Title of Invention

"SMALL INTERFERING RNA MOLECULES FOR INHIBITING EXPRESSION OF SYK KINASE"

Abstract The present invention relates to small interfering RNA (siRNA) molecules for inhibiting expression of Syk kinase in a cell. These siRNA molecules are independent of a liposome and are about 20-23 nucleotides in length. The siRNA molecules comprise two strands the anti-sense strands and sense strands, and at least 1 strand of these siRNA molecules has a 3'overhang of about 1 to 6 nucleotides in length.
Full Text TECHNICAL FIELD
The present invention relates, in general, to Syk kinase and, in particular, to small interfering RNA (siRNA) molecules for inhibiting expression of Syk kinase in a cell.
BACKGROUND
Double stranded RNA has been shown to be a powerful agent for interfering with gene expression in a number of organisms, including C. elegans and Drosophila, as well as plants (Bernstein et al, RNA 7: 1509-2151 (2001), McManus et al, Nat. Rev. Genet. 3: 737-747 (2992), Hutvagner et al, Curr. Opin. Genet. Dev. 12: 225-232 (2002), Zamore, Nat. Struct. Biol. 8 : 746-750 (2001) Tuschl et al, Genes Dev. 13: 3191-3197 (1999)). Early problems in silencing mammalian genes with double stranded RNA arose because the mammalian immune system destroys cells that contain double stranded RNA, through mechanisms such as the interferon response, evolved as defense against invading RNA viruses (Clarke et al, RNA 1: 7-20(1995)). It has been demonstrated, however, that

very short RNA fragments (e.g., 20-23nt in length), designated small interfering RNA (siRNA) , are able to escape the immune response. Thus introduced siRNAs can function well as gene silencing agents in mammalian cells (Elbashir et al, Nature 411:494-49 8 {2001}, Elbashir et al, Genes Dev." 15:188-200 {2001}, Paddison et al, Genes Dev. 16:948-958 (2002), Wianny et al, Nat. Cell Biol. 2:70-75 {2000}).
As it is presently understood, RNA interferanoe (RNAi) involves a multi-step process. Double stranded RNAs are cleaved by the endonuclease Dicer to generate 21-23 nucleotide fragments {siRNA). The siRNA duplex is resolved into 2 single stranded RNAs, one strand being incorporated into a protein-containing complex where it functions as guide RNA to direct cleavage of the target RNA (Schwarz et al, Mol. Cell. 10:537-548 (2002), Zaraore et al, Cell 101:25-33 (2000)), thus silencing a specific genetic message (see also Zeng et al, Proc. Natl. Acad. Sci. 100:9779 (2003)}.
Anti-sense DNA has also been widely used to inhibit gene expression (Roth et al, Annu. Rev. Biomed. Eng. 1:265-297 (1999)). Once inside the cell, anti-sense oligonucleotides (ASO) recognise, then bind tightly to complementary mRNA, thus preventing the mRNA from interacting with the protein translation machinery of the cell.
It has been demonstrated that inhibition of Syk kinase expression by Syk kinase mRNA ASO dramatically diminishes Fey receptor signaling

(Matsuda et al, Molec. Biol, of the Cell 7:1095-1106 (1996)), and that Syk kinase mRNA ASO introduced by aerosol into rat lungs protects against Feγ receptor-induced lung inflammation (Stenton et al, J. Immunol. 169:1028-1036 (2002)).
At least in certain systems, siRNA is more potent and reliable than ASO as an inhibitor of gene expression. The present invention results from studies designed to test the efficacy of siRNA as an inhibitor of Syk kinase expression.
SUMMARY OF THE INVENTION
The present invention relates generally to Syk kinase. In a preferred embodiment, the invention relates to a method of inhibiting Syk kinase expression using small interfering RNA (siRNA) and to therapeutic strategies based on such a method.
Objects and advantages of the present invention will be clear from the description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. The sense strand of each Syk kinase siRNA is the same sequence as the target sequence with the exception of the initial template adenine dimer and terminal overhang uridine dimer. The antisense strand of the siRNA is the reverse complement of the target sequence.

MV062 603). SiRNA can be produced from target DNA and T7 RNA polymerase promoter sequences using PCR based cloning. Following RNA transcription from the target sequence, recombinant Dicer can cleave the transcribed RNAi into 22 bp siRNAs.
Also as indicated above, siRNA molecules suitable for use in the present invention can also be recombinantly produced using methods known in the art. (See references and web sites cited above.) Recombinant technology permits in vivo transcription of siRNAs in mammalian cells. In accordance with this approach, vectors can be used that contain, for example, RNA polymerase III or 06 promoter sequences. Such vectors (including viral vectors and plasmid vectors (such as pSIREN) ) can be used as expression vectors or as shuttle vectors in conjunction with viral systems (e.g., adenoviral or retroviral systems) to introduce siRNA into mammalian cells. Vectors can be engineered to express sense and anti-sense strands of siRNAs that anneal in vivo to produce functional siRNAs. Alternatively, hairpin RNA can be expressed by inserting into a vector the sense strand (e.g., about 20 nt) of the target, followed by a short spacer (e.g., about 4 to about 10 nt), then the antisense strand of the target (e.g., about 20 nt) and, for example, about 5-6 U's as transcription terminator. The resulting RNA transcript folds back to form a stem-loop structure comprising, for example, about a 20 bp stem and about a 10 nt loop with 2-3 U's at the 3' end. (See also Paddison et

1) siKNA-1: Human, bp 296 to bp 316; Mouse and Rat,
bp 3 07 to bp 327.
sense 5' - gaagcccuucaaccggccc UU 3' antisense 3'- UU cuucgggaaguuggccggg 5'
2} siRNA-2: Human, bp 364 to bp 382; Mouse and Rat,
bp 37 5 to bp 393
sense 5' - ccucaucagggaauaugug UU 3'
antisense 3'- UU ggaguagucccuuauacac 5'
Trans fections
SiRNA was introduced into RBL-2H3 cells and into monocytes by transfection. For the
transfections, 5 X 104 RBL-2H3 cells or 1 X 105 monocytes were seeded into each well of a 24-well plate. Twenty-four hours later, the complete medium was replaced with 400 µ1 fresh medium lacking serum and antibiotic and siRNA/Lipofectamine 2000 complex was added to each well. For the RBL cells, the siRNA/Lipofectamine 2000 complex was formed by adding 3 µ1 of siRNA duplex (2 0/µM) and 3 µ1 of LipofcctaminG 2000 to 100 µ1 Opti-mero without serum or antibiotic according to the manufacturer's protocol. For monocytes, the siRNA/Lipofectamine 2000 complex was formed by adding 3µ1 of siRNA duplex (20/µM) and lµ1 of Lipofectamine 2000 to 100 µ1Opti-mem without serum or antibiotic. Cells were incubated at 37°C for 48 hours before examination of kinase protein expression by Western blotting .

then the sequences in rat and mouse Syk kinase mRNA were scanned in order to identify common Syk kinase mRNA target sequences in these species. Two appropriate target sequences were identified:
Targeted region (1} (cDNA): 5'aagaagcccttcaaccggccc; Targeted r eg ion {2) (cDNA); 5'aacctcatcagggaatatgtg.
The target sequences and Syk kinase siRNAs are shown in Figure 1. Duplexed RMA is not highly susceptible to nuclease degradation and the use of deoxynucleotides (thymidine (T) rather than uridine (U)) may affect the stability of the 3' overhang.
EXAMPLE 2 -
BS-24 cells (2xl05) were pre-treated with 3 µ1 of siRMA-1 (control) DNA Target: 5' AAGAAGCCCTTCAACCGGCCC
Sense siRNA 5' - gaagcccmicaaccggcec UU 3 ' Antisense siRNA 3'- UU cuucgggaaguuggccggg 5'
or siRMA-2
DNA Target: 5' AACCTCATCAGGGAATATGTG
sense 5' - ccucaucagggaauaugug UU 3'
antisense 3 - uy ggaguagucccuuauacac 5'
or Syk antisense, with Lipo fectamine 2000 in a 12-well plate for 24 or 48 hr, and stimulated by

Brown Norway rats were sensitized Lo OA i.p. as described {Laberge et al, Am. J. Respir. Crit. Care Med. 151-822 (1995)) and used on day 21 following sensitization.
The siRNA used in this Example is as follows: DNA Target: 5 ' AACCTCATCAGGGAATATGTG
sense 5' - ccucaucagggaauaugug uu 3' antisense 3'- uu ggaguagucccuuauacac 5'
The description siRNA 2M, used in Figures 7 and 8, refers to the above, modified by Dharmacon to provide additional stability. The designation siRNA-2, used in Figures 7 and 8, refers to the above sequence in unmodified form.
SiRNA was used alone without liposome or was used after formation of siRNA/liposome complexes. 1, 2 Dioleoyl-3-trimethyIammonium-propane (DQTAP) /dioleoyl-phosphatidyl-ethanol-amine (DOPE) liposomes were prepared as previously described (see Stenton et al, J. Immunol. 169:102 8 (2 002) and references cited therein). Cationic D0TAP:D0PE liposomes were incubated at a 2.S:1 ratio of the liposome to the siRNA and 125 micrograms of siRNA (with or without liposomes) was administered by-aerosol following nebulization.
The aerosolized administration of Syk kinase-targeted siRNA was as described by Stenton et al, J. Immunol. 164:3790 (2000). . Nine milliters of saline, siRNA or siRNA/liposome were administered per rat by nebulization for 45 min using a Sidestream nebulizer as described in Stenton et al, J. Immunol. 169:1028


We claim:
1. Small interfering RNA (siRNA) molecules for inhibiting expression of Syk kinase in a cell wherein said siRNA molecules are independent of a liposome and wherein said siRNA molecules are about 20-23 nucleotides in length; comprise two strands wherein said two strands are anti-sense strands and sense strands, and at least 1 strand of said siRNA molecules has a 3'overhang of about 1 to 6 nucleotides in length.
2. The siRNA molecules for inhibiting expression of Syk kinase as claimed in claim 1 wherein both strands of said siRNA molecules have 3'overhangs of about 2 to 3 nucleotides in length.
3. The siRNA molecules for inhibiting expression of Syk kinase as claimed in any of the preceding claims wherein said 3'overhangs comprise uridines.
4. The siRNA molecules for inhibiting expression of Syk kinase as claimed in any of the preceding claims wherein said siRNA molecules optionally have a stem loop structure.
5. The siRNA molecules for inhibiting expression of Syk kinase as claimed in claim 4 wherein said 3' overhang is about 2 to 3 nucleotides in length.

6. Small interfering RNA (siRNA) molecules for inhibiting expression of Syk kinase wherein the sense strands of said siRNA molecules
(Sequence Removed)
wherein said siRNA molecules direct cleavage of said Syk kinase mRNA sequence.
7. Small interfering RNA (siRNA molecules) as claimed in any preceding claim for use as a medicament for inhibiting expression of Syk kinase molecule in a cell wherein said siRNA molecules direct cleavage of a target Syk kinase mRNA sequence present in said cell thereby effecting inhibition.

8. The composition comprising the siRNA molecule as claimed in any preceding claim and a carrier or polymer.

Documents:

157-DELNP-2006-Abstract-(02-04-2009).pdf

157-DELNP-2006-Abstract-(16-04-2009).pdf

157-delnp-2006-abstract.pdf

157-delnp-2006-assignment.pdf

157-DELNP-2006-Claims-(02-04-2009).pdf

157-DELNP-2006-Claims-(15-04-2009).pdf

157-delnp-2006-claims.pdf

157-delnp-2006-correspondence-others 1.pdf

157-DELNP-2006-Correspondence-Others-(02-04-2009).pdf

157-DELNP-2006-Correspondence-Others-(09-04-2009).pdf

157-DELNP-2006-Correspondence-Others-(13-04-2009).pdf

157-DELNP-2006-Correspondence-Others-(15-04-2009).pdf

157-DELNP-2006-Correspondence-Others-(16-04-2009).pdf

157-DELNP-2006-Correspondence-Others-(21-12-2009).pdf

157-DELNP-2006-Correspondence-Others-02.04.2009.pdf

157-delnp-2006-correspondence-others.pdf

157-DELNP-2006-Description (Complete)-(02-04-2009).pdf

157-DELNP-2006-Description (Complete)-(16-04-2009).pdf

157-delnp-2006-description (complete).pdf

157-DELNP-2006-Drawings-(02-04-2009).pdf

157-DELNP-2006-Drawings-(08-04-2009).pdf

157-DELNP-2006-Drawings-(16-04-2009).pdf

157-delnp-2006-drawings.pdf

157-DELNP-2006-Form-1-(02-04-2009).pdf

157-delnp-2006-form-1.pdf

157-delnp-2006-form-13.pdf

157-delnp-2006-form-18.pdf

157-DELNP-2006-Form-2-(02-04-2009).pdf

157-delnp-2006-form-2.pdf

157-DELNP-2006-Form-3-(02-04-2009).pdf

157-DELNP-2006-Form-3-(13-04-2009).pdf

157-delnp-2006-form-3.pdf

157-delnp-2006-form-5.pdf

157-delnp-2006-gpa.pdf

157-delnp-2006-pct-101.pdf

157-DELNP-2006-Petition-137-(08-04-2009).pdf


Patent Number 233908
Indian Patent Application Number 157/DELNP/2006
PG Journal Number 18/2009
Publication Date 01-May-2009
Grant Date 21-Apr-2009
Date of Filing 10-Jan-2006
Name of Patentee THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA
Applicant Address UNIVERSITY OF PENNSYLVANIA, CENTRE FOR TECHNOLOGY TRANSFER, 3160 CHESTNUT STREET, SUITE 200, PHILADELPHIA, PA 19104, USA
Inventors:
# Inventor's Name Inventor's Address
1 SCHREIBER, ALAN, D. PHILADELPHIA, PA, USA.
2 INDIK, ZENA PHILADELPHIA, PA, USA
3 KIM, MOO-KYUNG PHILADELPHIA, PA, USA
PCT International Classification Number C07D
PCT International Application Number PCT/US2004/020990
PCT International Filing date 2004-07-01
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/484,299 2003-07-03 U.S.A.