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`US 20030027783Al
`
`(19) United States
`(12) Patent Application Publication
`Zernicka-Goetz et al.
`
`(10) Pub. No.: US 2003/0027783 Al
`Feb. 6, 2003
`(43) Pub. Date:
`
`(54)
`
`INHIBITING GENE EXPRESSION WITH
`DSRNA
`
`(76)
`
`Inventors: Magdalena Zernicka-Goetz,
`Cambridge (GB); Florence Wianny,
`Lyon (FR); Martin John Evans,
`Cardiff (GB); David Moore Glover,
`Bedfordshire (GB)
`
`Related U.S. Application Data
`
`(63) Continuation of application No. PCT/GB00/04404,
`filed on Nov. 17, 2000.
`
`(30)
`
`Foreign Application Priority Data
`
`Nov. 19, 1999
`
`(GB) ......................................... 9927444.1
`
`Correspondence Address:
`GARY CARY WARE & FRIENDENRICH LLP
`4365 EXECUTIVE DRIVE
`SUITE 1600
`SAN DIEGO, CA 92121-2189 (US)
`
`(21) Appl. No.:
`
`10/150,426
`
`(22) Filed:
`
`May 17, 2002
`
`Publication Classification
`
`Int. Cl.7 ............................. A61K 48/00; C12Q 1/68
`(51)
`(52) U.S. Cl. ................................ 514/44; 424/93.2; 435/6
`
`ABSTRACT
`(57)
`The present invention relates to the specific inhibition of
`gene expression in mammals by bringing the target into
`contact with double stranded RNA (dsRNA).
`
`Rigel Exhibit 1018
`Page 1 of 18
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`

`Patent Application Publication
`
`Feb. 6, 2003 Sheet 1 of 5
`
`US 2003/0027783 Al
`
`FIG. 1
`
`Rigel Exhibit 1018
`Page 2 of 18
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`

`

`Patent Application Publication
`
`Feb. 6, 2003 Sheet 2 of 5
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`US 2003/0027783 Al
`
`FIG. 2
`
`Rigel Exhibit 1018
`Page 3 of 18
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`

`

`Patent Application Publication
`
`Feb. 6, 2003 Sheet 3 of 5
`
`US 2003/0027783 Al
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`Rigel Exhibit 1018
`Page 4 of 18
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`

`

`Patent Application Publication
`
`Feb. 6, 2003 Sheet 4 of 5
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`Rigel Exhibit 1018
`Page 5 of 18
`
`

`

`Patent Application Publication
`
`Feb. 6, 2003 Sheet 5 of 5
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`US 2003/0027783 Al
`
`dsRNAi WORKS IN MOUSE EMBRYO
`IN A SPATIALLY DEFINED MANNER
`
`E cadherin ds
`+ GFP sense RNA
`
`-..
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`... ~• .... ~
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`Rigel Exhibit 1018
`Page 6 of 18
`
`

`

`US 2003/0027783 Al
`
`Feb.6,2003
`
`1
`
`INHIBITING GENE EXPRESSION WITH DSRNA
`
`RELATED APPLICATION DATA
`
`[0001] This application is a continuation of International
`Application PCT/GB00/04404, with an international filing
`date of Nov. 17, 2000, published in English under PCT
`Article 21(2), and now abandoned, which is incorporated
`herein by reference in its entirety.
`
`FIELD OF THE INVENTION
`
`[0002] The present invention relates to inhibiting gene
`expression. In particular, it relates to inhibiting gene expres(cid:173)
`sion in mammals using double stranded RNA (dsRNA).
`
`INHIBITING GENE EXPRESSION WITH dsRNA
`
`[0003] The benefits of being able to inhibit the expression
`of a specific gene or group of genes in mammals are obvious.
`Many diseases (such as cancer, endocrine disorders, immune
`disorders and so on) arise from the abnormal expression of
`a particular gene or group of genes within a mammal-the
`inhibition of the gene or group can therefore be used to treat
`these conditions. Similarly, disease can result through
`expression of a mutant form of protein, in which case it
`would be advantageous to eliminate the expression of the
`mutant allele. In addition, such gene specific inhibition may
`be used to treat viral diseases which are caused by for
`example retroviruses, such as HIV, in which viral genes are
`integrated into the genome of their host and expressed.
`
`[0004]
`In addition, the elimination or inhibition of expres(cid:173)
`sion of a specific gene can be used to study and manipulate
`early developmental events in the embryo. The most valu(cid:173)
`able information would be obtained if the function of the
`gene of interest could be disturbed in specific cells of the
`embryo and at defined times. In such a situation, in the
`mouse model, the classical techniques of gene "knockout"
`cannot be used, because they eliminate gene function uni(cid:173)
`versally throughout the embryo. Furthermore, if a gene is
`repeatedly used in space and time to direct developmental
`processes, elimination of its role by conventional gene
`"knockout" may deny an understanding of everything but
`the first event. Even when the interest is to study the very
`first time in development at which a gene functions, the
`contribution of maternal transcripts and their translation
`products can mask the effects of the gene knockout. Existing
`"knockout" technology is also extremely laborious. It neces(cid:173)
`sitates first making a disrupted gene segment that is suitably
`marked to enable the selection of homologous recombina(cid:173)
`tion events in cultured embryonic stem cells. Such cells must
`then be incorporated into blastocysts and the resulting
`chimaeric animals used to establish pure breeding lines
`before homozygous mutants can be obtained.
`
`[0005]
`It is known that expression of genes can be spe(cid:173)
`cifically inhibited by double stranded RNA in certain organ(cid:173)
`isms. Double stranded RNA interference (RNAi) of gene
`expression was first shown in Caenorhabditis elegans (Fire
`et al. Nature 391, 806-811 (1998); WO99/32619), has
`recently been shown to be effective in lower eukaryotes
`including Drosophila melanogaster
`(Kennerdell. &
`Carthew, Cell 95, 1017-1026 (1998)), Trypanosoma brucei
`(Ngo, et al. Proc Natl Acad Sci USA 95, 14687-14692
`(1998)), planarians (Sanchez Alvarado & Newmark, Proc
`Natl Acad Sci USA 96, 5049-5054 (1999)) and plants
`
`(Waterhouse, et al. Proc Natl Acad Sci USA 95, 13959-
`13964 (1998)). The application of this approach has also
`been demonstrated in Zebrafish embryos, but with limited
`success (Wargelius, et al. Biochem Biophys Res Commun
`263, 156-161 (1999)).
`
`[0006] To date, there has been no report that RNAi can be
`used in mammals and moreover there is a belief in the art
`that RNAi will not function in mammals. In this respect,
`concern has been expressed that the protocols used for
`invertebrate and plant systems are unlikely to be effective in
`mammals (reviewed by Fire (Fire Trends Genet 15, 358-363
`(1999)). This is because accumulation of dsRNA in mam(cid:173)
`malian cells can result in a general block to protein synthe(cid:173)
`sis. The accumulation of very small amounts of double
`stranded RNA ( dsRNA) in mammalian cells following viral
`infection results in the interferon response (Marcus, Inter(cid:173)
`feron 5, 115-180 (1983)) which leads to an overall block to
`translation and the onset of apoptosis (Lee & Esteban
`Virology 199, 491-496 (1994)). Part of the interferon
`response is the activation of a dsRNA responsive protein
`kinase (PKR) (Clemens, Int Biochem Cell Biol 29, 945-949
`(1997)). This enzyme phosphorylates and inactivates trans(cid:173)
`lation factor EIF2a in response to dsRNA. The consequence
`is a global suppression of translation, which in turn triggers
`apoptosis. Wagner & Sun. (Nature 391, 806-811 (1998))
`suggest that RNAi will not work in mammals because it has
`no effect when used as a control in experiments into anti(cid:173)
`sense RNA
`
`[0007] Anti-sense RNA has been attempted as a means of
`reducing gene expression in the embryos of a number of
`species. Whereas it has had considerable success in Droso(cid:173)
`phila, it has been disappointing in Zebrafish, Xenopus and
`mouse embryos. In Xenopus, there were some limitations in
`using the antisense approach. This is thought to be due to a
`prominent RNA melting activity (Bass, & Weintraub, Cell
`48, 607-613 (1987); Rebagliati & Melton, Cell 48, 599-605
`(1987)), exerted by the dsRNA specific adenosine deami(cid:173)
`nase (dsRAD), and suggests that RNAi is not likely to be
`successful.
`
`[0008]
`In the mouse embryo, anti-sense RNA has had
`inconsistent and limited success in reducing gene expres(cid:173)
`sion, particularly between the two-four cell stages (Bevil(cid:173)
`acqua, et al. Proc Natl Acad Sci USA 85,831-835(1988)).
`These authors were concerned that the partial inhibition of
`~-glucuronidase in their experiments might also reflect a
`melting activity acting upon sense/anti-sense duplexes, and
`so they examined the stability of ~-glucuronidase dsRNA
`microinjected into mouse blastomeres. They reported no
`effects on RNA stability, but this was only followed over a
`period of 5 hours. Thus, there is no suggestion in this paper
`that dsRNA can persist in mammalian cells long enough to
`interfere with gene expression. In addition, they reported no
`effects upon the expression of ~-glucuronidase following the
`injection of dsRNA. Thus, this paper does not suggest that
`dsRNA can inhibit gene expression in mammalian cells.
`
`[0009] WO99/32619 suggests that dsRNA can be used to
`inhibit gene expression in mammals. However, the only
`experimental evidence in this document shows that RNAi
`works in C. elegans; there is nothing to show that it could
`work in mammals. Indeed, later publications by the inven(cid:173)
`tors listed for WO99/32619 (Fire, Trends Genet 15, 358-363
`(1999); (Montgomery & Fire, Trends Genet 14, 255-258
`
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`(1998)) state that RNAi could only be made to work in
`mammals if the PKR response could be neutralised or some
`way avoided, although no suggestions are provided in
`WO99/32619 for how this might be achieved. These later
`publications indicate that the inventors of WO99/32619
`themselves believe that RNAi has not yet been ( and cannot
`be) made to work in mammals.
`[0010] Thus, there is a perception in the art that RNAi
`cannot be made to work in mammals. Contrary to this
`perception, the inventors have now shown that is possible to
`interfere with specific gene expression in the mouse oocyte
`and zygote following microinjection of the appropriate
`dsRNA. They have shown experimentally that RNAi can
`phenocopy the effects of disrupting the maternal expression
`of the c-mos gene in the oocyte to overcome the arrest of
`meiosis at metaphase II, or the zygotic expression of E-cad(cid:173)
`herin to prevent development of the blastocyst as observed
`in the corresponding knockout mice. The inventors have
`shown that the injection of a dsRNA is specific to the
`corresponding gene; it does not cause a general translational
`arrest, because embryos continue to develop and no signs of
`cell death can be observed. Thus, they have shown that
`RNAi can be effective in mammalian cells.
`
`SUMMARY OF THE INVENTION
`
`[0011] According to a first aspect of the present invention,
`there is provided a method for inhibiting the expression of
`a target gene in a mammalian cell, the method comprising:
`[0012]
`introducing into the cell an RNA comprising a
`double stranded structure having a nucleotide sequence
`which is substantially identical to at least a part of the target
`gene and which is derived from an endogenous template;
`and
`
`[0013] verifying inhibition of expression of the target
`gene.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`[0014] FIG. 1: MmGFP dsRNA specifically abrogates
`MmGFP expression in MmGFP transgenic embryos (a-c)
`Representative embryos out of 131 embryos obtained from
`eleven different matings between Fl females and MmGFP
`transgenic males. MmGFP transgenic 4-6 cell stage embryos
`(a), morula (b), blastocysts (c). A similar pattern of GFP
`expression was obtained after injection of antisense MmGFP
`RNA (d-t) Representative embryos out of 147 MmGFP
`transgenic embryos that had been injected with MmGFP
`dsRNA at the one cell stage. 4-6 cell stage embryos ( d),
`morula (e), blastocyst (t). (g-i) Representative embryos out
`of 18 MmGFP transgenic embryos that had been injected
`with c-mos dsRNAat the one cell stage. 6 cell stage embryos
`(g), morula (h), blastocyst (i). Scale bars represent 20 µm.
`The shading indicates green fluorescence.
`[0015] FIG. 2: Interference with expression of injected
`synthetic MmGFP mRNA. (a), Wild type morulae injected
`with MmGFP mRNA alone; (b), together with E-Cadherin
`dsRNA; and (c), together with MmGFP dsRNA, at the one
`cell stage. Scale bars represent 20 µm. The shading indicates
`green fluorescence.
`
`[0016] FIG. 3: Injection of E-cadherin dsRNA to the
`zygote reduces E-cadherin expression and perturbs the
`development of the injected embryos. (a), Immunofluores-
`
`cent stammg of E-cadherin in embryos injected at the
`one-cell stage with MmGFP dsRNA, and cultured for four
`days in vitro until the blastocyst stage. (b ), Immunofluores(cid:173)
`cent staining of E-cadherin in embryos injected at the
`one-cell stage with E-cadherin dsRNA, and cultured for four
`days in vitro. Note the altered development of these
`embryos. Scale bars represent 20 µm. (c), Western blot
`analysis of E-cadherin expression in zygotes, uninjected
`morulae ( collected at the one-cell stage and cultured in vitro
`for three days), morulae injected at the one-cell stage with
`2 mg m1- 1 of GFP dsRNA and cultured in vitro for three
`days, morulae injected at the one-cell stage with 2 mg m1- 1
`of E-cadherin dsRNA and cultured in vitro for three days. In
`each case, proteins were extracted from 15 embryos. This
`experiment has been repeated three times with the same
`result. The reduction of signal following E-cadherin dsRNA
`injection was approximately 6.5 fold. Scale bars represent
`20 µm. The shading indicates chemiluminescence.
`[0017] FIG. 4: Injection of c-mos dsRNA in immature
`oocyte inhibits c-mos expression and causes parthenoge(cid:173)
`netic activation. ( a-d) Examples of parthenogenetically acti(cid:173)
`vated eggs obtained after injection of c-mos dsRNA in
`germinal vesicle stage oocytes. (a), Control oocyte arrested
`in metaphase II; (b), one-cell embryo (white arrow points
`out the pronucleus); (c), two-cell embryo; (d), four cell
`embryo. Scale bars represent 20 µm. ( e ), Western blot
`in oocytes arrested
`in
`analysis of c-mos expression
`metaphase II, oocytes injected at the germinal vesicle stage
`with 2 mg m1- 1 of MmGFP dsRNA and cultured in vitro
`during 12 hours, oocytes injected at the germinal vesicle
`stage with 2 mg m1- 1 of c-mos dsRNA and cultured in vitro
`during 12 hours. In each case, proteins were extracted from
`35 oocytes. This experiment has been repeated two times
`with the same result.
`[0018] FIG. 5: Inhibition of gene expression following
`injection of double stranded RNA is restricted to the clonal
`lineage derived from the injected cell. Immunofluoresecent
`staining of E-cadherin in embryos injected in one cell at the
`two cell stage with E-cadherin dsRNA and synthetic mRNA
`for MmGFP. The left hand panels show single channel (red)
`fluorescence to reveal E-Cadherin. Note that the staining is
`markedly reduced in the progeny of the injected cell. These
`progeny cells are identified in the corresponding second
`(green) channels as cells expressing MmGFP.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`[0019] dsRNA useful in accordance with the invention is
`derived from an "endogenous template". Such a template
`may be all or part of a nucleotide sequence endogenous to
`the mammal; it may be a DNA gene sequence or a cDNA
`produced from an mRNA isolated from the mammal, for
`example by reverse transcriptase. When the template is all or
`a part of a DNA gene sequence, it is preferred if it is from
`one or more or all exons of the gene. Additionally, all or part
`of a viral gene may form an endogenous template, if it is
`expressed in the mammal in such a way that the interferon
`response is not induced, e.g. expression from a pro-virus
`integrated into the host cell chromosome. Thus, the dsRNA
`of the present invention is distinguished from viral dsRNA
`and synthetic pol yr IC, both of which have been observed to
`induce PKR which leads to apoptosis in mammalian cells.
`[0020] Whilst the dsRNA is derived from an endogenous
`template, there is no limitation on the manner in which it is
`
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`synthesised. Thus, it may synthesised in vitro or in vivo,
`using manual and/or automated procedures. In vitro synthe(cid:173)
`sis may be chemical or enzymatic, for example using cloned
`RNA polymerase ( e.g., T3, T7, SP6) for transcription of the
`endogenous DNA(or cDNA) template, or a mixture of both.
`
`[0021]
`In vivo, the dsRNA may be synthesised using
`recombinant techniques well known in the art (see e.g.,
`Sambrook, et al., MOLECULAR CLONING; A LABORA(cid:173)
`TORY MANUAL, SECOND EDITION (1989); DNA
`CLONING, VOLUMES I AND II (D. N Glover ed. 1985);
`OLIGONUCLEOTIDE SYNTHESIS (M. J. Gaited, 1984);
`NUCLEIC ACID HYBRIDISATION (B. D. Hames & S. J.
`Higgins eds. 1984); TRANSCRIPTION AND TRANSLA(cid:173)
`TION (B. D. Hames & S. J. Higgins eds. 1984); ANIMAL
`CELL CULTURE (R. I. Freshney ed. 1986); IMMOBIL(cid:173)
`ISED CELLS AND ENZYMES (IRL Press, 1986); B.
`Perbal,APRACTICALGUIDE TO MOLECULAR CLON(cid:173)
`ING (1984); the series, METHODS IN ENzyMOLOGY
`(Academic Press, Inc.); GENE TRANSFER VECTORS
`FOR MAMMALIAN CELLS (J. H. Miller and M. P. Calos
`eds. 1987, Cold Spring Harbor Laboratory), Methods in
`Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and
`Wu, eds., respectively), Mayer and Walker, eds. (1987),
`IMMUNOCHEMICAL METHODS
`IN CELL AND
`MOLECULAR BIOLOGY (Academic Press, London),
`Scopes, (1987), PROTEIN PURIFICATION: PRINCIPLES
`AND PRACTICE, Second Edition (Springer-Verlag, N.Y.),
`and HANDBOOK OF EXPERIMENTAL IMMUNOLOGY,
`VOLUMES I-IV (D. M. Weir and C. C. Blackwell eds
`1986).
`
`[0022] Thus, bacterial cells can be transformed with an
`expression vector which comprises the DNA template from
`which the dsRNAis to be derived. Alternatively, the cells of
`the mammal in which inhibition of gene expression is
`required may be transformed with an expression vector or by
`other means. Bidirectional transcription of one or more
`copies of the template may be by endogenous RNA poly(cid:173)
`merase of the transformed cell or by a cloned RNA poly(cid:173)
`merase (e.g., T3, T7, SP6) coded for by the expression
`vector or a different expression vector. The use and produc(cid:173)
`tion of an expression construct are known in the art (see
`WO98/32016; U.S. Pat. Nos. 5,593,874, 5,698,425, 5712,
`135, 5,789,214, and 5,804,693). Inhibition of gene expres(cid:173)
`sion may be targeted by specific transcription in an organ,
`tissue, or cell type; an environmental condition ( e.g. infec(cid:173)
`tion, stress, temperature, chemical); and/or engineering tran(cid:173)
`scription at a developmental stage or age, especially when
`the dsRNA is synthesised in vivo in the mammal. dsRNA
`may also be delivered to specific tissues or cell types using
`known gene delivery systems. Known eukaryotic vectors
`include pWLNEO, pSV2CAT, pOG44, pXTl and pSG
`available from Stratagene; and pSVK3, pBPV, pMSG and
`pSVL available from Pharmacia. These vectors are listed
`solely by way of illustration of the many commercially
`available and well known vectors that are available to those
`of skill in the art.
`
`[0023]
`If synthesised outside the mammalian cell, the
`RNA may be purified prior to introduction into the cell.
`Purification may be by extraction with a solvent (such as
`phenol/chloroform) or resin, precipitation (for example in
`ethanol), electrophoresis, chromatography, or a combination
`thereof. However, purification may result in loss of dsRNA
`and may therefore be minimal or not carried out at all. The
`
`RNA may be dried for storage or dissolved in an aqueous
`solution, which may contain buffers or salts to promote
`annealing, and/or stabilisation of the RNA strands.
`
`[0024] dsRNA useful in the present invention includes
`dsRNA which contains one or more modified bases, and
`dsRNA with a backbone modified for stability or for other
`reasons. For example, the phosphodiester linkages of natural
`RNA may be modified to include at least one of a nitrogen
`or sulphur heteroatom. Moreover, dsRNA comprising
`unusual bases, such as inosine, or modified bases, such as
`tritylated bases, to name just two examples, can be used in
`the invention. It will be appreciated that a great variety of
`modifications have been made to RNA that serve many
`useful purposes known to those of skill in the art. The term
`dsRNA as it is employed herein embraces such chemically,
`enzymatically or metabolically modified forms of dsRNA,
`provided that it is derived from an endogenous template.
`
`[0025] The double-stranded structure may be formed by a
`single self-complementary RNA strand or two separate
`complementary RNA strands. RNA duplex formation may
`be initiated either inside or outside the mammalian cell.
`
`[0026] The dsRNA comprises a double stranded structure,
`the sequence of which is "substantially identical" to at least
`a part of the target gene. "Identity", as known in the art, is
`the relationship between two or more polynucleotide (or
`polypeptide) sequences, as determined by comparing the
`sequences. In the art, identity also means the degree of
`sequence relatedness between polynucleotide sequences, as
`determined by the match between strings of such sequences.
`Identity can be readily calculated ( Computational Molecular
`Biology, Lesk, A M., ed., Oxford University Press, New
`York, 1988; Biocomputing: Informatics and Genome
`Projects, Smith, D. W., ed., Academic Press, New York,
`1993; Computer Analysis of Sequence Data, Part I, Griffin,
`A. M., and Griffin, H. G., eds., Humana Press, New Jersey,
`1994; Sequence Analysis in Molecular Biology, von Heinje,
`G., Academic Press, 1987; and Sequence Analysis Primer,
`Gribskov, M. and Devereux, J., eds., M Stockton Press, New
`York, 1991). While there exist a number of methods to
`measure identity between two polynucleotide sequences, the
`term is well known to skilled artisans (Sequence Analysis in
`Molecular Biology, von Heinje, G., Academic Press, 1987;
`Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
`eds., M Stockton Press, New York, 1991; and Carillo, H.,
`and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988).
`Methods commonly employed
`to determine
`identity
`between sequences include, but are not limited to those
`disclosed in Carillo, H., and Lipman, D., SIAM J. Applied
`Math., 48:1073 (1988). Preferred methods to determine
`identity are designed to give the largest match between the
`sequences tested. Methods to determine identity are codified
`in computer programs. Computer program methods to deter(cid:173)
`mine identity between two sequences include, but are not
`limited to, GCG program package (Devereux, J., et al.,
`Nucleic Acids Research 12(1): 387 (1984)), BLASTP,
`BLASTN, and PASTA (Atschul, S. F. et al., J. Malec. Biol.
`215: 403 (1990)). Another software package well known in
`the art for carrying out this procedure is the CLUSTAL
`program. It compares the sequences of two polynucleotides
`and finds the optimal alignment by inserting spaces in either
`sequence as appropriate. The identity for an optimal align(cid:173)
`ment can also be calculated using a software package such
`as BLASTx. This program aligns the largest stretch of
`
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`similar sequence and assigns a value to the fit. For any one
`pattern comparison several regions of similarity may be
`found, each having a different score. One skilled in the art
`will appreciate that two polynucleotides of different lengths
`may be compared over the entire length of the longer
`fragment. Alternatively small regions may be compared.
`Normally sequences of the same length are compared for a
`useful comparison to be made.
`
`[0027]
`It is preferred is there is 100% sequence identity
`between the inhibitory RNA and the part of the target gene.
`However, dsRNA having 70%, 80% or greater than 90% or
`95% sequence identity may be used in the present invention,
`and thus sequence variations that might be expected due to
`genetic mutation, strain polymorphism, or evolutionary
`divergence can be tolerated.
`
`[0028] The duplex region of the RNA may have a nucle(cid:173)
`otide sequence that is capable of hybridising with a portion
`of the target gene transcript (e.g., 400 mM NaCl, 40 mM
`PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridisation
`for 12-16 hours; followed by washing).
`
`[0029] Whilst the optimum length of the dsRNA may vary
`according to the target gene and experimental conditions, the
`duplex region of the RNA may be at least 25, 50, 100, 200,
`300, 400 or more bases long.
`
`[0030] As used herein "target gene" generally means a
`polynucleotide comprising a region that encodes a polypep(cid:173)
`tide, or a polynucleotide region that regulates replication,
`transcription or translation or other processes important to
`expression of the polypeptide, or a polynucleotide compris(cid:173)
`ing both a region that encodes a polypeptide and a region
`operably linked thereto that regulates expression. Target
`genes may be cellular genes present in the genome or viral
`and pro-viral genes that do not elicit the interferon response,
`such as retroviral genes. The target gene may be a protein(cid:173)
`coding gene or a non-protein coding gene, such as a gene
`which codes for ribosmal RNAs, splicosomal RNA, tRNAs,
`etc.
`
`[0031]
`It is preferred if the dsRNA is substantially iden(cid:173)
`tical to the whole of the target gene, i.e. the coding portion
`of the gene. However, the dsRNA can be substantially
`identical to a part of the target gene. The size of this part
`depends on the particular target gene and can be determined
`by those skilled in the art by varying the size of the dsRNA
`and observing whether expression of the gene has been
`inhibited.
`
`[0032]
`In the present invention, dsRNA can be used to
`inhibit a target gene which causes or is likely to cause
`disease, i.e. it can be used for the treatment or prevention of
`disease.
`
`[0033]
`In the prevention of disease, the target gene may be
`one which is required for initiation or maintenance of the
`disease, or which has been identified as being associated
`with a higher risk of contracting the disease.
`
`[0034]
`In the treatment of disease, the dsRNA can be
`brought into contact with the cells or tissue exhibiting the
`disease. For example, dsRNAsubstantially identical to all or
`part of a mutated gene associated with cancer, or one
`expressed at high levels in tumour cells, e.g. aurora kinase,
`may be brought into contact with or introduced into a
`cancerous cell or tumour gene. Examples of cancers which
`
`the present invention can be used to prevent or treat include
`solid tumours and leukaemias, including: apudoma, choris(cid:173)
`toma, branchioma, malignant carcinoid syndrome, carcinoid
`heart disease, carcinoma (e.g., Walker, basal cell, basosqua(cid:173)
`mous, Brown-Pearce, ductal, Ehrlich tumour, in situ, Krebs
`2, Merkel cell, mucinous, non-small cell lung, oat cell,
`papillary, scirrhous, bronchiolar, bronchogenic, squamous
`cell, and transitional cell), histiocytic disorders, leukaemia
`( e.g., B cell, mixed cell, null cell, T cell, T-cell chronic,
`HTLV-11-associated,
`lymphocytic
`acute,
`lymphocytic
`chronic, mast cell, and myeloid), histiocytosis malignant,
`Hodgkin disease, immunoproliferative small, non-Hodgkin
`lymphoma, plasmacytoma,
`reticuloendotheliosis, mela(cid:173)
`noma, chondroblastoma, chondroma, chondrosarcoma,
`fibroma, fibrosarcoma, giant cell tumours, histiocytoma,
`lipoma, liposarcoma, mesothelioma, myxoma, myxosar(cid:173)
`coma, osteoma, osteosarcoma, Ewing sarcoma, synovioma,
`adenofibroma, adenolymphoma, carcinosarcoma, chor(cid:173)
`doma, craniopharyngioma, dysgerminoma, hamartoma,
`mesenchymoma, mesonephroma, myosarcoma, ameloblas(cid:173)
`toma, cementoma, odontoma, teratoma, thymoma, tropho(cid:173)
`blastic tumour, adeno-carcinoma, adenoma, cholangioma,
`cholesteatoma, cylindroma, cystadenocarcinoma, cystad(cid:173)
`enoma,
`granulosa
`cell
`tumour,
`gynandroblastoma,
`hepatoma, hidradenoma, islet cell tumour, Leydig cell
`tumour, papilloma, Sertoli cell tumour, theca cell tumour,
`leiomyoma,
`leiomyosarcoma, myoblastoma, mymoma,
`myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependy(cid:173)
`moma, ganglioneuroma, glioma, medulloblastoma, menin(cid:173)
`gioma, neurilemmoma, neuroblastoma, neuroepithelioma,
`neurofibroma, neuroma, paraganglioma, paraganglioma
`nonchromaffin, angiokeratoma, angiolymphoid hyperplasia
`with eosinophilia, angioma sclerosing, angiomatosis, glo(cid:173)
`mangioma, hemangioendothelioma, hemangioma, heman(cid:173)
`giopericytoma, hemangiosarcoma,
`lymphangioma,
`lym(cid:173)
`phangiomyoma,
`lymphangiosarcoma,
`pinealoma,
`carcinosarcoma, chondrosarcoma, cystosarcoma, phyllodes,
`fibrosarcoma, hemangiosarcoma, leimyosarcoma, leukosar(cid:173)
`coma,
`liposarcoma,
`lymphangiosarcoma, myosarcoma,
`myxosarcoma, ovarian carcinoma, rhabdomyosarcoma, sar(cid:173)
`coma (e.g., Ewing, experimental, Kaposi, and mast cell),
`neoplasms (e.g., bone, breast, digestive system, colorectal,
`liver, pancreatic, pituitary, testicular, orbital, head and neck,
`central nervous system, acoustic, pelvic respiratory tract,
`and urogenital), neurofibromatosis, and cervical dysplasia,
`and other conditions in which cells have become immorta(cid:173)
`lised or transformed. The invention could be used in com(cid:173)
`bination with other treatments, such as chemotherapy, cryo(cid:173)
`therapy, hyperthermia, radiation therapy, and the like.
`
`[0035] The present invention may also be used in the
`treatment and prophylaxis of other diseases, especially those
`associated with expression or overexpression of a particular
`gene or genes. For example, expression of genes associated
`with the immune response could be inhibited to treat/prevent
`autoimmune diseases such as rheumatoid arthritis, graft(cid:173)
`versus-host disease, etc. In such treatment, the dsRNA may
`be used in conjunction with immunosuppressive drugs. The
`most commonly used immunosuppressive drugs currently
`include corticosteroids and more potent inhibitors like, for
`instance, methotrexate, sulphasalazine, hydroxychloro(cid:173)
`quine, 6-MP/azathioprine and cyclosporine. All of these
`treatments have severe side-effects related to toxicity, how(cid:173)
`ever, and the need for drugs that would allow their elimi(cid:173)
`nation from, or reduction in use is urgent. Other immuno-
`
`Rigel Exhibit 1018
`Page 10 of 18
`
`

`

`US 2003/0027783 Al
`
`Feb.6,2003
`
`5
`
`suppressive drugs include the gentler, but less powerful
`non-steroid treatments such as Aspirin and Ibuprofen, and a
`new class of reagents which are based on more specific
`immune modulator functions. This latter class includes
`interleukins, cytokines, recombinant adhesion molecules
`and monoclonal antibodies. The use of dsRNA to inhibit a
`gene associated with the immune response in an inmuno(cid:173)
`suppressive treatment protocol could increase the efficiency
`of immunosuppression, and particularly, may enable the
`administered amounts of other drugs, which have toxic or
`other adverse effects to be decreased.
`[0036] The following classes of possible target genes are
`examples of the genes which the present invention may used
`to inhibit: developmental genes (e.g., adhesion molecules,
`cyclin kinase inhibitors, Wnt family members, Pax family
`members, Winged helix family members, Hox family mem(cid:173)
`bers, cytokines/lymphokines and their receptors, growth/
`differentiation factors and their receptors, neurotransmitters
`and their receptors); oncogenes (e.g., ABU, BCLl, BCL2,
`BCL6, CBFA2, CBL, CSFIR, ERBA, ERBE, EBRB2,
`ETSl, ETSl, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN,
`KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLl,
`MYCN, NRAS, PIMl, PML, RET, SRC, TALl, TCL3 and
`YES);
`tumour suppresser genes (e.g., APC, BRCAl,
`BRCA2, MADH4, MCC, NFl, NF2, RBl, TP53 and WTl);
`and enzymes ( e.g., ACP desaturases and hydroxy lases,
`ADP-glucose pyrophorylases, ATPases, alcohol dehydroge(cid:173)
`nases, amylases, amyloglucosidases, catalases, cellulases,
`cyclooxygenases, decarboxylases, dextrinases, DNA and
`RNA polymerases, galactosidases, glucanases, glucose oxi(cid:173)
`dases, GTPases, helicases, hemicellulases, integrases, inver(cid:173)
`tases, isomerases, kinases, lactases, lipases, lipoxygenases,
`lysozymes, pectinesterases, peroxidases, phosphatases,
`phospholipases, phosphorylases, polygalacturonases, pro(cid:173)
`teinases and peptideases, pullanases, recombinases, reverse
`transcriptases, topoisomerases,