PROTIVA - EXHIBIT 2043
`Moderna Therapeutics, Inc. v. Protiva Biotherapeautics, Inc.
`IPR2018-00739
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`[0007] A safe andeffective nucleic acid delivery system is required for RNAito be
`therapeutically useful. Viral vectors are relatively efficient gene delivery systems, but suffer
`from a variety oflimitations, such as the potential for reversion to the wild-type as well as
`immune response concerns. Asa result, nonviral gene delivery systems are receiving
`increasing attention (Worgall et al., Human Gene Therapy, 8:37 (1997); Peeterset al.,
`Human Gene Therapy, 7:1693 (1996); Yeiet al., Gene Therapy, 1:192 (1994); Hopeet al.,
`Molecular Membrane Biology, 15:1 (1998)). Furthermore, viral systemsare rapidly cleared
`from the circulation, limiting transfection to “first-pass” organs such as the lungs,liver, and
`spleen. In addition, these systems induce immune responses that compromise delivery with
`subsequent injections.
`[0008]
`Plasmid DNA-cationic liposome complexesare currently the most commonly
`employed nonviral gene delivery vehicles (Felgner, Scientific American, 276:102 (1997);
`Chonn et 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 complexes are large, poorly defined systemsthat are not suited
`for systemic applications and can elicit considerable toxic side effects (Harrison ef al.,
`Biotechniques, 19:816 (1995); Li et al., The Gene, 4:891 (1997); Tam et al, Gene Ther.,
`7:1867 (2000)). As large, positively charged aggregates, lipoplexes are rapidly cleared when
`administered in vivo, with highest expression levels observedin first-pass organs, particularly
`the lungs (Huanget al., Nature Biotechnology, 15:620 (1997); Templetonet al., Nature
`Biotechnology, 15:647 (1997); Hoflandet al., Pharmaceutical Research, 14:742 (1997)).
`[0010] Other liposomal delivery systems include, for example, the use of reverse micelles,
`anionic liposomes, and polymer liposomes. Reverse micelles are disclosed in U.S. Patent No.
`6,429,200. Anionic liposomesare disclosed in U.S. Patent Publication No. 20030026831.
`Polymer liposomes that incorporate dextrin or glycerol-phosphocholine polymers are
`disclosed in U.S. Patent Publication Nos. 20020081736 and 20030082103, respectively.
`[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 oftime in order to achieve delivery to affected tissues.
`This requires a highly stable, serum-resistant nucleic acid-containing particle that does not
`interact with cells and other componentsof 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.
`[0012] Recent work has shown that nucleic acids can be encapsulated in small (e.g., about
`70 nm diameter) “stabilized plasmid-lipid particles” (SPLP) that consist of a single plasmid
`encapsulated within a bilayer lipid vesicle (Wheeler ef al., Gene Therapy, 6:271 (1999)).
`These SPLPstypically contain the “fusogenic”lipid dioleoylphosphatidylethanolamine
`(DOPE), low levels of cationic lipid, and are stabilized in aqueous media by the presence ofa
`poly(ethylene glycol) (PEG) coating. SPLPs have systemic application as they exhibit
`extended circulation lifetimes following intravenous(i.v.) injection, accumulate preferentially
`at distal tumor sites due to the enhanced vascular permeability in such regions, and can
`mediate transgene expression at these tumorsites. The levels of transgene expression
`observedat the tumorsite following i.v. injection of SPLPs containing the luciferase marker
`gene are superiorto the levels that can be achieved employing plasmid DNA-cationic
`liposome complexes (lipoplexes) or naked DNA.
`[0013] Thus, there remains a strong need in the art for novel and more efficient methods
`and compositions for introducing nucleic acids such as siRNAinto cells. In addition, there is
`a need in the art for methods of downregulating the expression of genesofinterest to treat or
`prevent diseases and disorders such as cancer and atherosclerosis. The present invention
`addresses these and other needs.
`
`BRIEF SUMMARYOF THE INVENTION
`[0014] The present invention provides novel, serum-stable lipid particles comprising one or
`more active agents or therapeutic agents, methods of making the lipid particles, and methods
`of delivering and/or administering the lipid particles (e.g., for the treatment of a disease or
`disorder).
`[0015]
`In preferred embodiments,the active agent or therapeutic agent is fully encapsulated
`within the lipid portion of the lipid particle such that the active agent or therapeutic agent in
`the lipid particle is resistant in aqueous solution to enzymatic degradation, e.g., by a nuclease
`or protease. In other preferred embodiments,the lipid particles are substantially non-toxic to
`mammals such as humans.
`(a) one or
`[0016]
`In one aspect, the present invention provideslipid particles comprising:
`more active agents or therapeutic agents; (b) one or morecationiclipids comprising from
`about 50 mol % to about 85 mol % ofthe total lipid present in the particle; (c) one or more
`non-cationic lipids comprising from about 13 mol % to about 49.5 mol % of the total lipid
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`present in the particle; and (d) one or more conjugated lipids that inhibit aggregation of
`particles comprising from about 0.5 mol % to about 2 mol % ofthe total lipid present in the
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`particle.
`[0017] More particularly, the present invention provides serum-stable nucleic acid-lipid
`particles (SNALP) comprising a nucleic acid (¢.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 disease or disorder).
`[0018]
`In certain embodiments, the nucleic acid-lipid particle (¢.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
`from about 13 mol % to about 49.5 mol % of the total lipid present in the particle; and (d) a
`conjugatedlipid that inhibits aggregation of particles comprising from about0.5 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 present in the particle; (c) cholesterol or a derivative thereof
`comprising from about 31.5 mol % to about 42.5 mol % of the total 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
`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
`mo! % ofthetotal lipid present in the particle; (c) a mixture ofa phospholipid and cholesterol
`or a derivative thereof comprising from about 36 mol % to about 47 mol % of the total lipid
`present in 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
`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 (¢.g., SNALP) and a pharmaceutically acceptable carrier.
`[0022]
`In anotheraspect, the present invention provides methods for introducing an active
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`agent or therapeutic agent (e.g., nucleic acid) intoacell, 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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`In yet another aspect, the present invention provides methods for the in vivo
`[0023]
`delivery of an active agentor therapeutic agent (e.g., nucleic acid), the method comprising
`administering to a mammalian subject a lipid particle described herein such as a nucleic acid-
`
`lipid particle (e.g., SNALP).
`[0024]
`Ina further aspect, the present invention provides methodsfor treating a disease or
`disorder in a mammalian subject in need thereof, the method comprising administering to the
`mammalian subject a therapeutically effective amountofa lipid particle described herein
`such as a nucleic acid-lipid particle (e.g., SNALP).
`[0025] Other objects, features, and advantages of the present invention will be apparent to
`one ofskill in the art from the following detailed description andfigures.
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`BRIEF DESCRIPTION OF THE DRAWINGS
`Figure | illustrates data demonstrating the activity of 1:57 SNALPcontaining Eg5
`[0026]
`siRNA in a human colon cancercell line.
`[0027]
`Figure 2 illustrates data demonstrating the activity of 1:57 SNALP containing ApoB
`siRNA following intravenous administration in mice.
`[0028]
`Figure 3 illustrates additional data demonstrating the activity of 1:57 SNALP
`containing ApoB siRNA following intravenous administration in mice. Each bar represents
`the group meanoffive animals. Error bars indicate the standard deviation.
`[0029]
`Figure 4 illustrates data demonstrating the activity of 1:57 and 1:62 SNALP
`containing ApoB siRNA following intravenous administration in mice.
`[0030]
`Figure 5 illustrates data demonstrating the activity of 1:62 SNALP containing ApoB
`siRNA following intravenous administration in mice.
`
`[0031] Figure6illustrates data demonstrating that the tolerability of 1:57 SNALP
`containing ApoB siRNA prepared by citrate buffer versus PBSdirect dilution did not differ
`significantly in terms of blood clinical chemistry parameters.
`[0032]
`Figure 7 illustrates data demonstrating that the efficacy of 1:57 SNALP containing
`ApoB siRNAprepared by gear pump wassimilar to the same SNALPprepared by syringe
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`press.
`Figure 8 illustrates data demonstrating that there wasverylittle effect on body
`[0033]
`weight 24 hours after administration of 1:57 SNALPcontaining ApoB siRNA.
`[0034]
`Figure 9 illustrates data demonstrating that there were no obvious changes in
`platelet count after administration of 1:57 SNALPcontaining ApoB siRNA.
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`Figure 10 illustrates data demonstrating that clinically significant liver enzyme
`[0035]
`elevations (3xULN) occurredat particular drug dosages of 1:57 SNALP containing ApoB
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`siRNA.
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`Figure 11 illustrates data demonstrating that the potency of the lower lipid:drug
`[0036]
`(L:D) 1:57 SNALP containing ApoB siRNA wasas good asthat 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
`levels were reduced to a similar extent by 1:57 SNALP containing ApoB siRNAat a 6:1
`input L:D ratio (final ratio of 7:1) and 1:57 SNALPat a 9:1 input L:D ratio (final ratio of
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`10:1).
`Figure 13 illustrates data demonstrating that a treatment regimen of 1:57 SNALP
`[0038]
`with siRNAtargeting PLK-1 is well tolerated with no apparentsignsof treatmentrelated
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`toxicity in mice bearing Hep3B liver tumors.
`[0039]
`Figure 14 illustrates data demonstrating that treatment with 1:57 SNALP containing
`PLK-1 siRNA causedasignificant increase in the survival of Hep3B tumor-bearing mice.
`15
`[0040]
`Figure 15 illustrates data demonstrating that treatment with 1:57 SNALPcontaining
`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.
`[0041]
`Figure 16 illustrates data demonstrating that a specific cleavage product of PLK-1
`mRNAwasdetectable by 5° RACE-PCRin mice treated with 1:57 SNALP containing PLK-1
`siRNA. 10 pl PCR product/well were loaded onto a 1.5% agarose gel. Lane Nos.:
`(1)
`molecular weight (MW) marker; (2) PBS mouse1; (3) PBS mouse 2; (4) PBS mouse3; (5)
`Luc SNALP mouse 1; (6) Luc SNALP mouse 2; (7) PLK SNALP mouse |; (8) PLK SNALP
`mouse 2; (9) PLK SNALP mouse3; and (10) no template control.
`[0042]
`Figure 17 illustrates data demonstrating that control (Luc) 1:57 SNALP-treated
`mice displayed normal mitoses in Hep3B tumors(top panels), whereas mice treated with 1:57
`SNALPcontaining PLK-1 siRNA exhibited numerousaberrant mitoses and tumorcell
`apoptosis in Hep3B tumors (bottom panels).
`[0043]
`Figure 18 illustrates data demonstrating that multiple doses of 1:57 PLK-1 SNALP
`containing PEG-cDSA inducedthe regression of established Hep3B subcutaneous(S.C.)
`tumors.
`Figure 19 illustrates data demonstrating PLK-1 mRNAsilencing using 1:57 PLK
`[0044]
`SNALPin S.C. Hep3B tumors following a single intravenous SNALP administration.
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`[0045]
`
`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
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`Hep3B intrahepatic tumors.
`[0047]
`Figure 22illustrates data demonstrating the blood clearance profile of 1:57 PLK-1
`SNALPcontaining either PEG-cDMA or PEG-cDSA.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`I.
`
`Introduction
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`[0048] The present invention is based, in part, upon the surprising discovery thatlipid
`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
`mol % of a lipid conjugate provide advantages when usedfor 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 Examplesherein, the present invention providesstable 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
`invention are stable in circulation, e.g., resistant to degradation by nucleases in serum, and
`are substantially non-toxic to mammals such as humans. As a non-limiting example, Figure 3
`of Example 4 shows that one SNALP embodimentofthe invention (“1:57 SNALP”) was
`more than 10 times as efficacious as compared to a nucleic acid-lipid particle previously
`described (“2:30 SNALP”) in mediating target gene silencing at a 10-fold lower dose.
`Similarly, Figure 2 of Example 3 shows that the “1:57 SNALP” formulation was substantially
`more effective at silencing the expression ofa target gene as comparedto nucleic acid-lipid
`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 improved lipid particle formulations ofthe invention are highly effective in
`downregulating the mRNA and/or protein levels of target genes. Furthermore, the Examples
`herein illustrate that the presence of certain molarratios of lipid components results in
`improved or enhancedactivity of these lipid particle formulations of the present invention.
`For instance, 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 and tolerability in vivo, are serum-stable, are substantially
`non-toxic, are capable of accessing extravascular sites, and are capable of reachingtargetcell
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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 methods for
`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, andtheir use to deliver therapeutic
`agents and modulate target gene and protein expression, are described in further detail below.
`
`Il.
`
`Definitions
`
`[0052] As used herein, the following terms have the meanings ascribed to them unless
`specified otherwise.
`[0053] The term “interfering RNA”or “RNAi”or “interfering RNA sequence”refers to
`single-stranded RNA(e.g., mature miRNA)or double-stranded RNA (i.e., duplex RNA such
`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 degradationor 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 thusrefers to the
`single-stranded RNA that is complementary to a target mRNAsequenceor to the double-
`stranded RNA formed by two complementary strandsor by a single, self-complementary
`strand. Interfering RNA may havesubstantial or complete identity to the target gene or
`sequence, or may comprise a region of mismatch (Z.e., a mismatch motif). The sequence of
`the interfering RNA can correspondto the full-length target gene, or a subsequencethereof.
`[0054]
`Interfering RNA includes “‘small-interfering RNA”or “siRNA,”e.g., interfering
`RNAof about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-
`30, 15-25, or 19-25 (duplex) nucleotides in length, and is preferably about 20-24, 21-22, or
`21-23 (duplex) nucleotides in length (e.g., each complementary sequence of the double-
`stranded siRNAis 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length,
`preferably about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded
`siRNA is 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 siRNAinclude, without limitation, a double-stranded
`
`polynucleotide molecule assembled from two separate stranded molecules, wherein one
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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 regions are linked by a nucleic acid-based or non-nucleic acid-based
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`linker; a double-stranded polynucleotide molecule with a hairpin secondary structure having
`
`self-complementary sense and antisense regions; and a circular single-stranded
`polynucleotide molecule with two or more loop structures and a stem havingself-
`complementary sense and antisense regions, where the circular polynucleotide can be
`processed in vivoor 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 RNaseIII or Dicer. These enzymesprocess the dsRNA into biologically active
`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 et al., Ambion TechNotes, 10(1):4-6
`(2003); Kawasaki et al., Nucleic Acids Res., 31:981-987 (2003); Knight et al., Science,
`293:2269-2271 (2001); and Robertsonef al., J. Biol. Chem., 243:82 (1968)). Preferably,
`dsRNAare at 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
`instances, siRNA may be encoded bya plasmid (e.g., transcribed as sequencesthat
`
`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)sequence that does not have 100 %
`complementarity to its target sequence. An interfering RNA mayhaveat least one, two,
`three, four, five, six, or more mismatch regions. The mismatch regions may be contiguousor
`may be separated by1, 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
`
`more nucleotides.
`
`[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 comparisonto 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 when the value obtained with an interfering RNA
`relative to the control is about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%,
`40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or O%. Suitable assays for measuring
`expressionofa target gene ortarget sequenceinclude, e.g., examination of protein or RNA
`levels using techniques known to thoseof skill in the art such as dotblots, northern blots, in
`situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic
`assays known to those ofskill in theart.
`[0058] By “decrease,” “decreasing,”“reduce,” or “reducing” of an immuneresponse by an
`interfering RNA is intended to mean a detectable decrease of an immuneresponseto a given
`interfering RNA (e.g., a modified interfering RNA). The amount of decrease of an immune
`response by a modified interfering RNA may be determined relative to the level of an
`immuneresponsein 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 immuneresponseto
`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 a mammalian subject after administration ofthe interfering RNA.
`[0059] As used herein, the term “responder cell” 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, ¢.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, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12,
`IL-13, TGF, and combinations thereof.
`[0060]
`“Substantial identity” refers to a sequence that hybridizes to a reference sequence
`understringent conditions, or to a sequencethat has a specified 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 of nucleic
`acids, but to no other sequences. Stringent conditionsare 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 found in 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 lowerthan the thermal
`melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm 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 presentin excess, at Tm, 50% of the probes are occupied at equilibrium).
`Stringent conditions may also be achieved with the addition of destabilizing agents such as
`formamide. Forselective or specific hybridization, a positive signal is at least two times
`background, preferably 10 times background hybridization.
`[0062] Exemplary stringent hybridization conditions can be as follows: 50% formamide,
`5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with
`wash in 0.2x SSC, and 0.1% SDS at 65°C. For PCR,a temperature of about 369°Cis typical
`for low stringency amplification, although annealing temperatures may vary between about
`320C and 48°C depending on primer length. For high stringency PCR amplification, a
`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 ofa nucleic acid is created using the maximum codon
`degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize
`under moderately stringent hybridization conditions. Exemplary “moderately stringent
`hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl,
`1% SDSat 37°C, and a wash in 1X SSC at 45°C. A positive hybridizationis at least twice
`background. Those of ordinary 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, Ausubelet 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 subsequencesthat 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 oneof the following sequence comparison algorithms or
`by manual alignmentand visual inspection. This definition, when the context indicates, also
`refers analogously to the complementof a sequence. Preferably, the substantial identity
`exists over a region thatis 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 comparisonalgorithm,test and
`reference sequencesare entered into a computer, subsequence coordinates are designated,if
`necessary, and sequence algorithm program parametersare designated. Default program
`parameters can be used,or alternative parameters can be designated. The sequence
`comparison algorithm then calculates the percent sequence identities for the test sequences
`relative to the reference sequence, based on the program parameters.
`[0066] A “comparison window,” as usedherein, includes reference to a segment of any one
`of a numberof contiguouspositions 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. Methodsof alignment of sequences
`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, Ady. Appl.
`Math., 2:482 (1981), by the homology alignmentalgorithm of Needleman 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 implementationsof these algorithms
`(GAP, BESTFIT, FASTA,and TFASTAin the Wisconsin Genetics Software Package,
`Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and
`visual inspection(see, e.g., Current Protocols in Molecular Biology, Ausubelef al., eds.
`(1995 supplement)).
`
`10
`
`15
`
`20
`
`25
`
`30
`
`12
`
`

`

`[0067] A preferred example of algorithmsthat are suitable for determining percent
`sequenceidentity 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 Altschul et 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.nim.nih.gow/).
`[0068] The BLASTalgorithm also performsa statistical analysis of the similarity between
`two sequences(see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci, USA, 90:5873-5787
`(1993)). One measure ofsimilarity 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 sequenceif the smallest sum probability in a comparison of the 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 polymercontaining at least two
`deoxyribonucleotidesor ribonucleotides in 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 PCR product, vectors (P1, PAC, BAC, YAC,artificial
`chromosomes), expression cassettes, chimeric sequences, chromosomal DNA,or derivatives
`and combinations of these groups. RNA maybein 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 orlinkages, which are synthetic, naturally
`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-methy] ribonucleotides, and peptide-nucleic acids (PNAS). Unless specifically limited, the
`term encompasses nucleic acids containing known analogues ofnatural nucleotides that have
`similar binding properties as the reference nucleic acid. Unless otherwise indicated, a
`particular nucleic acid sequence also implicitly encompasses conservatively modified
`variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and
`complementary sequencesas well as the sequence explicitly indicated. Specifically,
`degenerate codon substitutions may be achieved by generating sequences in which