT overhang at the 3(-end of the sense strand and a TG overhang at the *.

3(-end of the antisense strand. Targetl and Target2 encompass nucleotides +186 to +204 and +246 to +264, respectively, of Keapi cONK that encode amino acids in the BTB/POZ domain of Keapi (POZ = poxvirus and zinc finger).

Targeti and Target2 siRNAs were separately transfected into HaCaT cells at a dose of 100 nmol/I. After 48 h, Targeti reduced the steady-state levels of mRNA for Keapl to (30% of that observed in untreated HaCaT cells or those transfected with the scrambled siRNA (Fig. 1A). Target2 reduced Keapi mRNA to (50%. Given a transfection efficiency of 60-70%, Targeti appeared to abolish completely Keapl mRNA in transfected cells. As Targeti appeared to be slightly more effective than Target2 in reducing Keapl mRNA, the former siRNA was studied further.

Transfectjon of different amounts of siRNA into HaCaT cells showed that knockdown of Keapi mRNA occurred at a dose of only 25 nmol/l of Targeti, but that a greater reduction was observed using 100 nmol/l of the siRNA (Fig. 1 B). Study of the time-dependency of transient transfection with siRNA revealed that 24 h after transfection with 100 nmol/l of Targeti, the level of Keapl mRNA in HaCaT cells was reduced to about 30% (Fig. 1C).

Knockdown of Keapl was maintained up to 72 h. To determine whether the reduction in steady-state levels of Keapi mRNA translated into a loss of Keapi protein, western blotting experiments were conducted.

Attempts to detect endogenous Keapi protein in HaCaT cells met with variable success because of lack of suitable antibodies. Hence, the effect of 5iRNA on ectopically expressed N-terminally FLAG-epitope tagged human Keapi was investigated. We first demonstrated that N-terminally tagged Keapi protein produced from pCMVFLAG/hKeapl was functional in HaCaT cells, insofar as it could inhibit ARE-driven lucif erase activity (Fig. 2A).

Subsequently, western blotting for FLAG-tagged Keapi showed that synthesis of the ectopic protein was lowered to below the detection limit by transfection with the Targeti siRNA whereas the scrambled siRNA did not inhibit synthesis of tagged Keapi (Fig. 2B).

The specificity of the knockdown was investigated further using a V5tagged mouse Keapi expression vector. Within the region that Targeti was designed against, 2 mismatches occur between the human 5'-GGCCT1TGGCATCATGC-3' and mouse 5'- S.....

GGC I I I I GGCGTCATGAAC-3' sequences (with differences underlined). It was therefore expected that if the RNA interference process was specific, Targeti would not inhibit the * ectopic expression of mouse Keapi in HaCaT cells. As shown in Fig. 28, this prediction was:. .:.

confirmed.

Example 2 To demonstrate that transfection with Keapi sIRNA increases intracellular * *..

Nrf2 protein and ARE-driven reporter gene activity. : * Immunoblotting was performed to determine whether knockdown of Keapi affected by Targeti siRNA is sufficient to increase the intracellular level of Nrf2.

Transfection of HaCaT cells with Targeti caused a substantial increase in endogenous Nrf2 protein (Fig. 3A). The increase in Nrf2 was apparent 24 h post-transfection with Targeti. In these experiments, treatment with sulforaphane (Sul) was used as a positive control. The immuno-reactive Nt In order to determine whether the increase in Nrf2 protein elicited by the siRNA was sufficient to stimulate an increase in ARE-driven transcription, reporter gene assays were carried out. Co-transfection of HaCaT cells with the nqol-ARE-Iuciferase reporter and either Targeti or Scrambled siRNA showed a 2.3-fold increase in reporter gene activity in those cells exposed to the Keapi siRNA (Fig. 3B).

Example 3 Induction of antioxidant and detoxication genes by Keapi siRNA.

To allow exploration of whether knockdown of Keapi allows Nrf2 to induce gene transcription, the mRNA and protein levels of members of the AREgene battery were measured. The target genes examined included AKR1 Cl that is inducible by various blocking agents (36, 37), and NQO1 as it contains a functional ARE (38). In addition, GCLC and GCLM were studied because they also contain AREs (39, 40).

HaCaT cells were either mock transfected, transfected with Targeti, or treated with Sul. TaqMan( chemistry and western blotting showed that by comparison with mock transfection, the Targeti siRNA produced a 14-fold increase in AKR1 Cl mRNA and a 5-fold increase in AKR1 Cl protein (Fig. 4A, B). Transfection with scrambled S1RNA did not influence levels of AKR1C1 mRNA or protein. The amount of AKR1C1 in mock-transfected HaCaT cells appeared to be slightly elevated when compared with the level of AKR1 Cl in DMSO treated HaCaT cells. This may be due to the transfection procedure stimulating gene expression. Alternatively, DMSO can scavenge reactive oxygen species and has been found to repress gene induction (41).

Transfection with Targetl and treatment with Sul produced between 5-fold and 7-fold: ...:.

increases in mRNA for GCLC (Fig. 4C). Western blotting showed Targeti produced similar: increases in GCLC as measured for mRNA (Fig.4A). Likewise, Targeti and Sul induced GCLM between 2.5- and 3.5-fold (data not shown). By contrast, neither mock transfection, or:...:.

transfection with scrambled siRNA or treatment with DMSO produced increases in these:* : proteins. The level of GCLM was also increased by the siRNA (Fig. 4A).

The mRNA and protein levels of NOOl in HaCaT cells following treatment with siRNA.: . gave a modest maximal 3-fold induction when compared with results obtained for AKR1 Cl: I...

(Fig. 4A, D).

Example 4 To Demonstrate that Keapi siRNA can augment intracellular GSH levels.

We wished to test the hypothesis that increases in GCLC and GCLM protein produced by Targeti would result in an elevation in intracellular GSH. Transfection of HaCaT cells with Targeti siRNA was found to increase GSH levels in a time-dependent fashion. At 24 h post- transfection the level of GSH was increased by about 20% (data not shown), at 48 h posttransfection GSH was increased by 75% (Fig. 5). Mock transfection or transfection with scrambled siRNA did not alter the concentration of GSH (Fig. 5). The increase in GSH achieved by Targeti s1RNA is similar to that produced following treatment with Sul (data not shown).

SEQUENCES

Sequence SEQUENCE ID No Sequence Targetl for Keapl 1 5'-GGCCUUUGGCAUCAUGC.

3' (sense), minus overhang Target2 for Keapl 2 5'-GGUCAAGUACCAGGAUGCA_3' (sense), minus overhang AKRIC1 3 5'-CGAGAAGAACCATGGGTGGA.3' Forward Primer AKRIC1 4 5'-GGCACAAAGGACTGGGTCC.3' Reverse Primer AKRICI Probe 5 5'-CCMGAGCACCGGGGAGUCGG.3' GCLC 6 5'-TCTCTAATAJAAGAGATGAGCMCATGC.3' Forward Primer GCLC 7 5'-TTGACGATAGATWGAGATCTACG3' Reverse Primer GCLC Probe 8 5'-CAGGAGATGATCAJATGCCTTCCTGCC.3' GCLM 9 5'TAGAATCAMCTCUCATCATCMCTAGA3' Forward Primer GCLM 10 5'TCACAGAATCCAGCTGTGCM...3' Reverse Primer GCLM Probe 11 5'TGCAGUGACATGGCCTGUCAGTCC.3' Keapl 12 5'-TTCMGGCCATGTTCACCM.3' Forward Primer Keapl 13 5'-TGGATACCCTCMTGGACACC.3' Reverse Primer Keapi Probe 14 5'-TGCGGGAGCAGGGCATGG-3' NQO1 15 5'-GGAGAGTTTGCTTACACTTACGC.3' Forward Primer NQO1 16 5'-AGTGGTGATGGAAAGCACTGCCUC3' Reverse Primer NQO1 Probe 17 5'-CCATGTATGACMAGGACCCTTCCGGAG.3' References 1. Surh, Y.-J. (2003) Nat. Rev. Cancer 3, 768-780...DTD: 2. Manson, M.M. (2003) Trends Mol. Medicine 9, 11-18.

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Claims (14)

1. An siRNA comprising a sense RNA strand and an antisense RNA strand,

wherein the sense and antisense RNA strands form an RNA duplex, and wherein the sense ANA strand comprises a nucleotide sequence substantially identical to a target sequence of contiguous nucleotides in human Keapi mRNA, or an alternative splice form, variant or cognate thereof.

2. The molecule of claim 1 said molecule having a nucleotide sequence that comprises SEQ ID No 1 (SEQ ID No 1 = target 1 minus overhang) or a variant thereof.

3. The molecule of claim 1 said molecule having a nucleotide sequence that comprises SEQ ID No 2 (SEQ ID No 2 = target 2 minus overhang) or a variant thereof.

4. The sIRNA of claim 1, wherein the 5iRNA further comprises an addition, deletion, substitution or alteration of one or more nucleotides.

S

5. The siRNA of claim 1, further comprising a 3' overhang. . :. * *

S**IS *

6. The siRNA of claim 5, wherein the sense RNA strand comprises a first 3' overhang, and: the antisense RNA strand comprises a second 3' overhang. S... * * S...

7. A pharmaceutical composition comprising the siRNA molecule of claim 1 in an acceptable: **.

carrier or diluent.

8. The pharmaceutical composition of claim 7 wherein the siRNA molecule has a nucleotide sequence comprising SEQ ID No 1.

9. The pharmaceutical composition of claim 8 wherein the siRNA molecule has a nucleotide sequence further comprising SEQ ID No 2.

10. A medicament comprising the siRNA molecule of claim 1.

11. An active ingredient comprising the siRNA molecule of claim 1.

12. Use of a molecule according to claim 1 for inhibiting expression of human Keapi.

13. A recombinant plasmid comprising nucleic acid sequences for expressing an s1RNA comprising a sense RNA strand and an antisense RNA strand, wherein the sense and an antisense RNA strands form an RNA duplex, and wherein the sense RNA strand comprises a nucleotide sequence substantially identical to a target sequence of contiguous nucleotides in human Keapi mRNA, or an alternative splice form, variant or cognate thereof.

14. A recombinant viral vector comprising nucleic acid sequences for expressing an 5iRNA comprising a sense RNA strand and an antisense RNA strand, wherein the sense and an antisense RNA strands form an RNA duplex, and wherein the sense RNA strand comprises a nucleotide sequence substantially identical to a target sequence of contiguous nucleotides in human Keapi mRNA, or an alternative splice form, variant or cognate thereof. * *

S..... *...

S * S* * S

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S S...