PROTIVA - EXHIBIT 2045
`Moderna Therapeutics, Inc. v. Protiva Biotherapeautics, Inc.
`IPR2018-00739
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`[0006] Although the precise mechanism is still unclear, RNAi provides a potential new
`approach to downregulate or silence the transcription and translation of a gene ofinterest.
`For example, it is desirable to modulate (¢.g., reduce) the expression of certain genes for the
`treatment of neoplastic disorders such as cancer. It is also desirable to silence the expression
`of genes associated with liver diseases and disorders such as hepatitis. It is further desirable
`to reduce the expression of certain genes for the treatmentof atherosclerosis and its
`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 vectorsare 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
`immune response concerns. As a result, nonviral gene delivery systems are receiving
`increasing attention (Worgall et al, Human Gene Therapy, 8:37 (1997), Peeters ef al,
`Human Gene Therapy, 7:1693 (1996); Yei et al., Gene Therapy, 1:192 (1994); Hope e¢ al,
`Molecular Membrane Biology, 15:1 (1998)). Furthermore, viral systems are rapidly cleared
`from the circulation, limiting transfection to “first-pass” organs such as the lungs, liver, and
`spleen.
`In addition, these systems induce immuneresponsesthat compromise delivery with
`
`subsequentinjections.
`[0008]
`Plasmid DNA-cationic liposome complexes are currently the most commonly
`employed nonviral gene delivery vehicles (Felgner, Scientific American, 276:102 (1997);
`Chonnet al., Current Opinion in Biotechnology, 6:698 (1995)). For instance, cationic
`liposome complexes made of an amphipathic compound,a neutrallipid, and a detergent for
`transfecting insect cells are disclosed in U.S. Patent No. 6,458,382. Cationic liposome
`complexes are also disclosed in U.S. Patent Publication No. 20030073640.
`[0009] Cationic liposome complexes are large, poorly defined systems that are not suited
`for systemic applications and canelicit 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)). Aslarge, positively charged aggregates, lipoplexes are rapidly cleared when
`administered in vivo, with highest expression levels observed in first-pass organs, particularly
`the lungs (Huang ef al., Nature Biotechnology, 15:620 (1997); Templeton ef al., Nature
`Biotechnology, 15:647 (1997); Hoflandef al., Pharmaceutical Research, 14:742 (1997)).
`[0010] Other liposomal delivery systems include, for example, the use of reverse micelles,
`anionic liposomes, and polymerliposomes. Reverse micelles are disclosed in U.S. Patent No.
`6,429,200. Anionic liposomes are disclosed in U.S. Patent Publication No. 20030026831.
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`Polymerliposomesthat incorporate dextrin or glycerol-phosphocholine polymersare
`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 (7.e., less than about 100 nm diameter) and should remainintact 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 components of the vascular compartment. The particle should
`also readily interact with target cells at a disease site in orderto facilitate intracellular
`
`delivery of a desired nucleic acid.
`[8012] Recent work has shownthat 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 of a
`poly(ethylene glycol) (PEG) coating. SPLPs have systemic application as they exhibit
`extended circulation lifetimes following intravenous(i.v.) injection, accumulate preferentially
`at distal tumorsites 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 moreefficient 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 genes of interest 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 (¢.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 agentin
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`the lipid particle is resistant in aqueous solution to enzymatic degradation, ¢.g., by a nuclease
`or protease. In other preferred embodiments,the lipid particles are substantially non-toxic to
`
`mammals such as humans.
`
`In one aspect, the present invention provides lipid particles comprising: (a) one or
`[0016]
`more active agents or therapeutic agents; (b) one or more cationic lipids comprising from
`about 50 mol % to about $5 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
`presentin the particle; and (d) one or more conjugatedlipids that inhibit aggregation of
`particles comprising from about 0.5 mol % to about 2 mol % ofthe total lipid present in the
`
`particle,
`[0017] More particularly, 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 disease 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
`from about 13 mol % to about 49.5 mol % ofthetotal lipid present in the particle; and (d) a
`
`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 (¢.g., SNALP)
`comprises: (a) an siRNA;(b)a cationic lipid comprising from about 56.5 mol % to about
`66.5 mol % ofthe total lipid present in the particle; (c) cholesterol or a derivative thereof
`
`comprising from about 31.5 mol % to about 42.5 mol % ofthe 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 embodimentof nucleic acid-lipid particle
`
`is generally referred to herein as the “1:62” formulation.
`[0020]
`In another preferred embodiment, the nucleic acid-lipid particle (¢.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 % of the total lipid
`present in the particle; and (d) a PEG-lipid conjugate comprising from about 1 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.
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`[0021] The present invention also provides pharmaceutical compositions comprising a lipid
`particle described herein (e.g., SNALP) and a pharmaceutically acceptablecarrier.
`[0022]
`In another aspect, the present invention provides methods for introducing an active
`agentor 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.,
`
`SNALP).
`In yet another aspect, the present invention provides methods for the in vivo
`{0023]
`delivery of an active agent or therapeutic agent (e.g., nucleic acid), the method comprising
`administering to a mammalian subject a lipid particle described herein such as a nucleic acid-
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`lipid particle (e.g., SNALP).
`[0024]
`Ina further aspect, the present invention provides methods for treating a disease or
`disorder in a mammalian subject in need thereof, the method comprising administering to the
`mammalian subject a therapeutically effective amountof a lipid particle described herein
`such as 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
`one of skill in the art from the following detaited description and figures.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`Figure 1A (Samples 1-8) and Figure 1B (Samples 9-16) illustrate data
`[0026]
`demonstrating the activity of 1:57 SNALP containing Eg5 siRNA in a human colon cancer
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`cell line.
`
`[0027]
`
`Figure 2 illustrates data demonstrating the activity of 1:57 SNALP containing ApoB
`
`siRNA following intravenous administration in mice.
`[6028]
`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]
`Figure 6A (expressed as IU/L) and Figure 6B (expressed as x-Fold Upper Limit of
`Normal) illustrate data demonstrating that the tolerability of 1:57 SNALP containing ApoB
`siRNA prepared by citrate buffer versus PBS direct dilution did notdiffer significantly in
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`terms of blood clinical chemistry parameters.
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`Figure 7A (expressed as liver ApoB:GAPD mRNAratio), Figure 7B (expressed as
`[0032]
`relative plasma ApoB-100 concentration), and Figure 7C (expressed as plasmatotal
`cholesterol illustrate data demonstrating that the efficacy of 1:57 SNALP containing ApoB
`siRNA prepared by gear pump wassimilar to the same SNALP prepared by syringe press.
`[0033]
`Figure 8 illustrates data demonstrating that there was very little effect on body
`weight 24 hours after administration of 1:57 SNALP containing ApoB siRNA.
`(0034]
`Figure 9 illustrates data demonstrating that there were no obvious changesin
`platelet count after administration of 1:57 SNALP containing ApoB siRNA.
`[0035]
`Figure 10A (expressed as IU/L) and Figure 10B (expressed as x-Fold Upper Limit
`of Normal)illustrate data demonstrating that clinically significant liver enzyme elevations
`(3xULN)occurred at particular drug dosages of 1:57 SNALP containing ApoB siRNA.
`[0036]
`Figure 11A (expressed as liver ApoB:GAPD mRNA ratio) and Figure 11B
`(expressed as relative plasma ApoB-100 concentration) illustrate data demonstrating that the
`potency of the lower lipid:drug (L:D) 1:57 SNALP containing ApoB siRNA wasas goodas
`that of the higher L:D SNALPat the tested drug dosages.
`[0037]
`Figure 12 illustrates data demonstrating that ApoB protein andtotal 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 ratie (final ratio of
`
`10:1).
`Figure 13 illustrates data demonstrating that a treatment regimen of 1:57 SNALP
`[0038]
`with siRNA targeting PLK-1 is well tolerated with no apparent signs oftreatment related
`toxicity in mice bearing Hep3B liver tumors.
`[0039]
`Figure 14 illustrates data demonstrating that treatment with 1:57 SNALP containing
`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
`
`in mice 24 hours after SNALP administration.
`
`Figure 16 illustrates data demonstrating that a specific cleavage product of PLK-]
`[0041]
`mRNA was detectable by 5° RACE-PCR in mice treated with 1:57 SNALPcontaining PLK~-1
`
`siRNA. 10 yl PCR product/well were loaded onto a 1.5% agarose gel. Lane Nos.: (1)
`molecular weight (MW) marker; (2) PBS mouse 1; (3) PBS mouse 2; (4) PBS mouse3; (5)
`Luc SNALP mouse 1; (6) Luc SNALP mouse 2; (7) PLK SNALP mouse 1; (8) PLK SNALP
`mouse 2; (9) PLK SNALP mouse 3; and (10) no template control.
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`Figure 17 illustrates data demonstrating that control (Luc) 1:57 SNALP-treated
`j0042]
`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).
`[0043]
`Figure 18 illustrates data demonstrating that multiple doses of 1:57 PLK-1 SNALP
`containing PEG-cDSA induced the 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.
`[0045]
`Figure 20 illustrates data demonstrating that PLK-1 PEG-cDSA SNALPinhibited
`
`the growth oflarge 8.C. Hep3B tumors.
`[0046]
`Figure 21 illustrates data demonstrating tumor-derived PLK-1 mRNAsilencing in
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`Hep3B intrahepatic tumors.
`[0047]
`Figure 22 illustrates data demonstrating the blood clearance profile of 1:57 PLK-1
`SNALPcontaining either PEG-cDMAor PEG-cDSA.
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`DETAILED DESCRIPTION OF THE INVENTION
`
`I.
`
`Introduction
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`[0048] The present invention is based, in part, upon the surprising discovery that lipid
`particles comprising from about 50 mol % to about 85 mol % ofacationic 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 used for the i vifro 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 provides stable nucleic acid-lipid
`particles (SNALP) that advantageously impart increased activity of the encapsulated nucleic
`acid (e.¢., 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 showsthat one SNALP embodimentof the 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 showsthat the “1:57 SNALP” formulation was substantially
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`more effective at silencing the expression of a target gene as comparedto nucleic acid-lipid
`particles previously described (“2:40 SNALP”).
`[0049]
`In certain embodiments, the present invention provides improved compositionsfor
`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 mRNA and/orprotein levels of target genes. Furthermore, the Examples
`herein illustrate that the presence of certain molar ratios of lipid components results in
`improved or enhanced activity of these lipid particle formulations of the present invention.
`For instance, the “1:57 SNALP”and “1:62 SNALP” formulations described herein are
`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 targetcell
`
`populations.
`[0050]
`Thelipid particles and compositions ofthe 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 vive. 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 embodiments ofthe 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.
`
`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 (Z¢., 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 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 thusrefers to the
`single-stranded RNAthat is complementary to a target mRNA sequenceor to the double-
`stranded RNA formed by two complementary strands or by a single, self-complementary
`strand. Interfering RNA may have substantial or complete identity to the target gene or
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`sequence, or may comprise a region of mismatch (.e., a mismatch motif). The sequence of
`the interfering RNA can correspondto the full-length target gene, or a subsequence thereof.
`[0054]
`Interfering RNA includes “small-interfering RNA”or “siRNA,” ¢.g., interfering
`RNA ofabout 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 ofthe 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
`siRNAis about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferably
`about 18-22, 19-20, or 19-21 base pairs in length), siRNA duplexes may comprise 3°
`overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and 53’
`phosphate termini. Examples of siRNA include, without limitation, a double-stranded
`polynucleotide molecule assembled from two separate stranded molecules, wherein one
`strand is the sense strand and the other is the complementary antisense strand; a double-
`stranded polynucleotide molecule assembled from a single stranded molecule, where the
`sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based
`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 having self-
`complementary sense and antisense regions, where the circular polynucleotide can be
`processed in vive 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 III or Dicer. These enzymes process the dsRNA into biologically active
`siRNA(see, ¢.g., Yang et al., Proc. Natl. Acad. Sei. USA, 99:9942-9947 (2002); Calegariet
`al, Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom et af., Ambion TechNotes, 10(1):4-6
`(2003); Kawasaki et al., Nucleic Acids Res., 31:981-987 (2003); Knightet al., Science,
`293:2269-2271 (2001); and Robertson ef al., J. Biol. Chem., 243:82 (1968)). Preferably,
`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
`dsRNAcanencodefor an entire gene transcript or a partial gene transcript.
`In certain
`instances, siRNA may be encoded by a plasmid (e.g., transcribed as sequencesthat
`
`automatically fold into duplexes with hairpin loops).
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`[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 may haveat least one, two,
`three, four, five, six, or more mismatch regions. The mismatch regions may be contiguous or
`may be 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
`
`more nucleotides.
`
`[0057] An “effective amount”or “therapeutically effective amount” of an active agent or
`therapeutic agent such as an interfering RNA is an amount sufficient to produce the desired
`effect, e.g., an inhibition of expression of a target sequence in comparison to the normal
`expression level detected in the absence of an interfering RNA. Inhibition of expression of a
`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 0%. Suitable assays for measuring
`expression of a target gene or target sequence include, e.g., examination of protein or RNA
`levels using techniques knownto those of skill in the art such as dot blots, northern blots, i
`situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic
`
`assays knownto those of skill in the art.
`[0058] By “decrease,” “decreasing,”“reduce,” or “reducing” of an immuneresponse by an
`interfering RNA is intended to mean a detectable decrease of an immune responseto 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 determinedrelative to the level of an
`immune response 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.¢., 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 of the interfering RNA.
`[0059]
`Asused 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, ¢.g.,
`dendritic cells, macrophages, peripheral blood mononuclear cells (PBMCs), splenocytes, and
`the like. Detectable immune responses include, ¢.g., production of cytokines or growth
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`factors such as TNF-a, IFN-o, IFN-B, IFN-y, IL-1, IL-2, (1-3, IL-4, IL-5, IL-6, IL-10, TL-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 specified percent identity over a
`
`specified region of a reference sequence.
`[8061]
`The phrase “stringent hybridization conditions” refers to conditions under which a
`nucleic acid will hybridizeto its target sequence, typically in a complex mixture of nucleic
`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 found in Tijssen,
`Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes,
`“Overview ofprinciples 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 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 sequenceat equilibrium (as the
`target sequences are present in 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. For selective or specific hybridization, a positive signalis 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,
`
`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 36°Cis 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
`
`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 phaselasting 30 sec.-2
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`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, ¢.g., in Innis et al., PCR Protocols,
`A Guide to Methods and Applications, Academic Press, Inc. N.Y. (1990).
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`[0063] Nucleic acids that do not hybridize to each other understringent conditionsare still
`substantially identical if the polypeptides which they encode are 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 acids typically hybridize
`under moderately stringent hybridization conditions. Exemplary “moderately stringent
`hybridization conditions” include a hybridization in a buffer of 40% formamide, | M NaCl,
`1% SDS at 37°C, and a wash in 1X SSC at 45°C. A positive hybridization is at least twice
`background. Thoseofordinary skill will readily recognize that alternative hybridization and
`wash conditions can be utilized to provide conditions ofsimilar stringency. Additional
`guidelines for determining hybridization parameters are provided in numerous references,
`¢.g., Current Protocols in Molecular Biology, Ausubel et a/., 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 nucleotidesthat 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 oneofthe following sequence comparison algorithms or
`by manualalignment and visual inspection. This definition, when the context indicates, also
`refers analogously to the complement of a sequence. Preferably, the substantial identity
`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 sequence acts as a reference sequence,to
`which test sequences are compared. When using a sequence comparison algorithm,test and
`reference sequencesare entered into a computer, subsequence coordinates are designated,if
`necessary, and sequencealgorithm program parameters are designated. Default program
`parameters can be used,oralternative 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 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 compared to a reference sequence of the same number of contiguous
`positions after the two sequences are optimally aligned. Methods of alignment of sequences
`
`10
`
`20
`
`25
`
`30
`
`12
`
`

`

`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 alignment algorithm 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 implementations of 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, ¢.g., Current Protocols in Molecular Biology, Ausubeletal., eds.
`
`(1995 supplement)).
`[0067] A preferred example ofalgorithmsthat are suitable for determining percent
`sequence identity and sequence similarity are the BLAST and BLAST2.0 algorithms, which
`are described in Altschul et al., Nuc. Acids Res., 25:3389-3402 (1977) and Altschul e¢ ai, 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 BLAST analysesis 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 measureofsimilarity provided by the BLAST algorithm 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 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 polymer containing at least two
`deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and
`includes DNA and RNA. DNA maybein the form of, ¢.g., antisense molecules, plasmid
`DNA,pre-condensed DNA, a PCR product, vectors (P1, PAC, BAC, YAC,artificial
`chromosomes), expression cassettes, chimeric sequences, chromosomal DNA,orderivatives
`and combinations of these g