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 and its
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`manifestations, e. g, hypereholesterolemia, myocardial infarction, and thrombosis.
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`[0007] A safe and effective nucleic acid delivery system is required for RNAi to be
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`therapeutically useful. Viral vectors are relatively efficient gene delivery systems, but suffer
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`from a variety of limitations, such as the potential for reversion to the wild-type as well as
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`immune response concerns. As a result, nonviral gene delivery systems are receiving
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`increasing attention (Worgall el‘ al,, Human Gene Therapy, 8:37 (1997); Peeters et al,
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`Human Gene Therapy, 7: 1693 (1996); Yei eta/1,, 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, 2762102 (1997);
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`Chonn et al, Current Opinion in Biotechnology, 6:698 (1995)). For instance, cationic
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`liposome complexes made of an amphipathic compound, a neutral lipid, and a detergent for
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`transfecting insect cells are disclosed in US. Patent No. 6,458,382. Cationic liposome
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`complexes are also disclosed in US. Patent Publication No. 20030073 640.
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`[0009] Cationic liposome complexes are large, poorly defined systems that are not suited
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`for systemic applications and can elicit considerable toxic side effects (Harrison el al,
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`Biotechniques, 192816 (1995); Li et al., The Gene, 4:891 (1997); Tam et al, Gene Ther.,
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`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
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`the lungs (Huang et al, Nature Biotechnology, 15:620 (1997); Templeton et al, Nature
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`Biotechnology, 152647 (1997); Hofland et al,, Pharmaceutical Research, 14:742 (1997)).
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`[0010] Other liposomal delivery systems include, for example, the use of reverse micelles,
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`anionic liposomes, and polymer liposomes. Reverse micelles are disclosed in US. Patent No.
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`6,429,200. Anionic liposomes are disclosed in US. Patent Publication No. 20030026831.
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`Polymer liposomes that incorporate dextrin or glycerol—phosphocholine polymers are
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`disclosed in US. Patent Publication Nos. 20020081736 and 20030082103, respectively.
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`[0011] A gene delivery system containing an encapsulated nucleic acid for systemic
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`delivery should be small (i.e., less than about 100 nm diameter) and should remain intact in
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`the circulation for an extended period of time in order to achieve delivery to affected tissues.
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`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 (6. 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 (Wheeler er 511., Gene Therapy, 6:271 (1999)).
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`These SPLPs typically 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(cthylcnc 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 expression at these tumor sites. The levels of transgene expression
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`observed at the tumor site following iv. injection of SPLPS containing the luciferase marker
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`gene are superior to 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 of interest 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 SUMMARY OF 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, cg, 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 % of the total lipid present in the particle; (0) one or more
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`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
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`particles comprising from about 0.5 mol % to about 2 mol % ofthe total lipid present in the
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`particle.
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`[0017] More particularly, the present invention provides serum-stable nucleic acid-lipid
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`particles (SNALP) comprising a nucleic acid (e. g, one or more interfering RNA molecules
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`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 ofa disease or disorder).
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`[0018]
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`In certain embodiments, the nucleic acid—lipid particle (eg, SNALP) comprises: (a)
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`a nucleic acid (e.g., an interfering RNA); (b) a cationic lipid comprising from about 50 mol %
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`to about 85 mol % of the totai lipid present in the particle; (e) 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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`conjugated lipid that inhibits aggregation of particles comprising from about 0.5 mol % to
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`about 2 mol % of the total lipid present in the particle.
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`[0019]
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`In one preferred embodiment, the nucleic acid-lipid particle (e. g., SNALP)
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`comprises:
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`(a) an siRNA; (b) a cationic lipid comprising from about 56.5 mol % to about
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`66.5 mol % of the total lipid present in the particle; (c) cholesterol or a derivative thereof
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`comprising from about 31.5 mol % to about 42.5 mol % of the total lipid present in the
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`particle; and (d) a PEG—lipid conjugate comprising from about 1 mol % to about 2 mol % of
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`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.
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`[0020]
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`In another preferred embodiment, the nucleic acid—lipid particle (e.g., SNALP)
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`comprises:
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`(a) an siRNA; (b) a cationic lipid comprising from about 52 mol % to about 62
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`mol % of the total lipid present in the particle; (c) a mixture of a phospholipid and cholesterol
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`or a derivative thereof comprising from about 36 mol % to about 47 mol % of the total lipid
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`present in the particle; and (d) a PEG-lipid conjugate comprising from about 1 mol % to
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`about 2 mol % of the 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.
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`[0021] The present invention also provides pharmaceutical compositions comprising a lipid
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`particle described herein (e. g, SNALP) and a pharmaceutically acceptable carrier.
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`[0022]
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`In another aspect, the present invention provides methods for introducing an active
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`agent or therapeutic agent (e. g, nucleic acid) into a cell, the method comprising contacting
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`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 methods for the in vivo
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`delivery of an active agent or therapeutic agent (eg, 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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`In a 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
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`mammalian subject a therapeutically effective amount of a lipid particle described herein
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`such as a nucleic acid—lipid particle (6g, 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 l :57 SNALP containing Eg5
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`siRNA in a human colon cancer cell line.
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`[0027]
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`Figure 2 illustrates data demonstrating the activity of l :5 7 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 of five 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 l :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 siRNA prepared by citrate 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 l :57 SNALP containing
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`ApoB siRNA prepared by gear pump was similar to the same SNALP prepared 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 changes in
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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 (3 XULN) 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 lower lipid:drug
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`(LzD) 1:57 SNALP containing ApoB siRNA was as good as that ofthe higher L:D SNALP at
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`the tested drug dosages.
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`[0037]
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`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 siRNA at a 6:1
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`input L:D ratio (final ratio of 7:1) and 1:57 SNALP at a 9:1 input L:D ratio (final ratio 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 siRNA targeting PLK—l
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`is well tolerated with no apparent signs of treatment related
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`toxicity in mice bearing Hep3B liver tumors.
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`[0039]
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`Figure 14 illustrates data demonstrating that treatment with 1:57 SNALP containing
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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
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`PLK-1 siRNA reduced PLK-1 mRNA levels 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 was detectable by 5’ RACE—PCR in 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 mouse 3; (5)
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`Luc SNALP mouse 1; (6) Luc SNALP mouse 2; (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
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`SNALP containing PLK-1 siRNA exhibited numerous aberrant mitoses and tumor cell
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`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 mRNA silencing using 1:57 PLK
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`SNALP in SC. Hep3B tumors following a single intravenous SNALP administration.
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`[0045]
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`Figure 20 illustrates data demonstrating that PLK-l PEG—cDSA SNALP inhibited
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`the growth of large S.C. Hep3B tumors.
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`[0046]
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`Figure 21 illustrates data demonstrating tumor—derived PLK-l mRNA silencing in
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`Hep3B intrahepatic tumors.
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`[0047]
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`Figure 22 illustrates data demonstrating the blood clearance profile of 1:57 PLK-l
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`SNALP containing either PEG—cDMA or PEG-cDSA.
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`DETAILED DESCRIPTION OF THE INVENTION
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`I.
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`Introduction
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`[0048]
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`The present invention is based, in part, upon the surprising discovery that lipid
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`particles comprising from about 50 mol % to about 85 mol % ofa cationic lipid, from about
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`13 mol % to about 49.5 mol % ofa non—cationic lipid, and from about 0.5 mol % to about 2
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`mol % of a lipid conjugate provide advantages when used for the in vitro or in viva delivery
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`of an active agent, such as a therapeutic nucleic acid (eg, an interfering RNA). In particular,
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`as illustrated by the Examples herein, the present invention provides stable nucleic acid—lipid
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`particles (SNALP) that advantageously impart increased activity of the encapsulated nucleic
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`acid (e. g., an interfering RNA such as siRNA) and improved tolerability of the formulations
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`in vivo, resulting in a significant increase in the therapeutic index as compared to nucleic
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`acid—lipid particle compositions previously described. Additionally, the SNALP of the
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`invention are 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
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`of Example 4 shows that one SNALP embodiment of the invention (“1:57 SNALP”) was
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`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.
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`Similarly, Figure 2 of Example 3 shows that the “l :57 SNALP” formulation was substantially
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`more effective at silencing the expression of a target gene as compared to nucleic acid-lipid
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`particles previously described (“2:40 SNALP”).
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`[0049]
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`In certain embodiments, the present invention provides improved compositions for
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`the delivery of interfering RNA such as siRNA molecules. In particular, the Examples herein
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`illustrate that the improved lipid particle formulations of the invention are highly effective in
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`downregulating the mRNA and/or protein levels of target genes. Furthermore, the Examples
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`herein illustrate that the presence of certain molar ratios of lipid components results in
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`improved or enhanced activity of these lipid paiticle formulations of the present invention.
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`For instance, the “I :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
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`they provide improved efficacy and tolerability in vivo, are serum-stable, are substantially
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`non—toxic, are capable of accessing extravascular sites, and are capable of reaching target cell
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`populations.
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`[0050]
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`The lipid particles and compositions of the present invention may be used for a
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`variety of purposes, including the delivery of associated or encapsulated therapeutic agents to
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`cells, both in vitro and in vivo. Accordingly, the present invention provides methods for
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`treating diseases or disorders in a subject in need thereof, by contacting the subject with a
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`lipid particle described herein comprising one or more suitable therapeutic agents.
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`[0051] Various exemplary embodiments of the lipid particles of the invention, as well as
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`compositions and formulations comprising the same, and their use to deliver therapeutic
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`agents and modulate target gene and protein expression, are described in further detail below.
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`ll.
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`Definitions
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`[0052] As used herein, the following terms have the meanings ascribed 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
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`single-stranded RNA (e. g., mature miRNA) or double-stranded RNA (i.e., duplex RNA such
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`as siRNA, aiRNA, or pre-miRNA) that is capable of reducing or inhibiting the expression of
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`a target gene or sequence (e. g, by mediating the degradation or inhibiting the translation of
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`mRNAs which are complementary to the interfering RNA sequence) when the interfering
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`RNA is in the same cell 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 sequence or to the double—
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`stranded RNA formed by two complementary strands or by a single, self—complementary
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`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 (i.e., a mismatch motif). The sequence of
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`the interfering RNA can correspond to the full—length target gene, or a subsequence thereof.
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`[0054]
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`interfering RNA includes “small—interfering RNA” or “siRNA,” e.g,, interfering
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`RNA of about 15-60, 15-50, or l5-40 (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 (eg, each complementary sequence of the double—
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`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
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`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’
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`phosphate termini. Examples of siRNA include, without limitation, a double-stranded
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`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
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`self—complementary sense and antisense regions; and a circular single-stranded
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`polynucleotide molecule with two or more loop structures and a stem having self—
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`complementary sense and antisense regions, where the circular polynucleotide can be
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`processed in vivo or in vitro to generate an active double-stranded siRNA molecule.
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`[0055]
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`Preferably, siRNA are chemically synthesized. siRNA can also be generated by
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`cleavage of longer dsRNA (e. g., dsRNA greater than about 25 nucleotides in length) with the
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`E. coli RNase III or Dicer. These enzymes process the dsRNA into biologically active
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`siRNA (see, cg, Yang (2161]., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegari er
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`al., Proc. Natl. Acad. Sci. USA, 99: 14236 (2002); Byrom el al., Ambian TechNotes, 10(1):4-6
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`(2003); Kawasaki el al., Nucleic Acids Res, 31:981—987 (2003); Knight et al., Science,
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`293:2269—2271 (2001); and Robertson er £11., J. Biol. Chem, 243:82 (1968)). Preferably,
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`dsRNA are at least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotides in length.
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`A dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotides in length, or longer. The
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`dsRNA can encode for an entire gene transcript or a partial gene transcript. In certain
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`instances, siRNA may be encoded by a plasmid (e. g., transcribed as sequences that
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`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
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`of an interfering RNA (e. g, siRNA, aiRNA, miRNA) sequence that does not have 100 %
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`complementarity to its target sequence. An interfering RNA may have at least one, two,
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`three, four, five, six, or more mismatch regions. The mismatch regions may be contiguous or
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`may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides. The mismatch
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`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
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`therapeutic agent such as an interfering RNA is an amount sufficient to produce the desired
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`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 of a
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`target gene or target sequence is achieved when the value obtained with an interfering RNA
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`relative to the control is about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%,
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`40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring
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`expression of a target gene or target sequence include, e. g., examination of protein or RNA
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`levels using techniques known to those of skill in the art such as dot blots, northern blots, in
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`situ hybridization, ELISA, immunopreeipitation, enzyme function, as well as phenotypic
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`assays known to those of skill in the art.
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`[0058]
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`By “decrease,” “decreasing,” “reduce,” or “reducing” of an immune response by an
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`interfering RNA is intended to mean a detectable decrease of an immune response to a given
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`interfering RNA (e. g., a modified interfering RNA). The amount of decrease of an immune
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`response by a modified interfering RNA may be determined relative to the level of an
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`immune response in the presence of an unmodified interfering RNA. A detectable decrease
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`can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
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`75%, 80%, 85%, 90%, 95%, 100%, or more lower than the immune response detected in the
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`presence of the unmodified interfering RNA. A decrease in the immune response to
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`interfering RNA is typically measured by a decrease in cytokine production (e.g., IFNy,
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`IFNOL, TNFoc, IL-6, or IL— 12) by a responder cell in vitm or a decrease in cytokine production
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`in the sera of a mammalian subject after administration of the interfering RNA.
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`[0059] As used herein, the term “responder cell” refers to a cell, preferably a mammalian
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`ceEl, that produces a detectable immune response when contacted with an immunostimulatory
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`interfering RNA such as an unmodified siRNA. Exemplary responder cells include, e. g.,
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`dendritic cells, macrophages, peripheral blood mononuclear cells (PBMCS), splenocytes, and
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`the like. Detectable immune responses include, e.g., production of cytokines or growth
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`factors such as TNF-(x, lFN-OL, lFN—B, IFN—y, IL—l, lL—2, IL—3, IL—4, IL—5, IL-6, IL-10, IL—12,
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`IL—l3, TGF, and combinations thereof.
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`[0060]
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`“Substantial identity” refers to a sequence that hybridizes to a reference sequence
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`under stringent conditions, or to a sequence that has a specified percent identity over a
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`specified region of a reference sequence.
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`[0061]
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`The phrase “stringent hybridization conditions” refers to conditions under which a
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`nucleic acid will hybridize to its target sequence, typically in a complex mixture of nucleic
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`acids, but to no other sequences. Stringent conditions are sequence-dependent and will be
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`different in different circumstances. Longer sequences hybridize specifically at higher
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`temperatures. An extensive guide to the hybridization of nucleic acids is found in Tij ssen,
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`Techniques in Biochemistry and Molecular Biology-~Hybridization with Nucleic Probes,
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`“Overview of principles of hybridization and the strategy of nucleic acid assays” (1993).
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`Generally, stringent conditions are selected to be about 5—100C lower than the thermal
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`melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the
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`temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of
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`the probes complementary to the target hybridize to the target sequence at equilibrium (as the
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`target sequences are present in excess, at Tm, 50% (of the probes are occupied at equilibrium).
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`Stringent conditions may also be achieved with the addition of destabilizing agents such as
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`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 420C, or, 5x SSC, 1% SDS, incubating at 650C, with
`
`wash in 0.2x SSC, and 0.1% SDS at 650C. For PCR, a temperature of about 360C is typical
`
`for low stringency amplification, although annealing temperatures may vary between about
`
`32°C and 480C depending on primer length. For high stringency PCR amplification, a
`
`temperature of about 620C is typical, although high stringency annealing temperatures can
`
`range from about 500C 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 900C-950C for 30 see—2 min., an annealing phase lasting 30 sec-2
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`min., and an extension phase of about 720C for 1-2 min. Protocols and guidelines for low
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`and high stringency amplification reactions are provided, 6. g., in Innis et al, PCR Protocols,
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`A Guide to Methods and Applications, Academic Press, Inc. NY. (1990).
`
`[0063] Nucleic acids that do not hybridize to each other under stringent conditions are still
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`substantially identical if the polypeptides which they encode are substantially identical. This
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`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% SDS at 370C, and a wash in 1X SSC at 45°C. A positive hybridization is 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 numerous references,
`
`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 (116., 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
`
`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 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
`
`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 number of 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
`
`for comparison are well known in 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 homology alignment algorithm ofNeedleman and Wunsch, J.
`
`Mol. Biol, 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc.
`
`Natl. Acad. Sci. USA, 8522444 (1988), by computerized implementations of these algorithms
`
`(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,
`
`Genetics Computer Group, 575 Science D12, Madison, WI), or by manual alignment and
`
`visual inspection (see, eg, Current Protocols in Molecular Biology, Ausubel et al., eds.
`
`(1995 supplement)).
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`[0067] A preferred example of algorithms that are suitable for determining percent
`
`sequence identity and sequence similarity are the BLAST and BLAST 2,0 algorithms, which
`
`are described in Altschul el 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 BLAST analyses is publicly available through the
`
`National Center for Biotechnology Information (http://wwwncbi.nlm.nih.gov/).
`
`[0068]
`The BLAST algorithm also performs a 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 of similarity 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 doubie—stranded form and
`
`includes DNA and RNA. DNA may be in the form of, e.g., antiscnsc molecules, plasmid
`
`DNA, pre—condensed DNA, a PCR product, vectors (Pl, 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 backbone residues or linkages, which are synthetic, naturally
`
`occurring, and non—naturally occurring, and which have simi