PROTIVA - EXHIBIT 2044
`Moderna Therapeutics, Inc. v. Protiva Biotherapeautics, Inc.
`IPR2018-00739
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`to reduce the expression of certain genes for the treatment of atherosclerosis andits
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`manifestations, e.g., hypercholesterolemia, myocardial infarction, and thrombosis.
`[0007] A safe and effective nucleic acid delivery system is required for RNAi to be
`therapeutically useful. Viral vectors are relatively efficient gene delivery systems, but suffer
`from a variety of limitations, such as the potential for reversion to the wild-type as well as
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`immuneresponse concerns. Asa result, nonviral gene delivery systems are receiving
`increasing attention (Worgall ez al., Human Gene Therapy, 8:37 (1997); Peeters ef al.,
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`Human Gene Therapy, 7:1693 (1996); Yei et al., Gene Therapy, 1:192 (1994); Hope et al.,
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`Molecular Membrane Biology, 15:1 (1998)). Furthermore, viral systems are rapidly cleared
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`from the circulation, limiting transfection to “first-pass” organs such as the lungs, liver, and
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`spleen. In addition, these systems induce immune responses that compromise delivery with
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`subsequent injections.
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`[0008]
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`Plasmid DNA-cationic liposome complexes are currently the most commonly
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`employed nonviral gene delivery vehicles (Felgner, Scientific American, 276:102 (1997);
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`Chonnet al., Current Opinion in Biotechnology, 6:698 (1995)). For instance, cationic
`liposome complexes made of an amphipathic compound,a neutral lipid, and a detergent for
`transfecting insect cells are disclosed in U.S. Patent No. 6,458,382. Cationic liposome
`
`complexesare also disclosed in U.S. Patent Publication No. 20030073640.
`[0009] Cationic liposome complexesare large, poorly defined systemsthat are not suited
`for systemic applications and can elicit considerable toxic side effects (Harrison et al.,
`Biotechniques, 19:816 (1995); Li et al., The Gene, 4:891 (1997); Tam etal, Gene Ther.,
`7:1867 (2000)). As large, positively charged aggregates, lipoplexes are rapidly cleared when
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`administered in vivo, with highest expression levels observed in first-pass organs, particularly
`
`the lungs (Huanget al., Nature Biotechnology, 15:620 (1997); Templeton ef al., Nature
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`Biotechnology, 15:647 (1997); Hofland et al., Pharmaceutical Research, 14:742 (1997)).
`
`[0010] Other liposomaldelivery systems include, for example, the use of reverse micelles,
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`anionic liposomes, and polymer liposomes. Reverse micelles are disclosed in U.S. Patent No.
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`6,429,200. Anionic liposomesare disclosed in U.S. Patent Publication No. 20030026831.
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`Polymer liposomesthat incorporate dextrin or glycerol-phosphocholine polymersare
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`disclosed in U.S. Patent Publication Nos. 20020081736 and 20030082103, respectively.
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`[0011] A gene delivery system containing an encapsulated nucleic acid for systemic
`delivery should be small (i.e., less than about 100 nm diameter) and should remain intact in
`the circulation for an extended period of time in order to achieve delivery to affected tissues.
`
`This requires a highly stable, serum-resistant nucleic acid-containing particle that does not
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`interact with cells and other components of the vascular compartment. The particle should
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`also readily interact with target cells at a disease site in order to facilitate intracellular
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`delivery of a desired nucleic acid.
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`[0012] Recent work has shown that nucleic acids can be encapsulated in small (e.g., about
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`70 nm diameter) “stabilized plasmid-lipid particles” (SPLP) that consist of a single plasmid
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`encapsulated within a bilayer lipid vesicle (Wheeleret al., Gene Therapy, 6:271 (1999)).
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`These SPLPstypically contain the “fusogenic” lipid dioleoylphosphatidylethanolamine
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`(DOPE), low levels of cationic lipid, and are stabilized in aqueous media by the presence ofa
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`poly(ethylene glycol) (PEG) coating. SPLPs have systemic application as they exhibit
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`extended circulation lifetimes following intravenous (i.v.) injection, accumulate preferentially
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`at distal tumor sites due to the enhanced vascular permeability in such regions, and can
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`mediate transgene expressionat these tumorsites. The levels of transgene expression
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`observed at the tumorsite following i.v. injection of SPLPs containing the luciferase marker
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`gene are superiorto the levels that can be achieved employing plasmid DNA-cationic
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`liposome complexes (lipoplexes) or naked DNA.
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`[0013]
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`Thus, there remains a strong need in the art for novel and more efficient methods
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`and compositions for introducing nucleic acids such as siRNA into cells. In addition, there is
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`a need in the art for methods of downregulating the expression of genes ofinterest to treat or
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`prevent diseases and disorders such as cancer and atherosclerosis. The present invention
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`addresses these and other needs.
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`BRIEF SUMMARYOF THE INVENTION
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`[0014]
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`The present invention provides novel, scrum-stable lipid particles comprising one or
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`more active agents or therapeutic agents, methods of making the lipid particles, and methods
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`of delivering and/or administering the lipid particles (e.g., for the treatment of a disease or
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`disorder).
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`[0015]
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`In preferred embodiments, the active agent or therapeutic agent is fully encapsulated
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`within the lipid portion of the lipid particle such that the active agent or therapeutic agent in
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`the lipid particle is resistant in aqueous solution to enzymatic degradation, e.g., by a nuclease ‘
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`or protease. In other preferred embodiments, the lipid particles are substantially non-toxic to
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`mammals such as humans.
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`[0016]
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`In one aspect, the present invention provides lipid particles comprising:
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`(a) one or
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`more active agents or therapeutic agents; (b) one or more cationic lipids comprising from
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`about 50 mol % to about 85 mol % ofthe total lipid present in the particle; (c) one or more
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`non-cationic lipids comprising from about 13 mol % to about 49.5 mol % of the total lipid
`presentin the particle; and (d) one or more conjugated lipids that inhibit aggregation of
`particles comprising from about 0.5 mol % to about 2 mol % ofthetotal lipid present in the
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`particle.
`[0017] Moreparticularly, the present invention provides serum-stable nucleic acid-lipid
`particles (SNALP) comprising a nucleic acid (e.g., one or more interfering RNA molecules
`such as siRNA, aiRNA, and/or miRNA), methods of making the SNALP, and methods of
`delivering and/or administering the SNALP (e.g., for the treatment of a discase or disorder).
`[0018]
`In certain embodiments, the nucleic acid-lipid particle (e.g., SNALP) comprises:
`(a)
`a nucleic acid (e.g., an interfering RNA); (b) a cationic lipid comprising from about 50 mol %
`to about 85 mol % ofthetotal lipid present in the particle; (c) a non-cationic lipid comprising
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`from about 13 mol % to about 49.5 mol % ofthe total lipid present in the particle; and (d) a
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`conjugatedlipid that inhibits aggregation of particles comprising from about 0.5 mol % to
`about 2 mol % ofthe total lipid present in the particle.
`[0019]
`In one preferred embodiment, the nucleic acid-lipid particle (e.g., SNALP)
`comprises:
`(a) an siRNA;(b) a cationic lipid comprising from about 56.5 mol % to about
`66.5 mol % ofthetotal lipid presentin the particle; (c) cholesterol or a derivative thereof
`comprising from about 31.5 mol %to about 42.5 mol % of thetotal lipid present in the
`particle; and (d) a PEG-lipid conjugate comprising from about 1 mol % to about 2 mol % of
`the total lipid present in the particle. This preferred embodiment of nucleic acid-lipid particle
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`is generally referred to herein as the “1:62” formulation.
`[0020]
`In another preferred embodiment, the nucleic acid-lipid particle (e.g., SNALP)
`comprises:
`(a) an siRNA; (b) a cationic lipid comprising from about 52 mol % to about 62
`mol % ofthe total lipid present in the particle; (c) a mixture of a phospholipid and cholesterol
`or a derivative thereof comprising from about 36 mol % to about 47 mol % ofthetotal lipid
`presentin the particle; and (d) a PEG-lipid conjugate comprising from about | mol % to
`about 2 mol % ofthe total lipid present in the particle. This preferred embodiment of nucleic
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`acid-lipid particle is generally referred to herein as the “1:57” formulation.
`[0021]
`‘The present invention also provides pharmaceutical compositions comprising a lipid
`particle described herein (e.g., SNALP) and a pharmaceutically acceptable carrier.
`[0022]
`In anotheraspect, the present invention provides methods for introducing an active
`agent or therapeutic agent (e.g., nucleic acid) into a cell, the method comprising contacting
`the cell with a lipid particle described herein such as a nucleic acid-lipid particle (e.g.,
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`SNALP).
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`[0023]
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`In yet another aspect, the present invention provides methodsfor the in vivo
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`delivery of an active agent or therapeutic agent (e.g., nucleic acid), the method comprising
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`administering to a mammalian subject a lipid particle described herein such as a nucleic acid-
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`lipid particle (e.g, SNALP).
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`[0024]
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`Ina further aspect, the present invention provides methods for treating a disease or
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`disorder in a mammalian subject in need thereof, the method comprising administering to the
`mammalian subject a therapeutically effective amount ofa lipid particle described herein
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`suchas a nucleic acid-lipid particle (e¢.g., SNALP).
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`[0025] Other objects, features, and advantages of the present invention will be apparent to
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`one of skill in the art from the following detailed description and figures.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`(0026]
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`Figure 1 illustrates data demonstrating the activity of 1:57 SNALP containing Eg5
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`siRNA in a human colon cancercell line.
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`[0027]
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`Figure 2 illustrates data demonstrating the activity of 1:57 SNALP containing ApoB
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`siRNA following intravenous administration in mice.
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`[0028]
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`Figure 3 illustrates additional data demonstrating the activity of 1:57 SNALP
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`containing ApoB siRNA following intravenous administration in mice. Each bar represents
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`the group mean offive animals, Error bars indicate the standard deviation.
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`[0029]
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`Figure 4 illustrates data demonstrating the activity of 1:57 and 1:62 SNALP
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`containing ApoB siRNA following intravenous administration in mice.
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`[0030]
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`Figure 5 illustrates data demonstrating the activity of 1:62 SNALP containing ApoB
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`siRNA following intravenous administration in mice.
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`[0031]
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`Figure 6 illustrates data demonstrating that the tolerability of 1:57 SNALP
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`containing ApoB siRNAprepared bycitrate buffer versus PBS direct dilution did not differ
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`significantly in terms of blood clinical chemistry parameters.
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`[0032]
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`Figure 7 illustrates data demonstrating that the efficacy of 1:57 SNALP containing
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`ApoB siRNAprepared by gear pump wassimilar to the same SNALPprepared by syringe
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`press.
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`{0033]
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`Figure 8 illustrates data demonstrating that there was very little effect on body
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`weight 24 hours after administration of 1:57 SNALP containing ApoB siRNA.
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`[0034]
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`Figure 9 illustrates data demonstrating that there were no obvious changesin
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`platelet count after administration of 1:57 SNALP containing ApoB siRNA.
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`[0035]
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`Figure 10 illustrates data demonstrating that clinically significant liver enzyme
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`elevations (3xULN) occurred at particular drug dosages of 1:57 SNALP containing ApoB
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`siRNA.
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`[0036]
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`Figure 11 illustrates data demonstrating that the potency of the lowerlipid:drug
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`(L:D) 1:57 SNALPcontaining ApoB siRNA wasas goodasthat of the higher L:D SNALPat
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`the tested drug dosages.
`[0037]
`Figure 12 illustrates data demonstrating that ApoB protein and total cholesterol]
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`levels were reduced to a similar extent by 1:57 SNALP containing ApoB siRNAata 6:1
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`input L:D ratio (final ratio of 7:1) and 1:57 SNALPat a 9:1 input L:D ratio (finalratio of
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`10:1).
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`[0038]
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`Figure 13 illustrates data demonstrating that a treatment regimen of 1:57 SNALP
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`with siRNAtargeting PLK-1 is well tolerated with no apparent signs of treatment related
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`toxicity in mice bearing Hep3B liver tumors.
`[0039]
`Figure 14 illustrates data demonstrating that treatment with 1:57 SNALPcontaining
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`PLK-1 siRNA caused a significant increase in the survival of Hep3B tumor-bearing mice.
`[0040]
`Figure 15 illustrates data demonstrating that treatment with 1:57 SNALP containing
`PLK-1 siRNA reduced PLK-1 mRNAlevels by 50% in intrahepatic Hep3B tumors growing
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`in mice 24 hours after SNALP administration.
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`[0041]
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`Figure 16 illustrates data demonstrating that a specific cleavage product of PLK-1
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`mRNA wasdetectable by 5’ RACE-PCRin mice treated with 1:57 SNALP containing PLK-1
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`siRNA. 10 ul PCR product/well were loaded onto a 1.5% agarose gel. Lane Nos.:
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`(1)
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`molecular weight (MW) marker; (2) PBS mouse 1; (3) PBS mouse 2; (4) PBS mouse3; (5)
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`Luc SNALP mouse1; (6) Luc SNALP mouse2; (7) PLK SNALP mouse 1; (8) PLK SNALP
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`mouse 2; (9) PLK SNALP mouse 3; and (10) no template control.
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`[0042]
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`Figure 17 illustrates data demonstrating that control (Luc) 1:57 SNALP-treated
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`mice displayed normal mitoses in Hep3B tumors (top panels), whereas mice treated with 1:57
`SNALPcontaining PLK-1 siRNA exhibited numerous aberrant mitoses and tumorcell
`apoptosis in Hep3B tumors (bottom panels).
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`[0043]
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`Figure 18 illustrates data demonstrating that multiple doses of 1:57 PLK-1 SNALP
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`containing PEG-cDSA induced the regression of established Hep3B subcutaneous(S.C.)
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`tumors.
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`[0044]
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`Figure 19 illustrates data demonstrating PLK-1 mRNAsilencing using 1:57 PLK
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`SNALPin S.C. Hep3B tumors following a single intravenous SNALP administration.
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`[0045]
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`Figure 20 illustrates data demonstrating that PLK-1 PEG-cDSA SNALPinhibited
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`the growth of large S.C. Hep3B tumors.
`[0046]
`Figure 21 illustrates data demonstrating tumor-derived PLK-1 mRNAsilencing in
`Hep3B intrahepatic tumors.
`[0047]
`Figure 22 illustrates data demonstrating the blood clearance profile of 1:57 PLK-1
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`SNALPcontaining either PEG-cDMA or PEG-cDSA.
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`DETAILED DESCRIPTION OF THE INVENTION
`
`I.
`
`Introduction
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`The present invention is based, in part, upon the surprising discovery that lipid
`[0048]
`particles comprising from about 50 mol % to about 85 mol % of a cationic lipid, from about
`13 mol % to about 49.5 mol % of a non-cationic lipid, and from about 0.5 mol % to about 2
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`mol % of a lipid conjugate provide advantages whenusedfor the in vitro or in vivo delivery
`of an active agent, such as a therapeutic nucleic acid (e.g., an interfering RNA). In particular,
`as illustrated by the Examples herein, the present invention provides stable nucleic acid-lipid
`particles (SNALP) that advantageously impart increased activity of the encapsulated nucleic
`acid (e.g., an interfering RNA such as siRNA) and improvedtolerability of the formulations
`in vivo, resulting in a significant increase in the therapeutic index as compared to nucleic
`acid-lipid particle compositions previously described. Additionally, the SNALP ofthe
`inventionare stable in circulation, e.g., resistant to degradation by nucleases in serum, and
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`are substantially non-toxic to mammals such as humans. As a non-limiting example, Figure 3
`of Example 4 shows that one SNALP embodimentof the invention (“1:57 SNALP”) was
`more than 10 times as efficacious as compared to a nucleic acid-lipid particle previously
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`described (“2:30 SNALP”) in mediating target gene silencing at a 10-fold lower dose.
`Similarly, Figure 2 of Example 3 showsthat the “1:57 SNALP” formulation was substantially
`moreeffective at silencing the expression of a target gene as comparedto nucleic acid-lipid
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`particles previously described (“2:40 SNALP”).
`[0049]
`In certain embodiments, the present invention provides improved compositions for
`the delivery of interfering RNA such as siRNA molecules. In particular, the Examples herein
`illustrate that the improvedlipid particle formulations of the invention are highly effective in
`downregulating the mRNAand/or protein levels of target genes. Furthermore, the Examples
`herein illustrate that the presence of certain molar ratios of lipid componentsresults in
`improved or enhancedactivity of these lipid particle formulations of the present invention.
`Forinstance, the “1:57 SNALP”and “1:62 SNALP”formulations described herein are
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`exemplary formulations of the present invention that are particularly advantageous because
`they provide improved efficacy andtolerability in vivo, are serum-stable, are substantially
`non-toxic, are capable of accessing extravascular sites, and are capable of reaching target ccll
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`populations.
`[0050]
`Thelipid particles and compositions of the present invention may be used for a
`variety of purposes, including the delivery of associated or encapsulated therapeutic agents to
`cells, both in vitro and in vivo. Accordingly, the present invention provides methodsfor
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`treating diseases or disorders in a subject in need thereof, by contacting the subject with a
`lipid particle described herein comprising one or more suitable therapeutic agents.
`[0051] Various exemplary embodimentsofthe lipid particles of the invention, as well as
`compositions and formulations comprising the same, and their use to deliver therapeutic
`agents and modulate target gene and protein expression, are described in further detail below.
`
`II.
`
`Definitions
`
`[0052] As used herein, the following terms have the meaningsascribed to them unless
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`specified otherwise.
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`[0053] The term “interfering RNA” or “RNAi”or “interfering RNA sequence” refers to
`single-stranded RNA (e.g., mature miRNA)or double-stranded RNA (7.e., duplex RNA such
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`as siRNA, aiRNA,or pre-miRNA)that is capable of reducing or inhibiting the expression of
`a target gene or sequence(e.g., by mediating the degradation or inhibiting the translation of
`mRNAswhich are complementary to the interfering RNA sequence) whenthe interfering
`RNAis in the samecell as the target gene or sequence. Interfering RNA thus refers to the
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`single-stranded RNA that is complementary to a target mRNA sequenceorto the double-
`stranded RNA formed by two complementary strands or by a single, sclf-complementary
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`strand. Interfering RNA may have substantial or complete identity to the target gene or
`sequence, or may comprise a region of mismatch(7.e., a mismatch motif). The sequence of
`the interfering RNA can correspondto the full-length target gene, or a subsequence thereof.
`[0054]
`Interfering RNAincludes “small-interfering RNA”or“siRNA,”e.g., interfering
`RNAofabout 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-
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`30, 15-25, or 19-25 (duplex) nucleotides in length, and is preferably about 20-24, 21-22, or
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`21-23 (duplex) nucleotides in length (e.g., each complementary sequence of the double-
`stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length,
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`preferably about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded
`siRNAis about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferably
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`about 18-22, 19-20, or 19-21 base pairs in length). siRNA duplexes may comprise 3’
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`overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and 5’
`phosphate termini. Examples of siRNA include, without limitation, a double-stranded
`polynucleotide molecule assembled from two separate stranded molecules, wherein onc
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`strand is the sense strand and the other is the complementary antisense strand; a double-
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`stranded polynucleotide molecule assembled from a single stranded molecule, where the
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`sense and antisense regionsare linked by a nucleic acid-based or non-nucleic acid-based
`linker; a double-stranded polynucleotide molecule with a hairpin secondary structure having
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`self-complementary sense and antisense regions; and a circular single-stranded
`polynucleotide molecule with two or more loop structures and a stem having self-
`complementary sense and antisense regions, where the circular polynucleotide can be
`processed in vivo or in vitro to generate an active double-stranded siRNA molecule.
`[0055]
`Preferably, siRNA are chemically synthesized. siRNA can also be generated by
`cleavage of longer dsRNA(e.g., dsRNA greater than about 25 nucleotides in length) with the
`E. coli RNase IU or Dicer. These enzymes process the dsRNAinto biologically active
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`siRNA(see, e.g., Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegari et
`al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom er al., Ambion TechNotes, 10(1):4-6
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`(2003); Kawasaki et al., Nucleic Acids Res., 31:981-987 (2003); Knight et al., Science,
`293:2269-2271 (2001); and Robertsonet al., J. Biol. Chem., 243:82 (1968)). Preferably,
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`dsRNAareat least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotides in length.
`
`A dsRNA maybeas long as 1000, 1500, 2000, 5000 nucleotides in length, or longer. The
`dsRNAcan encodefor an entire gene transcript or a partial gene transcript. In certain
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`instances, siRNA may be encodedby a plasmid (e.g., transcribed as sequences that
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`automatically fold into duplexes with hairpin loops).
`[0056] As used herein, the term “mismatch motif” or “mismatch region”refers to a portion
`of an interfering RNA (e.g., sIRNA, aiRNA, miRNA) sequencethat does not have 100 %
`complementarityto its target sequence. An interfering RNA may haveat least one, two,
`three, four, five, six, or more mismatch regions. The mismatch regions may be contiguous or
`maybe separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides. The mismatch
`motifs or regions may comprise a single nucleotide or may comprise two, three, four, five, or
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`more nucleotides.
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`[0057] An “effective amount”or “therapeutically effective amount” of an active agent or
`therapeutic agent such as an interfering RNA is an amountsufficient to produce the desired
`effect, e.g., an inhibition of expression of a target sequence in comparison to the normal
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`expression level detected in the absence of an interfering RNA. Inhibition of expression ofa
`target gene or target sequence is achieved whenthe value obtained with an interfering RNA
`relative to the control is about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%,
`
`A0%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring
`expressionof a target gene ortarget sequenceinclude, e.g., examination of protein or RNA
`levels using techniques known to those ofskill in the art such as dot blots, northern blots, in
`situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic
`assays knownto those ofskill in the art.
`[0058]
`By “decrease,”“decreasing,” “reduce,”or “reducing” of an immune response by an
`interfering RNAis intended to mean a detectable decrease of an immune response to a given
`interfering RNA (e.g., a modified interfering RNA). The amountof decrease of an immune
`response by a modified interfering RNA may be determinedrelative to the level of an
`immuneresponse in the presence of an unmodified interfering RNA. A detectable decrease
`can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
`75%, 80%, 85%, 90%, 95%, 100%, or more lower than the immune response detected in the
`presence of the unmodified interfering RNA. A decrease in the immune response to
`interfering RNAis typically measured by a decrease in cytokine production (e.g., IFNy,
`IFNa, TNFa, IL-6, or IL-12) by a respondercell in vitro or a decrease in cytokine production
`in the sera of amammalian subject after administration of the interfering RNA.
`[0059] As used herein, the term “respondercell” refers to a cell, preferably a mammalian
`cell, that produces a detectable immune response when contacted with an immunostimulatory
`interfering RNA such as an unmodified siRNA. Exemplary respondercells include, e.g.,
`dendritic cells, macrophages, peripheral blood mononuclear cells (PBMCs), splenocytes, and
`the like. Detectable immune responsesinclude, e.g., production of cytokines or growth
`factors such as TNF-a, IFN-a, IFN-B, IFN-y, IL-1, 0L-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12,
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`IL-13, TGF, and combinations thereof.
`[0060]
`“Substantial identity” refers to a sequence that hybridizes to a reference sequence
`under stringent conditions, or to a sequence that has a specificd percent identity over a
`
`specified region of a reference sequence.
`[0061]
`‘The phrase “‘stringent hybridization conditions” refers to conditions under which a
`nucleic acid will hybridize to its target sequence, typically in a complex mixture ofnucleic
`acids, but to no other sequences. Stringent conditions are sequence-dependent and will be
`different in different circumstances. Longer sequences hybridize specifically at higher
`temperatures. An extensive guide to the hybridization of nucleic acids is foundin Tijssen,
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`Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes,
`“Overview of principles of hybridization and the strategy of nucleic acid assays” (1993).
`
`Generally, stringent conditions are selected to be about 5-10°C lower than the thermal
`melting point (T,,) for the specific sequence at a defined ionic strength pH. The Ty is the
`temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of
`the probes complementary to the target hybridize to the target sequence at equilibrium (as the
`target sequencesare present in excess, at T;,, 50% of the probes are occupied at equilibrium).
`Stringent conditions may also be achieved with the addition of destabilizing agents such as
`formamide. For selective or specific hybridization, a positive signal is at least two times
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`background, preferably 10 times background hybridization.
`[0062]
`Exemplary stringent hybridization conditions can be as follows: 50% formamide,
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`5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with
`washin 0.2x SSC, and 0.1% SDS at 65°C. For PCR, a temperature of about 36°C is typical
`
`for low stringency amplification, although annealing temperatures may vary between about
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`32°C and 48°C depending on primer length. For high stringency PCR amplification, a
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`temperature of about 62°Cis typical, although high stringency annealing temperatures can
`
`range from about 50°C to about 65°C, depending on the primer length and specificity.
`Typical cycle conditions for both high and low stringency amplifications include a
`
`denaturation phase of 90°C-95°C for 30 sec.-2 min., an annealing phase lasting 30 sec.-2
`
`min., and an extension phase of about 72°C for 1-2 min. Protocols and guidelines for low
`and high stringency amplification reactions are provided, e.g., in Innis et al., PCR Protocols,
`A Guide to Methods and Applications, Academic Press, Inc. N.Y. (1990).
`[0063] Nucleic acids that do not hybridize to each other under stringent conditionsarestill
`substantially identical if the polypeptides which they encodeare substantially identical. This
`occurs, for example, when a copy of a nucleic acid is created using the maximum codon
`degeneracy permitted by the genetic code. In such cases, the nucleic acidstypically hybridize
`under moderately stringent hybridization conditions. Exemplary “moderately stringent
`hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl,
`
`1% SDS at 37°C, and a wash in 1X SSC at 45°C. A positive hybridization is at least twice
`background. Those ofordinary skill will readily recognize that alternative hybridization and
`wash conditions can be utilized to provide conditions of similar stringency. Additional
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`guidelines for determining hybridization parameters are provided in numerousreferences,
`
`e.g,, Current Protocols in Molecular Biology, Ausubel et al., eds.
`
`[0064]
`
`The terms “substantially identical” or “substantial identity,” in the context of two or
`
`more nucleic acids, refer to two or more sequences or subsequences that are the same or have
`
`a specified percentage of nucleotides that are the same (i.e., at least about 60%, preferably at
`least about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region), when
`
`compared and aligned for maximum correspondence over a comparison window,or
`
`designated region as measured using one of the following sequence comparison algorithms or
`by manual alignment and visual inspection. This definition, when the context indicates,also
`refers analogously to the complement of a sequence. Preferably, the substantial identity
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`exists over a region that is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60
`
`nucleotides in length.
`
`[0065]
`
`For sequence comparison, typically one sequenceacts as a reference sequence, to
`
`which test sequences are compared. When using a sequence comparison algorithm, test and
`
`reference sequences are entered into a computer, subsequence coordinates are designated, if
`
`necessary, and sequence algorithm program parameters are designated. Default program
`
`parameters can be used,or alternative parameters can be designated. The sequence
`
`comparisonalgorithm then calculates the percent sequence identities for the test sequences
`
`relative to the reference sequence, based on the program parameters.
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`[0066] A “comparison window,”as used herein, includes reference to a segment of any one
`
`of a numberof contiguous positions selected from the group consisting of from about 5 to
`
`about 60, usually about 10 to about 45, more usually about 15 to about 30, in which a
`
`sequence may be comparedto a reference sequence of the same numberof contiguous
`
`positions after the two sequencesare optimally aligned. Methods of alignment of sequences
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`
`for comparison are well knownin the art. Optimal alignment of sequences for comparison
`
`can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl.
`
`Math., 2:482 (1981), by the homologyalignmentalgorithm of Necdleman and Wunsch,J.
`
`Mol. Biol., 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc.
`
`Natl. Acad, Sci. USA, 85:2444 (1988), by computerized implementations of these algorithms
`
`30
`
`(GAP, BESTFIT, FASTA, and TFASTAin the Wisconsin Genetics Software Package,
`
`Genetics Computer Group, 575 Science Dr., Madison, WD), or by manual alignment and
`visual inspection (see, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds.
`
`(1995 supplement)).
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`

`[0067] A preferred example of algorithmsthat are suitable for determining percent
`sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which
`are described in Altschul et al., Nuc. Acids Res., 25:3389-3402 (1977) and Altschulet al., J.
`
`Mol. Biol., 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the
`parameters described herein, to determine percent sequence identity for the nucleic acids of
`the invention. Software for performing BLASTanalysesis publicly available through the
`
`National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
`
`The BLASTalgorithm also performs a statistical analysis of the similarity between
`[0068]
`two sequences(see, ¢.g., Karlin and Altschul, Proc, Natl. Acad. Sci, USA, 90:5873-5787
`(1993)). One measureof similarity provided by the BLASTalgorithm is the smallest sum
`probability (P(N)), which provides an indication of the probability by which a match between
`two nucleotide sequences would occur by chance. For example, a nucleic acid is considered
`similar to a reference sequence if the smallest sum probability in a comparison ofthe test
`
`nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than
`
`about 0.01, and most preferably less than about 0.001.
`[0069]
`The term “nucleic acid” as used herein refers to a polymer containingat least two
`deoxyribonucleotides or ribonucleotidesin either single- or double-stranded form and
`includes DNA and RNA. DNA maybein the form of, e.g., antisense molecules, plasmid
`DNA,pre-condensed DNA, a PCRproduct, vectors (PI, PAC, BAC, YAC, artificial
`chromosomes), expression cassettes, chimeric sequences, chromosomal DNA,or derivatives
`
`and combinations of these groups. RNA may be in the form of siRNA, asymmetrical
`
`interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA,viral RNA
`
`(vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known
`nucleotide analogs or modified backboneresidues or linkages, which are synthetic, naturally
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`i5
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`occurring, and non-naturally occurring, and which have similar binding properties as the
`
`reference nucleic acid. Examples of such analogs include, without limitation,
`
`phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2’-
`O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically limited, the
`term encompasses nucleic acids containing known analogues of natural nucleotides that have
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`
`similar binding propert