WO2012177639A2

WO2012177639A2 - Bioprocessing and bioproduction using avian cell lines

Info

Publication number: WO2012177639A2
Application number: PCT/US2012/043148
Authority: WO
Inventors: Gregory Hinkle; Brian Bettencourt
Original assignee: Alnylam Pharmaceuticals, Inc.
Priority date: 2011-06-22
Filing date: 2012-06-19
Publication date: 2012-12-27
Also published as: WO2012177639A3

Abstract

The invention relates to compositions and methods of modulating the status, activity or expression of avian transcripts in the process of bioproduction. In one aspect, the invention provides for a method for producing a biological product from an avian host cell.

Description

BIOPROCESSING AND BIOPRODUCTION USING AVIAN CELL LINES

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 61/499,757, filed June 22, 201 1, entitled "BIOPROCESSING AND BIOPRODUCTION USING AVIAN CELL LINES" and U.S. Provisional Patent Application No. 61/639,333, filed April 27, 2012, entitled "BIOPROCESSING AND BIOPRODUCTION USING AVIAN CELL LINES" the contents, each of which is incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The instant application contains a "lengthy" Sequence Listing which has been submitted via CD-R in lieu of a printed paper copy, and is hereby incorporated by reference in its entirety. This sequence listing text file which is part of the originally filed subject matter has the following features: File name: 200212pc.TXT; File size:

622,641,152 Bytes; Date created: May 22, 2012. These CD-Rs are labeled "CRF," "Copy 1,", "Copy 2," and "Copy 3" respectively, and each contains only one identical file, as identified immediately above. REFERENCE TO LENGTHY TABLE

The specification includes Lengthy Table 1. Lengthy Table 1 has been submitted via EFS-Web in electronic format as follows: File name: AvianTranscripts.PDF; Date created: June 19, 2012; File size: 1,928,702 Bytes, and is incorporated herein by reference in its entirety. Please refer to the end of the specification for access

instructions.

FIELD OF THE INVENTION

The invention relates generally to avian transcriptomes, organized transcriptomes and systems and methods (including methods of doing business) using transcriptomes for the design of targeting constructs useful in the production of biomolecules in avian cells and avian cell cultures. The invention further relates to engineering avian cells and cell lines for more effective and efficient production of biological products and biomolecules. BACKGROUND OF THE INVENTION

Cell culture and bioprocessing techniques are used to manufacture a wide range of biological products, including biopharmaceuticals, biofuels, metabolites, vitamins and nutraceuticals. A number of strategies have been developed to enhance productivity, yield, efficiency, and other aspects of cell culture bioprocesses in order to facilitate industrial scale production and meet applicable standards for product quality and consistency. Traditional strategies for optimizing cell culture bioprocesses involve adjusting physical and biochemical parameters, such as culture media (e.g., pH, nutrients) and conditions (e.g., temperature, duration), and selecting host cells having desirable phenotypes. More recently, genetic approaches have been developed for optimizing cell culture bioprocesses by introducing recombinant DNA into host cells, where the DNA encodes an exogenous protein that influences production of a biological product or regulates expression of an endogenous protein that influences production of the biological product. However, such methods require costly and time-consuming laboratory manipulations and can be incompatible with certain genes, proteins, host cells, and biological products.

Avian cells have been used in the production of biomolecules, particularly complex proteins such as antibodies. However, little is known about the coordinate expression and modulation of genes in these cells, and thus, optimized and targeted modulation of bioprocesses of these cells is unavailable presently.

Accordingly, there is a need in the art for new genetic approaches for optimizing cell culture bioprocesses involving a wide range of host cells, including avian cells, and biological products produced in these cells.

SUMMARY OF THE INVENTION

The invention is based at least in part on the surprising discovery that Targeting Constructs (TCs) can be applied to cells in culture to effect potent, durable modulation of gene expression, such that the quality and quantity of biological product that is produced by an avian host cell can be improved without the need for extensive cell line

engineering. As such, in a first aspect, the invention provides compositions and methods for producing a biological product from a host cell. In one embodiment the host cell is an avian cell or cell derived from avian cells. In various embodiments, the biological product is a polypeptide (including enzymes), a metabolite, a nutraceutical, a chemical intermediate, a biofuel, a food additive, or an antibiotic (e.g., antiviral, antibacterial, antifungal, and the like, etc). The methods of the present invention also find utility in the fields of chemical manufacture, textiles and electronics.

The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods for producing a biological product in an avian host cell, the methods including the steps of contacting the cell with at least one or more Targeting Construct (TC), maintaining the avian cell for a time sufficient to modulate expression of the target gene or alter a cellular process, wherein the modulation and/or alteration enhances production of the biological product, and recovering the biological product from the cell, cell culture or supernatant. The description provided herein discloses how to make and use targeting constructs (TCs) to produce a biological product in an avian host cell or cell culture according to methods provided herein. Also disclosed are cell culture reagents and compositions comprising the targeting constructs and kits for carrying out the disclosed methods.

Also described herein are compositions and methods of modulation the status, activity, or expression of Avian Transcripts (ATs) in an avian cell (or cell culture), tissue or organism. Further provided are compositions and methods for treating pathological conditions and diseases in a mammal using a biological product or targeting construct of the present invention. Further disclosed are screening methods, kits and assays which are designed to utilize the cellular pathways and systems associated with avian transcripts and their manipulation in bioprocessing and bioproduction. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the targeting constructs (TC) and methods featured in the invention, suitable methods and materials are described below.

The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

As used herein and in the claims, the singular forms include the plural reference and vice versa unless the context clearly indicates otherwise. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about."

All patents, oligonucleotide sequences identified by gene identification numbers (whether GenelD, SEQ ID, transcript identifier, etc), and other publications identified herein are expressly incorporated by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art to which this invention pertains. Although human gene symbols are typically designated by uppercase letters, in the present specification the use of either upper-case or lower-case gene symbols may be used interchangeably and include both human and/or non-human species. In other words, the upper-case or lower-case letters in a particular gene symbol do not limit the scope of the gene or gene target to human or non-human species.

This application describes a variety of genes, transcripts, proteins, etc. using known names for the nucleic acid sequence. To the extent a specific sequence identifier is not cross-referenced to such a name, the artisan can readily do so by known means. For example, there are numerous searchable sites such as GeneCards (a collaborative searchable, integrated, database of human genes that provides concise genomic, transcriptomic, genetic, proteomic, functional and disease related information on all known and predicted human genes; database developed at the Crown Human Genome Center, Department of Molecular Genetics, the Weizmann Institute of Science), and publications that form the basis of such sites. One can readily use the name to locate the sequence and using such sequence cross-reference the Sequence ID No. used herein.

Throughout the specification, in some cases we have given the gene abbreviation or alias of the avian target gene and corresponding SEQ ID NOs for that gene or transcript. In some cases we have given the full gene name of the target gene, the corresponding SEQ ID No for the target gene (e.g., transcript sequence) as well as example nucleic acid based targeting construct SEQ ID NOs directed against the target gene. In various embodiments of the invention, the targeting construct is a siRNA that comprises an antisense strand comprising at least 16 contiguous nucleotides of a siRNA nucleotide sequence of any of the siRNA sequences identified herein by SEQ ID NO.

I. Avian transcript targets

Targets of the present invention include nucleic acid transcripts of avian cells. The avian nucleic acid transcripts include the genomic DNA genes and RNA transcripts (whether transcribed from either strand of chromosome and whether or not translated).

The avian transcript targets may be derived from the cells, cell cultures, tissue or hybridomas of any Superorder or Order of birds (Aves). Superorders include both the Palaeognathae and Neognathae. Orders of birds within the Paleaegnathae include the Struthioniformes (ostriches, emus, kiwis) and Tinamiformes (tinamous) any of whose cells may encode avian transcripts as targets of the invention. The superorder Neognathae includes the orders Anseriformes (waterfowl), Galliformes (fowl),

Charadriiformes (gulls, button-quails, plover), Gaviiformes (loons), Podicipediformes (grebes), Procellariiformes (albatrosses, petrels), Sphenisciformes (penguins),

Pelecaniformes (pelicans), Phaethontiformes (tropicbirds), Ciconiiformes (storks), Cathartiformes (New World vultures), Phoenicopteriformes (flamingos), Falconiformes (falcons, eagles, hawks), Gruiformes (cranes), Pteroclidiformes (sandgrouse),

Columbiformes (doves and pigeons), Psittaciformes (parrots), Cuculiformes (cuckoos and turacos), Opisthocomiformes (hoatzin), Strigiformes (owls), Caprimulgiformes

(nightjars), Apodiformes (swifts and hummingbirds), Coraciiformes (kingfishers), Piciformes (woodpeckers), Trogoniformes (trogons), Coliiformes (mousebirds) and Passeriformes (passerines).

In one embodiment, the avian transcript targets are those of the Order

Anseriformes (waterfoul) and in particular those of ducks. Ducks whose transcripts, cells, cell cultures or tissues which may be targeted by the targeting constructs of the present invention include Abacot Ranger (also known as Streicher), Allier Duck (Blanc dAllier), Ancona duck, Aylesbury Duck, Bali Duck, Black East Indian, Blue Swedish duck, Buff Orpington Duck, Call Duck, Challans, Cayuga Duck, Chara Chemballi Duck, Crested Duck, Danish Duck, Duclair, Dutch Hookbill (kromsnaveleend), East Indie Duck, Forest Duck (Eend van Vorst), Gimbsheimer, Golden Cascade, Gressingham Duck (Wild Mallard crossed with Pekin), Huttengem Duck, Indian Runner Duck, Magpie Duck,

Majorcan Duck, Muscovy duck, Orpington duck, Pekin Duck (also known as Long Island duck), Rouen Duck, Saxony Duck, Semois, Silver Appleyard Duck, Silver Bantam Duck, Termonde Duck, Venetian Duck (Germanata Veneta), Welsh Harlequin Duck, Wood Duck and the Swedish Duck.

In one embodiment the transcripts, cells or cell cultures are from the Pekin duck.

Cells of the Pekin duck can be obtained from ATCC (Mannassas, VA; number CCL-141; Anas platyrhynchus domesticus). Other duck cells which may be used in the methods of the present invention include those disclosed in US Publications 20090081251 ,

20100062489 and 20100226912, each of which is incorporated herein in its entirety. For example, cells derived by the methods in these publications such as EB12, EB24, or EB66 may serve as suitable cell lines for the present invention. Targeting constructs which are useful in, for example, the CCL-1 line may also be applied to any of the EB- series of cell lines, or derivatives or hybrids thereof.

Avian genes may be as small as lkb (kilobase) or as large as lOOkb (kilobases) while avian transcripts may range in size from 50 nucleotides to 20kb.

Avian transcripts may be at least 50, 75, 100, 150, 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, at least 900 nucleotides, at least 1000 nucleotides, at least 5000 nucleotides, at least 10,000 nucleotides or at least 20,000 nucleotides, and range from 250-300 nucleotides, 300-400 nucleotides, 400-500 nucleotides, 500-600 nucleotides, 600-700 nucleotides, 700-800 nucleotides, 800-900 nucleotides, 900-1000 nucleotides, 1000-5000 nucleotides, 5000-10,000 nucleotides, or 10,000-20,000 nucleotides in length.

As used herein, the term "avian gene" refers to a gene which is encoded within an avian genome or in a genomic construct (whether natural or synthetic). Endogenous avian genes, e.g., those encoded within or engineered to be encoded by a host cell genome, may be intronic or intergenic and may encode proteins or other structural or functional R As. Avian genes include non-coding genes including those for lincR A (long non coding RNAs) and the like.

"Intronic avian genes" are those found to be encoded substantially within an intron of a gene.

"Intergenic avian genes" are those found to be encoded between two different genes.

As used herein, the term "avian transcript," or "avian transcript target" refers to an RNA transcript encoded by an "avian gene." Avian transcripts may encode peptides of 50 amino acids or less. It should be understood that avian transcripts may be synthesized as avian transcript variants, which may be engineered to encode a variety of peptides or proteins.

Other avian transcripts which may be targets of the invention includes primary microRNAs (pri-miRs) which are processed to produce micro-RNAs or small RNA species. In this context, either the pri-miRs or the pre-miRs may be avian transcript targets.

Representative avian transcript targets are listed in Lengthy Table 1. Certain avian genes of the present invention were identified from the Ensembl database

(www.ensembl.org) and their respective RNA transcripts were extracted (SEQ ID NOs: 1-17169, having transcript names ENSAPLT00000000001 through

ENSAPLT00000017169). Other microRNA avian transcripts were idenfied via internal sequencing efforts and these are also listed in Lengthy Table 1 (SEQ ID NOs: 17170 through 18039). To be clear, Table 1 lists the Ensembl gene identifier of each avian transcript with the prefix, E SAPLT.

Also reported in Lengthy Table 1 are the length of the transcript, type and description of the transcript, SEQ ID NO of the transcript and the SEQ ID NOs of the Targeting Constructs designed to target each avian transcript. Where no targeting constructs were designed, the entry "NA" appears in the table. However, the selection of nucleic acid targeting constructs which would hybridize to the avian transcripts provided in Table 1 (and the sequence listing) is within the skill of one in the art.

As used herein, "target gene" refers to a gene that affects one or more aspects of the production of a biological product by a host cell, such that modulating expression of the gene enhances production of the biological product. Target genes may or may not encode proteins. Target genes can be derived from the host cell, may be latent in the host cell, may be endogenous to the host cell (present in the host cell genome), may be transgenes (gene constructs inserted at ectopic sites in the host cell genome), or may be derived from a pathogen (e.g., a virus, fungus or bacterium) that is capable of infecting the host cell or the subject who will use the biological product or derivatives thereof (e.g., humans). Additionally, in some embodiments, a "target gene"refers to a gene that regulates expression of a nucleic acid (i.e., non-encoding genes) that affects one or more aspects of the production of a biological product by a cell, such that modulating expression of the gene enhances production of the biological product.

In one embodiment, target genes are avian transcripts. By "target gene R A" or "target R A" is meant R A transcribed from the target gene. Hence, a target RNA may comprise a coding region, an introns, an exon, an introns-exon junction, a promoter region, a 3' untranslated region (3'-UTR), and/or a 5'- UTR of the target gene.

A target gene RNA that encodes a polypeptide is more commonly known as messenger RNA (mRNA). In some embodiments, the target gene encodes a protein that affects one or more aspects of post-translational modification, e.g., peptide glycosylation, by an avian host cell.

In some embodiments, the target gene encodes an avian non-coding RNA

(ncRNA), such as an untranslated region. As used herein, a ncRNA refers to a target gene RNA that is not translated into a protein. The ncRNA can also be referred to as non- protein-coding RNA (npcRNA), non-messenger RNA (nmRNA), small non-messenger RNA (snmRNA), and functional RNA (fRNA) in the art. The target gene from which a ncRNA is transcribed as the end product is also referred to as a RNA gene or ncRNA gene. ncRNA genes include highly abundant and functionally important RNAs such as transfer RNA (tRNA) and ribosomal RNA (rRNA), long intervening or intergenic RNAs (IncRNAs), as well as RNAs such as snoRNAs, microRNAs, siRNAs, and piRNAs.

In some embodiments, the target gene is an endogenous gene of the avian host cell. For example, the target gene can encode the biological product or a portion thereof when the biological product is a polypeptide. The target gene can also encode an avian host cell protein that directly or indirectly affects one or more aspects of the production of the biological product. Examples of target genes that affect the production of polypeptides include genes encoding proteins involved in the secretion, folding or post- translational modification of polypeptides (e.g., glycosylation, deamidation, disulfide bond formation, methionine oxidation, or pyroglutamation); genes encoding proteins that influence a property or phenotype of the host cell (e.g., growth, viability, cellular pH, cell cycle progression, apoptosis, carbon metabolism or transport, lactate formation, cytoskeletal structure (e.g., actin dynamics), susceptibility to viral infection or RNAi uptake, activity or efficacy); and genes encoding proteins that impair the production of a biological product by the host cell (e.g., a protein that binds or co-purifies with the biological product).

In some embodiments, production of a biological product is enhanced by targeting the expression of a protein that binds to the biological product. For example, in producing a growth factor, a hormone, or a cell signaling protein, it can be advantageous to reduce or inhibit expression of its receptor/ligand so that its production in the cell does not elicit a biological response. A receptor can be a cell surface receptor or an internal (e.g., nuclear) receptor. The expression of the binding partner can be modulated by contacting the host cell with a targeting construct targeting the receptor target gene according to methods described herein.

In some embodiments, the target gene encodes an avian host cell protein that indirectly affects the production of the biological product such that inhibiting expression of the target gene enhances production of the biological product. For example, the target gene can encode an abundantly expressed avian host cell protein that does not directly influence production of the biological product, but indirectly decreases its production, for example by utilizing cellular resources that could otherwise enhance production of the biological product.

Consequently, the production of biomolecules and biological products can be affected by modulation of the expression of an avian transcript target. The term

"modulates expression of and the like, in so far as it refers to a target gene, herein refers to the alteration of expression of a target gene, as manifested by a change (e.g., an increase or a decrease) in the amount of target gene R A, mR A, or protein that can be isolated from or detected in cells in which a target gene is transcribed (and/or translated) and that have been treated such that the expression of a target gene is modulated, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but that have not been so treated (control cells). Avian cells are preferred cells of the invention.

The degree of modulation can be given in terms of a percent change of control or in terms of a parameter that is functionally linked to target gene expression, e.g., the amount of protein encoded by a target gene, or the number of cells displaying a certain phenotype, e.g., stabilization of microtubules. In principle, target gene modulation can be determined in any avian host cell expressing the target gene, either constitutively or by genomic engineering, and by any appropriate assay known in the art.

In certain instances, expression of a target gene is inhibited. For example, expression of a target gene is inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of a targeting construct provided herein. In some embodiments, a target gene is inhibited by at least about 60%, 70%, or 80% by administration of a targeting construct. In some embodiments, a target gene is inhibited by at least about 85%, 90%, or 95% or more by administration of a targeting construct as described herein. In other instances, expression of a target gene is activated by at least about 10%, 20%, 25%, 50%, 100%, 200%, 400% or more by administration of a targeting construct provided herein. In some embodiments, the modulation of expression is a partial inhibition. In some aspects, the partial inhibition is no greater than a percent inhibition selected from the group consisting of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and 85%.

It is expected that modulation or alteration of avian transcript function or levels (modulation of expression) can be used to regulate chromatin status, gene expression, transcription, translation, post-translational events and global biomolecular trafficking in the cell.

Methods of designing, modulating or targeting avian transcripts may be either structure-based or sequence based. Traditionally, methods of targeting nucleic acid molecules in the cell have been sequenced based and have depended in some form on harnessing the hybridization or base pairing of two complementary molecules.

Sequence-based methods of modulating or altering avian transcript function and levels are described herein.

Also described are herein structure -based methods. As used herein, "structure based methods" are those methods of altering or modulating an avian transcript function or level that depends on the determination or knowledge of the higher order structure of at least a portion of an avian transcript target. "Higher order structures" include but are not limited to the overall secondary, tertiary or quarternary structure of a molecule, e.g., hairpin structures, bulges, etc. These structures may be determined informatically with prediction algorithms based on thermodynamic parameters and energy calculations. Preferably, secondary structure prediction is performed using either M-fold or RNA Structure algorithm. Programs for secondary structure determination are freely available online. Structures may also be determined by NMR, Mass Spectroscopy or by crystallographic methods. For RNA molecules, methods of determining overall structure or structures of portions of the RNA molecule are known in the art.

In US Patent 6,221 ,587, incorporated herein by reference in its entirety, are methods of identifying secondary structures in eukaryotic and prokaryotic RNA molecules termed "molecular interaction sites." Molecular interaction sites are small, usually less than 30 nucleotides, independently folded, functional subdomains contained within a larger RNA molecule. These methods may be used to determine molecular interaction sites on avian transcripts.

Avian transcript targets may also be subjected to mimicry design. Disclosed in US Patent 6,368,863 incorporated herein by reference in its entirety, are methods of identifying protein interacting sites on an RNA molecule and then designing an oligonucleotide that mimics that portion of the larger RNA molecules. These methods may be used in the present invention to design small avian transcript target mimics which will bind proteins.

Unlike sequence-based or hybridization driven targeting, which must rely on access of the targeting molecule to the target in order for base pairing to occur, structure- based targeting embraces a larger portion of the avian transcript target such as one or more features.

"Features," when referring to avian transcripts, are defined as distinct nucleic acid-based components of the molecule. Features of the avian transcripts of the present invention may be structural features and may include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini or any combination thereof. When designing avian transcript variant molecules, the starting molecule may be one selected from Table 1 or known in the cell as the wild type molecule. Alternatively, a series of modifications may be made in which the starting molecule may be referred to simply as the parent molecule. Structural features of the present invention may be at least 200 nucleotides in length or from about 200 to about 500 nucleotides in length or from about 200 to about 300 nucleotides in length or from about 50 to about 100 nucleotides in length. They may also comprise the whole or any part of a defined structural feature. Structural features may be 4-10, 5-15, 10-20, 10-30, or 20-50 nucleotides in length. These may be represented in increments of the triplet code and therefore may be any multiple of three. For example, features may be from 15-18, 15-30, 15-36, 15-60, 30-60, 30-90, 30-120 or larger.

As used herein when referring to avian transcripts the term "surface

manifestation" refers to a nucleic acid based component of an avian transcript appearing on an outermost surface of the avian transcript.

As used herein when referring to avian transcripts the term "local conformational shape" means a nucleic acid based structural manifestation of an avian transcript which is located within a definable space of the avian transcript.

As used herein when referring to avian transcripts the term "fold" means the resultant conformation of a nucleic acid sequence upon energy minimization. A fold may occur at the secondary or tertiary level of the folding process. Examples of secondary level folds include hairpins, loops and bulges. Examples of tertiary folds include domains and regions formed due to aggregation or separation of energetic forces. Regions formed in this way include hydrophobic and hydrophilic pockets, and the like.

As used herein the term "turn" as it relates to avian transcript conformation means a bend which alters the direction of the backbone of a poly- or oligonucleotide and may involve one, two, three or more nucleotides.

As used herein when referring to avian transcripts the term "loop" refers to a structural feature of a poly- or oligonucleotide which reverses the direction of the backbone of a poly- or oligonucleotide and comprises four or more nucleotides.

As used herein when referring to avian transcripts the term "half-loop" refers to a portion of an identified loop having at least half the number of nucleotides as the loop from which it is derived. It is understood that loops may not always contain an even number of nucleotides. Therefore, in those cases where a loop contains or is identified to comprise an odd number of nucleotides, a half-loop of the odd-numbered loop will comprise the whole number portion or next whole number portion of the loop (number of nucleotides of the loop/2+/-0.5 nucleotides). For example, a loop identified as a 7 nucleotide loop could produce half-loops of 3 nucleotides or 4 nucleotides (7/2=3.5+/-0.5 being 3 or 4).

As used herein when referring to avian transcripts the term "domain" refers to a motif of a poly- or oligonucleotide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions, etc).

As used herein when referring to avian transcripts the term "half-domain" means portion of an identified domain having at least half the number of nucleotides as the domain from which it is derived. It is understood that domains may not always contain an even number of nucleotides. Therefore, in those cases where a domain contains or is identified to comprise an odd number of nucleotides, a half-domain of the odd-numbered domain will comprise the whole number portion or next whole number portion of the domain (number of nucleotides of the domain/2+/-0.5 nucleotides). It is also understood that sub-domains may be identified within domains or half-domains, these subdomains possessing less than all of the structural or functional properties identified in the domains or half domains from which they were derived. It is also understood that the nucleotides that comprise any of the domain types herein need not be contiguous along the backbone of the poly- or oligonucleotide (i.e., nonadjacent nucleotides may fold structurally to produce a domain, half-domain or subdomain).

As used herein when referring to avian transcripts the term "site" represents a location for targeting an avian transcript. A site represents a position within a poly- or oligonucleotide that may be modified, manipulated, altered, derivatized or varied within the polypeptide based molecules of the present invention. In one embodiment, "sites" of targeting can represent hundreds to thousands of nucleotides and may include nucleotides very distal in sequence location. For example, upon folding, avian transcripts may present surfaces, domains or sites which comprise nucleotides which ony appear juxtaposed due to the folded nature of the avian transcript. In one embodiment of the invention, targeting constructs may target any site on an avian transcript.

As used herein the terms "termini or terminus" when referring to avian transcripts refers to an extremity of a poly- or oligonucleotide. Such extremity is not limited only to the first or final site of the poly- or oligonucleotide but may include additional nucleotides in the terminal regions. The poly- or oligonucleotide based molecules of the present invention may be characterized as having both a 5' and a 3' terminus. Poly- or oligonucleotides of the invention are in some cases made up of multiple chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers or dendrimers). These sorts of poly- or oligonucleotides will have multiple 5' and 3'- termini. Alternatively, the termini of the poly- or oligonucleotide may be modified such that they begin or end, as the case may be, with a non- poly- or oligonucleotide based moiety such as a conjugate.

Once any of the features have been identified or defined as a component of an avian transcript of the invention, any of several manipulations and/or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating them to produce additional avian transcript variants.

In one embodiment an avian transcript transcript variant is designed to encode a targeting construct. For example, an avian transcript may comprise the sequence of a shRNA or other nucleic acid based targeting construct. When encoded in the avian transcript variant the targeting construct (nucleic acid based) may be one that targets a different site on the avian transcript in which is is encoded or it may target the RNA transcript of a coding gene or any nucleic acid based transcript to which it will either hybridize (sequence based targeting) or form an interation with (structure based targeting).

Furthermore, it is understood that manipulation of features may result in the same outcome as a modification to the molecules of the invention. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a full length molecule would. Modifications and manipulations can be accomplished by methods known in the art. The resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.

In one embodiment of the invention, a feature of an avian transcript is removed to produce an avian transcript variant.

In one embodiment of the invention, a feature of an avian transcript is duplicated to produce an avian transcript variant.

In one embodiment of the invention, a feature of an avian transcript is swapped with a second feature of an avian transcript to produce an avian transcript variant. In some embodiments the second feature is from the same or a different avian transcript.

In one embodiment, hairpin features of an avian transcript are altered or modified. Hairpin structures of a first avian transcript may be inserted into a second avian transcript. They may also be removed from an avian transcript. Where a feature is found to have a biological activity such as an interface for binding, or as a signal for

localization, the feature may be reproduced in isolation by chemical or synthethic methods and used as targeting construct of the invention.

In one embodiment of the invention, avian transcripts may be targeted to alter cellular memory, or cell identity. Without wishing to be bound by theory, it is believed that the avian transcripts may contribute to cellular memory and play a determinative role in the cells ability to produce daughter cells of the same lineage or RNA population signature thereby maintaining the identity of the cells during cell divisions. As used herein, the "RNA population signature" of a cell is the qualitative complement of RNA transcripts present in a cell at a particular time or timeframe that distinguishes the cell from other cell types. It should be understood that an RNA population signature of a cell does not necessarily comprise the sum total of all RNAs present in a cell but a set or subset of transcripts which may be used to identify one cell type from another cell type. RNA population signatures may comprise "nuclear signatures", "cytoplasmic signatures", "organelle associated signatures," "tissue-associated signatures," or combinations thereof. They may also comprise the set of avian genes or transcripts or subsets thereof. For example, the RNA nuclear population signature of an avian cell may comprise the set or subset of RNA transcripts present in the nucleus of the cell at a particular time or developmental phase such that this signature can be compared to other cells in order to determine whether the cells are of the same type or along the same path of lineage. Methods of measuring the presence of RNA in a cell are well known in the art. Methods of determining the cell type of a specific cell are known in the art and include for example the measurement of cell type specific markers such as proteins or protein or ligand expression, receptor or ligand presence or secretion.

In one embodiment of the present invention, the RNA population signature of an avian cell may be measured or identified and compared to another cell. The comparison will reveal differences between the two signatures. Differences in the avian transcript components of the signature may then be assessed and a cellular RNA population may be supplemented or reduced to effect a similar signature in the target cell population.

Avian cells of the present invention include, but are not limited to, cells that are derived primarily from endoderm (gland cells, exocrine secretory epithelial cells, hormone secreting cells, epithelial cells lining closed internal body cavities); cells derived primarily from ectoderm (integumentary system, keratinizing epithelial cells, wet stratified barrier epithelial cells, nervous system cells, sensory transducer cells, autonomic neuron cells, sense organ and peripheral neuron supporting cells, central nervous system neurons and glial cells, lens cells); cells derived primarily from mesoderm (metabolism and storage cells, barrier function cells (Lung, Gut, Exocrine Glands and Urogenital Tract), kidney; extracellular matrix secretion cells, contractile cells, blood and immune system cells, pigment cells, nurse cells and interstitial cells). Avian stem cells include, but are not limited to adult, embryonic, pluripotent, totipotent, and induced pluripotent.

In one embodiment of the invention are methods of controlling or reprogramming the cellular memory of a cell by adminsitering to said cell or introducing into said cell a targeting construct which alters the function of an avian transcript. Alnternatively, the RNA population signature may be altered by adding back one or more avian genes or transcripts. By altering the level of avian transcripts, cellular processes are altered such that the cell may differentiate along a different path to alter the phenotype of the cell or to mirror the RNA population signature of a target cell. In one embodiment, alteration of cellular processs improves production of a biological product from an avian cell.

Alternatively, it is contemplated that the RNA population signature of an avian cell may be altered by administering a targeting construct or an exogenously prepared avian gene or transcript, the outcomes of which would result in the alteration of the cellular phenotype. In one example, avian cells may be treated to alter the lineage or differentiation state of for example stem cells or cells of early developmental lineage.

These methods and compositions have utility in the areas of not only cellular regulation but in the field of stem cell technology and the guided evolution of avian cellular pheno types including the processes of bioproduction.

In one embodiment of the invention, the RNA population signature of an avian cell, cell line or tissue may be used in screening applications. According to the present invention, methods are provided for the use of the RNA population signature, more specifically the avian transcript RNA population signature of an avian cell or tissue, in screening applications. It is also the case that avian transcripts may be supplemented in cells or cell populatations that lack the normative complement of avian genes or transcripts. Supplementation may be from a donor cell which is avian. The donor cell may also be non-avian.

In one embodiment, the milieu of a cell or tissue may be used to provide a signaling environment for the study or exploitation of avian transcript regulation. As used herein, the term "milieu of a cell or tissue" means the supernatant or "soup" of a cell population or an extract of the cell system.

According to the present invention, a first population of cells may be incubated in the milieu of a second population of cells in order to provide an environment which alters the expression levels or RNA population signature of the first population of cells. At least one of the first or second cell populations may be avian. To the milieu may be added one or more avian genes, transcripts or targeting constructs of the present invention. As a consequence of this addition the development, differentiation or overall gene expression profile of the first population of cells may be changed. Cells which may be used to provide the incubating milieu or which may be incubated in the milieu include but are not limited to somatic or gamete, stem cells, pluripotetent cells, cells of primary origin, cells of any avian tissue, etc.

In one embodiment of the invention, chromatin inactivation or activation may be effected by the administration of one or more targeting constructs (whether sequence based or structure based) or the administration of one or more avian genes, avian transcripts or variant avian transcripts. According to the present invention, avian transcripts may be added to cells or cell systems to alter the epigenetic landscape of a cell or tissue.

In one embodiment of the invention, avian transcript cassettes may be added to or administered to a cell or tissue. As used herein an "avian transcript cassette" is a polynucleotide that encodes one or more avian transcripts. Avian transcript cassettes may endcode a full length wild type avian transcript or may be designed to encode a modified avian transcript. The term "modified avian transcript" means an avian transcript which differs from the wild type sequence of the avian transcript in question.

Modifications to avian transcripts include those modifications to the exonic structure of the avian transcript and include those having shuffled exon structures, omitted exons and additional exons.

According to the present invention, the avian transcript cassette may also contain a modified promoter from that which is found in the wild type avian transcript. Promoters may be swapped with those of protein coding genes or other avian genes. They may also be modified by addition, deletion or shuffling of promoter components. Synthetic avian genes or transcripts may be modified before contacting or administration to cells and the promoters may have temporary or permanent tags or transcription factors pre-associated with them. Targeting constructs of the present invention may also be designed to target the promoters of avian genes. In this design, the targeting constructs may be linked to, conjutaged, associated or complexed with factors that target the targeting construct to the site of the avian transcript in the cell.

Where avian transcripts are found to encode or engineered to encode proteins or peptides, these proteins or peptides or their locus within the avian transcript may be a target of the invention. Consequently, methods of regulating avian transcripts of the present invention may also regulate their encoded peptides.

As used herein "synthetic" refers to a state of having been created or man-made, e.g., not of natural origin. The targeting constructs or avian transcripts of the present invention may be synthesized using chemical or enzymatic or recombinant methods. They may then be isolated from the synthetic mixture. Compounds of the present invention may also be isolated from a natural source.

Targeting constructs or avian transcripts of the present invention may be associated with chromatin modifying complexes, nucleosome components, proteins or enzymes. They may also be modified to localize to either the nucleus or cytoplasm of the cells. In this manner, the targeting constructs or avian transcripts of the present invention may be guided to specific sites in a cell or tissue and may affect cellular processes such as gene expression, imprinting, aging, epigenetic signatures and the like.

II. Targeting constructs

The compositions of the present invention are those which may be used to regulate, control, manipulate, perturb or otherwise alter the expression, levels, activity or status of avian transcripts which in turn may alter processes in avian cells, cell culture or avian tissues. As such, the compositions of the present invention are termed "targeting constructs" or "TC."

The targeting constructs of the present invention broadly include, but are not limited to, oligonucleotides, polynucleotides, iR A agents, antisense molecules, ribozymes, aptamers, small molecules, amino acid-based constructs (antibodies, peptides, proteins, enzymes or fragments thereof), and vitamins.

Preferably, the targeting constructs useful for the methods and compositions featured in the invention specifically target RNAs (primary or processed) of the avian transcript target. Compositions and methods for inhibiting the expression of avian transcripts using iR As can be prepared and performed as described herein. In one embodiment, a composition containing a targeting construct, where the targeting construct includes a nucleotide sequence that is complementary to at least a part of an R A or DNA transcript of the avian transcript is administered to an avian cell.

In one embodiment, a targeting construct as described herein effects modulation, inhibition or activation of avian transcript expression. As used herein, the term "modulate the expression of," refers to an at least partial "inhibition" or partial "activation" of an avian gene or transcript expression in a cell contacted with a targeting construct composition as described herein compared to the expression of an avian gene or transcript in an untreated cell. Modulation of expression may be determined not only by direct measurement of an avian transcript level after contacting with the targeting construct, but also be inference by associating a known phenotypic outcome which correlates to said contacting.

The term "expression" as used herein is intended to mean the transcription to a RNA and/or translation to one or more polypeptides from a target gene (e.g., the avian transcripts) coding for the sequence of the RNA and/or the polypeptide. The terms "silence," "inhibit the expression of," "down-regulate the expression of," "suppress the expression of," and the like, in so far as they refer to an avian gene or transcript, herein refer to the at least partial suppression of the expression of avian transcript, as manifested by a reduction of the amount of the avian transcript in whole or in part which may be isolated from or detected in a first cell or group of cells in which an avian transcript is transcribed and which has or have been treated such that the expression of an avian transcript is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).

Alternatively, inhibition or the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to avian transcript expression, e.g., the amount of protein encoded by a mRNA that itself is controlled by an avian transcript, or the number of cells displaying a certain phenotype, e.g., lack of or decreased cytokine production or the status of a cell, e.g., the epigenetic profile or signature of a cell which is altered upon modulation of one or more avian transcript targets.

In principle, avian transcript silencing may be determined in any cell expressing avian transcripts, either constitutively or by genomic engineering, and by any appropriate assay.

For example, in certain instances, expression of an avian transcript is suppressed by a targeting construct by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of a targeting construct featured in the invention. In some embodiments, an avian transcript is suppressed by at least about 60%, 70%, or 80% by administration of a targeting construct featured in the invention. In some embodiments, an avian transcript is suppressed by at least about 85%, 90%, 95%, 98%, 99%, or more by administration of a targeting construct as described herein.

The terms "activate," "enhance," "up-regulate the expression of," "increase the expression of," and the like, in so far as they refer to an avian gene or transcript, herein refer to the at least partial activation of the expression of an avian transcript, as manifested by an increase in the amount of avian transcript in whole or in part, which may be isolated from or detected in a first cell or group of cells in which an avian transcript is transcribed and which has or have been treated such that the expression of an avian transcript is increased, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).

In one embodiment, expression of an avian transcript is activated by a targeting construct by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of a targeting construct as described herein. In some embodiments, an avian transcript is activated by at least about 60%, 70%, or 80% by administration of a targeting construct featured in the invention. In some embodiments, expression of an avian transcript is activated by at least about 85%, 90%, or 95% or more by

administration of a targeting construct as described herein. In some embodiments, the avian transcript expression is increased by at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000 fold or more in cells treated with a targeting construct as described herein compared to the expression in an untreated cell. Activation of expression of coding mRNAs by small dsR As is described, for example, in Li et al., 2006 Proc. Natl. Acad. Sci. U.S.A.

103: 17337-42, and in US200701 11963 and US2005226848, each of which is

incorporated herein by reference. It is believed that constructs that activate the expression of coding R A transcripts will also activate the expression of non-coding RNA transcripts such as the avian transcripts of the present invention.

Targeting constructs may be designed to target regions or sites along an avian transcript which correlate to hypersensitivity sites (HS) found on the corresponding DNA encoding the avian transcript. Hypersensitivity sites (HS) and methods of identification are described in PCT Publication WO/2004/053106, the contents of which are incorporated by reference herein in its entirety.

In some embodiments, the "region" when used in reference to a targeting construct comprises a portion of a targeting construct. Where the targeting construct is an oligonucleotide, one of skill in the art can vary the length of the "region" that is complementary to the target gene or arranged in a duplex, such that a targeting construct having desired characteristics (e.g., inhibition of a target gene or stability) is produced. In one embodiment, the targeting constructs of the present invention may target, mimic, bind to, replace or alter the levels or function of a product of an avian transcript. As used herein "avian transcript products" include any molecule engineered to be a product of an avian transcript either by transcription, translation, cleavage, splicing, or other mechanism that produces a derivative of an avian transcript. Examples of avian transcript products include, but are not limited to, peptides or proteins engineered to be coded by the avian transcript or fragments of the avian transcript.

Targeting constructs may be oligonucleotides or nucleic acid molecules. In the context of this invention, the term "oligonucleotide" or "nucleic acid molecule" encompasses not only nucleic acid molecules as expressed or found in nature, but also analogs and derivatives of nucleic acids comprising one or more ribo- or deoxyribo- nucleotide/nucleoside analogs or derivatives as described herein or as known in the art. Such analogs or derivatives (including modified and/or substituted oligonucleotides) are often used over natural (native or wild type) forms because of properties such as, for example, enhanced cellular uptake, increased stability in the presence of nucleases, and the like, discussed further herein. A "nucleoside" includes a nucleoside base and a ribose sugar, and a "nucleotide" is a nucleoside with one, two or three phosphate moieties. The terms "nucleoside" and "nucleotide" can be considered to be equivalent as used herein. An oligonucleotide can be modified in the nucleobase structure or in the sugar-phosphate backbone structure, e.g., as described herein, including the modification of a RNA nucleotide into a D A nucleotide.

As non-limiting examples, an oligonucleotide can also include at least one modified nucleoside including but not limited to a 2'-0-methyl modified nucleoside, a nucleoside comprising a 5' phosphorothioate group, a terminal nucleoside linked to a cholesterol derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2'-deoxy-2'-fiuoro modified nucleoside, a 2'-amino-modified nucleoside, 2'-alkyl-modified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof. Alternatively, an oligonucleotide can comprise at least two modified nucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or more, up to the entire length of the oligonucleotide. The modifications need not be the same for each of such a plurality of modified nucleosides in an oligonucleotide. When the targeting construct (TC) is double stranded oligonucleotide, each strand can be independently modified as to number, type and/or location of the modified nucleosides. In one embodiment, modified oligonucleotides contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA via a RISC pathway.

The terms "ribonucleoside", "ribonucleotide", "nucleotide", or

"deoxyribonucleotide" can also refer to a modified nucleotide, as further detailed herein, or a surrogate replacement moiety. A ribonucleotide comprising a thymine base is also referred to as 5 -methyl uridine and a deoxyribonucleotide comprising a uracil base is also referred to as deoxy-Uridine in the art. Guanine, cytosine, adenine, thymine and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the

compositions and methods featured in the invention.

Similarly, the skilled artisan will recognize that the term "RNA molecule" or "ribonucleic acid molecule" encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more

ribonucleotide or ribonucleoside analogs or derivatives as described herein or as known in the art.

In one aspect, a targeting construct can include a deoxyribonucleoside residue. In such an instance, a targeting construct agent can comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang(s), or one or more deoxynucleosides within the double stranded portion of a dsRNA.

In some embodiments, a plurality of targeting constructs is used to modulate expression of one or more target genes. A "plurality" refers to at least 2 or more targeting constructs e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 80, 100 targeting constructs or more. "Plurality" can also refer to at least 2 or more target genes, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 target genes or more.

Targeting constructs of the present invention are administered to the avian host cell in a variety of ways. Cells may be simply contacted and/or the targeting constructs may be taken up by the cells. As used herein the term "contacting a host cell" refers to the treatment of a host cell with an agent such that the agent is introduced into the cell. Typically the host cell is in culture, e.g., using at least one Targeting Construct (e.g., an siRNA), often prepared in a composition comprising a delivery agent that facilitates targeting construct uptake into the cell e.g., to contact the cell in culture by adding the composition to the culture medium. In one embodiment the host cell is contacted with a vector that encodes a targeting construct, e.g., an integrating or non-integrating vector. In one embodiment the cell is contacted with a vector that encodes a targeting construct prior to culturing the host cell for biological production, e.g., by transfection or transduction.

In one embodiment, contacting a host cell includes contacting an avian host cell with a vector that encodes a targeting construct, e.g., added to the host cell culture during the process of producing a biological product. The step of contacting a host cell in culture with a targeting construct(s) can be repeated more than once (e.g., twice, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, l lx, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, lOOx or more). In one embodiment, the cell is contacted such that the target gene is modulated only transiently, e.g., by addition of a targeting construct composition to the cell culture medium used for the production of a biological product where the presence of the targeting constructs dissipates over time, i.e., the targeting construct is not constitutively expressed in the cell. "Introducing into a cell", when referring to a targeting construct, means facilitating or effecting uptake or absorption into the avian cell, as is understood by those skilled in the art. Absorption or uptake of a targeting construct can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. For example, introducing into a cell means contacting a host cell with at least one targeting construct, or means the treatment of a cell with at least one targeting construct and an agent that facilitates or effects uptake or absorption into the cell, often prepared in a composition comprising the targeting construct and delivery agent that facilitates targeting construct uptake (e.g., a transfection reagent, an emulsion, a cationic lipid, a non-cationic lipid, a charged lipid, a liposome, an anionic lipid, a penetration enhancer, or a modification to the targeting construct to attach, e.g., a ligand, a targeting moiety, a peptide, a lipophillic group etc.). In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or known in the art.

Compositions comprising the targeting constructs of the present invention are also provided. As used herein, a "targeting construct composition" includes an effective amount of a targeting construct and an acceptable carrier. As used herein, "effective amount" refers to that amount of a targeting construct effective to produce an effect (e.g., modulatory effect) on a bioprocess for the production of a biological product. In one embodiment, the targeting construct composition comprises a reagent that facilitates targeting construct uptake (e.g., a transfection reagent, an emulsion, a cationic lipid, a non-cationic lipid, a charged lipid, a liposome, an anionic lipid, a penetration enhancer, or a modification to the targeting construct to attach e.g., a ligand, a targeting moiety, a peptide, a lipophillic group, etc.).

The term "acceptable carrier" refers to a carrier for administration of a targeting construct to avian cells. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. In one embodiment the term "acceptable carrier" specifically excludes cell culture medium.

In one embodiment, the method includes administering a targeting construct composition as described herein to an avian cell such that expression of the avian transcript is increased by e.g., at least 10% compared to an untreated cells. In some embodiments, the activation of avian transcripts occurs over an extended duration, e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, four weeks, or more. Without wishing to be bound by theory, a targeting construct can activate avian transcript expression by stabilizing the avian transcript, interacting with a promoter in the genome, and/or inhibiting an inhibitor of avian transcript expression.

Amino acid-based targeting constructs

The targeting constructs of the present invention may also be amino acid based molecules. These molecules may be "peptides," "polypeptides," or "proteins" whether structural (antibodies) or catalytic (enzymes). While it is known in the art that these terms imply relative size, these terms as used herein should not be considered limiting with respect to the size of the various polypeptide based molecules referred to herein and which are encompassed within this invention.

The terms "amino acid" and "amino acids" refer to all naturally occurring L- alpha-amino acids. The amino acids are identified by either the one-letter or three-letter designations as follows: aspartic acid (Asp:D), isoleucine (Ile:I), threonine (Thr:T), leucine (Leu:L), serine (Ser:S), tyrosine (Tyr:Y), glutamic acid (Glu:E), phenylalanine (Phe:F), proline (Pro:P), histidine (His:H), glycine (Gly:G), lysine (Lys:K), alanine (Ala:A), arginine (Arg:R), cysteine (Cys:C), tryptophan (Trp:W), valine (Val:V), glutamine (Gln:Q) methionine (Met:M), asparagines (Asn:N), where the amino acid is listed first followed parenthetically by the three and one letter codes, respectively.

The amino acid sequences of the targeting constructs of the invention may comprise naturally occurring amino acids and as such may be considered to be proteins, peptides, polypeptides, or fragments thereof. Alternatively, the targeting constructs may comprise both naturally and non-naturally occurring amino acids or only non-naturally occurring amino acids.

The term "amino acid sequence variant" refers to molecules with some differences in their amino acid sequences as compared to a native sequence. The amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence. Ordinarily, variants will possess at least about 70% homology to a native sequence, and preferably, they will be at least about 80%, more preferably at least about 90% homologous to a native sequence.

"Homology" as it applies to amino acid sequences is defined as the percentage of residues in the candidate amino acid sequence that are identical with the residues in the amino acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well known in the art. It is understood that homology depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation.

By "ho mo logs" as it applies to amino acid sequences is meant the corresponding sequence of other species having substantial identity to a second sequence of a second species.

"Analogs" is meant to include polypeptide variants which differ by one or more amino acid alterations, e.g., substitutions, additions or deletions of amino acid residues that still maintain the properties of the parent polypeptide.

The term "derivative" is used synonymously with the term "variant" and refers to a molecule that has been modified or changed in any way relative to a reference molecule or starting molecule.

The present invention contemplates several types of targeting constructs which are amino acid based including variants and derivatives. These include substitutional, insertional, deletion and covalent variants and derivatives. As such, included within the scope of this invention are polypeptide based molecules containing substitutions, insertions and/or additions, deletions and covalently modifications. For example, sequence tags or amino acids, such as one or more lysines, can be added to the peptide sequences of the invention (e.g., at the N-terminal or C-terminal ends). Sequence tags can be used for peptide purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble, or linked to a solid support.

"Substitutional variants" when referring to proteins are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.

As used herein the term "conservative amino acid substitution" refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.

"Insertional variants" when referring to proteins are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native or starting sequence. "Immediately adjacent" to an amino acid means connected to either the alpha-carboxy or alpha-amino functional group of the amino acid.

"Deletional variants" when referring to proteins are those with one or more amino acids in the native or starting amino acid sequence removed. Ordinarily, deletional variants will have one or more amino acids deleted in a particular region of the molecule.

"Covalent derivatives" when referring to proteins include modifications of a native or starting protein with an organic proteinaceous or non-proteinaceous derivatizing agent, and post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of the protein with an organic derivatizing agent that is capable of reacting with selected side-chains or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected recombinant host cells. The resultant covalent derivatives are useful in programs directed at identifying residues important for biological activity, for immunoassays, or for the preparation of anti-protein antibodies for immunoaffinity purification of the recombinant glycoprotein. Such modifications are within the ordinary skill in the art and are performed without undue experimentation.

Certain post-translational modifications are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues may be present in the proteins used in accordance with the present invention.

Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the .alpha.-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)).

Covalent derivatives specifically include fusion molecules in which proteins of the invention are covalently bonded to a non-proteinaceous polymer. The non- proteinaceous polymer ordinarily is a hydrophilic synthetic polymer, i.e. a polymer not otherwise found in nature. However, polymers which exist in nature and are produced by recombinant or in vitro methods are useful, as are polymers which are isolated from nature. Hydrophilic polyvinyl polymers fall within the scope of this invention, e.g.

polyvinylalcohol and polyvinylpyrrolidone. Particularly useful are polyvinylalkylene ethers such a polyethylene glycol, polypropylene glycol. The proteins may be linked to various non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

"Features" when referring to proteins are defined as distinct amino acid sequence- based components of a molecule. Features of the proteins of the present invention include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini or any combination thereof.

As used herein when referring to proteins the term "surface manifestation" refers to a polypeptide based component of a protein appearing on an outermost surface.

As used herein when referring to proteins the term "local conformational shape" means a polypeptide based structural manifestation of a protein which is located within a definable space of the protein.

As used herein when referring to proteins the term "fold" means the resultant conformation of an amino acid sequence upon energy minimization. A fold may occur at the secondary or tertiary level of the folding process. Examples of secondary level folds include beta sheets and alpha helices. Examples of tertiary folds include domains and regions formed due to aggregation or separation of energetic forces. Regions formed in this way include hydrophobic and hydrophilic pockets, and the like.

As used herein the term "turn" as it relates to protein conformation means a bend which alters the direction of the backbone of a peptide or polypeptide and may involve one, two, three or more amino acid residues.

As used herein when referring to proteins the term "loop" refers to a structural feature of a peptide or polypeptide which reverses the direction of the backbone of a peptide or polypeptide and comprises four or more amino acid residues. Oliva et al. have identified at least 5 classes of protein loops (J. Mol Biol 266 (4): 814-830; 1997).

As used herein when referring to proteins the term "half-loop" refers to a portion of an identified loop having at least half the number of amino acid resides as the loop from which it is derived. It is understood that loops may not always contain an even number of amino acid residues. Therefore, in those cases where a loop contains or is identified to comprise an odd number of amino acids, a half-loop of the odd-numbered loop will comprise the whole number portion or next whole number portion of the loop (number of amino acids of the loop/2+/-0.5 amino acids). For example, a loop identified as a 7 amino acid loop could produce half-loops of 3 amino acids or 4 amino acids (7/2=3.5+/-0.5 being 3 or 4).

As used herein when referring to proteins the term "domain" refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions.

As used herein when referring to proteins the term "half-domain" means portion of an identified domain having at least half the number of amino acid resides as the domain from which it is derived. It is understood that domains may not always contain an even number of amino acid residues. Therefore, in those cases where a domain contains or is identified to comprise an odd number of amino acids, a half-domain of the odd- numbered domain will comprise the whole number portion or next whole number portion of the domain (number of amino acids of the domain/2+/-0.5 amino acids). For example, a domain identified as a 7 amino acid domain could produce half-domains of 3 amino acids or 4 amino acids (7/2=3.5+/-0.5 being 3 or 4). It is also understood that sub- domains may be identified within domains or half-domains, these subdomains possessing less than all of the structural or functional properties identified in the domains or half domains from which they were derived. It is also understood that the amino acids that comprise any of the domain types herein need not be contiguous along the backbone of the polypeptide (i.e., nonadjacent amino acids may fold structurally to produce a domain, half-domain or subdomain).

As used herein when referring to proteins the terms "site" as it pertains to amino acid based embodiments is used synonymous with "amino acid residue" and "amino acid side chain". A site represents a position within a peptide or polypeptide that may be modified, manipulated, altered, derivatized or varied within the polypeptide based molecules of the present invention.

As used herein the terms "termini or terminus" when referring to proteins refers to an extremity of a peptide or polypeptide. Such extremity is not limited only to the first or final site of the peptide or polypeptide but may include additional amino acids in the terminal regions. The polypeptide based molecules of the present invention may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins of the invention are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These sorts of proteins will have multiple N- and C-termini. Alternatively, the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide based moiety such as an organic conjugate.

Once any of the features have been identified or defined as a component of a molecule of the invention, any of several manipulations and/or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating. Furthermore, it is understood that manipulation of features may result in the same outcome as a modification to the molecules of the invention. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a full length molecule would.

Modifications and manipulations can be accomplished by methods known in the art such as site directed mutagenesis. The resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.

Antibodies

In one embodiment of the present invention, antibodies and antibody technology may be employed to affect the expression or role of avian transcripts in a cell. As discussed elsewhere herein, antibodies may also represent the biological product or biomolecule to be produced by the methods of the present invention.

As used herein, the term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g.

bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

"Antibody fragments" comprise a portion of an intact antibody, preferably comprising the antigen binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')₂, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')₂ fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

For the purposes herein, an "intact antibody" is one comprising heavy and light variable domains as well as an Fc region.

"Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V_H) followed by a number of constant domains. Each light chain has a variable domain at one end (V_L) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

The term "variable domains" in reference to antibodies refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen.

"Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl , IgG2, IgG3, IgG4, IgA, and IgA2.

"Single-chain Fv" or "scFv" antibody fragments comprise the V_H and V_L domains of antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the V_H and V_L domains that enables the scFv to form the desired structure for antigen binding.

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain V_H connected to a light chain variable domain V_L in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen- binding sites.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants

(epitopes), each monoclonal antibody is directed against a single determinant on the antigen

The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. The monoclonal antibodies herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.

"Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.

The term "hypervariable region" when used herein in reference to antibodies refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from a "complementarity determining region.

In one embodiment of the invention, antibodies which alter the levels or function of avian transcripts may be designed to directly or indirectly affect avian transcript targets. The preparation of antibodies, whether monoclonal or polyclonal, is know in the art. Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane "Using Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, 1999.

Antibodies of the present invention may be designed to directly affect the levels of avian transcript targets by binding directly to the avian transcripts. Alternatively, antibodies can be designed to target the proteins encoded by the nearest neighbor (those immediately upstream or downstream on a chromosome) genes of avian transcripts, thereby altering the expression of the avian transcript.

Other proteins as targeting constructs

In one embodiment, artificial avian nucleosome proteins are provided. These proteins are only "artificial" in the sense that they are not identical to the wild-type proteins of the nucleosomes of the cells, or histones.

It is known in the art that nucleosomes undergo dynamic and necessary changes in order to facilitate presentation of genomic loci to the transcriptional machinery. This opening and closing of this chromosome packaging structure or "nucleosome respiration" is also accompanied by changes in the histone proteins at the nucleosome core.

Taking advantage of this cellular process, the present invention provides compositions and methods for the manipulation and/or control of avian gene expression and cellular regulation via the substitution or alteration of the protein components of avian nucleosomes.

More specifically, the present invention provides amino acid based compositions which replace one or more of the proteins contained in an avian nucleosome, but in doing so, alter the overall function of the nucleosome in a manner that alters the expression of one or more avian genes. The avian gene affected in this manner can be one whose DNA is packaged around the artificial nucleosome (e.g., the nucleosome harboring the protein variant) or one lying up or downstream of the artificial nucleosome whose transcription is altered as a result of the changes in the wild-type nucleosome.

In one embodiment are methods of altering the gene expression pattern of avian genes within a cell comprising contacting the cells with artificial nucleosome proteins.

In one embodiment are methods of altering the epigenetic signature of avian cells comprising contacting the cell with one or more artificial nucleosome proteins.

The artificial nucleosome proteins of the present invention include, but are not limited to analogs, homologs, variants or derivatives of wild- type avian histone proteins.

In one embodiment, the avian epigenetic signature, a component of which is the modificatoin pattern of the histones, e.g, methylation, may be changed by replacing wild type nucleosome proteins or histones with artificial histones engineered to have sites which are blocked such that cellular enzymes may no longer access or effect

modifications to the proteins. These modifications may be permanent or transient. For example, blocking access of histones by histone methyl transferases, or other modifying enzymes, can alter the status of a cells resulting in gene expression being

"transcriptionally on" or "transcriptionally off. This embodiment is an attractive alternative to the small molecules being developed to modulate cellular enzymes which act to acetylate, methylate, ubiquitinate, etc, DNA or nucleosome components. Other artificial proteins, not contained within the nucleosome, are also provided which may alter the expression of an avian gene. These include variants or derivatives of nucleic acid binding proteins (e.g., RNA binding proteins, DNA binding proteins, chaperones, and the like) either localized in or which traffic to or within the nucleus.

iRNA agents

"iRNA agents" of the present invention include, but are not limited to, small interfering RNAs (siRNA), double stranded RNAs (dsRNAs), inverted repeats, short hairpin RNAs (shRNAs), small temporally regulated RNAs (stRNA), clustered inhibitory RNAs (cRNAs), including radial clustered inhibitory RNA, asymmetric clustered inhibitory RNA, linear clustered inhibitory RNA, and complex or compound clustered inhibitory RNA, dicer substrates, DNA-directed RNAi (ddRNAi), single-stranded RNAi (ssRNAi), microRNA (miRNA) antagonists, microRNA mimics, microRNA agonists, blockmirs (a.k.a. Xmirs), microRNA mimetics, microRNA addbacks, supermiRs, the oligomeric constructs disclosed in PCT Publication WO/2005/013901 the contents of which are incorporated herein in its entirety, tripartite RNAi constructs such as those disclosed in US Publication 20090131360, the contents of which are incorporated herein in its entirety, the solo-rxRNA constructs disclosed in PCT Publication

WO/2010/01 1346, the contents of which are incorporated herein by reference in its entirety; the sd-rxRNA constructs disclosed in PCT Publication WO/2010/033247 the contents of which are incorporated herein by reference in its entirety, dual acting RNAi constructs which reduce RNA levels and also modulate the immune response as disclosed in PCT Publications WO/2010/002851 and WO/2009/141146 the contents of which are incorporated herein by reference in their entirety and antigene RNAs (agRNA) or small activiating RNAs (saRNAs) which increase expression of the target to which they are designed disclosed in PCT Publications WO/2006/130201 , WO/2007/086990,

WO/2009/046397, WO/2009/149182, WO/2009/086428 the contents of which are incorporated herein by reference in their entirety.

As used herein, the term "iRNA" refers to an agent that comprises at least an oligonucleotide component (e.g., nucleic acid, either RNA or DNA or modifications thereof), and which is capable of functioning one or more hybridization and/or binding mechanisms. In some embodiments, the iR A agent mediates the targeted cleavage of an R A transcript via an R A-induced silencing complex (RISC) pathway. In one embodiment, an iRNA agent as described herein effects inhibition of avian transcript expression. Alternatively, in another embodiment, an iRNA agent as described herein activates avian transcript expression. Alternatively, in one embodiment, an iRNA agent sterically blocks access to at least a portion of the avian transcript target. Such blocking can result in the modulation of avian transcript expression, levels or function.

It is also understood that iRNA agents may act via binding but not trigger any cleavage event, but exert an effect on the function of the avian transcript target by steric means. For example, the agent may block the site of another moiety which normally would bind to the avian transcript to itself effect cleavage.

In one embodiment, the iRNA agent will comprise nucleic acid and non-nucleic acid components and the nucleic acid component may be responsible for the binding but not directly for the alteration in function of the avian transcript target. For example, conjugates of iRNA agents may have two or more functions with the nucleic acid component providing at least the hybridization function, while second, third or additional components provide functional effect to the targeting construct.

As used herein when referring to iRNA agents, "target sequence" refers to a contiguous portion of the nucleotide sequence of a DNA molecule of an avian gene or RNA sequence formed during the transcription of an avian transcript, including the avian transcript that is a product of RNA processing of a primary transcription product. Where the targeting construct is an iRNA agent, the target sequence will be at least long enough to serve as a substrate for iRNA-directed activity ( e.g., cleavage, blocking, etc) at or near that portion. For example, the target sequence will generally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all sub-ranges therebetween. As non- limiting examples, the target sequence can be from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides,20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides.

Smaller target sequences are also contemplated by the present invention. In one embodiment the target sequence can be from 9-15 nucleotides, 10-12 nucleotides, 9 nucleotides, 10 nucleotides, 1 1 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, or 15 nucleotides.

As used herein, the term "strand comprising a sequence" refers to an

oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, or cell or tissue or cell culture can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary sequences within a targeting construct, e.g., within an iR A agent as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over a portion of one or both nucleotide sequences. When base-pairing is over the entire length of both sequences, such sequences can be referred to as "fully complementary" with respect to each other herein. However, where a first sequence is referred to as "substantially complementary" with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., modulation of gene expression via a RNAi pathway. For the case of longer sequences, (>30nt), mismatches may be as many as 10, 20, 30 or more up to 25% of the molecule. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, an iR A agent (e.g., dsRNA) comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as "fully complementary" for the purposes described herein.

"Complementary" sequences, as used herein, may also include, or be formed entirely from, non- Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non- Watson-Crick base pairs includes, but are not limited to, G:U Wobble or Hoogstein base pairing.

The terms "complementary," "fully complementary" and "substantially complementary" herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA or siRNA, or between the antisense strand of a targeting construct and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is "substantially complementary to at least part of an avian transcript refers to a polynucleotide that is substantially complementary to a contiguous portion of the avian transcript of interest (e.g., an avian transcript or gene). For example, a polynucleotide is complementary to at least a part of an avian transcript if the sequence is substantially complementary to a non-interrupted portion of an avian transcript or gene. "G," "C," "A," "T" and "U" each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. The skilled person is also aware that a representation of an oligonucleotide as DNA may also be construed as R A if the "T" nucleotides of the DNA are replaced in the sequence representation by "U".

In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target avian transcript. US Patent 7,732,593 describes constructs forming G-Uwobble base pairs and is incorporated herein by reference in its entirety. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

ssRNAi

In one aspect, an RNA interference agent (iRNA agent) includes a single stranded RNA that interacts with an avian transcript to direct the cleavage of the avian transcript. Thus, in one aspect the invention relates to a single stranded RNA that promotes silencing of the avian transcript, i.e., ssRNAi.

A "single strand iRNA agent" as used herein, is an iRNA agent which is made up of a single molecule. It may include a duplexed region, formed by intra-strand pairing, e.g., it may be, or include, a hairpin or pan-handle structure. Single strand iRNA agents are preferably antisense with regard to the target molecule. In preferred embodiments single strand iRNA agents are 5' phosphorylated or include a phosphoryl analog at the 5' prime terminus. 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5 '-monophosphate ((HO)2(0)P-0-5^*); 5 '-diphosphate ((HO)2(0)P-0-P(HO)(0)-0-5^*); 5^*-triphosphate ((HO)2(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5^*); 5^*-guanosine cap (7 -methylated or non- methylated) (7m-G-0-5^*-(HO)(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5^*); 5^*-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure ( -0-5'-(HO)(0)P-0- (HO)(0)P-0-P(HO)(0)-0-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P-0- 5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-0-5'), 5'- phosphorothiolate ((HO)2(0)P-S-5'); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((ΗΟ)2(0)Ρ-ΝΗ-5',

(ΗΟ)( Η2)(0)Ρ-0-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(0)-0-5^*-, (OH)2(0)P-5^*-CH2-), 5^*-alkyletherphosphonates

(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(0)-0-5'-). (These modifications can also be used with the antisense strand of a double stranded iRNA.) If the iRNA agent is a single strand it is particularly preferred that it include a 5 ' modification which includes one or more phosphate groups or one or more analogs of a phosphate group.

A single strand iRNA agent should be sufficiently long that it can enter the RISC and participate in RISC mediated cleavage of a target avian transcript. A single strand iRNA agent is at least 14, and more preferably at least 15, 20, 25, 29, 35, 40, or 50 nnucleotides in length. It is preferably less than 200, 100, or 60 nucleotides in length.

Hairpin i single strand RNA agents will have a duplex region equal to or at least

17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region will preferably be equal to or less than 200, 100, or 50, in length. Preferred ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length. The hairpin will preferably have a single strand overhang or terminal unpaired region, preferably the 3', and preferably of the antisense side of the hairpin. Preferred overhangs are 2-3 nucleotides in length.

pdRNA

In some embodiments, the targeting construct is a promoter-directed RNA (pdRNA) which is substantially complementary to at least a portion of a noncoding region of an mRNA transcript of an avian target gene. The pdRNA can be substantially complementary to at least a portion of the promoter region of the avian target gene mR A at a site located upstream from the transcription start site, e.g., more than 100, more than 200, or more than 1 ,000 bases upstream from the transcription start site. Also, the pdRNA can substantially complementary to at least a portion of the 3'-UTR of an avian target gene mRNA transcript. For example, the pdRNA comprises dsRNA of 18 to 28 bases optionally having 3' di- or tri-nucleotide overhangs on each strand. The dsRNA is substantially complementary to at least a portion of the promoter region or the 3 ' UTR region of a target gene mRNA transcript. In another embodiment, the pdRNA comprises a gapmer consisting of a single stranded polynucleotide comprising a DNA sequence which is substantially complementary to at least a portion of the promoter or the 3'-UTR of a target gene mRNA transcript, and flanking the polynucleotide sequences (e.g., comprising the five terminal bases at each of the 5' and 3' ends of the gapmer) comprising one or more modified nucleotides, such as 2'MOE, 2'OMe, or Locked Nucleic Acid bases (LNA), which protect the gapmer from cellular nucleases.

pdRNA can be used to selectively increase, decrease, or otherwise modulate expression of an avian target gene. Without being limited to theory, pdRNAs may modulate expression of target genes by binding to endogenous antisense RNA transcripts which overlap with noncoding regions of a target gene mRNA transcript, and recruiting Argonaute proteins (in the case of dsRNA) or host cell nucleases (e.g., RNase H) (in the case of gapmers) to selectively degrade the endogenous antisense RNAs. In some embodiments, the endogenous antisense RNA negatively regulates expression of the target gene and the pdtargeting construct activates expression of the avian target gene. Thus, in some embodiments, pdRNAs can be used to selectively activate the expression of an avian target gene by inhibiting the negative regulation of target gene expression by endogenous antisense RNA. Methods for identifying antisense transcripts encoded by promoter sequences of target genes and for making and using promoter-directed RNAs are known. See, e.g., WO 2009/046397; the contents of which are incorporated herein by reference in their entirety.

eiRNA Expressed interfering RNA (eiRNA) can be used to selectively increase, decrease, or otherwise modulate expression of an avian target gene. Typically with eiRNA, the dsRNA is expressed in the first transfected cell from an expression vector. In such a vector, the sense strand and the antisense strand of the dsRNA can be transcribed from the same nucleic acid sequence using e.g., two convergent promoters at either end of the nucleic acid sequence or separate promoters transcribing either a sense or antisense sequence. Alternatively, two plasmids can be cotransfected, with one of the plasmids designed to transcribe one strand of the dsRNA while the other is designed to transcribe the other strand. Methods for making and using eiRNA targeting constructs are known in the art. See, e.g., WO 2006/033756; U.S. Patent Pubs. No. 2005/0239728 and No.

2006/0035344.

piRNA

In some embodiments, the targeting construct comprises a small single-stranded Piwi-interacting RNA (piRNA) which is substantially complementary to at least a portion of an avian target gene, as defined herein, and which selectively binds to proteins of the Piwi or Aubergine subclasses of Argonaute proteins. Without being limited to a particular theory, it is believed that piRNA interact with RNA transcripts of target genes and recruit Piwi and/or Aubergine proteins to form a ribonucleoprotein (RNP) complex that induces transcriptional and/or post-transcriptional gene silencing of target genes. A piRNA can be about 10 to 50 nucleotides in length, about 25 to 39 nucleotides in length, or about 26 to 31 nucleotides in length. See, e.g., U.S. Patent Application Pub. No. 2009/0062228.

miRNA

MicroRNAs are a highly conserved class of small RNA molecules that are transcribed from DNA in the genomes of plants and animals, but are not translated into protein. Pre-microRNAs are processed into miRNAs. Processed micro RNAs are single stranded ~17 to 25 nucleotide (nt) RNA molecules that become incorporated into the RNA-induced silencing complex (RISC) and have been identified as key regulators of development, cell proliferation, apoptosis and differentiation. They are believed to play a role in regulation of gene expression by binding to the 3 '-untranslated region of specific mRNAs. MicroRNAs cause post-transcriptional silencing of specific target genes, e.g., by inhibiting translation or initiating degradation of the targeted mRNA. In some embodiments, the miRNA is completely complementary with the avian target nucleic acid. In other embodiments, the miRNA has a region of noncomplementarity with the target nucleic acid, resulting in a "bulge" at the region of non-complementarity. In some embodiments, the region of noncomplementarity (the bulge) is flanked by regions of sufficient complementarity, e.g., complete complementarity, to allow duplex formation. For example, the regions of complementarity are at least 8 to 10 nucleotides long (e.g., 8, 9, or 10 nucleotides long).

miRNA can inhibit gene expression by, e.g., repressing translation, such as when the miRNA is not completely complementary to the target nucleic acid, or by causing target RNA degradation, when the miRNA binds its target with perfect or a high degree of complementarity. In further embodiments, the targeting construct can include an oligonucleotide agent which targets an endogenous avian miRNA or pre-miRNA. For example, the targeting construct can target an endogenous miRNA which negatively regulates expression of an avian target gene, such that the targeting construct alleviates miRNA-based inhibition of the target gene. The targeting construct can include naturally occurring nucleobases, sugars, and covalent internucleotide (backbone) linkages and/or oligonucleotides having one or more non-naturally-occurring features that confer desirable properties, such as enhanced cellular uptake, enhanced affinity for the endogenous miRNA target, and/or increased stability in the presence of nucleases. In some embodiments, a targeting construct designed to bind to a specific endogenous miRNA has substantial complementarity, e.g., at least 70%, 80%, 90%, or 100% complementary, with at least 10, 20, or 25 or more bases of the target miRNA.

Exemplary targeting constructs that target miRNAs and pre-miRNAs are described, for example, in U.S. Patent Pubs. No. 20090317907, No. 20090298174, No. 20090291907, No. 20090291906, No. 20090286969, No. 20090236225, No. 20090221685, No.

20090203893, No. 20070049547, No. 20050261218, No. 20090275729, No.

20090043082, No. 20070287179, No. 20060212950, No. 20060166910, No.

20050227934, No. 20050222067, No. 20050221490, No. 20050221293, No.

20050182005, and No. 20050059005. Antagomirs

In some embodiments, the targeting construct comprises an antagomir.

Antagomirs are single stranded, double stranded, partially double stranded or hairpin structures that target a microRNA. An antagomir consists essentially of or comprises at least 10 or more contiguous nucleotides substantially complementary to an endogenous miR A and more particularly a target sequence of an miRNA or pre-miRNA nucleotide sequence. Antagomirs preferably have a nucleotide sequence sufficiently complementary to a miRNA target sequence of about 12 to 25 nucleotides, such as about 15 to 23 nucleotides, to allow the antagomir to hybridize to the target sequence. More preferably, the target sequence differs by no more than 1 , 2, or 3 nucleotides from the sequence of the antagomir. In some embodiments, the antagomir includes a non-nucleotide moiety, e.g., a cholesterol moiety, which can be attached, e.g., to the 3' or 5' end of the oligonucleotide agent.

In some embodiments, antagomirs are stabilized against nucleolytic degradation by the incorporation of a modification, e.g., a nucleotide modification. For example, in some embodiments, antagomirs contain a phosphorothioate comprising at least the first, second, and/or third internucleotide linkages at the 5' or 3' end of the nucleotide sequence. In further embodiments, antagomirs include a 2'-modified nucleotide, e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0- aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0- dimethylaminopropyl (2'-0-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0- DMAEOE), or 2'-0-N-methylacetamido (2'-0-NMA). In some embodiments, antagomirs include at least one 2'-0-methyl-modified nucleotide.

Antisense

In one embodiment of the present invention, antisense molecules or compounds are used as targeting constructs. The design, synthesis and use of compounds utilitzing RNaseH as a cleavage mechanisms are known in the art and described in "Antisense drug technology: principles, strategies, and applications" by Stanley T. Crooke, 2007, Marcel Dekker, New York. Given a coding strand sequence (e.g., the sequence of a sense strand of a cDNA molecule), antisense nucleic acids can be designed according to the rules of Watson- Crick base pairing. The antisense nucleic acid can be complementary to a portion of the coding or noncoding region of a R A, e.g., the region surrounding the translation start site of a pre-mRNA or mRNA, e.g., the 5' UTR. An antisense oligonucleotide can be, for example, about 10 to 25 nucleotides in length (e.g., 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 nucleotides in length). In some embodiments, the antisense oligonucleotide comprises one or more modified nucleotides, e.g., phosphorothioate derivatives and/or acridine substituted nucleotides, designed to increase its biological stability of the molecule and/or the physical stability of the duplexes formed between the antisense and target nucleic acids. Antisense oligonucleotides can comprise

ribonucleotides only, deoxyribonucleotides only (e.g., oligodeoxynucleotides), or both deoxyribonucleotides and ribonucleotides. For example, an antisense agent consisting only of ribonucleotides can hybridize to a complementary RNA and prevent access of the translation machinery to the target RNA transcript, thereby preventing protein synthesis. An antisense molecule including only deoxyribonucleotides, or deoxyribonucleotides and ribonucleotides, can hybridize to a complementary RNA and the RNA target can be subsequently cleaved by an enzyme, e.g., RNAse H, to prevent translation. The flanking RNA sequences can include 2' O methylated nucleotides, and phosphorothioate linkages, and the internal DNA sequence can include phosphorothioate internucleotide linkages. The internal DNA sequence is preferably at least five nucleotides in length when targeting by RNAseH activity is desired.

Ribozymes

Targeting constructs of the present invention may also be designed to be catalytic nucleic acids or ribozymes. The design, synthesis and use of ribozyme technology is known in the art and described in "Intracellular Ribozyme Applications: Principles and Protocols", by Rossi and Couture, 1999; Taylor & Francis, Inc. In one embodiment avian transcript variants are designed to encode one or more ribozymes or ribozyme moieties. These ribozyme or ribozyme moieties are either active as encoded in the avian transcript variant or when cleaved from the parent avian transcript variant. Aptamers

In some embodiments, the targeting construct comprises an aptamer which binds to a non-nucleic acid ligand, such as a small organic molecule or protein, e.g., a transcription or translation factor, and subsequently modifies (e.g., inhibits) activity. The design, synthesis and use of aptamer technology is known in the art and described in "The Aptamer Handbook: Functional Oligonucleotides and Their Applications" by Klussman, 2006, Wiley VCH. In one embodiment, aptamers may be designed to target one or more features of an avian transcript, transcript variant or biological product. An aptamer can fold into a specific structure that directs the recognition of a targeted binding site on the non-nucleic acid ligand. Aptamers can contain any of the modifications described herein.

Double-stranded ribonucleic acid (dsRNA)

Described herein are targeting constructs that inhibit the expression of one or more avian transcripts. In one embodiment, the targeting construct includes double - stranded ribonucleic acid (dsRNA) molecules for inhibiting or activating the expression of an avian gene or transcript in a cell. Expression of an avian transcript in cell culture can be assayed by measuring avian R A levels, such as by bDNA or TaqMan assay, or by measuring protein levels of an associated protein coding gene (e.g., one indicative of avian transcript levels), such as by immunofluorescence analysis, using, for example, Western Blotting or flowcytometric techniques.

The term "double-stranded RNA" or "dsRNA," as used herein, refers to an targeting construct that includes an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti-parallel and substantially

complementary nucleic acid strands, which will be referred to as having "sense" and "antisense" orientations with respect to a target RNA. The duplex region can be of any length that permits specific degradation of a desired target RNA through a RISC pathway, but will typically range from 9 to 50 base pairs in length, e.g., 15-30 base pairs in length. Where relevant, a "part" of an avian transcript target is a contiguous sequence of an avian transcript of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsR As having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed R A cleavage.

In another embodiment, a targeting construct agent useful to target avian transcript expression is generated in the target cell by cleavage of a larger dsR A.

Considering a duplex between 9 and 50 base pairs, the duplex can be any length in this range, for example, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 45, 46, 47, 48, 49 or 50 and any sub-range therein between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. The two strands forming the duplex structure can be from a single RNA molecule having at least one self- complementary region, or can be formed from two or more separate RNA molecules. Where the duplex region is formed from two strands of a single molecule, the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a "hairpin loop") between the 3'-end of one strand and the 5'-end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucleotide; in some embodiments the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than a hairpin loop, the connecting structure is referred to as a "linker." The term "siRNA" while being an iRNA agent may also used herein to refer to a dsRNA as described above. A dsRNA as described herein may further include one or more single-stranded nucleotide overhangs. The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of a targeting construct, e.g., a dsRNA, siRNA or generally an iRNA agent. For example, when a 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three

nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) may be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5' end, 3' end or both ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide overhang at the 3' end and/or the 5' end. In one embodiment, the sense strand of a dsR A has a 1-10 nucleotide overhang at the 3' end and/or the 5' end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

In one embodiment, the sense strand of a dsRNA is connected with a biocleavable or biostable 1-25 nucleotide overhang at the 3' end and/or the 5' end capable of activating RNAse H. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleotide sequence functioning as an immunostimuatory agent or as an aptamer. In another embodiment, the 5 '-end of the sense strand or antisense stand or both strands carry a triphosphate capable of activating RIG-I protein.

The terms "blunt" or "blunt ended" as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a "blunt ended" dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double- stranded over its entire length.

The term "antisense strand" or "guide strand" refers to the strand of an iRNA agent, e.g., a dsRNA or siRNA, which includes a region that is substantially

complementary to a target sequence. As used herein, the term "region of

complementarity" refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and/or 3' terminus. However, mismatches may be located in the internal positions of the molecule and on either strand of a dsRNA molecule.

The term "sense strand" or "passenger strand" as used herein, refers to the strand of a iRNA agent that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

In specific embodiments, the first sequence is a sense strand of a dsRNA that includes a sense sequence disclosed herein, and the second sequence is selected from the group consisting of the corresponding antisense sequences disclosed herein, the pairs of which are reported along with SEQ ID Nos of each sense: antisense pair in the accompanying sequence listing. Pairs are listed with the sense strand first and then the antisense strand. For example, for the sense: antisense pair, (18040,18041), SEQ ID NO: 18040 is the sense strand and SEQ ID NO: 18041 is the antisense strand. Each avian transcript from which the dsRNA are designed are also disclosed in Lengthy Table 1.

Alternative dsRNA agents that target elsewhere in the target sequence provided in Table 1 can readily be determined using the target sequence and the flanking avian sequence.

In addition, the avian transcript targeting constructs provided identify a site that is susceptible to RISC-mediated cleavage. As such, the present invention further features dsR As that target within one of such sequences. As used herein, an iR A agent (e.g., dsRNA) is said to target within a particular site of an avian RNA transcript if the iRNA agent promotes cleavage of the transcript anywhere within that particular site. Such an iRNA agent will generally include at least 15 contiguous nucleotides from one of the sequences provided here coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in an avian transcript. Given the identification of the sites for targeting provided by the sequences disclosed here, it is also within the scope of the present invention for a dsRNA to target substantially the same location or site.

While an avian target site is generally 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given avian target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a "window" or "mask" of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively

(including, e.g., in silico) placed on the target sequence to identify sequences in the size range that may serve as target sequences. By moving the sequence "window" progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those sequences that, when targeted with a dsRNA, mediate the best inhibition of target gene expression. Thus, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively "walking the window" one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those and sequences generated by walking a window of the longer or shorter size up or down the target avian transcript from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of dsR As based on those target sequences in an inhibition assay as known in the art or as described herein can lead to further improvements in the efficiency of inhibition.

Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) as an expression inhibitor.

A dsR A as described herein can contain one or more mismatches to the target sequence. In one embodiment, a dsRNA as described herein contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5' or 3' end of the region of complementarity. For example, for a 23 nucleotide antisense strand which is complementary to a region of an avian transcript, the antisense strand generally does not contain any mismatch within the central 13 nucleotides.

The methods described herein or methods known in the art can be used to determine whether an iRNA agent containing a mismatch to a target sequence is effective in inhibiting the expression of an avian transcript. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of an avian transcript is important, especially if the particular region of complementarity in an avian transcript is known to have polymorphic sequence variation within the population.

Vector encoded dsRNAs In another aspect, targeting constructs (e.g., iRNAs, proteins or peptides) targeting the avian transcript can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et ah, TIG. (1996), 12:5-10; Skillern, A., et ah, International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/221 14, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et ah, Proc. Natl. Acad. Sci. USA (1995) 92: 1292).

The individual strand or strands of a targeting construct (when RNA or DNA) can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

Expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of a targeting construct as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of targeting construct expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell. Targeting construct expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Trans it-TKO™). Multiple lipid transfections for targeting construct- mediated knockdowns targeting different regions of an avian transcript target over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno- associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication- defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct may be incorporated into vectors capable of episomal replication, e.g EPV and EBV vectors. Constructs for the recombinant expression of a targeting construct will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the targeting construct in target cells. Other aspects to consider for vectors and constructs are further described below.

Vectors useful for the delivery of a targeting construct will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the targeting construct in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression. Expression of the targeting construct can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et ah, 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D 1 - thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the transgene.

In a specific embodiment, viral vectors that contain nucleic acid sequences encoding a targeting construct can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding a targeting construct are cloned into one or more vectors, which facilitates delivery of the nucleic acid into a cell, tissue or patient. More detail about retroviral vectors can be found, for example, in

Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83: 1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4: 129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3: 110-1 14 (1993).

Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Patent Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.

In one embodiment, the targeting constructs of the present invention may be delivered via a bacterial delivery approach as disclosed in PCT Publication

WO/2008/156702, the contents of which are incorporated herein in its entirety.

Adenoviruses are also contemplated for use in delivery of nucleic acid based targeting constructs. Adenoviruses are especially attractive vehicles. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. A suitable AV vector for expressing a targeting construct featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

Use of Adeno-associated virus (AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the targeting construct can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or HI RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61 : 3096-3101 ; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641 , the entire disclosures of which are herein incorporated by reference.

Another preferred viral vector is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

Modifications

The nucleic acid based molecules featured in the invention may be synthesized and/or modified by methods well established in the art, such as those described in

"Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference.

Modifications to nucleic acid based targeting constructs are discussed in more detail below under iRNA agent structure. Briefly, modifications may include, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation (mono-, di- and tri-), conjugation, inverted linkages, etc.) 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the

phosphodiester linkages. Specific examples of modified compounds useful in this invention include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the modified RNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus- containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863;

4,476,301 ; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;

5,321,131 ; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126;

5,536,821 ; 5,541,316; 5,550,11 1; 5,563,253; 5,571 ,799; 5,587,361 ; 5,625,050; 6,028,188;

6,124,445; 6,160,109; 6,169,170; 6,172,209; 6, 239,265; 6,277,603; 6,326,199;

6,346,614; 6,444,423; 6,531 ,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;

6,878,805; 7,015,315; 7,041 ,816; 7,273,933; 7,321,029; and US Pat RE39464, each of which is herein incorporated by reference.

Modified R A backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O,

S and C¾ component parts.

Representative U.S. patents that teach the preparation of the above

oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141 ; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257;

5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561 ,225; 5,596,086; 5,602,240; 5,608,046;

5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.

In other RNA mimetics suitable or contemplated for use in iRNAs, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331 ; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et ah, Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs with

phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular --CH₂~NH--CH₂~, ~CH₂--N(CH₃)~0~CH₂-- [known as a methylene

(methylimino) or MMI backbone], -CH₂--0-N(CH₃)--CH₂--, --CH₂-N(CH₃)--N(CH₃)-- CH₂~ and ~N(CH₃)~CH₂~CH₂~[wherein the native phosphodiester backbone is represented as --0— P--0— CH₂--] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified RNAs may also contain one or more substituted sugar moieties. The targeting constructs, e.g., dsRNAs, featured herein can include one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O- alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cio alkyl or C₂ to Cio alkenyl and alkynyl. Exemplary suitable modifications include 0[(CH₂)_nO] _mCH₃, 0(CH₂)._nOCH₃, 0(CH₂)_nNH₂, 0(CH₂) _nCH₃, 0(CH₂)_nONH₂, and 0(CH₂)_nON[(CH₂)_nCH₃)]₂, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2' position: Ci to Cio lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, CI, Br, CN, CF₃, OCF₃, SOCH₃, S0₂CH₃, ON0₂, N0₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, amino alkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iR A agent, or a group for improving the pharmacodynamic properties of an iRNA agent, and other substituents having similar properties. In some

embodiments, the modification includes a 2'-methoxyethoxy (2'-0--ΟΗ₂θ¾ΟΟΗ₃, also known as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et al, Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'- dimethylaminooxyethoxy, i.e., a 0(CH₂)₂0N(CH₃)₂ group, also known as 2'-DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-0~CH₂~0~CH₂~ N(CH₃)₂, also described in examples herein below.

Other modifications include 2'-methoxy (2'-OCH₃), 2'-aminopropoxy (2'- OCH₂CH₂CH₂NH₂) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the RNA of a targeting construct, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5' position of 5' terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl, cyclohexenyl (CeNA), Hexose (HNA), FHNA moieties in place of the pentofuranosyl sugar.

Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981 ,957; 5,118,800; 5,319,080;

5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,81 1; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

A modified compounds may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5- propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5- substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3- deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley- VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et ah, Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA

Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5- substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711 ; 5,552,540; 5,587,469; 5,594,121 , 5,596,091; 5,614,617; 5,681 ,941 ; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference. Nucleic acid based targeting construct can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in the 3'-endo structural conformation. Also within the present invention are the use of bicyclic nucleic acids, carbocyclic LNAs and amino LNAs. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al, (2005) Nucleic Acids Research 33(l):439-447; Mook, OR. et al, (2007) Mol Cane Ther 6(3):833-843; Grunweller, A. et al, (2003) Nucleic Acids Research 31(12):3185-3193).

Representative U.S. Patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461 ; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is herein incorporated by reference in its entirety. iRNA agent structure

An iRNA agent can include a single strand or can include more than one strands, e.g., it can be a double stranded iRNA agent. If the iRNA agent is a single strand it is particularly preferred that it include a 5' modification which includes one or more phosphate groups or one or more analogs of a phosphate group.

In addition to homology to target RNA and the ability to down regulate a target gene, an iRNA agent will preferably have one or more of the following properties:

(1) it will be of the Formula 1, 2, 3, or 4 set out below;

(2) if single stranded it will have a 5' modification which includes one or more phosphate groups or one or more analogs of a phosphate group;

(3) it will, despite modifications, even to a very large number, or all of the nucleosides, have an antisense strand that can present bases (or modified bases) in the proper three dimensional framework so as to be able to form correct base pairing and form a duplex structure with a homologous target RNA which is sufficient to allow down regulation of the target, e.g., by cleavage of the target RNA; (4) it will, despite modifications, even to a very large number, or all of the nucleosides, still have "RNA-like" properties, i.e., it will possess the overall structural, chemical and physical properties of an R A molecule, even though not exclusively, or even partly, of ribonucleotide-based content. For example, an iRNA agent can contain, e.g., a sense and/or an antisense strand in which all of the nucleotide sugars contain e.g., 2' fluoro in place of 2' hydroxyl. This deoxyribonucleotide-containing agent can still be expected to exhibit RNA-like properties. While not wishing to be bound by theory, the electronegative fluorine prefers an axial orientation when attached to the C2' position of ribose. This spatial preference of fluorine can, in turn, force the sugars to adopt a C₃ - endo pucker. This is the same puckering mode as observed in RNA molecules and gives rise to the RNA-characteristic A-family-type helix. Further, since fluorine is a good hydrogen bond acceptor, it can participate in the same hydrogen bonding interactions with water molecules that are known to stabilize RNA structures. (Generally, it is preferred that a modified moiety at the 2' sugar position will be able to enter into H- bonding which is more characteristic of the OH moiety of a ribonucleotide than the H moiety of a deoxyribonucleotide. A preferred iRNA agent will: exhibit a Cy-endo pucker in all, or at least 50, 75,80, 85, 90, or 95 % of its sugars; exhibit a Cy-endo pucker in a sufficient amount of its sugars that it can give rise to a the RNA-characteristic A- family-type helix; will have no more than 20, 10, 5, 4, 3, 2, orl sugar which is not a C₃ - endo pucker structure. These limitations are particularly preferably in the antisense strand;

(5) regardless of the nature of the modification, and even though the RNA agent can contain deoxynucleotides or modified deoxynucleotides, particularly in overhang or other single strand regions, it is preferred that DNA molecules, or any molecule in which more than 50, 60, or 70 % of the nucleotides in the molecule, or more than 50, 60, or 70 % of the nucleotides in a duplexed region are deoxyribonucleotides, or modified deoxyribonucleotides which are deoxy at the 2' position, are excluded from the definition of RNA agent.

RNA agents discussed herein include otherwise unmodified RNA as well as RNA which have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et ah, (1994) Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183-2196. Such rare or unusual RNAs, often termed modified RNAs (apparently because the are typically the result of a post transcriptionally modification) are within the term unmodified RNA, as used herein. Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, preferably different from that which occurs in the human body. While they are referred to as modified "RNAs," they will of course, because of the modification, include molecules which are not RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to the presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone. Examples of all of the above are discussed herein.

Much of the discussion below refers to single strand molecules. In many embodiments of the invention a double stranded iRNA agent, e.g., a partially double stranded iRNA agent, is required or preferred. Thus, it is understood that that double stranded structures (e.g. where two separate molecules are contacted to form the double stranded region or where the double stranded region is formed by intramolecular pairing (e.g., a hairpin structure)) made of the single stranded structures described below are within the invention. Preferred lengths are described elsewhere herein.

As nucleic acids are polymers of subunits or monomers, many of the

modifications described below occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or the a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many, and infact in most cases it will not. By way of example, a modification may only occur at a 3' or 5' terminal position, may only occur in a terminal regions, e.g. at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of an R A. E.g., a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal regions, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5' end or ends can be phosphorylated.

In some embodiments it is particularly preferred, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5' or 3' overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3' or 5' overhang will be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2' OH group of the ribose sugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine, instead of ribonucleotides, and modifications in the phosphate group, e.g., phosphothioate modifications. Overhangs need not be homologous with the target sequence.

Further modifications and nucleotide surrogates are discussed below.

FORMULA 1

The scaffold presented above in Formula 1 represents a portion of a ribonucleic acid. The basic components are the ribose sugar, the base, the terminal phosphates, and phosphate internucleotide linkers. Where the bases are naturally occurring bases, e.g., adenine, uracil, guanine or cytosine, the sugars are the unmodified 2' hydroxyl ribose sugar (as depicted) and W, X, Y, and Z are all O, Formula 1 represents a naturally occurring unmodified oligoribonucleotide.

Unmodified oligoribonucleotides may be less than optimal in some applications, e.g., unmodified oligoribonucleotides can be prone to degradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleic acid phosphodiester bonds. However, chemical modifications to one or more of the above RNA components can confer improved properties, and, e.g., can render oligoribonucleotides more stable to nucleases. Umodified oligoribonucleotides may also be less than optimal in terms of offering tethering points for attaching ligands or other moieties to an iRNA agent.

Modified nucleic acids and nucleotide surrogates can include one or more of:

(i) alteration, e.g., replacement, of one or both of the non-linking (X and Y) phosphate oxygens and/or of one or more of the linking (W and Z) phosphate oxygens (When the phosphate is in the terminal position, one of the positions W or Z will not link the phosphate to an additional element in a naturally occurring ribonucleic acid.

However, for simplicity of terminology, except where otherwise noted, the W position at the 5' end of a nucleic acid and the terminal Z position at the 3' end of a nucleic acid, are within the term "linking phosphate oxygens" as used herein.);

(ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar, or wholesale replacement of the ribose sugar with a structure other than ribose, e.g., as described herein;

(iii) wholesale replacement of the phosphate moiety (bracket I) with "dephospho" linkers;

(iv) modification or replacement of a naturally occurring base;

(v) replacement or modification of the ribose-phosphate backbone (bracket II); (vi) modification of the 3' end or 5' end of the RNA, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, e.g. a fluorescently labeled moiety, to either the 3' or 5' end of RNA.

The terms replacement, modification, alteration, and the like, as used in this context, do not imply any process limitation, e.g., modification does not mean that one must start with a reference or naturally occurring ribonucleic acid and modify it to produce a modified ribonucleic acid bur rather modified simply indicates a difference from a naturally occurring molecule.

It is understood that the actual electronic structure of some chemical entities cannot be adequately represented by only one canonical form (i.e. Lewis structure).

While not wishing to be bound by theory, the actual structure can instead be some hybrid or weighted average of two or more canonical forms, known collectively as resonance forms or structures. Resonance structures are not discrete chemical entities and exist only on paper. They differ from one another only in the placement or "localization" of the bonding and nonbonding electrons for a particular chemical entity. It can be possible for one resonance structure to contribute to a greater extent to the hybrid than the others. Thus, the written and graphical descriptions of the embodiments of the present invention are made in terms of what the art recognizes as the predominant resonance form for a particular species. For example, any phosphoroamidate (replacement of a nonlinking oxygen with nitrogen) would be represented by X = O and Y = N in the above figure. Specific modifications are discussed in more detail below.

The Phosphate Group

The phosphate group is a negatively charged species. The charge is distributed equally over the two non-linking oxygen atoms (i.e., X and Y in Formula 1 above). However, the phosphate group can be modified by replacing one of the oxygens with a different substituent. One result of this modification to RNA phosphate backbones can be increased resistance of the oligoribonucleotide to nucleolytic breakdown. Thus, while not wishing to be bound by theory, it can be desirable in some embodiments to introduce alterations which result in either an uncharged linker or a charged linker with

unsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate,

phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen

phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.

Phosphorodithioates have both non-linking oxygens replaced by sulfur. Unlike the situation where only one of X or Y is altered, the phosphorus center in the

phosphorodithioates is achiral which precludes the formation of oligoribonucleotides diastereomers. Diastereomer formation can result in a preparation in which the individual diastereomers exhibit varying resistance to nucleases. Further, the

hybridization affinity of RNA containing chiral phosphate groups can be lower relative to the corresponding unmodified RNA species. Thus, while not wishing to be bound by theory, modifications to both X and Y which eliminate the chiral center, e.g.

phosphorodithioate formation, may be desirable in that they cannot produce diastereomer mixtures. Thus, X can be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl). Thus

Y can be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl). Replacement of X and/or Y with sulfur is preferred.

The phosphate linker can also be modified by replacement of a linking oxygen

(i.e., W or Z in Formula 1) with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at a terminal oxygen (position W (3') or position Z (5'). Replacement of W with carbon or Z with nitrogen is preferred.

The Sugar Group

A modified RNA can include modification of all or some of the sugar groups of the ribonucleic acid. E.g., the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents. While not being bound by theory, enhanced stability is expected since the hydroxyl can no longer be deprotonated to form a 2' alkoxide ion. The 2' alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom. Again, while not wishing to be bound by theory, it can be desirable to some embodiments to introduce alterations in which alkoxide formation at the 2' position is not possible.

Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar);

polyethyleneglycols (PEG), 0(CH₂CH₂0)_nCH₂CH₂OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl is connected, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; O-AMINE (AMINE = NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino) and aminoalkoxy, 0(CH₂)_nAMINE, (e.g., AMINE = NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino). It is noteworthy that oligonucleotides containing only the methoxyethyl group (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nuclease stabilities comparable to those modified with the robust phosphorothioate modification.

"Deoxy" modifications include hydrogen (i.e. deoxyribose sugars, which are of particular relevance to the overhang portions of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH₂CH₂NH)_nCH₂CH₂- AMINE (AMINE = NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino), -NHC(0)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality. Preferred substitutents are 2'-methoxyethyl, 2'-OCH3, 2'-0-allyl, 2'-C- allyl, and 2'-fiuoro.

The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified RNA can include nucleotides containing e.g., arabinose, as the sugar. Modified R A's can also include "abasic" sugars, which lack a nucleobase at C- . These abasic sugars can also be further contain modifications at one or more of the constituent sugar atoms.

To maximize nuclease resistance, the 2' modifications can be used in combination with one or more phosphate linker modifications (e.g., phosphorothioate). The so-called "chimeric" oligonucleotides are those that contain two or more different modifications.

The modificaton can also entail the wholesale replacement of a ribose structure with another entity at one or more sites in the iRNA agent. These modifications are described in section entitled Ribose Replacements for RRMSs.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containing connectors (cf. Bracket I in Formula 1 above). While not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center in nucleolytic degradation, its replacement with neutral structural mimics should impart enhanced nuclease stability. Again, while not wishing to be bound by theory, it can be desirable, in some embodiment, to introduce alterations in which the charged phosphate group is replaced by a neutral moiety.

Examples of moieties which can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino,

methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and

methyleneoxymethylimino. Preferred replacements include the methylenecarbonylamino and methylenemethylimino groups.

Replacement of Ribophosphate Backbone

Oligonucleotide- mimicking scaffolds can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates (see Bracket II of Formula 1 above). While not wishing to be bound by theory, it is believed that the absence of a repetitively charged backbone diminishes binding to proteins that recognize poly anions (e.g. nucleases). Again, while not wishing to be bound by theory, it can be desirable in some embodiment, to introduce alterations in which the bases are tethered by a neutral surrogate backbone.

Examples include the mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. A preferred surrogate is a PNA surrogate.

Terminal Modifications

The 3' and 5' ends of an oligonucleotide can be modified. Such modifications can be at the 3' end, 5' end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group. E.g., the 3' and 5' ends of an oligonucleotide can be conjugated to other functional molecular entities such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester). The functional molecular entities can be attached to the sugar through a phosphate group and/or a spacer. The terminal atom of the spacer can connect to or replace the linking atom of the phosphate group or the C-3' or C-5' O, N, S or C group of the sugar. Alternatively, the spacer can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs). These spacers or linkers can include e.g., - (CH₂)_n-, -(CH₂)_nN-, -(CH₂)_nO-, -(CH₂)_nS-, 0(CH₂CH₂0)_nCH₂CH₂OH (e.g., n = 3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents. When a

spacer/phosphate-functional molecular entity-spacer/phosphate array is interposed between two strands of iRNA agents, this array can substitute for a hairpin RNA loop in a hairpin-type RNA agent. The 3 ' end can be an -OH group. While not wishing to be bound by theory, it is believed that conjugation of certain moieties can improve transport, hybridization, and specificity properties. Again, while not wishing to be bound by theory, it may be desirable to introduce terminal alterations that improve nuclease resistance. Other examples of terminal modifications include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid,

dihydrotestosterone, l ,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group,

hexadecylglycerol, borneol, menthol, 1 ,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine- imidazole conjugates, Eu3+ complexes of tetraazamacrocycles).

Terminal modifications can be added for a number of reasons, including as discussed elsewhere herein to modulate activity or to modulate resistance to degradation. Terminal modifications useful for modulating activity include modification of the 5 ' end with phosphate or phosphate analogs. E.g., in preferred embodiments iR A agents, especially antisense strands, are 5 ' phosphorylated or include a phosphoryl analog at the 5' prime terminus. 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5 '-monophosphate ((HO)2(0)P-0-5*); 5 '-diphosphate ((HO)2(0)P-0-P(HO)(0)-0-5*); 5*-triphosphate ((HO)2(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5*); 5*-guanosine cap (7-methylated or non- methylated) (7m-G-0-5*-(HO)(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5*); 5*-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure ( -0-5'-(HO)(0)P-0- (HO)(0)P-0-P(HO)(0)-0-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P-0- 5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-0-5'), 5'- phosphorothiolate ((HO)2(0)P-S-5'); any additional combination of oxgen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((ΗΟ)2(0)Ρ-ΝΗ-5',

(ΗΟ)( Η2)(0)Ρ-0-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(0)-0-5*-, (OH)2(0)P-5*-CH2-), 5*-alkyletherphosphonates

(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(0)-0-5'-). Terminal modifications useful for increasing resistance to degradation include Terminal modifications can also be useful for monitoring distribution, and in such cases the preferred groups to be added include fluorophores, e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross-linking an R A agent to another moiety; modifications useful for this include mitomycin C.

The Bases

Adenine, guanine, cytosine and uracil are the most common bases found in RNA. These bases can be modified or replaced to provide RNA's having improved properties. E.g., nuclease resistant oligoribonucleotides can be prepared with these bases or with synthetic and natural nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any one of the above modifications.

Alternatively, substituted or modified analogs of any of the above bases, e.g., "unusual bases" and "universal bases," can be employed. Examples include without limitation 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5- substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,

dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5- alkyl cytosine,7-deazaadenine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino- allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3- nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2- thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N⁴- acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio- N6-isopentenyladenine, N-methylguanines, or O-alkylated bases. Further purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859,

Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et ah, Angewandte Chemie, International Edition, 1991 , 30, 613.

Generally, base changes are less preferred for promoting stability, but they can be useful for other reasons, e.g., some, e.g., 2,6-diaminopurine and 2 amino purine, are fluorescent. Modified bases can reduce target specificity. This should be taken into consideration in the design of iRNA agents.

Evaluation of iRNA agents

One can evaluate a candidate iRNA agent, e.g., a modified RNA, for a selected property by exposing the agent or modified molecule and a control molecule to the appropriate conditions and evaluating for the presence of the selected property. For example, resistance to a degradent can be evaluated as follows. A candidate modified iRNA (and preferably a control molecule, usually the unmodified form) can be exposed to degradative conditions, e.g., exposed to a milieu, which includes a degradative agent, e.g., a nuclease. E.g., one can use a biological sample, e.g., one that is similar to a milieu, which might be encountered, in therapeutic use, e.g., blood or a cellular fraction, e.g., a cell-free homogenate or disrupted cells. The candidate and control could then be evaluated for resistance to degradation by any of a number of approaches. For example, the candidate and control could be labeled, preferably prior to exposure, with, e.g., a radioactive or enzymatic label, or a fluorescent label, such as Cy3 or Cy5. Control and modified iRNA's can be incubated with the degradative agent, and optionally a control, e.g., an inactivated, e.g., heat inactivated, degradative agent. A physical parameter, e.g., size, of the modified and control molecules are then determined. They can be determined by a physical method, e.g., by polyacrylamide gel electrophoresis or a sizing column, to assess whether the molecule has maintained its original length, or assessed functionally. Alternatively, Northern blot analysis can be used to assay the length of an unlabeled modified molecule. A functional assay can also be used to evaluate the candidate iR A agent. A functional assay can be applied initially or after an earlier non-functional assay, (e.g., assay for resistance to degradation) to determine if the modification alters the ability of the molecule to silence avian transcript expression. For example, a cell, e.g., a mammalian cell, such as a mouse or human cell, can be co-transfected with a plasmid expressing a fluorescent protein, e.g., GFP, and a candidate R A agent homologous to the transcript encoding the fluorescent protein (see, e.g., WO 00/44914). For example, a modified dsRNA homologous to the GFP mRNA can be assayed for the ability to inhibit GFP expression by monitoring for a decrease in cell fluorescence, as compared to a control cell, in which the transfection did not include the candidate dsRNA, e.g., controls with no agent added and/or controls with a non-modified RNA added. Efficacy of the candidate agent on gene expression can be assessed by comparing cell fluorescence in the presence of the modified and unmodified dsRNA agents.

Preferred iRNA Agents

Preferred RNA agents have the following structure (see Formula 2 below):

FORMULA 2

Referring to Formula 2 above, R¹, R², and R³ are each, independently, H, (i.e. abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2- aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5- alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6- dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5- oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5- methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino- 3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2- thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6- isopentenyladenine, N-methylguanines, or O-alkylated bases.

R⁴, R⁵, and R⁶ are each, independently, OR⁸, 0(CH₂CH₂0)_mCH₂CH₂OR⁸;

0(CH₂)_nR⁹; 0(CH₂)_nOR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂;

NH(CH₂CH₂NH)_mCH₂CH₂NHR⁹; NHC(0)R⁸; ; cyano; mercapto, SR⁸; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido; or R⁴, R⁵, or R⁶ together combine with R⁷ to form an [-0-CH₂-] covalently bound bridge between the sugar 2' and 4' carbons.

A¹ is:

; H; OH; OCH₃; W ; an abasic nucleotide; or absent;

(a preferred Al , especially with regard to anti-sense strands, is chosen from 5'- monophosphate ((HO)₂(0)P-0-5^*), 5 '-diphosphate ((HO)₂(0)P-0-P(HO)(0)-0-5^*), 5^*- triphosphate ((HO)₂(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5^*), 5^*-guanosine cap (7- methylated or non-methylated) (7m-G-0-5^*-(HO)(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5^*), 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure ( -O- 5'-(HO)(0)P-0-(HO)(0)P-0-P(HO)(0)-0-5'), 5'-monothiophosphate (phosphorothioate; (HO)₂(S)P-0-5^*), 5^*-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-0-5^*), 5^*- phosphorothiolate ((HO)₂(0)P-S-5'); any additional combination of oxgen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5'-alpha-thiotriphosphate, 5'- gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((ΗΟ)₂(0)Ρ-ΝΗ-5', (HO)(NH₂)(0)P- 0-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g.

RP(OH)(0)-0-5^*-, (OH)₂(0)P-5^*-CH₂-), 5^*-alkyletherphosphonates

(R=alkylether=methoxymethyl (MeOCH₂-), ethoxymethyl, etc., e.g. RP(OH)(0)-0-5'-)). A² is:

A³ is:

; and

A⁴ is:

Zi

X₄^=P Y₄

-1 Zi

X₄^=P Y₄ X₄^=P Y₄

or

W₄

Zi Zi

or

X₄^=P Y₄ X₄^=P Y₄ X₄^=P Y₄ z₄ z₄

; H; Z⁴; an inverted nucleotide; an abasic nucleotide; or absent.

W¹ is OH, (CH₂)_nR¹⁰, (CH₂)_nNHR¹⁰, (CH₂)_n OR¹⁰, (CH₂)_n SR¹⁰; 0(CH₂)_nR¹⁰; 0(CH₂)_nOR¹⁰, 0(CH₂)_nNR¹⁰, 0(CH₂)_nSR¹⁰; 0(CH₂)_nSS(CH₂)_nOR¹⁰, 0(CH₂)_nC(0)OR¹⁰, NH(CH₂)_nR¹⁰; NH(CH₂)_nNR¹⁰ ;NH(CH₂)_nOR¹⁰, NH(CH₂)_nSR¹⁰; S(CH₂)_nR¹⁰,

S(CH₂)_nNR¹⁰, S(CH₂)_nOR¹⁰, S(CH₂)_nSR¹⁰ 0(CH₂CH₂0)_mCH₂CH₂OR¹⁰;

0(CH₂CH₂0)_mCH₂CH₂NHR¹⁰ , NH(CH₂CH₂NH)_mCH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R¹⁰ N-Q- R¹⁰, S-Q-R¹⁰ or -0-. W⁴ is O, CH₂, NH, or S.

X^!, X², X³, and X⁴ are each, independently, O or S.

Y¹, Y², Y³, and Y⁴ are each, independently, OH, 0^~, OR⁸, S, Se, BH₃ , H, NHR⁹, N(R⁹)₂ alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted.

Z², and Z³ are each independently O, CH₂, NH, or S. Z⁴ is OH, (CH₂)_nR¹⁰, (CH₂)_nNHR¹⁰, (CH₂)_n OR¹⁰, (CH₂)_n SR¹⁰; 0(CH₂)_nR¹⁰; 0(CH₂)_nOR¹⁰, 0(CH₂)_nNR¹⁰, 0(CH₂)_nSR¹⁰, 0(CH₂)_nSS(CH₂)_nOR¹⁰, 0(CH₂)_nC(0)OR¹⁰; NH(CH₂)_nR¹⁰;

NH(CH₂)_nNR¹⁰ ;NH(CH₂)_nOR¹⁰, NH(CH₂)_nSR¹⁰; S(CH₂)_nR¹⁰, S(CH₂)_nNR¹⁰, S(CH₂)_nOR^IU, S(CH₂)_nSR^IU 0(CH₂CH₂0)_mCH₂CH₂OR^IU, 0(CH₂CH₂0)_mCH₂CH₂NHR^IU , NH(CH₂CH₂NH)_mCH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R¹⁰ N-Q-R¹⁰, S-Q-R¹⁰.

x is 5-100, chosen to comply with a length for an RNA agent described herein. R⁷ is H; or is together combined with R⁴, R⁵, or R⁶ to form an [-0-CH₂-] covalently bound bridge between the sugar 2' and 4' carbons.

R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid; and R¹⁰ is H; fluorophore (pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes); sulfur, silicon, boron or ester protecting group; intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipohilic carriers (cholesterol, cholic acid, adamantane acetic acid, 1 -pyrene butyric acid,

dihydrotestosterone, l ,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group,

hexadecylglycerol, borneol, menthol, 1 ,3-propanediol, heptadecyl group, palmitic acid,myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino; alkyl, cycloalkyl, aryl, aralkyl, heteroaryl; radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacro cycles); or an RNA agent, m is 0-1 ,000,000, and n is 0-20. Q is a spacer selected from the group consisting of abasic sugar, amide, carboxy, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, biotin or fluorescein reagents.

85 Preferred iRNA agents in which the entire phosphate group has been replaced have the following structure (see Formula 3 below):

FORMULA 3

Referring to Formula 3, A -A is L-G-L; A and/or A may be absent, in which L is a linker, wherein one or both L may be present or absent and is selected from the group consisting of CH₂(CH₂)_g; (CH₂)_g; 0(CH₂)_g; S(CH₂)_g. G is a functional group selected from the group consisting of siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

R , R , and R^JL are each, independently, H, (i.e. abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5- halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5- amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7- methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7- alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6- dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5- oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5- methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino- 3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2- thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6- isopentenyladenine, N-methylguanines, or O-alkylated bases.

R⁴⁰, R⁵⁰, and R⁶⁰ are each, independently, OR⁸, 0(CH₂CH₂0)_mCH₂CH₂OR⁸; 0(CH₂)_nR⁹; 0(CH₂)_nOR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂; NH(CH₂CH₂NH)_mCH₂CH₂R⁹;

NHC(0)R 8 ;; cyano; mercapto, SR 7 ; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl,

alkoxycarbonyl, carboxy, hydroxy alkyl, alkanesulfonyl, alkanesulfonamido,

arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups; or R , R , or R together combine with R to form an [-O-CH₂-] covalently bound bridge between the sugar 2' and 4' carbons.

x is 5-100 or chosen to comply with a length for an RNA agent described herein.

R⁷⁰ is H; or is together combined with R⁴⁰, R⁵⁰, or R⁶⁰ to form an [-O-CH₂-] covalently bound bridge between the sugar 2' and 4' carbons.

R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; and R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid, m is 0-1,000,000, n is 0-20, and g is 0-2.

Preferred nucleoside surrogates have the following structure (see Formula 4 below):

SLR¹⁰⁰-(M-SLR²⁰⁰)_X-M-SLR³⁰⁰ FORMULA 4

S is a nucleoside surrogate selected from the group consisting of mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid. L is a linker and is selected from the group consisting of CH₂(CH₂)_g; N(CH₂)_g; 0(CH₂)_g; S(CH₂)_g; -C(0)(CH₂)_n-or may be absent. M is an amide bond; sulfonamide; sulfinate; phosphate group; modified phosphate group as described herein; or may be absent.

R¹⁰⁰, R²⁰⁰, and R³⁰⁰ are each, independently, H (i.e., abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5- halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5- amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7- methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7- alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6- dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1, 2, 4,-triazoles, 2-pyridinones, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5- oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5- methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino- 3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2- thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6- isopentenyladenine, N-methylguanines, or O-alkylated bases.

x is 5-100, or chosen to comply with a length for an RNA agent described herein; and g is 0-2.

Nuclease resistant monomers

In one aspect, the invention features a nuclease resistant monomer, or a an iRNA agent which incorporates a nuclease resistant monomer (NRM), such as those described herein and those described in copending, co-owned United States Provisional Application Serial No. 60/469,612, filed on May 9, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents having a NRM and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a NRM.

An iRNA agent can include monomers which have been modifed so as to inhibit degradation, e.g., by nucleases, e.g., endonucleases or exonucleases, found in the body of a subject. These monomers are referred to herein as NRM's, or nuclease resistance promoting monomers or modifications. In many cases these modifications will modulate other properties of the iRNA agent as well, e.g., the ability to interact with a protein, e.g., a transport protein, e.g., serum albumin, or a member of the RISC (RNA-induced Silencing Complex), or the ability of the first and second sequences to form a duplex with one another or to form a duplex with another sequence, e.g., a target molecule.

While not wishing to be bound by theory, it is believed that modifications of the sugar, base, and/or phosphate backbone in an iRNA agent can enhance endonuclease and exonuclease resistance, and can enhance interactions with transporter proteins and one or more of the functional components of the RISC complex. Preferred modifications are those that increase exonuclease and endonuclease resistance and thus prolong the halflife of the iRNA agent prior to interaction with the RISC complex, but at the same time do not render the iRNA agent resistant to endonuclease activity in the RISC complex.

Again, while not wishing to be bound by any theory, it is believed that placement of the modifications at or near the 3' and/or 5' end of antisense strands can result in iRNA agents that meet the preferred nuclease resistance criteria delineated above. Again, still while not wishing to be bound by any theory, it is believed that placement of the modifications at e.g., the middle of a sense strand can result in iRNA agents that are relatively less likely to undergo off-targeting.

Modifications described herein can be incorporated into any double-standed RNA and RNA-like molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more of the modifications described herein. The anti sense strand may include modifications at the 3' end and/or the 5' end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. The sense strand may include modifications at the 3' end and/or the 5' end and/or at any one of the intervening positions between the two ends of the strand. The iRNA agent may also include a duplex comprising two hybridized antisense strands. The first and/or the second antisense strand may include one or more of the modifications described herein. Thus, one and/or both antisense strands may include modifications at the 3' end and/or the 5' end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. Particular configurations are discussed below. Modifications that can be useful for producing iRNA agents that meet the preferred nuclease resistance criteria delineated above can include one or more of the following chemical and/or stereochemical modifications of the sugar, base, and/or phosphate backbone:

(i) chiral (Sp) thioates. Thus, preferred NRM's include nucleotide dimers with an enriched or pure for a particular chiral form of a modified phosphate group containing a heteroatom at the nonbridging position, e.g., Sp or Rp, at the position X, where this is the position normally occupied by the oxygen. The atom at X can also be S, Se, Nr₂, or Br₃. When X is S, enriched or chirally pure Sp linkage is preferred. Enriched means at least 70, 80, 90, 95, or 99% of the preferred form. Such NRM's are discussed in more detail below;

(ii) attachment of one or more cationic groups to the sugar, base, and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety. Thus, preferred NRM's include monomers at the terminal position derivitized at a cationic group. As the 5 ' end of an antisense sequence should have a terminal -OH or phosphate group this NRM is preferraly not used at th 5' end of an anti-sense sequence. The group should be attached at a position on the base which minimizes intererence with H bond formation and hybridization, e.g., away form the face which intereacts with the complementary base on the other strand, e.g, at the 5' position of a pyrimidine or a 7- position of a purine. These are discussed in more detail below;

(iii) nonphosphate linkages at the termini. Thus, preferred NRM's include Non- phosphate linkages, e.g., a linkage of 4 atoms which confers greater resistance to cleavage than does a phosphate bond. Examples include 3 ' CH2-NCH₃-0-CH2-5 ' and 3' CH2-NH-(0=)-CH2-5 '.;

(iv) 3 '-bridging thiophosphates and 5 '-bridging thiophosphates. Thus, preferred

NRM's can inlcuded these structures;

(v) L-RNA, 2 '-5' likages, inverted linkages, a-nucleosides. Thus, other preferred NRM's include: L nucleosides and dimeric nucleotides derived from L-nucleosides; 2'- 5' phosphate, non-phosphate and modified phosphate linkages (e.g., thiophospahtes, phosphoramidates and boronophosphates); dimers having inverted linkages, e.g., 3'-3' or 5 '-5' linkages; monomers having an alpha linkage at the 1 ' site on the sugar, e.g., the structures described herein having an alpha linkage;

(vi) conjugate groups. Thus, preferred NRM's can include e.g., a targeting moiety or a conjugated ligand described herein conjugated with the monomer, e.g., through the sugar , base, or backbone ;

(vi) abasic linkages. Thus, preferred NRM's can include an abasic monomer, e.g., an abasic monomer as described herein (e.g., a nucleobaseless monomer); an aromatic or heterocyclic or polyheterocyclic aromatic monomer as described herein.; and

(vii) 5'-phosphonates and 5 '-phosphate prodrugs. Thus, preferred NRM's include monomers, preferably at the terminal position, e.g., the 5' position, in which one or more atoms of the phosphate group is derivatized with a protecting group, which protecting group or groups, are removed as a result of the action of a component in the subject's body, e.g, a carboxyesterase or an enzyme present in the subject's body. E.g., a phosphate prodrug in which a carboxy esterase cleaves the protected molecule resulting in the production of a thioate anion which attacks a carbon adjacent to the O of a phosphate and resulting in the production of an uprotected phosphate.

One or more different NRM modifications can be introduced into an iRNA agent or into a sequence of an iRNA agent. An NRM modification can be used more than once in a sequence or in an iRNA agent. As some NRM's interfere with hybridization the total number incorporated, should be such that acceptable levels of iRNA agent duplex formation are maintainted.

In some embodiments NRM modifications are introduced into the terminal the cleavage site or in the cleavage region of a sequence (a sense strand or sequence) which does not target a desired sequence or gene in the subject. This can reduce off- target silencing.

Chiral Sp Thioates

A modification can include the alteration, e.g., replacement, of one or both of the non-linking (X and Y) phosphate oxygens and/or of one or more of the linking (W and Z) phosphate oxygens. Formula X below depicts a phosphate moiety linking two sugar/sugar surrogate -base moities, SBi and SB₂.

FORMULA X

In certain embodiments, one of the non-linking phosphate oxygens in the phosphate backbone moiety (X and Y) can be replaced by any one of the following: S, Se, BR₃ (R is hydrogen, alkyl, aryl, etc.), C (i.e., an alkyl group, an aryl group, etc.), H, NR₂ (R is hydrogen, alkyl, aryl, etc.), or OR (R is alkyl or aryl). The phosphorus atom in an unmodified phosphate group is achiral. However, replacement of one of the non- linking oxygens with one of the above atoms or groups of atoms renders the phosphorus atom chiral; in other words a phosphorus atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorus atom can possess either the "R" configuration (herein R_P) or the "S" configuration (herein Sp). Thus if 60% of a population of stereogenic phosphorus atoms have the Rp configuration, then the remaining 40% of the population of stereogenic phosphorus atoms have the Sp configuration.

In some embodiments, iR A agents, having phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms, may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the Sp configuration. Alternatively, iR A agents having phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the Rp configuration. In other embodiments, the population of stereogenic phosphorus atoms may have the Sp configuration and may be substantially free of stereogenic phosphorus atoms having the Rp configuration. In still other embodiments, the population of stereogenic phosphorus atoms may have the Rp configuration and may be substantially free of stereogenic phosphorus atoms having the Sp configuration. As used herein, the phrase "substantially free of stereogenic phosphorus atoms having the Rp configuration" means that moieties containing stereogenic phosphorus atoms having the RP configuration cannot be detected by conventional methods known in the art (chiral HPLC, 1H NMR analysis using chiral shift reagents, etc.). As used herein, the phrase "substantially free of stereogenic phosphorus atoms having the Sp configuration" means that moieties containing stereogenic phosphorus atoms having the Sp configuration cannot be detected by conventional methods known in the art (chiral HPLC, ^!H NMR analysis using chiral shift reagents, etc.).

In a preferred embodiment, modified iRNA agents contain a phosphorothioate group, i.e., a phosphate groups in which a phosphate non-linking oxygen has been replaced by a sulfur atom. In an especially preferred embodiment, the population of phosphorothioate stereogenic phosphorus atoms may have the S_P configuration and be substantially free of stereogenic phosphorus atoms having the Rp configuration.

Phosphorothioates may be incorporated into iRNA agents using dimers e.g., formulas X-l and X-2. The former can be used to introduce phosphorothioate

X-1 X-2 at the 3' end of a strand, while the latter can be used to introduce this modification at the 5' end or at a position that occurs e.g., 1 , 2, 3, 4, 5, or 6 nucleotides from either end of the strand. In the above formulas, Y can be 2-cyanoethoxy, W and Z can be O, Rr can be, e.g., a substituent that can impart the C-3 endo configuration to the sugar (e.g., OH, F, OCH₃), DMT is dimethoxytrityl, and "BASE" can be a natural, unusual, or a universal base.

X- 1 and X-2 can be prepared using chiral reagents or directing groups that can result in phosphorothioate-containing dimers having a population of stereogenic phosphorus atoms having essentially only the Rp configuration (i.e., being substantially free of the Sp configuration) or only the Sp configuration (i.e., being substantially free of the Rp configuration). Alternatively, dimers can be prepared having a population of stereogenic phosphorus atoms in which about 50% of the atoms have the Rp

configuration and about 50% of the atoms have the Sp configuration. Dimers having stereogenic phosphorus atoms with the Rp configuration can be identified and separated from dimers having stereogenic phosphorus atoms with the Sp configuration using e.g., enzymatic degradation and/or conventional chromatography techniques.

Cationic Groups

Modifications can also include attachment of one or more cationic groups to the sugar, base, and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety. A cationic group can be attached to any atom capable of substitution on a natural, unusual or universal base. A preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions needed for base pairing. A cationic group can be attached e.g., through the C2' position of a sugar or analogous position in a cyclic or acyclic sugar surrogate. Cationic groups can include e.g., protonated amino groups, derived from e.g., O-AMINE (AMINE = NH₂;

alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); aminoalkoxy, e.g., 0(CH₂)_nAMINE, {e.g., AMINE = NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); amino {e.g. NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or NH(CH₂CH₂NH)_nCH₂CH₂- AMINE (AMINE = NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino).

Nonphosphate Linkages

Modifications can also include the incorporation of nonphosphate linkages at the 5' and/or 3' end of a strand. Examples of nonphosphate linkages which can replace the phosphate group include methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and

methyleneoxymethylimino. Preferred replacements include the methyl phosphonate and hydroxylamino groups. 3 '-bridging thiophosphates and 5 '-bridging thiophosphates ; locked-RNA, 2 '-5 ' likages, inverted linkages, a-nucleosides ; conjugate groups; abasic linkages; and 5 '- phosphonates and 5 '-phosphate prodrugs

Referring to formula X above, modifications can include replacement of one of the bridging or linking phosphate oxygens in the phosphate backbone moiety (W and Z). Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of iRNA agents containing a stereogenic phosphorus atom..

Modifications can also include linking two sugars via a phosphate or modified phosphate group through the 2' position of a first sugar and the 5' position of a second sugar. Also contemplated are inverted linkages in which both a first and second sugar are eached linked through the respective3' positions. Modified RNA's can also include "abasic" sugars, which lack a nucleobase at C- . The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified iRNA agent can include nucleotides containing e.g., arabinose, as the sugar. In another subset of this modification, the natural, unusual, or universal base may have the a-configuration. Modifcations can also include L-RNA.

Modifications can also include 5 '-phosphonates, e.g., P(0)(0^~)₂-X-C⁵ -sugar (X= CH2, CF2, CHF and 5 '-phosphate prodrugs, e.g., P(0)[OCH2CH2SC(0)R]₂CH₂C^5'- sugar. In the latter case, the prodrug groups may be decomposed via reaction first with carboxy esterases. The remaining ethyl thiolate group via intramolecular S 2

displacement can depart as episulfide to afford the underivatized phosphate group.

Modification can also include the addition of conjugating groups described elseqhere herein, which are prefereably attached to an iRNA agent through any amino group available for conjugation. Nuclease resistant modifications include some which can be placed only at the terminus and others which can go at any position. Generally the modifications that can inhibit hybridization so it is preferably to use them only in terminal regions, and preferrable to not use them at the cleavage site or in the cleavage region of a sequence which targets a subject sequence or gene. The can be used anywhere in a sense sequence, provided that sufficient hybridization between the two sequences of the iRNA agent is maintained. In some embodiments it is desirabable to put the NRM at the cleavage site or in the cleavage region of a sequence which does not target a subject sequence or gene, as it can minimize off-target silencing.

In most cases, the nuclease-resistance promoting modifications will be distributed differently depending on whether the sequence will target a sequence in the subject (often referred to as an anti-sense sequence) or will not target a sequence in the subject (often referred to as a sense sequence). If a sequence is to target a sequence in the subject, modifications which interfer with or inhibit endonuclease cleavage should not be inserted in the region which is subject to RISC mediated cleavage, e.g., the cleavage site or the cleavage region (As described in Elbashir et ah, 2001 , Genes and Dev. 15: 188, hereby incorporated by reference, cleavage of the target occurs about in the middle of a 20 or 21 nt guide RNA, or about 10 or 11 nucleotides upstream of the first nucleotide which is complementary to the guide sequence. As used herein cleavage site refers to the nucleotide on either side of the cleavage site, on the target or on the iRNA agent strand which hybridizes to it. Cleavage region means a nucleotide with 1, 2, or 3 nucletides of the cleave site, in either direction.)

Such modifications can be introduced into the terminal regions, e.g., at the terminal position or with 2, 3, 4, or 5 positions of the terminus, of a sequence which targets or a sequence which does not target a sequence in the subject.

An iRNA agent can have a first and a second strand chosen from the following: a first strand which does not target a sequence and which has an NRM

modification at or within 1 , 2, 3, 4, 5 , or 6 positions from the 3' end;

a first strand which does not target a sequence and which has an NRM

modification at or within 1 , 2, 3, 4, 5 , or 6 positions from the 5' end; a first strand which does not target a sequence and which has an NRM

modification at or within 1 , 2, 3, 4, 5 , or 6 positions from the 3 ' end and which has a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end;

a first strand which does not target a sequence and which has an NRM

modification at the cleavage site or in the cleavage region;

a first strand which does not target a sequence and which has an NRM

modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1 , 2, 3, 4, 5 , or 6 positions from the 3 ' end, a NRM

modification at or within 1 , 2, 3, 4, 5 , or 6 positions from the 5 ' end, or NRM

modifications at or within 1, 2, 3, 4, 5 , or 6 positions from both the 3' and the 5' end; and

a second strand which targets a sequence and which has an NRM modification at or within 1 , 2, 3, 4, 5 , or 6 positions from the 3' end;

a second strand which targets a sequence and which has an NRM modification at or within 1 , 2, 3, 4, 5 , or 6 positions from the 5' end (5' end NRM modifications are preferentially not at the terminus but rather at a position 1 , 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand);

a second strand which targets a sequence and which has an NRM modification at or within 1 , 2, 3, 4, 5 , or 6 positions from the 3' end and which has a NRM modification at or within 1 , 2, 3, 4, 5 , or 6 positions from the 5' end;

a second strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region;

a second strand which targets a sequence and which does not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1 , 2, 3, 4, 5 , or 6 positions from the 3 ' end, a NRM

modifications at or within 1 , 2, 3, 4, 5 , or 6 positions from both the 3 ' and the 5' end(5' end NRM modifications are preferentially not at the terminus but rather at a position 1 , 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand). An iRNA agent can also target two sequences and can have a first and second strand chosen from:

a first strand which targets a sequence and which has an NRM modification at or within 1 , 2, 3, 4, 5 , or 6 positions from the 3' end;

a first strand which targets a sequence and which has an NRM modification at or within 1 , 2, 3, 4, 5 , or 6 positions from the 5' end (5' end NRM modifications are preferentially not at the terminus but rather at a position 1 , 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand);

a first strand which targets a sequence and which has an NRM modification at or within 1 , 2, 3, 4, 5 , or 6 positions from the 3' end and which has a NRM modification at or within 1 , 2, 3, 4, 5 , or 6 positions from the 5' end;

a first strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region;

a first strand which targets a sequence and which dose not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1 , 2, 3, 4, 5 , or 6 positions from the 3 ' end, a NRM

modifications at or within 1 , 2, 3, 4, 5 , or 6 positions from both the 3 ' and the 5' end(5' end NRM modifications are preferentially not at the terminus but rather at a position 1 , 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand) and

a second strand which targets a sequence and which has an NRM modification at or within 1 , 2, 3, 4, 5 , or 6 positions from the 3' end and which has a NRM modification at or within 1 , 2, 3, 4, 5 , or 6 positions from the 5' end; a second strand which targets a sequence and which preferably does not have an an NPvM modification at the cleavage site or in the cleavage region;

a second strand which targets a sequence and which dose not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1 , 2, 3, 4, 5 , or 6 positions from the 3 ' end, a NRM

modifications at or within 1 , 2, 3, 4, 5 , or 6 positions from both the 3 ' and the 5' end(5' end NRM modifications are preferentially not at the terminus but rather at a position 1 , 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand).

Ribose Mimics

In one aspect, the invention features a ribose mimic, or an iRNA agent which incorporates a ribose mimic, such as those described herein and those described in copending co-owned United States Provisional Application Serial No. 60/454,962, filed on March 13, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents having a ribose mimic and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a ribose mimic.

Thus, an aspect of the invention features an iRNA agent that includes a secondary hydroxyl group, which can increase efficacy and/or confer nuclease resistance to the agent. Nucleases, e.g., cellular nucleases, can hydrolyze nucleic acid phosphodiester bonds, resulting in partial or complete degradation of the nucleic acid. The secondary hydroxy group confers nuclease resistance to an iRNA agent by rendering the iRNA agent less prone to nuclease degradation relative to an iRNA which lacks the

modification. While not wishing to be bound by theory, it is believed that the presence of a secondary hydroxyl group on the iRNA agent can act as a structural mimic of a 3 ' ribose hydroxyl group, thereby causing it to be less susceptible to degradation.

The secondary hydroxyl group refers to an "OH" radical that is attached to a carbon atom substituted by two other carbons and a hydrogen. The secondary hydroxyl group that confers nuclease resistance as described above can be part of any acyclic carbon-containing group. The hydroxyl may also be part of any cyclic carbon-containing group, and preferably one or more of the following conditions is met (1) there is no ribose moiety between the hydroxyl group and the terminal phosphate group or (2) the hydroxyl group is not on a sugar moiety which is coupled to a base.. The hydroxyl group is located at least two bonds (e.g., at least three bonds away, at least four bonds away, at least five bonds away, at least six bonds away, at least seven bonds away, at least eight bonds away, at least nine bonds away, at least ten bonds away, etc.) from the terminal phosphate group phosphorus of the iRNA agent. In preferred embodiments, there are five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group.

Preferred iRNA agent delivery modules with five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group have the following structure (see formula Y below):

(Y) Referring to formula Y, A is an iRNA agent, including any iR A agent described herein. The iRNA agent may be connected directly or indirectly (e.g., through a spacer or linker) to "W" of the phosphate group. These spacers or linkers can include e.g., - (CH₂)„-, -(CH₂)„N-, -(CH₂)„0-, -(CH₂)„S-, 0(CH₂CH₂0)_nCH₂CH₂OH (e.g., n = 3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents.

The iRNA agents can have a terminal phosphate group that is unmodified (e.g., W, X, Y, and Z are O) or modified. In a modified phosphate group, W and Z can be independently NH, O, or S; and X and Y can be independently S, Se, BH₃ ^~, Ci-C₆ alkyl, C₆-Cio aryl, H, O, O^", alkoxy or amino (including alkylamino, arylamino, etc.).

Preferably, W, X and Z are O and Y is S.

Ri and R₃ are each, independently, hydrogen; or Ci-Cioo alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl.

R₂ is hydrogen; Ci-Cioo alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1 , R₂ may be taken together with with R4 or R₆ to form a ring of 5- 12 atoms.

R4 is hydrogen; C1-C100 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1 , R4 may be taken together with with R₂ or R5 to form a ring of 5- 12 atoms.

R5 is hydrogen, C1-C100 alkyl optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1 , R5 may be taken together with with R4 to form a ring of 5-12 atoms. R6 is hydrogen, C1-C100 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl, or, when n is 1 , R₆ may be taken together with with R₂ to form a ring of 6-10 atoms;

R₇ is hydrogen, C1-C100 alkyl, or C(0)(CH₂)_qC(0)NHR₉; T is hydrogen or a functional group; n and q are each independently 1-100; Rs is C1-C10 alkyl or C₆-Cio aryl; and R9 is hydrogen, CI -CIO alkyl, C6-C10 aryl or a solid support agent. Preferred embodiments may include one of more of the following subsets of iR A agent delivery modules.

In one subset of R Ai agent delivery modules, A can be connected directly or indirectly through a terminal 3' or 5 ' ribose sugar carbon of the R A agent.

In another subset of RNAi agent delivery modules, X, W, and Z are O and Y is S.

In still yet another subset of RNAi agent delivery modules, n is 1 , and R₂ and R₆ are taken together to form a ring containing six atoms and R4 and R5 are taken together to form a ring containing six atoms. Preferably, the ring system is a trans-decalin. For example, the RNAi agent delivery module of this subset can include a compound of Formula (Y-l):

The functional group can be, for example, a targeting group (e.g., a steroid or a carbohydrate), a reporter group (e.g., a fluorophore), or a label (an isotopically labelled moiety). The targeting group can further include protein binding agents, endothelial cell targeting groups (e.g., RGD peptides and mimetics), cancer cell targeting groups (e.g., folate Vitamin B 12, Biotin), bone cell targeting groups (e.g., bisphosphonates, polyglutamates, polyaspartates), multivalent mannose (for e.g., macrophage testing), lactose, galactose, N-acetyl-galactosamine, monoclonal antibodies, glycoproteins, lectins, melanotropin, or thyrotropin.

As can be appreciated by the skilled artisan, methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art.The synthesized compounds can be separated from a reaction mixture and further purified by a method such as column chromatography, high pressure liquid chromatography, or recrystallization. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

Ribose Replacement Monomer Subunits (RRMS)

iRNA agents can be modified in a number of ways which can optimize one or more characteristics of the iRNA agent. In one aspect, the invention features a ribose replacement monomer subunit (RRMS), or a an iRNA agent which incorporates a RRMS, such as those described herein and those described in one or more of United States Provisional Application Serial No. 60/493,986, filed on August 8, 2003, which is hereby incorporated by reference; United States Provisional Application Serial No. 60/494,597, filed on August 1 1, 2003, which is hereby incorporated by reference; United States Provisional Application Serial No. 60/506,341 , filed on September 26, 2003, which is hereby incorporated by reference; and in United States Provisional Application Serial No. 60/158,453, filed on November 7, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents having a RRMS and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an archtecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a RRMS. The ribose sugar of one or more ribonucleotide subunits of an iRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The carriers further include (i) at least two "backbone attachment points" and (ii) at least one "tethering attachment point." A "backbone attachment point" as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A "tethering attachment point" as used herein refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a ligand, e.g., a targeting or delivery moiety, or a moiety which alters a physical property, e.g., lipophilicity, of an iRNA agent. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, it will include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

Incorporation of one or more RRMSs described herein into an RNA agent, e.g., an iRNA agent, particularly when tethered to an appropriate entity, can confer one or more new properties to the RNA agent and/or alter, enhance or modulate one or more existing properties in the RNA molecule. E.g., it can alter one or more of lipophilicity or nuclease resistance. Incorporation of one or more RRMSs described herein into an iRNA agent can, particularly when the RRMS is tethered to an appropriate entity, modulate, e.g., increase, binding affinity of an iRNA agent to a target avian transcripts, change the geometry of the duplex form of the iRNA agent, alter distribution or target the iRNA agent to a particular part of the body, or modify the interaction with nucleic acid binding proteins (e.g., during RISC formation and strand separation).

Accordingly, in one aspect, the invention features, an iR A agent preferably comprising a first strand and a second strand, wherein at least one subunit having a formula (R-1) is incorporated into at least one of said strands.

(R-1)

Referring to formula (R-1), X is N(CO)R⁷, NR⁷ or CH₂; Y is NR⁸, O, S, CR⁹R¹⁰, or absent; and Z is CR 11 R 12 or absent.

Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR^a, OR^b, (CH₂)„OR^a, or (CH₂)_nOR^b, provided that at least one of R¹, R², R³, R⁴, R⁹, and R¹⁰ is OR^a or OR^b and that at least one of R¹, R², R³, R⁴, R⁹, and R¹⁰ is (CH₂)_nOR^a, or (CH₂)_nOR^b (when the RRMS is terminal, one of R¹, R², R³, R⁴, R⁹, and R¹⁰ will include R^a and one will include R^b; when the RRMS is internal, two of R¹ , R², R³, R⁴, R⁹, and R¹⁰ will each include an R^b); further provided that preferably OR^a may only be present with (CH₂)_nOR^b and (CH₂)_nOR^a may only be present with OR^b.

Each of R⁵, R⁶, R¹¹, and R¹² is, independently, H, Ci-C₆ alkyl optionally substituted with 1-3 R¹³, or C(0)NHR⁷; or R⁵ and R¹¹ together are C3-C8 cycloalkyl optionally substituted with R¹⁴.

R⁷ is Ci-C₂o alkyl substituted with NR°R^d; R⁸ is Ci-C₆ alkyl; R¹³ is hydroxy, d- C₄ alkoxy, or halo; and R¹⁴ is NR°R⁷.

R^a is:

; and

R is:

A

^■3 O Strand

C

Each of A and C is, independently, O or S.

B is OH, O^", or

O O

o- -o- -OH

O^" O^"

R° is H or C1-C6 alkyl; R^d is H or a ligand; and n is 1-4.

In a preferred embodiment the ribose is replaced with a pyrroline scaffold, and X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is absent.

In other preferred embodiments the ribose is replaced with a piperidine scaffold, and X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CRⁿR¹².

In other preferred embodiments the ribose is replaced with a piperazine scaffold, and X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CRⁿR¹². In other preferred embodiments the ribose is replaced with a morpholino scaffold, and X is N(CO)R⁷ or NR⁷, Y is O, and Z is CRⁿR¹² .

In other preferred embodiments the ribose is replaced with a decalin scaffold, and X isCH₂; Y is CR⁹R¹⁰; and Z is CRⁿR¹²; and R⁵ and R¹¹ together are C⁶ cycloalkyl.

In other preferred embodiments the ribose is replaced with a decalin/indane scafold and, and X is CH₂; Y is CR⁹R¹⁰; and Z is CRⁿR¹²; and R⁵ and R¹¹ together are C⁵ cycloalkyl.

In other preferred embodiments, the ribose is replaced with a hydroxyproline scaffold.

RRMSs described herein may be incorporated into any double-stranded RNA-like molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more of the RRMSs described herein. An RRMS can be introduced at one or more points in one or both strands of a double-stranded iRNA agent. An RRMS can be placed at or near (within 1 , 2, or 3 positions) of the 3' or 5' end of the sense strand or at near (within 2 or 3 positions of) the 3' end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5' end of the antisense strand. An RRMS can be internal, and will preferably be positioned in regions not critical for antisense binding to the target.

In an embodiment, an iRNA agent may have an RRMS at (or within 1 , 2, or 3 positions of) the 3' end of the antisense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3' end of the antisense strand and at (or within 1 , 2, or 3 positions of) the 3' end of the sense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3' end of the antisense strand and an RRMS at the 5' end of the sense strand, in which both ligands are located at the same end of the iRNA agent.

In certain embodiments, two ligands are tethered, preferably, one on each strand and are hydrophobic moieties. While not wishing to be bound by theory, it is believed that pairing of the hydrophobic ligands can stabilize the iRNA agent via intermolecular van der Waals interactions. In an embodiment, an iR A agent may have an RRMS at (or within 1 , 2, or 3 positions of) the 3' end of the antisense strand and an RRMS at the 5' end of the sense strand, in which both RRMSs may share the same ligand (e.g., cholic acid) via connection of their individual tethers to separate positions on the ligand. A ligand shared between two proximal RRMSs is referred to herein as a "hairpin ligand."

In other embodiments, an iRNA agent may have an RRMS at the 3' end of the sense strand and an RRMS at an internal position of the sense strand. An iRNA agent may have an RRMS at an internal position of the sense strand; or may have an RRMS at an internal position of the antisense strand; or may have an RRMS at an internal position of the sense strand and an RRMS at an internal position of the antisense strand.

In preferred embodiments the iRNA agent includes a first and second sequences, which are preferably two separate molecules as opposed to two sequences located on the same strand, have sufficient complementarity to each other to hybridize (and thereby form a duplex region), e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme.

It is preferred that the first and second sequences be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, a ds iRNA agent contains first and second sequences, preferable paired to contain an overhang, e.g., one or two 5' or 3' overhangs but preferably a 3' overhang of 2-3 nucleotides. Most embodiments will have a 3' overhang. Preferred sRNA agents will have single-stranded overhangs, preferably 3' overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. 5' ends are preferably phosphorylated.

Preferred carriers have the general formula (R-3) provided below. (In that structure preferred backbone attachment points can be chosen from R¹ or R²; R³ or R⁴; or R⁹ and R¹⁰ if Y is CR⁹R¹⁰ (two positions are chosen to give two backbone attachment points, e.g., R¹ and R⁴, or R⁴ and R⁹. Preferred tethering attachment points include R⁷; R⁵ or R⁶ when X is CH₂. The carriers are described below as an entity, which can be incorporated into a strand. Thus, it is understood that the structures also encompass the situations wherein one (in the case of a terminal position) or two (in the case of an internal position) of the attachment points, e.g., R¹ or R²; R³ or R⁴; or R⁹ or R¹⁰ (when Y is CR⁹R¹⁰), is connected to the phosphate, or modified phosphate, e.g., sulfur containing, backbone. E.g., one of the above-named R groups can be -CH2-, wherein one bond is connected to the carrier and one to a backbone atom, e.g., a linking oxygen or a central phosphorus atom.)

(R-3)

X is N(CO)R⁷, NR⁷ or CH₂; Y is NR⁸, O, S, CR⁹R¹⁰; and Z is CRⁿR¹² or absent.

Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR^a, or (CH₂)„OR^b, provided that at least two of R¹, R², R³, R⁴, R⁹, and R¹⁰ are OR^a and/or (CH₂)_nOR^b.

Each of R⁵, R⁶, R¹¹, and R¹² is, independently, a ligand, H, Ci-C₆ alkyl optionally substituted with 1-3 R¹³, or C(0)NHR⁷; or R⁵ and R¹¹ together are C₃-C₈ cycloalkyl optionally substituted with R¹⁴.

R⁷ is H, a ligand, or Ci-C₂o alkyl substituted with NR°R^d; R⁸ is H or Ci-C₆ alkyl; R¹³ is hydroxy, C1-C4 alkoxy, or halo; R¹⁴ is NR°R⁷; R¹⁵ is Ci-C₆ alkyl optionally substituted with cyano, or C₂-C₆ alkenyl; R¹⁶ is C1-C10 alkyl; and R¹⁷ is a liquid or solid phase support reagent.

L is -C(0)(CH₂)_qC(0)-, or -C(0)(CH₂)_qS-; R^a is CAr₃; R^b is P(0)(0 )H,

P(OR¹⁵)N(R¹⁶)₂ or L-R¹⁷; R^c is H or Ci-Qs alkyl; and R^d is H or a ligand.

I l l Each Ar is, independently, C₆-Cio aryl optionally substituted with C1-C4 alkoxy; n is 1-4; and q is 0-4.

Exemplary carriers include those in which, e.g., X is N(CO)R⁷ or NR⁷, Y is CRV°, and Z is absent; or X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR^NR¹²; or X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR^NR¹²; or X is N(CO)R⁷ or NR⁷, Y is O, and Z is CR^NR¹²; or X is CH₂; Y is CR⁹R¹⁰; Z is CR^NR¹², and R⁵ and R^{1 1} together form C₆ cycloalkyl (H, z = 2), or the indane ring system, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR^NR¹², and R⁵ and R^{1 1} together form C₅ cycloalkyl (H, z = 1).

In certain embodiments, the carrier may be based on the pyrroline ring system or the 3-hydroxyproline ring system, e.g., X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is absent (D). OFG¹ is preferably attached to a primary carbon, e.g., an exocyclic alkylene

D group, e.g., a methylene group, connected to one of the carbons in the five-membered ring (-CH2OFG¹ in D). OFG² is preferably attached directly to one of the carbons in the five-membered ring (-OFG² in D). For the pyrroline -based carriers, -CH2OFG¹ may be attached to C-2 and OFG² may be attached to C-3; or -CH2OFG¹ may be attached to C-3 and OFG² may be attached to C-4. . In certain embodiments, CH2OFG¹ and OFG² may be geminally substituted to one of the above-referenced carbons. For the 3-

1 2

hydroxyproline-based carriers, -CH₂OFG may be attached to C-2 and OFG may be attached to C-4. The pyrroline- and 3 -hydroxyproline-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH₂OFG¹ and OFG may be cis or trans with respect to one another in any of the pairings delineated above Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.

In certain embodiments, the carrier may be based on the piperidine ring system (E), e.g., X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CRⁿR¹². OFG¹ is preferably

E attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group (n=l) or ethylene group (n=2), connected to one of the carbons in the six-membered ring [-(CH₂)_nOFG¹ in E]. OFG² is preferably attached directly to one of the carbons in the six-membered ring (-OFG² in E). -(CH₂)_nOFG¹ and OFG² may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C- 3, or C-4. Alternatively, -(CH₂)_nOFG¹ and OFG² may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., - (CH₂)„OFG¹ may be attached to C-2 and OFG² may be attached to C-3; -(CH₂)„OFG¹

2 1

may be attached to C-3 and OFG may be attached to C-2; -(CH₂)_nOFG may be attached

2 1

to C-3 and OFG may be attached to C-4; or -(CH₂)_nOFG may be attached to C-4 and OFG² may be attached to C-3. The piperidine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, -

1 2

(CH₂)_nOFG and OFG may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.

In certain embodiments, the carrier may be based on the piperazine ring system

(F), e.g., X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CRⁿR¹², or the morpholine ring system (G), e.g., X is N(CO)R⁷ or NR⁷, Y is O, and Z is CRⁿR¹². OFG¹ is preferably

F G

attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group,

1 · 2 connected to one of the carbons in the six-membered ring (-CH₂OFG in F or G). OFG is preferably attached directly to one of the carbons in the six-membered rings (-OFG in F or G). For both F and G, -CH₂OFG¹ may be attached to C-2 and OFG² may be attached to C-3; or vice versa. In certain embodiments, CH₂OFG¹ and OFG² may be geminally substituted to one of the above-referenced carbons.The piperazine- and morpholine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the

1 2

presence of a ring. Thus, CH₂OFG and OFG may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. R'" can be, e.g., Ci-C₆ alkyl, preferably CH₃. The tethering attachment point is preferably nitrogen in both F and G.

In certain embodiments, the carrier may be based on the decalin ring system, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CRⁿR¹², and R⁵ and R¹¹ together form C₆ cycloalkyl (H, z = 2), or the indane ring system, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CRⁿR¹², and R⁵ and R¹¹ together form C5 cycloalkyl (H, z = 1). OFG¹ is preferably attached to a primary carbon,

e.g., an exocyclic methylene group (n=l) or ethylene group (n=2) connected to one of C- 2, C-3, C-4, or C-5 [-(CH₂)_nOFG¹ in H]. OFG² is preferably attached directly to one of C-2, C-3, C-4, or C-5 (-OFG² in H). -(CH^OFG¹ and OFG² may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, C-4, or C-5. Alternatively, -(CH^OFG¹ and OFG² may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon

1 2

atoms, e.g., -(CH₂)_nOFG may be attached to C-2 and OFG may be attached to C-3; - (CH^OFG¹ may be attached to C-3 and OFG² may be attached to C-2; -(CH^OFG¹ may be attached to C-3 and OFG² may be attached to C-4; or -(CH₂)_nOFG¹ may be

2 1 attached to C-4 and OFG may be attached to C-3; -(CH₂)_nOFG may be attached to C-4 and OFG² may be attached to C-5; or -(CH₂)_nOFG¹ may be attached to C-5 and OFG² may be attached to C-4. The decalin or indane-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, - (CH₂)_nOFG¹ and OFG² may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. In a preferred embodiment, the substituents at C-l and C-6 are trans with respect to one another. The tethering attachment point is preferably C-6 or C-7.

Other carriers may include those based on 3-hydroxyproline (J). Thus, - (CH₂)_nOFG¹ and OFG² may be cis or trans with respect to one another. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers

J

and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen. In certain embodiments, a moiety, e.g., a ligand may be connected indirectly to the carrier via the intermediacy of an intervening tether. Tethers are connected to the carrier at the tethering attachment point (TAP) and may include any C₁-C₁₀₀ carbon- containing moiety, (e.g. C1-C75, C1-C50, C1-C20, C1-C10, Ci-C₆), preferably having at least one nitrogen atom. In preferred embodiments, the nitrogen atom forms part of a terminal amino group on the tether, which may serve as a connection point for the ligand.

Preferred tethers (underlined) include TAP-(CH_z)_nNH_z; TAP-C(0)(CH₂)_aNH_z; or TAP- NR" "(CIDnNH?. in which n is 1-6 and R" " is Ci-C₆ alkyl. and R^d is hydrogen or a ligand. In other embodiments, the nitrogen may form part of a terminal oxyamino group, e.g., -ONH₂, or hydrazino group, -NHNH₂. The tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S. Preferred tethered ligands may include, e.g., TAP-(CH?)nNH(LIGAND).

TAP-C(Q)(CH7)nNH(LIGAND). or TAP-NR' ' "(CIDnNIKLIGAND);

TAP-(CH?)nONH (LIGAND). TAP-C(Q)(CH₂)nONH(LIGAND). or

TAP-NR' ' ' ^,(CH7)nONH(LIGAND); TAP-(CH7)nNHNH7(LIGAND).

TAP-C(0)(CH7)nNHNH7(LIGAND). or TAP-NR' ' ' '(CIDnNHNILILIGAND).

In other embodiments the tether may include an electrophilic moiety, preferably at the terminal position of the tether. Preferred electrophilic moieties include, e.g., an aldehyde, alkyl halide, mesylate, tosylate, nosylate, or brosylate, or an activated carboxylic acid ester, e.g. an NHS ester, or a pentafluorophenyl ester. Preferred tethers (underlined) include TAP-(CH7)_nCHO; TAP-C(0)(CH7)nCHO; or TAP- NR" "(CIDnCHO. in which n is 1-6 and R" " is Ci-Qs alkyl; or TAP₌

(CH7)nC(0)ONHS; ΤΑΡ-0(0¥ΟΗ₇) nC(Q)ONHS; or

TAP-NR' ' "(CH7) nC(Q)ONHS. in which n is 1 -6 and R" " is Ci-C₆ alkyl;

TAP-(CH7 nC(Q OCfiFs: TAP-C(0¥CH7)„C(Q) OC£ or TAP-NR" "(CH7)„C(0) OGiFs. in which n is 1-6 and R" " is Ci-C₆ alkyl; or -(CIDnCILLG; TAP- C(0)(CH7)nCH7LG; or TAP-NR' ' " CH7)_nCH7LG. in which n is 1-6 and R" " is Ci-C₆ alkyl (LG can be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate). Tethering can be carried out by coupling a nucleophilic group of a ligand, e.g., a thiol or amino group with an electrophilic group on the tether.

Tethered Entities

A wide variety of entities can be tethered to an iRNA agent, e.g., to the carrier of an RRMS. Examples are described below in the context of an RRMS but that is only preferred, entities can be coupled at other points to an iRNA agent.

Preferred moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether, to the RRMS carrier. In preferred embodiments, the ligand is attached to the carrier via an intervening tether. As discussed above, the ligand or tethered ligand may be present on the RRMS monomer when the RRMS monomer is incorporated into the growing strand. In some embodiments, the ligand may be incorporated into a "precursor" RRMS after a "precursor" RRMS monomer has been incorporated into the growing strand. For example, an RRMS monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., TAP-(CH₂)_n Fl₂ may be incorporated into a growing sense or antisense strand. In a subsequent operation, i.e., after incorporation of the precursor monomer into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor RRMS by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor RRMS tether.

In preferred embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g, molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.

Preferred ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified

oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides. Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; and nuclease-resistance conferring moieties. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine.

Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, bone cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl- gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B 12, biotin, or an RGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol, cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, 1 ,3-Bis- 0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1 ,3- propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of

tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.

The ligand can be a substance, e.g, a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

The ligand can increase the uptake of the iRNA agent into the cell by activating an inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNF alpha), interleukin- 1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid- based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non- kidney target tissue of the body. Preferably, the target tissue is the liver, preferably parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a seru protein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell- permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D- amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or pepti do mimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF. An RFGF analogue containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein and the Drosophila Antennapedia protein have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic tethered to an iRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 50 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an iRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an iRNA agent to a tumor cell expressing αγβ₃ (Haubner et al, Jour. Nucl. Med., 42:326-336, 2001).

Peptides that target markers enriched in proliferating cells can be used. E.g., RGD containing peptides and peptidomimetics can target cancer cells, in particular cells that exhibit an Ι_νθ₃ integrin. Thus, one could use RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the Ι_ν-θ₃ integrin ligand. Generally, such ligands can be used to control proliferating cells and

angiogeneis. Preferred conjugates of this type include an iRNA agent that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene described herein.

A "cell permeation peptide" is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an a-helical linear peptide (e.g., LL-37 or Ceropin PI), a disulfide bond-containing peptide (e.g., a -defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal ( LS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV- 1 gp41 and the NLS of SV40 large T antigen (Simeoni et al, Nucl. Acids Res. 31 :2717-2724, 2003).

In one embodiment, a targeting peptide tethered to an RRMS can be an amphipathic a- helical peptide. Exemplary amphipathic a-helical peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H₂A peptides, Xenopus peptides, esculentinis-1 , and caerins. A number of factors will preferably be considered to maintain the integrity of helix stability. For example, a maximum number of helix stabilization residues will be utilized {e.g., leu, ala, or lys), and a minimum number helix destabilization residues will be utilized {e.g., proline, or cyclic monomeric units. The capping residue will be considered (for example Gly is an exemplary N- capping residue and/or C-terminal amidation can be used to provide an extra H-bond to stabilize the helix. Formation of salt bridges between residues with opposite charges, separated by i ± 3, or i ± 4 positions can provide stability. For example, cationic residues such as lysine, arginine, homo-arginine, ornithine or histidine can form salt bridges with the anionic residues glutamate or aspartate.

Peptide and petidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; α, β, or γ peptides; N-methyl peptides;

azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.

Methods for making iRNA agents

iRNA agents can include modified or non-naturally occuring bases, e.g., bases described in copending and coowned United States Provisional Application Serial No. 60/463,772, filed on April 17, 2003, which is hereby incorporated by reference and/or in copending and coowned United States Provisional Application Serial No. 60/465,802, filed on April 25, 2003, which is hereby incorporated by reference. Monomers and iRNA agents which include such bases can be made by the methods found in United States Provisional Application Serial No. 60/463,772, filed on April 17, 2003, and/or in United States Provisional Application Serial No. 60/465,802, filed on April 25, 2003.

In addition, the invention includes iRNA agents having a modified or non- naturally occuring base and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a modified or non-naturally occuring base.

The synthesis and purification of oligonucleotide peptide conjugates can be performed by established methods. See, for example, Trufert et ah, Tetrahedron, 52:3005, 1996; and Manoharan, "Oligonucleotide Conjugates in Antisense Technology," in Antisense Drug Technology, ed. S.T. Crooke, Marcel Dekker, Inc., 2001.

In one embodiment of the invention, a peptidomimetic can be modified to create a constrained peptide that adopts a distinct and specific preferred conformation, which can increase the potency and selectivity of the peptide. For example, the constrained peptide can be an azapeptide (Gante, Synthesis, 405-413, 1989). An azapeptide is synthesized by replacing the a-carbon of an amino acid with a nitrogen atom without changing the structure of the amino acid side chain. For example, the azapeptide can be synthesized by using hydrazine in traditional peptide synthesis coupling methods, such as by reacting hydrazine with a "carbonyl donor," e.g., phenylchloroformate.

In one embodiment of the invention, a peptide or peptidomimetic {e.g., a peptide or peptidomimetic tethered to an RRMS) can be an N-methyl peptide. N-methyl peptides are composed of N-methyl amino acids, which provide an additional methyl group in the peptide backbone, thereby potentially providing additional means of resistance to proteolytic cleavage. N-methyl peptides can by synthesized by methods known in the art (see, for example, Lindgren et ah, Trends Pharmacol. Sci. 21 :99, 2000; Cell Penetrating Peptides: Processes and Applications. Langel, ed., CRC Press, Boca Raton, FL, 2002; Fische et ah, Bioconjugate. Chem. 12: 825, 2001; Wander et ah, J. Am. Chem. Soc, 124: 13382, 2002). For example, an Ant or Tat peptide can be an N-methyl peptide.

In one embodiment of the invention, a peptide or peptidomimetic {e.g., a peptide or peptidomimetic tethered to an RRMS) can be a β-peptide. β-peptides form stable secondary structures such as helices, pleated sheets, turns and hairpins in solutions. Their cyclic derivatives can fold into nanotubes in the solid state, β-peptides are resistant to degradation by proteolytic enzymes, β-peptides can be synthesized by methods known in the art. For example, an Ant or Tat peptide can be a β-peptide.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be a oligocarbamate. Oligocarbamate peptides are internalized into a cell by a transport pathway facilitated by carbamate transporters. For example, an Ant or Tat peptide can be an oligocarbamate.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be an oligourea conjugate (or an

oligothiourea conjugate), in which the amide bond of a peptidomimetic is replaced with a urea moiety. Replacement of the amide bond provides increased resistance to

degradation by proteolytic enzymes, e.g., proteolytic enzymes in the gastrointestinal tract. In one embodiment, an oligourea conjugate is tethered to an iR A agent for use in oral delivery. The backbone in each repeating unit of an oligourea peptidomimetic can be extended by one carbon atom in comparison with the natural amino acid. The single carbon atom extension can increase peptide stability and lipophilicity, for example. An oligourea peptide can therefore be advantageous when an iRNA agent is directed for passage through a bacterial cell wall, or when an iRNA agent must traverse the blood- brain barrier, such as for the treatment of a neurological disorder. In one embodiment, a hydrogen bonding unit is conjugated to the oligourea peptide, such as to create an increased affinity with a receptor. For example, an Ant or Tat peptide can be an oligourea conjugate (or an oligothiourea conjugate).

The siRNA peptide conjugates of the invention can be affiliated with,

e.g., tethered to, RRMSs occurring at various positions on an iRNA agent. For example, a peptide can be terminally conjugated, on either the sense or the antisense strand, or a peptide can be bisconjugated (one peptide tethered to each end, one conjugated to the sense strand, and one conjugated to the antisense strand). In another option, the peptide can be internally conjugated, such as in the loop of a short hairpin iRNA agent. In yet another option, the peptide can be affiliated with a complex, such as a peptide-carrier complex.

A peptide-carrier complex consists of at least a carrier molecule, which can encapsulate one or more iRNA agents (such as for delivery to a biological system and/or a cell), and a peptide moiety tethered to the outside of the carrier molecule, such as for targeting the carrier complex to a particular tissue or cell type. A carrier complex can carry additional targeting molecules on the exterior of the complex, or fusogenic agents to aid in cell delivery. The one or more iRNA agents encapsulated within the carrier can be conjugated to lipophilic molecules, which can aid in the delivery of the agents to the interior of the carrier.

A carrier molecule or structure can be, for example, a micelle, a liposome (e.g., a cationic liposome), a nanoparticle, a microsphere, or a biodegradable polymer. A peptide moiety can be tethered to the carrier molecule by a variety of linkages, such as a disulfide linkage, an acid labile linkage, a peptide-based linkage, an oxyamino linkage or a hydrazine linkage. For example, a peptide-based linkage can be a GFLG peptide.

Certain linkages will have particular advantages, and the advantages (or disadvantages) can be considered depending on the tissue target or intended use. For example, peptide based linkages are stable in the blood stream but are susceptible to enzymatic cleavage in the lysosomes.

Asymmetric Modifications

In one aspect, the invention features an iRNA agent which can be asymmetrically modified as described herein.

In addition, the invention includes iRNA agents having asymmetrical

modifications and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iR A agent formulated as described herein, which also incorporates an asymmetrical modification.

iRNA agents of the invention can be asymmetrically modified. An

asymmetrically modified iRNA agent is one in which a strand has a modification which is not present on the other strand. An asymmetrical modification is a modification found on one strand but not on the other strand. Any modification, e.g., any modification described herein, can be present as an asymmetrical modification. An asymmetrical modification can confer any of the desired properties associated with a modification, e.g., those properties discussed herein. E.g., an asymmetrical modification can: confer resistance to degradation, an alteration in half life; target the iRNA agent to a particular target, e.g., to a particular tissue; modulate, e.g., increase or decrease, the affinity of a strand for its complement or target sequence; or hinder or promote modification of a terminal moiety, e.g., modification by a kinase or other enzymes involved in the RISC mechanism pathway. The designation of a modification as having one property does not mean that it has no other property, e.g., a modification referred to as one which promotes stabilization might also enhance targeting.

While not wishing to be bound by theory or any particular mechanistic model, it is believed that asymmetrical modification allows an iRNA agent to be optimized in view of the different or "asymmetrical" functions of the sense and antisense strands. For example, both strands can be modified to increase nuclease resistance, however, since some changes can inhibit RISC activity, these changes can be chosen for the sense strand. In addition, since some modifications, e.g., targeting moieties, can add large bulky groups that, e.g., can interfere with the cleavage activity of the RISC complex, such

modifications are preferably placed on the sense strand. Thus, targeting moieties, especially bulky ones (e.g. cholesterol), are preferentially added to the sense strand. In one embodiment, an asymmetrical modification in which a phosphate of the backbone is substituted with S, e.g., a phosphorothioate modification, is present in the antisense strand, and a 2' modification, e.g., 2' OMe is present in the sense strand. A targeting moiety can be present at either (or both) the 5' or 3' end of the sense strand of the iRNA agent. In a preferred example, a P of the backbone is replaced with S in the antisense strand, 2'OMe is present in the sense strand, and a targeting moiety is added to either the 5' or 3' end of the sense strand of the iR A agent.

In a preferred embodiment an asymmetrically modified iRNA agent has a modification on the sense strand which modification is not found on the antisense strand and the antisense strand has a modification which is not found on the sense strand.

Each strand can include one or more asymmetrical modifications. By way of example: one strand can include a first asymmetrical modification which confers a first property on the iRNA agent and the other strand can have a second asymmetrical modification which confers a second property on the iRNA. E.g., one strand, e.g., the sense strand can have a modification which targets the iRNA agent to a tissue, and the other strand, e.g., the antisense strand, has a modification which promotes hybridization with the target gene sequence.

In some embodiments both strands can be modified to optimize the same property, e.g., to increase resistance to nucleolytic degradation, but different

modifications are chosen for the sense and the antisense strands, e.g., because the modifications affect other properties as well. E.g., since some changes can affect RISC activity these modifications are chosen for the sense strand.

In an embodiment one strand has an asymmetrical 2' modification, e.g., a 2' OMe modification, and the other strand has an asymmetrical modification of the phosphate backbone, e.g., a phosphorothioate modification. So, in one embodiment the antisense strand has an asymmetric 2' OMe modification and the sense strand has an asymmetrical phosphorothioate modification (or vice versa). In a particularly preferred embodiment the RNAi agent will have asymmetrical 2'-0 alkyl, preferably, 2'-OMe modifications on the sense strand and asymmetrical backbone P modification, preferably a phosphothioate modification in the antisense strand. There can be one or multiple 2 '-OMe modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunits of the sense strand can be so modified. There can be one or multiple phosphorothioate modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunits of the antisense strand can be so modified. It is preferable to have an iRNA agent wherein there are multiple 2 '-OMe modifications on the sense strand and multiple phophorothioate modifications on the antisense strand. All of the subunits on one or both strands can be so modified. A particularly preferred embodiment of multiple asymmetric modification on both strands has a duplex region about 20-21 , and preferably 19, subunits in length and one or two 3' overhangs of about 2 subunits in length.

Asymmetrical modifications are useful for promoting resistance to degradation by nucleases, e.g., endonuc leases. iR A agents can include one or more asymmetrical modifications which promote resistance to degradation. In preferred embodiments the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target. Most if not all sites on a strand are vulnerable, to some degree, to degradation by endo nucleases. One can determine sites which are relatively vulnerable and insert asymmetrical modifications which inhibit degradation. It is often desirable to provide asymmetrical modification of a UA site in an iRNA agent, and in some cases it is desirable to provide the UA sequence on both strands with asymmetrical modification. Examples of modifications which inhibit endonucleolytic degradation can be found herein. Particularly favored

modifications include: 2' modification, e.g., provision of a 2' OMe moiety on the U, especially on a sense strand; modification of the backbone, e.g., with the replacement of an O with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification, on the U or the A or both, especially on an antisense strand; replacement of the U with a C5 amino linker; replacement of the A with a G (sequence changes are preferred to be located on the sense strand and not the antisense strand); and modification of the at the 2', 6', 7', or 8' position. Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.

Asymmetrical modification can be used to inhibit degradation by exo nucleases. Asymmetrical modifications can include those in which only one strand is modified as well as those in which both are modified. In preferred embodiments the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target. Some embodiments will have an asymmetrical modification on the sense strand, e.g., in a 3' overhang, e.g., at the 3' terminus, and on the antisense strand, e.g., in a 3' overhang, e.g., at the 3' terminus. If the modifications introduce moieties of different size it is preferable that the larger be on the sense strand. If the modifications introduce moieties of different charge it is preferable that the one with greater charge be on the sense strand.

Examples of modifications which inhibit exonucleolytic degradation can be found herein. Particularly favored modifications include: 2' modification, e.g., provision of a 2' OMe moiety in a 3' overhang, e.g., at the 3' terminus (3' terminus means at the 3' atom of the molecule or at the most 3' moiety, e.g., the most 3' P or 2' position, as indicated by the context); modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P in a 3' overhang, e.g., at the 3' terminus; combination of a 2' modification, e.g., provision of a 2' O Me moiety and modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P, in a 3' overhang, e.g., at the 3' terminus; modification with a 3' alkyl; modification with an abasic pyrolidine in a 3' overhang, e.g., at the 3' terminus; modification with naproxene, ibuprofen, or other moieties which inhibit degradation at the 3' terminus.

Modifications, e.g., those described herein, which affect targeting can be provided as asymmetrical modifications. Targeting modifications which can inhibit silencing, e.g., by inhibiting cleavage of a target, can be provided as asymmetrical modifications of the sense strand. A biodistribution altering moiety, e.g., cholesterol, can be provided in one or more, e.g., two, asymmetrical modifications of the sense strand. Targeting

modifications which introduce moieties having a relatively large molecular weight, e.g., a molecular weight of more than 400, 500, or 1000 daltons, or which introduce a charged moiety (e.g., having more than one positive charge or one negative charge) can be placed on the sense strand.

Modifications, e.g., those described herein, which modulate, e.g., increase or decrease, the affinity of a strand for its compliment or target, can be provided as asymmetrical modifications. These include: 5 methyl U; 5 methyl C; pseudouridine, Locked nucleic acids, 2 thio U and 2-amino-A. In some embodiments one or more of these is provided on the antisense strand. Asymmetrical modification can be used to optimize the activity of an iR A agent structure, e.g., by being placed selectively within the iRNA. In preferred embodiments asymmetrical modifications which result in one or more of the following are used:

modifications of the 5' end of the sense strand which inhibit kinase activation of the sense strand, including, e.g., attachments of conjugates which target the molecule or the use modifications which protect against 5 ' exonucleolytic degradation; or modifications of either strand, but preferably the sense strand, which enhance binding between the sense and antisense strand and thereby promote a "tight" structure at this end of the molecule.

The end region of the iRNA agent defined by the 3' end of the sense strand and the 5 'end of the antisense strand is also important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3' overhang. Preferred embodiments include asymmetrical modifications of either strand, but preferably the sense strand, which decrease binding between the sense and antisense strand and thereby promote an "open" structure at this end of the molecule. Such modifications include placing conjugates which target the molecule or modifications which promote nuclease resistance on the sense strand in this region.

Exemplary modifications for asymmetrical placement in the sense strand include the following:

(a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S- P=S); these modifications can be used to promote nuclease resistance;

(b) 2'-0 alkyl, e.g., 2'-OMe, 3'-0 alkyl, e.g., 3'-OMe (at terminal and/or internal positions); these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand, the 3' modifications can be used at the 5' end of the sense strand to avoid sense strand activation by RISC;

(c) 2 '-5' linkages (with 2'-H, 2' -OH and 2'-OMe and with P=0 or P=S) these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5' end of the sense strand to avoid sense strand activation by RISC; (d) L sugars (e.g., L ribose, L-arabinose with 2'-H, 2'-OH and 2'-OMe); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5' end of the sense strand to avoid sense strand activation by RISC;

(e) modified sugars (e.g., locked nucleic acids (LNA's), hexose nucleic acids

(HNA's) and cyclohexene nucleic acids (CeNA's)); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5' end of the sense strand to avoid sense strand activation by RISC;

(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines), these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand;

(g) cationic groups and Zwitterionic groups (preferably at a terminus), these modifications can be used to promote nuclease resistance;

(h) conjugate groups (preferably at terminal positions), e,g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates; these modifications can be used to promote nuclease resistance or to target the molecule, or can be used at the 5' end of the sense strand to avoid sense strand activation by RISC.

Exemplary modifications for asymmetrical placement in the antisense strand include the following:

(a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S- P=S);

(b) 2'-0 alkyl, e.g., 2'-OMe, (at terminal positions);

(c) 2'-5' linkages (with 2'-H, 2'-OH and 2'-OMe) e.g., terminal at the 3' end); e.g., with P=0 or P=S preferably at the 3 '-end, these modifications are preferably excluded from the 5' end region as they may interfere with RISC enzyme activity such as kinase activity;

(d) L sugars (e.g, L ribose, L-arabinose with 2'-H, 2'-OH and 2'-OMe); e.g., terminal at the 3' end; e.g., with P=0 or P=S preferably at the 3 '-end, these modifications are preferably excluded from the 5' end region as they may interfere with kinase activity; (e) modified sugars (e.g., LNA's, HNA's and CeNA's); these modifications are preferably excluded from the 5 ' end region as they may contribute to unwanted enhancements of paring between the sense and antisense strands, it is often preferred to have a "loose" structure in the 5' region, additionally, they may interfere with kinase activity;

(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines);

(g) cationic groups and Zwitterionic groups (preferably at a terminus);

conjugate groups (preferably at terminal positions), e,g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates, but bulky groups or generally groups which inhibit RISC activity should are less preferred.

The 5'-OH of the antisense strand should be kept free to promote activity. In some preferred embodiments modifications that promote nuclease resistance should be included at the 3' end, particularly in the 3' overhang.

In another aspect, the invention features a method of optimizing, e.g., stabilizing, an iR A agent. The method includes selecting a sequence having activity, introducing one or more asymmetric modifications into the sequence, wherein the introduction of the asymmetric modification optimizes a property of the iRNA agent but does not result in a decrease in activity.

The decrease in activity can be less than a preselected level of decrease. In preferred embodiments decrease in activity means a decrease of less than 5, 10, 20, 40, or 50 % activity, as compared with an otherwise similar iRNA lacking the introduced modification. Activity can, e.g., be measured in vivo, or in vitro, with a result in either being sufficient to demonstrate the required maintenance of activity.

The optimized property can be any property described herein and in particular the properties discussed in the section on asymmetrical modifications provided herein. The modification can be any asymmetrical modification, e.g., an asymmetric modification described in the section on asymmetrical modifications described herein. Particularly preferred asymmetric modifications are 2'-0 alkyl modifications, e.g., 2'-OMe modifications, particularly in the sense sequence, and modifications of a backbone O, particularly phosphorothioate modifications, in the antisense sequence.

In a preferred embodiment a sense sequence is selected and provided with an asymmetrical modification, while in other embodiments an antisense sequence is selected and provided with an asymmetrical modification. In some embodiments both sense and antisense sequences are selected and each provided with one or more asymmetrical modifications.

Multiple asymmetric modifications can be introduced into either or both of the sense and antisense sequence. A sequence can have at least 2, 4, 6, 8, or more modifications and all or substantially all of the monomers of a sequence can be modified.

Z-X-Y Architecture

In one aspect, the invention features an iR A agent which can have a Z-X-Y architecture or structure such as those described herein and those described in copending, co-owned United States Provisional Application Serial No. 60/510,246, filed on October 9, 2003, which is hereby incorporated by reference, and in copending, co-owned United States Provisional Application Serial No. 60/510,318, filed on October 10, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents having a Z-X-Y structure and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a Z-X-Y architecture.

The invention provides an iRNA agent having a first segment, the Z region, a second segment, the X region, and optionally a third region, the Y region:

Z— X— Y. It may be desirable to modify subunits in one or both of Zand/or Y on one hand and X on the other hand. In some cases they will have the same modification or the same class of modification but it will more often be the case that the modifications made in Z and/or Y will differ from those made in X.

The Z region typically includes a terminus of an iRNA agent. The length of the Z region can vary, but will typically be from 2-14, more preferably 2-10, subunits in length. It typically is single stranded, i.e., it will not base pair with bases of another strand, though it may in some embodiments self associate, e.g., to form a loop structure. Such structures can be formed by the end of a strand looping back and forming an intrastrand duplex. E.g., 2, 3, 4, 5 or more intra-strand bases pairs can form, having a looped out or connecting region, typically of 2 or more subunits which do not pair. This can occur at one or both ends of a strand. A typical embodiment of a Z region is a single strand overhang, e.g., an over hang of the length described elsewhere herein. The Z region can thus be or include a 3 ' or 5 ' terminal single strand. It can be sense or antisense strand but if it is antisense it is preferred that it is a 3- overhang. Typical inter-subunit bonds in the Z region include: P=0; P=S; S-P=S; P-NR₂; and P-BR₂. Chiral P=X, where X is S, N, or B) inter-subunit bonds can also be present. (These inter-subunit bonds are discussed in more detail elsewhere herein.) Other preferred Z region subunit modifications (also discussed elsewhere herein) can include: 3'-OR, 3'SR, 2'-OMe, 3'-OMe, and 2ΌΗ modifications and moieties; alpha configuration bases; and 2' arabino modifications.

The X region will in most cases be duplexed, in the case of a single strand iRNA agent, with a corresponding region of the single strand, or in the case of a double stranded iRNA agent, with the corresponding region of the other strand. The length of the X region can vary but will typically be between 10-45 and more preferably between 15 and 35 subunits. Particularly preferred region X's will include 17, 18, 19, 29, 21 , 22, 23, 24, or 25 nucleotide pairs, though other suitable lengths are described elsewhere herein and can be used. Typical X region subunits include 2'-OH subunits. In typical embodiments phosphate inter-subunit bonds are preferred while phophorothioate or non-phosphate bonds are absent. Other modifications preferred in the X region include: modifications to improve binding, e.g., nucleobase modifications; cationic nucleobase modifications; and C-5 modified pyrimidines, e.g., allylamines. Some embodiments have 4 or more consecutive 2ΌΗ subunits. While the use of phosphorothioate is sometimes non preferred they can be used if they connect less than 4 consecutive 2ΌΗ subunits.

The Y region will generally conform to the the parameters set out for the Z regions. However, the X and Z regions need not be the same, different types and numbers of modifications can be present, and infact, one will usually be a 3' overhang and one will usually be a 5' overhang.

In a preferred embodiment the iR A agent will have a Y and/or Z region each having ribonucleosides in which the 2'-OH is substituted, e.g., with 2'-OMe or other alkyl; and an X region that includes at least four consecutive ribonucleoside subunits in which the 2'-OH remains unsubstituted.

The subunit linkages (the linkages between subunits) of an iRNA agent can be modified, e.g., to promote resistance to degradation. Numerous examples of such modifications are disclosed herein, one example of which is the phosphorothioate linkage. These modifications can be provided bewteen the subunits of any of the regions, Y, X, and Z. However, it is preferred that their occureceis minimized and in particular it is preferred that consecutive modified linkages be avoided.

In a preferred embodiment the iRNA agent will have a Y and Z region each having ribonucleosides in which the 2'-OH is substituted, e.g., with 2'-OMe; and an X region that includes at least four consecutive subunits, e.g., ribonucleoside subunits in which the 2'-OH remains unsubstituted.

As mentioned above, the subunit linkages of an iRNA agent can be modified, e.g., to promote resistance to degradation. These modifications can be provided between the subunits of any of the regions, Y, X, and Z. However, it is preferred that they are minimized and in particular it is preferred that consecutive modified linkages be avoided.

Thus, in a preferred embodiment, not all of the subunit linkages of the iRNA agent are modified and more preferably the maximum number of consecutive subunits linked by other than a phospodiester bond will be 2, 3, or 4. Particulary preferred iRNA agents will not have four or more consecutive subunits, e.g., 2'-hydroxyl ribonucleoside subunits, in which each subunits is joined by modified linkages - i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA.

It is particularly preferred to minimize the occurrence in region X. Thus, in preferred embodiments each of the nucleoside subunit linkages in X will be

phosphodiester linkages, or if subunit linkages in region X are modified, such modifications will be minimized. E.g., although the Y and/or Z regions can include inter subunit linkages which have been stabilized against degradation, such modifications will be minimized in the X region, and in particular consecutive modifications will be minimized. Thus, in preferred embodiments the maximum number of consecutive subunits linked by other than a phospodiester bond will be 2, 3, or 4. Parti culary preferred X regions will not have four or more consecutive subunits, e.g., 2'-hydroxyl ribonucleoside subunits, in which each subunits is joined by modified linkages - i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA.

In a preferred embodiment Y and /or Z will be free of phosphorothioate linkages, though either or both may contain other modifications, e.g., other modifications of the subunit linkages.

In a preferred embodiment region X, or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical 2' moieties.

In a preferred embodiment region X, or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical subunit linkages.

In a preferred embodiment one or more phosphorothioate linkages (or other modifications of the subunit linkage) are present in Y and/or Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2'

modification, e.g., a 2'-0-alkyl moiety. E.g., any adjacent 2'-0-alkyl moieties in the Y and/or Z, are connected by a linkage other than a a phosphorothioate linkage.

In a preferred embodiment each of Y and/or Z independently has only one phosphorothioate linkage between adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., 2'-0-alkyl nucleosides. If there is a second set of adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., 2'-0-alkyl nucleosides, in Y and/or Z that second set is connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage.

In a prefered embodiment each of Y and/orZ independently has more than one phosphorothioate linkage connecting adjacent pairs of subunits, e.g., nucleosides, having a 2' modification, e.g., 2'-0-alkyl nucleosides, but at least one pair of adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., 2'-0-alkyl nucleosides, are be connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage.

In a prefered embodiment one of the above recited limitation on adjacent subunits in Y and or Z is combined with a limitation on the subunits in X. E.g., one or more phosphorothioate linkages (or other modifications of the subunit linkage) are present in Y and/or Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., a 2'-0-alkyl moiety. E.g., any adjacent 2'-0- alkyl moieties in the Y and/or Z, are connected by a linkage other than a phosporothioate linkage. In addition, the X region has no more than 3 or no more than 4 identical subunits, e.g., subunits having identical 2' moieties or the X region has no more than 3 or no more than 4 subunits having identical subunit linkages.

A Y and/or Z region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein. Such modifications can be chosen, independently, from any modification described herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety. In a preferred embodiment more than 1 of such subunits can be present but in some emobodiments it is prefered that no more than 1, 2, 3, or 4 of such modifications occur, or occur

consecutively. In a preferred embodiment the frequency of the modification will differ between Y and /or Z and X, e.g., the modification will be present one of Y and/or Z or X and absent in the other.

An X region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein. Such modifications can be chosen, independently, from any modification desribed herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety. In a preferred embodiment more than 1 of such subunits can b present but in some emobodiments it is prefered that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively.

An RRMS (described elswhere herein) can be introduced at one or more points in one or both strands of a double-stranded iR A agent. An RRMS can be placed in a Y and/or Z region, at or near (within 1 , 2, or 3 positions) of the 3' or 5' end of the sense strand or at near (within 2 or 3 positions of) the 3 ' end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1 , 2, or 3 positions of) the 5' end of the antisense strand. An RRMS can be positioned in the X region, and will preferably be positioned in the sense strand or in an area of the antisense strand not critical for antisense binding to the target.

Differential Modification of Terminal Duplex Stability (DMTDS)

In one aspect, the invention features an iRNA agent which can have differential modification of terminal duplex stability (DMTDS).

In addition, the invention includes iRNA agents having DMTDS and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates DMTDS.

iRNA agents can be optimized by increasing the propensity of the duplex to disassociate or melt (decreasing the free energy of duplex association), in the region of the 5' end of the antisense strand duplex. This can be accomplished, e.g., by the inclusion of subunits which increase the propensity of the duplex to disassociate or melt in the region of the 5' end of the antisense strand. It can also be accomplished by the attachment of a ligand that increases the propensity of the duplex to disassociate of melt in the region of the 5 'end . While not wishing to be bound by theory, the effect may be due to promoting the effect of an enzyme such as helicase, for example, promoting the effect of the enzyme in the proximity of the 5 ' end of the antisense strand.

The inventors have also discovered that iRNA agents can be optimized by decreasing the propensity of the duplex to disassociate or melt (increasing the free energy of duplex association), in the region of the 3' end of the antisense strand duplex. This can be accomplished, e.g., by the inclusion of subunits which decrease the propensity of the duplex to disassociate or melt in the region of the 3' end of the antisense strand. It can also be accomplished by the attachment of ligand that decreases the propensity of the duplex to disassociate of melt in the region of the 5 'end.

Modifications which increase the tendency of the 5' end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which decrease the tendency of the 3' end of the duplex to dissociate. Likewise, modifications which decrease the tendency of the 3' end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which increase the tendency of the 5' end of the duplex to dissociate.

Decreasing the stability of the AS 5 ' end of the duplex

Subunit pairs can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation:

A:U is preferred over G:C;

G:U is preferred over G:C;

I:C is preferred over G:C (I=inosine); mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings;

pairings which include a universal base are preferred over canonical pairings.

A typical ds iR A agent can be diagrammed as follows: s 5' Ri Ni N₂ N₃ N₄ N₅

[N] N_5 N_4 N_3 N_2 N.i R₂ 3'

AS 3' R₃ Ni N₂ N₃ N₄ N₅

[N] N_5 N_4 N_3 N_2 N.i R4 5'

S:AS Pi P₂ P₃ P₄

5'

S indicates the sense strand; AS indicates antisense strand; Ri indicates an optional (and nonpreferred) 5 ' sense strand overhang; R₂ indicates an optional (though preferred) 3 ' sense overhang; R₃ indicates an optional (though preferred) 3 ' antisense sense overhang; R4 indicates an optional (and nonpreferred) 5 ' antisense overhang; N indicates subunits; [N] indicates that additional subunit pairs may be present; and P_x, indicates a paring of sense N_x and antisense N_x. Overhangs are not shown in the P diagram. In some embodiments a 3' AS overhang corresponds to region Z, the duplex region corresponds to region X, and the 3' S strand overhang corresponds to region Y, as described elsewhere herein. (The diagram is not meant to imply maximum or minimum lengths, on which guidance is provided elsewhere herein.)

It is preferred that pairings which decrease the propensity to form a duplex are used at 1 or more of the positions in the duplex at the 5 ' end of the AS strand. The terminal pair (the most 5 ' pair in terms of the AS strand) is designated as P_i, and the subsequent pairing positions (going in the 3 ' direction in terms of the AS strand) in the duplex are designated, P_-2, P_₃, P_4, P_5, and so on. The preferred region in which to modify to modulate duplex formation is at P_5 through P_i , more preferably P_4 through P_ i , more preferably P_₃ through P_i . Modification at P_i , is particularly preferred, alone or with modification(s) other position(s), e.g., any of the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions be chosen independently from the group of: (A:U), (G:U), (I:C), mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base.

In preferred embodiments the change in subunit needed to achieve a pairing which promotes dissociation will be made in the sense strand, though in some embodiments the change will be made in the antisense strand.

In a preferred embodiment the at least 2, or 3, of the pairs in P_i , through P_4, are pairs which promote disociation.

In a preferred embodiment the at least 2, or 3, of the pairs in P_i , through P_4, are

A:U.

G:U.

I:C.

In a preferred embodiment the at least 2, or 3, of the pairs in P_i , through P_4, are mismatched pairs, e.g., non-canonical or other than canonical pairings pairings.

In a preferred embodiment the at least 2, or 3, of the pairs in P_i , through P_4, are pairings which include a universal base.

Increasing the stability of the AS 3 ' end of the duplex

Subunit pairs can be ranked on the basis of their propensity to promote stability and inhibit dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting duplex stability: G:C is preferred over A:U. Watson-Crick matches (A:T, A:U, G:C) are preferred over non-canonical or other than canonical pairings. Analogs that increase stability are preferred over Watson-Crick matches (A:T, A:U, G:C). 2-amino-A:U is preferred over A:U. 2-thio U or 5 Me-thio-U: A are preferred over U: A. G-clamp (an analog of C having 4 hydrogen bonds):G is preferred over C:G. Guanadinium-G- clamp:G is preferred over C:G. Psuedo uridine:A is preferred over U:A. Sugar modifications, e.g., 2' modifications, e.g., 2'F, ENA, or LNA, which enhance binding are preferred over non-modified moieties and can be present on one or both strands to enhance stability of the duplex. It is preferred that pairings which increase the propensity to form a duplex are used at 1 or more of the positions in the duplex at the 3' end of the AS strand. The terminal pair (the most 3' pair in terms of the AS strand) is designated as Pi, and the subsequent pairing positions (going in the 5' direction in terms of the AS strand) in the duplex are designated, P₂, P₃, P4, P5, and so on. The preferred region in which to modify to modulate duplex formation is at P5 through Pi, more preferably P4 through Pi , more preferably P₃ through Pi. Modification at Pi, is particularly preferred, alone or with mdification(s) at other position(s), e.g., any of the positions just identified. It is preferred that at least 1 , and more preferably 2, 3, 4, or 5 of the pairs of the recited regions be chosen independently from the group of: G:C, a pair having an analog that increases stability over Watson-Crick matches (A:T, A:U, G:C), 2-amino-A:U, 2-thio U or 5 Me-thio-U:A, G-clamp (an analog of C having 4 hydrogen bonds):G, guanadinium- G-clamp:G, psuedo uridine:A, a pair in which one or both subunits has a sugar modification, e.g., a 2' modification, e.g., 2'F, ENA, or LNA, which enhance binding.

In a preferred embodiment the at least 2, or 3, of the pairs in P_i, through P_4, are pairs which promote duplex stability.

In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P4, are G:C.

In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P4, are a pair having an analog that increases stability over Watson-Crick matches.

In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P4, are 2- amino-A:U. In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P4, are 2- thio U or 5 Me-thio-U:A.

In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P4, are G- clamp:G.

In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P4, are guanidinium-G-clamp:G.

In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P4, are psuedo uridine:A.

In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P4, are a pair in which one or both subunits has a sugar modification, e.g., a 2' modification, e.g., 2'F, ENA, or LNA, which enhances binding.

G-clamps and guanidinium G-clamps are discussed in the following references: Holmes and Gait, "The Synthesis of 2'-0-Methyl G-Clamp Containing Oligonucleotides and Their Inhibition of the HIV-1 Tat- TAR Interaction," Nucleosides, Nucleotides & Nucleic Acids, 22: 1259-1262, 2003; Holmes et al., "Steric inhibition of human immunodeficiency virus type-1 Tat-dependent trans-activation in vitro and in cells by oligonucleotides containing 2'-0-methyl G-clamp ribonucleoside analogues," Nucleic Acids Research, 31 :2759-2768, 2003; Wilds, et al., "Structural basis for recognition of guanosine by a synthetic tricyclic cytosine analogue: Guanidinium G-clamp," Helvetica Chimica Acta, 86:966-978, 2003; Rajeev, et al, "High-Affinity Peptide Nucleic Acid Oligomers Containing Tricyclic Cytosine Analogues," Organic Letters, 4:4395-4398, 2002; Ausin, et al., "Synthesis of Amino- and Guanidino-G-Clamp PNA Monomers," Organic Letters, 4:4073-4075, 2002; Maier et al., "Nuclease resistance of

oligonucleotides containing the tricyclic cytosine analogues phenoxazine and 9-(2- aminoethoxy)-phenoxazine ("G-clamp") and origins of their nuclease resistance properties," Biochemistry, 41 : 1323-7, 2002; Flanagan, et ah, "A cytosine analog that confers enhanced potency to antisense oligonucleotides," Proceedings Of The National Academy Of Sciences Of The United States Of America, 96:3513-8, 1999.Gclamps may also comprise a locked nucleic acid. Methods of synthesizing such LNA-Gclamps are shown in the following schematics.



Compound 900 is synthesized utilizing the literature procedure (Tetrahedron Lett. ,49, 7168, 2008). Treatment of 900 with acetic anhydride in pyridine followed by reaction with N-bromosuccinimide gives the protected 5-bromo LNA 902. This on reaction with phosphorus oxychloride and 1 ,2,4-triazole in acetonitrile gives the triazolyl derivative 903 which on treatment with aminophenol is converted to the corresponding N-substituted cytidine derivative 904 . Cyclization of 904 is accomplished by treating with triethylamine in ethanol to give the protected LNA- Phenoxazine or G-Clamp. Removal of the protecting groups is accomplished by treating with ammonium hydroxide at room temperature to give 906. This on reaction with DMTr-Cl gives the 5'-DMTr derivative 907 which on phosphitylation gives the target phosphoramidite 908.

The dimethoxytrityl derivative 907 is converted to the corresponding succinate 909 by the treatment with succinic anhydride in pyridine in the presence of DMAP. This is activated using HBTU in DMF in the presence of Hunig's base and the activated ester is coupled with long chain alkyl amine-CPG to give 910.

lopyrimidine Gclamp.

Compound 225 A: To a suspension of 223A (lOg) in dry methanol (20 ml) was added ICl (lOg) and the mixture was heated under reflux for 20 h. The solvent was evaporated and the crude was purified by silica gel column chromatography using a gradient 0-25% methanol in dichloromethane to give 11.2 g of 224A. To a cold solution of 224A (1 g, 2.69 mmol) in dry pyridine (20 ml) was added benzoyl chloride (1.26 ml, 10.8 mmol) and the mixture was stirred at room temperature overnight. Reaction was quenched by the addition of water. The mixture was diluted with dichloromethane and washed with sat. sodium bicarbonate solution. Organic layer was evaporated and coevaporated with toluene. The residue was purified by silica gel column

chromatography using a gradient 0-5% methanol in dichloromethane to give 1.6 g of 225A.

Compound 227A: A solution of 225A (4.2g, 6.1 4mmol) and 226 (2.3 g, 8.8 mmol) in anhydrous DMF (60 ml) and triethylamine (30 ml) was degassed by bubbling argon. To this solution Dichloro bis (triphenylphosphine) palladium (0.42 g) and copper iodide (0.23 g) were added and the mixture was heated at 48°C for 18h. The solvent was evaporated and the residue was dissolved in methanol (300 ml). The reaction mixture was heated under reflux for 18 h. The solvent was evaporated and the residue was purified by silica gel column chromatography. The product was eluted using a gradient of 0-5% methanol in dichloromethane. Evaporation of the appropriate fractions containing the product gave 3.8 g of 227A.

Compound 228 A: To a cold (ice bath) solution of 227A (3.8 g) in a mixture of pyridine (60 ml) and methanol (60 ml) was added 1 N NaOH (11 ml). After stirring the mixture at 0-5°C for lh, the reaction was quenched by the addition of dilute HCl (IN, 11 ml). Methanol was evaporated and the solution was diluted with dichloromethane (250 ml) and washed with water (50 ml). Organic layer was dried over sodium sulfate and evaporated. The residue was co-evaporated with toluene and purified by silica gel column chromatography to give 1.8 g of 228A.

1H NMR (400 MHz, DMSO) δ 11.47 (s, 1H), 8.71 (s, 1H), 7.78 - 7.66 (m, 1H), 7.38 - 7.25 (m, 1H), 7.1 1 (t, J = 6.6, 2H), 7.00 (t, J = 7.5, 1H), 6.76 (s, 1H), 6.04 (d, J = 17.5, 1H), 5.57 (d, J = 6.5, 1H), 5.29 (t, J = 5.0, 1H), 4.93 (dd, J= 52.9, 4.0, 1H), 4.22 - 4.05 (m, 3H), 3.97 (d, J= 8.7, 1H), 3.94 - 3.82 (m, 1H), 3.75 - 3.62 (m, 1H), 3.38 (dd, J = 1 1.1 , 5.5, 2H), 1.34 (s, 9H). ^iyF NMR (376 MHz, DMSO) δ -203.50 (m).

MS: Calcd: 505 Found: 504 (M-l)-

Compound 229A: A solution of compound 228A (0.8g) in 50% TFA- dichloromethane (20 ml) was stirred at 0°C for 2h. After this time the reaction mixture was evaporated. The residue was co-evaporated with toluene (25 ml) followed by anhydrous pyridine (20 ml). This was dissolved in dry pyridine (15 ml), the solution was cooled in an ice bath and trifluoroacetic anhydride (2 ml) was added. The reaction mixture was stirred at 0-5°C for 2h. Reaction was quenched by the addition of methanol, diluted with dichloro methane (150 ml) and washed with water (50 ml). Organic layer was dried over sodium sulfate and evaporated. The residue was co-evaporated with toluene (20 ml) and purified by silica gel column chromatography. The product was eluted using a gradient of 0-10% methanol in dichloro methane. Appropriate fractions containing the product were evaporated to give 0.4 g of pure 229A.

Compound 230A: To a solution of 229A (0.4 g) in anhydrous pyridine was added 4,4'-DMT-Cl ( 0.4 g) and the mixture was stirred at room temperature for 5h. The reaction mixture was diluted with dichloro methane (100 ml) and washed with water (50 ml). Organic layer was evaporated and the residue was co-evaporated with toluene. The product was purified using silica gel column chromatography using a gradient of 0-5% methanol in dichloromethane to give 0.36 g of 230A.

!H NMR (400 MHz, DMSO) δ 11.39 (s, 1H), 9.57 (t, J= 5.3, 1H), 8.54 (s, 1H),

7.64 - 7.54 (m, 1H), 7.43 (d, J = 7.5, 3H), 7.36 - 7.26 (m, 9H), 7.21 (t, J = 7.2, 1H), 7.13 (d, J = 8.3, 1H), 7.02 (t, J = 7.6, 1H), 6.86 (dd, J = 8.9, 7.4, 5H), 6.02 (d, J= 18.6, 1H), 5.77 - 5.68 (m, 1H), 5.01 (dd, J = 52.9, 3.9, 1H), 4.55 - 4.37 (m, 1H), 4.14 (dd, J = 13.3, 7.4, 4H), 4.00 (q, J = 7.1 , 1H), 3.70 - 3.60 (m, 8H), 3.57 - 3.42 (m, 4H), 3.34 (d, J = 11.1, 2H). ¹⁹F NMR (376 MHz, DMSO) δ -77.22 (s), -202.65 (m).

Compound 230 B (2'OMe PC-GClamp): In a similar manner as described for 230A, compound 230B was synthesized starting from Tri-benzoyl-2'-OMe-5- iodocytidine and 226.

^!H NMR (400 MHz, DMSO) δ 11.35 (s, 1H), 9.56 (t, J= 5.4, 1H), 8.57 (s, 1H), 7.64 - 7.54 (m, 1H), 7.43 (d, J = 7.5, 2H), 7.38 - 7.26 (m, 8H), 7.21 (t, J = 7.2, 1H), 7.13 (d, J = 8.3, 1H), 7.02 (t, J = 7.5, 1H), 6.86 (t, J = 8.2, 4H), 5.94 (s, 1H), 5.87 (s, 1H), 5.22 (d, J = 7.5, 1H), 4.32 (td, J = 8.1 , 5.1 , 1H), 4.14 (t, J = 5.5, 2H), 4.06 (d, J= 7.5, 1H), 3.73 (d, J = 4.8, 1H), 3.66 (s, 3H), 3.63 (s, 3H), 3.53 (s, 3H). ¹⁹F NMR (376 MHz, DMSO) 5 -77.15 (s).

Compound 231 : To a solution of 230 (lmmol) in dichloromethane (10ml) is added 2-cyanoethyl-tetraisopropylphosphoramidite (1.3 mmol) and dicyano imidazole (0.9 mmol). The mixture is stirred at room temperature for 6 h, diluted with

dichloromethane and washed with sodium bicarbonate solution. Organic layer is dried over sodium sulfate and evaporated. The residue is subjected to column chromatography to give compound 231.

Solid support derivatization: The dimethoxytrityl derivative 230 is converted to the corresponding succinate 232 by the treatment with succinic anhydride in pyridine in the presence of DMAP. This is activated using HBTU in DMF in the presence of Hunig's base and the activated ester is coupled with long chain alkyl amine-CPG to give 232B.

l

Tribenzoyl-5-iodocytidine 235 on reaction with 3 -ethynylpyridine (or 4- ethynylpyridine) in DMF in the presence of bis(triphenylphosphine)palladium(II) dichloride and Cul gave 236. Deprotection of 236 with sodium hydroxide in a mixture of methanol and pyridine gave 237 which on reaction with DMTr-Cl in pyridine gives the corresponding 5'-dimethoxytrityl derivative 238. This on phosphitylation with 2- cyanoethyl N,N-diisopropylchlorophosphoramidite in dichloromethane in the presence of Hunig's base gives the target monomer 239.

Derivatization of the solid support: The dimethoxytrityl derivative 238 is converted to the corresponding succinate 240 by the treatment with succinic anhydride in pyridine in the presence of DMAP. This is activated using HBTU in DMF in the presence of Hunig's base and the activated ester is coupled with long chain alkyl amine - CPG to give 241.

Simultaneously decreasing the stability of the AS 5 'end of the duplex and increasing the stability of the AS 3 ' end of the duplex

As is discussed above, an iR A agent can be modified to both decrease the stability of the AS 5 'end of the duplex and increase the stability of the AS 3' end of the duplex. This can be effected by combining one or more of the stability decreasing modifications in the AS 5' end of the duplex with one or more of the stability increasing modifications in the AS 3' end of the duplex. Accordingly a preferred embodiment includes modification in P_5 through P_i, more preferably P_4 through P_i and more preferably P_₃ through P_i. Modification at P_i, is particularly preferred, alone or with other position, e.g., the positions just identified. It is preferred that at least 1 , and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions of the AS 5' end of the duplex region be chosen independently from the group of: (A:U), (G:U), (I:C), mismatched pairs, e.g., non-canonical or other than canonical pairings which include a universal base; and a modification in P5 through Pi, more preferably P4 through Pi and more preferably P₃ through Pi. Modification at Pi, is particularly preferred, alone or with other position, e.g., the positions just identified. It is preferred that at least 1 , and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions of the AS 3' end of the duplex region be chosen independently from the group of: G:C, a pair having an analog that increases stability over Watson-Crick matches (A:T, A:U, G:C), 2-amino-A:U, 2-thio U or 5 Me-thio-U:A, G-clamp (an analog of C having 4 hydrogen bonds):G, guanadinium-G-clamp:G, psuedo uridine:A, a pair in which one or both subunits has a sugar modification, e.g., a 2' modification, e.g., 2'F, ENA, or LNA, which enhance binding.

The invention also includes methods of selecting and making iR A agents having DMTDS, e.g., when screening a target sequence for candidate sequences for use as iRNA agents one can select sequences having a DMTDS property described herein or one which can be modified, preferably with as few changes as possible, especially to the AS strand, to provide a desired level of DMTDS.

The invention also includes, providing a candidate iRNA agent sequence, and modifying at least one P in P_5 through P_i and/or at least one P in P5 through Pi to provide a DMTDS iRNA agent.

DMTDS iRNA agents can be used in any method described herein, e.g., to silence any gene disclosed herein, to treat any disorder described herein, in any formulation described herein, and generally in and/or with the methods and compositions described elsewhere herein. DMTDS iRNA agents can incorporate other modifications described herein, e.g., the attachment of targeting agents or the inclusion of modifications which enhance stability, e.g., the inclusion of nuclease resistant monomers or the inclusion of single strand overhangs (e.g., 3' AS overhangs and/or 3' S strand overhangs) which self associate to form intrastrand duplex structure.

III. Delivery of Targeting Constructs

A targeting construct (such as an iRNA agent) can be linked, e.g., noncovalently linked to a polymer for the efficient delivery of the targeting construct to an avian cell or cell culture. The targeting construct can, for example, be complexed with cyclodextrin. Cyclodextrins have been used as delivery vehicles of therapeutic compounds.

Cyclodextrins can form inclusion complexes with drugs that are able to fit into the hydrophobic cavity of the cyclodextrin. In other examples, cyclodextrins form non- covalent associations with other biologically active molecules such as oligonucleotides and derivatives thereof. The use of cyclodextrins creates a water-soluble drug delivery complex that can be modified with targeting or other functional groups. Cyclodextrin cellular delivery system for oligonucleotides described in U.S. Pat. No. 5,691,316, which is hereby incorporated by reference, are suitable for use in methods of the invention. In this system, an oligonucleotide is noncovalently complexed with a cyclodextrin, or the oligonucleotide is covalently bound to adamantine which in turn is non-covalently associated with a cyclodextrin.

The delivery molecule can include a linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer having at least one ligand bound to the cyclodextrin copolymer. Delivery systems, as described in U.S. Patent No. 6,509,323, herein incorporated by reference, are suitable for use in methods of the invention. A targeting construct can be bound to the linear cyclodextrin copolymer and/or a linear oxidized cyclodextrin copolymer. Either or both of the cyclodextrin or oxidized cyclodextrin copolymers can be crosslinked to another polymer and/or bound to a ligand.

A composition for iRNA delivery can employ an "inclusion complex," a molecular compound having the characteristic structure of an adduct. In this structure, the "host

molecule" spatially encloses at least part of another compound in the delivery vehicle. The enclosed compound (the "guest molecule") is situated in the cavity of the host molecule without affecting the framework structure of the host. A "host" is preferably cyclodextrin, but can be any of the molecules suggested in U.S. Patent Publ.

2003/0008818, herein incorporated by reference in its entirety.

Cyclodextrins can interact with a variety of ionic and molecular species, and the resulting inclusion compounds belong to the class of "host-guest" complexes. Within the host-guest relationship, the binding sites of the host and guest molecules should be complementary in the stereoelectronic sense. A composition of the invention can contain at least one polymer and at least one therapeutic agent, generally in the form of a particulate composite of the polymer and therapeutic agent, e.g., the targeting construct. The targeting construct can contain one or more complexing agents. At least one polymer of the particulate composite can interact with the complexing agent in a host- guest or a guest-host interaction to form an inclusion complex between the polymer and the complexing agent. The polymer and, more particularly, the complexing agent can be used to introduce functionality into the composition. For example, at least one polymer of the particulate composite has host functionality and forms an inclusion complex with a complexing agent having guest functionality. Alternatively, at least one polymer of the particulate composite has guest functionality and forms an inclusion complex with a complexing agent having host functionality. A polymer of the particulate composite can also contain both host and guest functionalities and form inclusion complexes with guest complexing agents and host complexing agents. A polymer with functionality can, for example, facilitate cell targeting and/or cell contact (e.g., targeting or contact to a liver cell), intercellular trafficking, and/or cell entry and release.

Upon forming the particulate composite, the targeting construct may or may not retain its biological or therapeutic activity. Upon release from the composition, specifically, from the polymer of the particulate composite, the activity of the targeting construct is restored. Accordingly, the particulate composite advantageously affords the targeting construct protection against loss of activity due to, for example, degradation and offers enhanced bioavailability. Thus, a composition may be used to provide stability, particularly storage or solution stability, to a targeting construct or any active chemical compound. The targeting construct may be further modified with a ligand prior to or after particulate composite or therapeutic composition formation. The ligand can provide further functionality. For example, the ligand can be a targeting moiety.

Physiological Effects

The targeting constructs described herein can be designed such that determining therapeutic toxicity is made easier by the complementarity of the targeting construct with both a human and a non-human animal sequence. By these methods, a nucleic acid based targeting construct can consist of a sequence that is fully complementary to a nucleic acid sequence from a human and a nucleic acid sequence from at least one non-human animal, e.g., a non-human mammal, such as a rodent, ruminant or primate or avian species. By determining the toxicity of the targeting construct in the non-human mammal, one can extrapolate the toxicity of the targeting construct in a human. For a more strenuous toxicity test, the targeting construct can be complementary to a human and more than one, e.g., two or three or more, non-human animals. The methods described herein can be used to correlate any physiological effect of a targeting construct on a human, e.g., any unwanted effect, such as a toxic effect, or any positive, or desired effect.

In one aspect, the invention features a delivery conjugate or module, such as those described herein and those described in copending, co-owned United States Provisional Application Serial No. 60/454,265, filed on March 12, 2003, which is hereby

incorporated by reference.

In addition, the invention includes targeting constructs which are iRNA agents and are described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, an iRNA agent having a chemical modification described herein, e.g., a modification which enhances resistance to degradation, an iRNA agent having an architecture or structure described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, combined with, associated with, and delivered by such a drug delivery conjugate or module.

The iRNA agents can be complexed to a delivery agent that features a modular complex. The complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of): (a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic or electrostatic interactions); (b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane, e.g., an endosome membrane); and (c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type.

A targeting construct, e.g., iRNA agent or siRNA agent described herein, can be linked, e.g., coupled or bound, to the modular complex. The iRNA agent can interact with the condensing agent of the complex, and the complex can be used to deliver an iRNA agent to a cell, e.g., in vitro or in vivo.

The fusogenic agent and the condensing agent can be different agents or the one and the same agent. For example, a polyamino chain, e.g., polyethyleneimine (PEI), can be the fusogenic and/or the condensing agent. The delivery agent can be a modular complex. For example, the complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of):

(a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic interaction),

(b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through an avian cell membrane, e.g., an endosome membrane), and

(c) a targeting group, e.g., an avian cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type.

A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N- acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, poly aspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, Neproxin, or an RGD peptide or RGD peptide mimetic.

The carrier agent of a modular complex described herein can be a substrate for attachment of one or more of: a condensing agent, a fusogenic agent, and a targeting group. The carrier agent would preferably lack an endogenous enzymatic activity. The agent would preferably be a biological molecule, preferably a macromolecule.

Polymeric biological carriers are preferred. It would also be preferred that the carrier molecule be biodegradable.

The carrier agent can be a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or lipid. The carrier molecule can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Other useful carrier molecules can be identified by routine methods.

A carrier agent can be characterized by one or more of: (a) is at least 1 Da in size; (b) has at least 5 charged groups, preferably between 5 and 5000 charged groups; (c) is present in the complex at a ratio of at least 1 : 1 carrier agent to fusogenic agent; (d) is present in the complex at a ratio of at least 1 : 1 carrier agent to condensing agent; (e) is present in the complex at a ratio of at least 1 : 1 carrier agent to targeting agent.

A fusogenic agent of a modular complex described herein can be an agent that is responsive to, e.g., changes charge depending on, the pH environment. Upon

encountering the pH of an endosome, it can cause a physical change, e.g., a change in osmotic properties which disrupts or increases the permeability of the endosome membrane. Preferably, the fusogenic agent changes charge, e.g., becomes protonated, at pH lower than physiological range. For example, the fusogenic agent can become protonated at pH 4.5-6.5. The fusogenic agent can serve to release the iR A agent into the cytoplasm of an avian cell after the complex is taken up, e.g., via endocytosis, by the cell, thereby increasing the cellular concentration of the iRNA agent in the cell.

In one embodiment, the fusogenic agent can have a moiety, e.g., an amino group, which, when exposed to a specified pH range, will undergo a change, e.g., in charge, e.g., protonation. The change in charge of the fusogenic agent can trigger a change, e.g., an osmotic change, in a vesicle, e.g., an endocytic vesicle, e.g., an endosome. For example, the fusogenic agent, upon being exposed to the pH environment of an endosome, will cause a solubility or osmotic change substantial enough to increase the porosity of (preferably, to rupture) the endosomal membrane.

The fusogenic agent can be a polymer, preferably a polyamino chain, e.g., polyethyleneimine (PEI). The PEI can be linear, branched, synthetic or natural. The PEI can be, e.g., alkyl substituted PEI, or lipid substituted PEI.

In other embodiments, the fusogenic agent can be polyhistidine, polyimidazole, polypyridine, polypropyleneimine, mellitin, or a polyacetal substance, e.g., a cationic polyacetal. In some embodiment, the fusogenic agent can have an alpha helical structure. The fusogenic agent can be a membrane disruptive agent, e.g., mellittin. A fusogenic agent can have one or more of the following characteristics: (a) is at least IDa in size; (b) has at least 10 charged groups, preferably between 10 and 5000 charged groups, more preferably between 50 and 1000 charged groups; (c) is present in the complex at a ratio of at least 1 : 1 fusogenic agent to carrier agent; (d) is present in the complex at a ratio of at least 1 : 1 fusogenic agent to condensing agent; (e) is present in the complex at a ratio of at least 1 : 1 fusogenic agent to targeting agent.

Other suitable fusogenic agents can be tested and identified by a skilled artisan. The ability of a compound to respond to, e.g., change charge depending on, the pH environment can be tested by routine methods, e.g., in a cellular assay. For example, a test compound is combined or contacted with a cell, and the cell is allowed to take up the test compound, e.g., by endocytosis. An endosome preparation can then be made from the contacted cells and the endosome preparation compared to an endosome preparation from control cells. A change, e.g., a decrease, in the endosome fraction from the contacted cell vs. the control cell indicates that the test compound can function as a fusogenic agent. Alternatively, the contacted cell and control cell can be evaluated, e.g., by microscopy, e.g., by light or electron microscopy, to determine a difference in endosome population in the cells. The test compound can be labeled. In another type of assay, a modular complex described herein is constructed using one or more test or putative fusogenic agents. The modular complex can be constructed using a labeled nucleic acid instead of the targeting construct. The ability of the fusogenic agent to respond to, e.g., change charge depending on, the pH environment, once the modular complex is taken up by the cell, can be evaluated, e.g., by preparation of an endosome preparation, or by microscopy techniques, as described above. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to respond to, e.g., change charge depending on, the pH environment; and a second assay evaluates the ability of a modular complex that includes the test compound to respond to, e.g., change charge depending on, the pH environment.

The condensing agent of a modular complex described herein can interact with (e.g., attracts, holds, or binds to) a targeting construct and act to (a) condense, e.g., reduce the size or charge of the iRNA agent and/or (b) protect the targeting construct, e.g., protect the iRNA agent against degradation. The condensing agent can include a moiety, e.g., a charged moiety, that can interact with a nucleic acid, e.g., an iRNA agent, e.g., by ionic interactions. The condensing agent would preferably be a charged polymer, e.g., a polycationic chain. The condensing agent can be a polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quarternary salt of a polyamine, or an alpha helical peptide.

A condensing agent can have the following characteristics: (a) at least IDa in size; (b) has at least 2 charged groups, preferably between 2 and 100 charged groups; (c) is present in the complex at a ratio of at least 1 : 1 condensing agent to carrier agent; (d) is present in the complex at a ratio of at least 1 : 1 condensing agent to fusogenic agent; (e) is present in the complex at a ratio of at least 1 : 1 condensing agent to targeting agent.

Other suitable condensing agents can be tested and identified by a skilled artisan, e.g., by evaluating the ability of a test agent to interact with a targeting construct, e.g., an iRNA agent. The ability of a test agent to interact with a targeting construct, e.g., an iRNA agent, e.g., to condense or protect the iRNA agent, can be evaluated by routine techniques. In one assay, a test agent is contacted with a nucleic acid, and the size and/or charge of the contacted nucleic acid is evaluated by a technique suitable to detect changes in molecular mass and/or charge. Such techniques include non-denaturing gel electrophoresis, immunological methods, e.g., immunoprecipitation, gel filtration, ionic interaction chromatography, and the like. A test agent is identified as a condensing agent if it changes the mass and/or charge (preferably both) of the contacted nucleic acid, compared to a control. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to interact with, e.g., bind to, e.g., condense the charge and/or mass of, a nucleic cid; and a second assay evaluates the ability of a modular complex that includes the test compound to interact with, e.g., bind to, e.g., condense the charge and/or mass of, a targeting construct. Amphipathic Delivery Agents

In one aspect, the invention features an amphipathic delivery conjugate or module, such as those described herein and those described in copending, co-owned United States Provisional Application Serial No. 60/455,050, filed on March 13, 2003, which is hereby incorporated by reference.

In addition, the invention include a targeting construct described herein, e.g., a palindromic iRNA agent, an iRNA agent hving a non canonical pairing, an iRNA agent which targets a gene described herein, an iRNA agent having a chemical modification described herein, e.g., a modification which enhances resistance to degradation, an iRNA agent having an architecture or structure described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, combined with, associated with, and delivered by such an amphipathic delivery conjugate.

An amphipathic molecule is a molecule having a hydrophobic and a hydrophilic region. Such molecules can interact with (e.g., penetrate or disrupt) lipids, e.g., a lipid bylayer of a cell. As such, they can serve as delivery agent for an associated (e.g., bound) iRNA (e.g., an iRNA or sRNA described herein). A preferred amphipathic molecule to be used in the compositions described herein (e.g., the amphipathic iRNA constructs descriebd herein) is a polymer. The polymer may have a secondary structure, e.g., a repeating secondary structure.

One example of an amphipathic polymer is an amphipathic polypeptide, e.g., a polypeptide having a secondary structure such that the polypeptide has a hydrophilic and a hybrophobic face. The design of amphipathic peptide structures (e.g., alpha-helical polypeptides) is routine to one of skill in the art.

Another example of an amphipathic polymer is a polymer made up of two or more amphipathic subunits, e.g., two or more subunits containing cyclic moieties (e.g., a cyclic moiety having one or more hydrophilic groups and one or more hydrophobic groups). For example, the subunit may contain a steroid, e.g., cholic acid; or an aromatic moiety. Such moieties preferably can exhibit atropisomerism, such that they can form opposing hydrophobic and hydrophilic faces when in a polymer structure. The ability of a putative amphipathic molecule to interact with a lipid membrane, e.g., a cell membrane, can be tested by routine methods, e.g., in a cell free or cellular assay. For example, a test compound is combined or contacted with a synthetic lipid bilayer, a cellular membrane fraction, or a cell, and the test compound is evaluated for its ability to interact with, penetrate or disrupt the lipid bilayer, cell membrane or cell. The test compound can labeled in order to detect the interaction with the lipid bilayer, cell membrane or cell. In another type of assay, the test compound is linked to a reporter molecule or a targeting construct (e.g., an iRNA or siRNA described herein) and the ability of the reporter molecule or targeting construct to penetrate the lipid bilayer, cell membrane or cell is evaluated. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to interact with a lipid bilayer, cell membrane or cell; and a second assay evaluates the ability of a construct (e.g., a construct described herein) that includes the test compound and a reporter or targeting construct to interact with a lipid bilayer, cell membrane or cell.

An amphipathic polymer useful in the compositions described herein has at least

2, preferably at least 5, more preferably at least 10, 25, 50, 100, 200, 500, 1000, 2000, 50000 or more subunits (e.g., amino acids or cyclic subunits). A single amphipathic polymer can be linked to one or more, e.g., 2, 3, 5, 10 or more targeting constructs (e.g., iRNA or siRNA agents described herein). In some embodiments, an amphipathic polymer can contain both amino acid and cyclic subunits, e.g., aromatic subunits.

The invention features a composition that includes a targeting construct (e.g., an iRNA or siRNA described herein) in association with an amphipathic molecule. Such compositions may be referred to herein as "amphipathic iRNA constructs." Such compositions and constructs are useful in the delivery or targeting of targeting constructs, e.g., delivery or targeting of iRNA agents to an avian cell. While not wanting to be bound by theory, such compositions and constructs can increase the porosity of, e.g., can penetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g., to allow entry of the targeting construct into a cell.

In one aspect, the invention relates to a composition comprising an targeting construct (e.g., an iRNA or siRNA agent described herein) linked to an amphipathic molecule. The targeting construct and the amphipathic molecule may be held in continuous contact with one another by either covalent or noncovalent linkages.

The amphipathic molecule of the composition or construct is preferably other than a phospholipid, e.g., other than a micelle, membrane or membrane fragment.

The amphipathic molecule of the composition or construct is preferably a polymer. The polymer may include two or more amphipathic subunits. One or more hydrophilic groups and one or more hydrophobic groups may be present on the polymer. The polymer may have a repeating secondary structure as well as a first face and a second face. The distribution of the hydrophilic groups and the hydrophobic groups along the repeating secondary structure can be such that one face of the polymer is a hydrophilic face and the other face of the polymer is a hydrophobic face.

The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an a-helical conformation as its secondary structure.

In one embodiment, the amphipathic polymer includes one or more subunits containing one or more cyclic moiety (e.g., a cyclic moiety having one or more hydrophilic groups and/or one or more hydrophobic groups). In one embodiment, the polymer is a polymer of cyclic moieties such that the moieties have alternating hydrophobic and hydrophilic groups. For example, the subunit may contain a steroid, e.g., cholic acid. In another example, the subunit may contain an aromatic moiety. The aromatic moiety may be one that can exhibit atropisomerism, e.g., a 2,2'-bis(substituted)- l-l '-binaphthyl or a 2,2'-bis(substituted) biphenyl. A subunit may include an aromatic moiety of Formula (M):

Referring to Formula M, Ri is C1-C100 alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; Ci-Cioo perfluoroalkyl; or OR5.

R₂ is hydroxy; nitro; sulfate; phosphate; phosphate ester; sulfonic acid; OR₆; or Ci-Cioo alkyl optionally substituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester, substituted or unsubstituted aryl, carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(O), S0₂, alkenyl, or alkynyl.

R3 is hydrogen, or when taken together with R4 froms a fused phenyl ring.

R4 is hydrogen, or when taken together with R3 froms a fused phenyl ring.

R5 is Ci-Cioo alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; or C1-C100 perfluoroalkyl; and R₆ is C1-C100 alkyl optionally substituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester, substituted or unsubstituted aryl, carboxyl, carboxylate, amino carbonyl, or

alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(O), S0₂, alkenyl, or alkynyl. Increasing cellular uptake of dsRNAs

A method of the invention that can include the administration of a targeting construct and a drug that affects the uptake of the targeting construct into the cell. The drug can be administered before, after, or at the same time that the targeting construct is administered. The drug can be covalently linked to the targeting construct. The drug can have a transient effect on the cell.

The drug can increase the uptake of the targeting construct into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latranculin A, phalloidin, swinholide A, indanocine, or myoservin.

The drag can also increase the uptake of the targeting constract into the cell by activating an inflammatory response, for example. Exemplary drag's that would have such an effect include tumor necrosis factor alpha (TNF alpha), interleukin- 1 beta, or gamma interferon.

IV. Formulations

Nucleic acid lipid particles

In one embodiment, an avian targeting constract, e.g., a dsRNA, featured in the invention is fully encapsulated in a lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term "SNALP" refers to a stable nucleic acid-lipid particle, including SPLP. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA agent or a plasmid from which a iRNA agent is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and in

International Application No. WO 2009082817. These applications are incorporated herein by reference in their entirety. As used herein, the term "SPLP" refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include "pSPLP," which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 1 10 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid- lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease.

Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Patent Nos. 5,976,567; 5,981 ,501 ; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964, each of which is incorporated herein by reference in its entirety.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to targeting construct, e.g., dsRNA ratio) will be in the range of from about 1 : 1 to about 50: 1 , from about 1 : 1 to about 25: 1 , from about 3: 1 to about 15: 1 , from about 4: 1 to about 10: 1 , from about 5: 1 to about 9: 1 , or about 6: 1 to about 9: 1.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I - (2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I -(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3- dio ley loxy propylamine (DODMA), 1 ,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1 ,2- Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1 ,2-Dilinoleyoxy-3- (dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), l ,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), l ,2-Dilinoleylthio-3- dimethylaminopropane (DLin-S-DMA), l-Linoleoyl-2-linoleyloxy-3- dimethylaminopropane (DLin-2-DMAP), 1 ,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1 ,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), l ,2-Dilinoleyloxy-3-( -methylpiperazino)propane (DLin-MPZ), or 3- ( ,Ν-Dilinoleylamino)- 1 ,2-propanediol (DLinAP), 3-( ,N-Dioleylamino)- 1 ,2- propanedio (DOAP), l,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin- EG-DMA), l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl- 4-dimethylaminomethyl-[l ,3]-dioxolane (DLin-K-DMA) or analogs thereof,

(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH- cyclopenta[d][l ,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- tetraen-19-yl 4-(dimethylamino)butanoate (MC3), l,l'-(2-(4-(2-((2-(bis(2- hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l- yl)ethylazanediyl)didodecan-2-ol (Tech Gl), or a mixture thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-

[l,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2- Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane is described in United States provisional patent application number 61/107,998 filed on October 23, 2008, which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0 ± 20 nm and a 0.027 siRNA/Lipid Ratio.

The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylchohne (DSPC), dioleoylphosphatidylchohne (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-( -maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE),

dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1 -stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG- dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (Ci₆), or a PEG- distearyloxypropyl (C]s). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

LNP01

In one embodiment, the lipidoid D98-4HCl (MW 1487) (see U.S. Patent Application No. 12/056,230, filed 3/26/2008, which is herein incorporated by reference), Cholesterol (Sigma- Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG- Ceramide CI 6, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide CI 6 stock solutions can then be combined in a, e.g., 42:48: 10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1 , about pH 7.2, about pH 7.3, or about pH 7.4.

Formula 1

LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid-dsRNA formulations are as follows:

Table 2 Lipid Nanop article formulations

Lipid:siRNA: 10:1 C12-200/DSPC/Chol/PEG-DSG

LNP21 C12-200 50/10/38.5/1.5

Lipid:siRNA: 7:1

XTC/DSPC/Chol/PEG-DSG

LNP22 XTC 50/10/38.5/1.5

Lipid:siRNA: 10:1

DSPC: distearoylphosphatidylcholine

DPPC: dipalmitoylphosphatidylcholine

PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000)

PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000)

PEG-cDMA: PEG-carbamoyl-l ,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)

SNALP (l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed April 15, 2009, which is hereby incorporated by reference in its entirety.

XTC comprising formulations are described, e.g., in U.S. Provisional Serial No. 61/239,686, filed September 3, 2009 as well as PCT/US 10/22614 filed January 29, 2010 each of which is hereby incorporated by reference in its entirety. Further XTC formulations useful in the present invention are disclosed in PCT/US08/088588 filed 31- Dec-2008 and PCT/US08/88587 filed 31-Dec-2008 and PCT/US09/041442 filed 22-Apr- 2009 and PCT/US09/061897 filed 23-Oct-2009 and PCT/US 10/38224 filed June 10, 2010, each of which is hereby incorporated by reference in its entirety.

MC3 comprising formulations are described, e.g., in U.S. Provisional Serial No. 61/244,834, filed September 22, 2009, and U.S. Provisional Serial No. 61/185,800, filed June 10, 2009, and PCT/US09/63933 filed November 10, 2009 and

PCT/US09/63927 filed 10-Nov-2009 and PCT/US09/63931 filed 10-Nov-2009 and PCT/US09/63897 filed 10-Nov-2009, each of which are hereby incorporated by reference in its entirety.

ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on November 10, 2009, which is hereby incorporated by reference in its entirety. The synthesis is outlined in Example 8. C12-200 comprising formulations are described in U.S. Provisional Serial No. 61/175,770, filed May 5, 2009, as well as PCT/US 10/33777 which are hereby incorporated by reference in its entirety. The synthesis is outlined in Example 8.

Transfection reagents useful in the present invention are disclosed in US provisional 61/267,419 filed December 7, 2009, which is hereby incorporated by reference in its entirety.

Formulations for targeting immune cells useful in the present invention are disclosed in PCT/US 10/033747 filed May 5, 2010, which is hereby incorporated by reference in its entirety.

Pyrrolidine cationic lipids useful in the formulations of the present invention are disclosed in USSN 12/123,922 filed May 20, 2008 which is hereby incorporated by reference in its entirety.

In one embodiment, the reagent that facilitates targeting construct uptake used herein comprises a cationic lipid as described in e.g., U.S. Application Ser.

No. 61/267,419, filed 7 December 2009, and U.S. Application Ser. No. 61/334,398, filed 13 May 2010. In various embodiments, the targeting construct composition described herein comprisescomprises a cationic lipid selected from the group consisting of: "Lipid H", "Lipid K"; "Lipid L", "Lipid M"; "Lipid P"; or "Lipid R", whose formulas are indicated as follows:

Also contemplated herein are various formulations of the lipids described above, such as, e.g., K8, P8 and L8 which refer to formulations comprising Lipid K, P, and L, respectively. Some exemplary lipid formulations for use with the methods and compositions described herein are found in e.g., Table 3:

Table 3. Example lipid formulations

In another embodiment, the targeting construct composition described herein further comprises a lipid formulation comprising a lipid selected from the group consisting of Lipid H, Lipid K, Lipid L, Lipid M, Lipid P, and Lipid R, and further comprises a neutral lipid and a sterol. In particular embodiments, the lipid formulation comprises between approximately 25 mol % - 100 mol% of the lipid. In another embodiment, the lipid formulation comprises between 0 mol% - 50 mol% cholesterol. In still another embodiment, the lipid formulation comprises between 30 mol% - 65 mol% of a neutral lipid. In particular embodiments, the lipid formulation comprises the relative mol% of the components as listed in Table 4 as follows: Table 4. Example lipid formulae

Other particles

In vivo delivery can also be by a beta-glucan delivery system, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677, and U.S. Publication No.

2005/0281781, which are hereby incorporated by reference in their entirety. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.

Chitosan and minicell nanoparticles may also be utilized.

Liposomal formulations

There are many organized surfactant structures that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up in vivo.

Ill It is desirable to use a liposome which is highly deformable and able to pass through fine pores with a diameter less than 50 nm, under the influence of a suitable gradient.

Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical

administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al, Biochem. Biophys. Res. Commun, 1987, 147, 980-985). Liposomes which are pH-sensitive or negatively charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl

phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or

phosphatidylcholine and/or cholesterol.

Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_MI, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG- derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al, FEBS Letters, 1987, 223, 42; Wu et al, Cancer Research, 1993, 53, 3765). Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_MI, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_MI or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al) discloses liposomes comprising sphingomyelin. Liposomes comprising 1 ,2-sn- dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C_I2ISG, that contains a PEG moiety. Ilium et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols {e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising

phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta,

1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 Bl and WO 90/04384 to Fisher. Liposome compositions containing 1 -20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos.

5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 Bl). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al) and in WO 94/20073 (Zalipsky et al) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al) and U.S. Pat. No. 5,556,948 (Tagawa et al) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein- bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations of targeting constructs. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which may

conveniently be presented in unit dosage form, may be prepared according to

conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the

pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.

Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Emulsions

The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μηι in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1 , p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et ah, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms,

Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1 , p. 285; Idson, in

Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1 , p. 285).

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1 , p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid.

Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,

Marcel Dekker, Inc., New York, N.Y., volume 1 , p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1 , p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

In one embodiment of the present invention, the compositions of targeting constructs are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and

thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms,

Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1 , p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water- insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously. Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with co surfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1 -propanol, and 1 -butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated

polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Patent Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099;

Constantinides et ah, Pharmaceutical Research, 1994, 1 1, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Patent Nos. 6,191 ,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al, Pharmaceutical Research, 1994, 11 , 1385; Ho et al, J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile targeting construct drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of nucleic acid based targeting constructs, e.g., iRNAs and nucleic acids, from the gastrointestinal tract, as well as improve the local cellular uptake.

Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the targeting constructs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non- chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991 , p. 92). Each of these classes has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of targeting constructs, particularly iRNAs to avian cells. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail. Surfactants: In connection with the present invention, surfactants (or "surface- active agents") are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of targeting constructs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et ah, Critical Reviews in Therapeutic Drug Carrier Systems, 1991 , p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al, J. Pharm. Pharmacol, 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, l-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, Ci-20 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di- glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991 , p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al, J. Pharm. Pharmacol, 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed.,

Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term "bile salts" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et ah, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92;

Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et ah, J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et ah, J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of targeting constructs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium

ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5- methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N- amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991 , page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al, J. Control Rel, 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of targeting constructs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1- alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991 , page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm.

Pharmacol, 1987, 39, 621-626).

Agents that enhance uptake of targeting constructs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et ah, PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen;

Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™ (Invitrogen;

Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX (Invitrogen; Carlsbad, CA), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, CA), Lipofectamine™ (Invitrogen; Carlsbad, CA), RNAiMAX (Invitrogen; Carlsbad, CA), Oligofectamine™ (Invitrogen; Carlsbad, CA), Optifect™ (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal

Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, WI), TransFast™ Transfection Reagent (Promega; Madison, WI), Tfx™-20 Reagent (Promega; Madison, WI), Tfx™-50 Reagent (Promega; Madison, WI), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^a Dl Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec™/LipoGen™ (Invivogen; San Diego, CA, USA), PerFectin Transfection Reagent (Genlantis; San Diego, CA, USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER Transfection reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin Transfection Reagent (Genlantis; San Diego, CA, USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, CA, USA ), RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B- Bridge International; Mountain View, CA, USA), or HiFect™ (B-Bridge International, Mountain View, CA, USA), among others.

Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation.

Excipients

In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Other Components

The compositions of the present invention may additionally contain other adjunct components. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation. Aqueous suspensions may contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In addition to their administration, as discussed above, the targeting constructs featured in the invention can be administered in combination with other known agents. Further, toxicity and therapeutic efficacy of compounds of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.

V. Uses for biological products and biomolecules

The invention relates in particular to the use of a targeting construct to modulate the expression of one or more avian transcripts in the bioprocessing or bioproduction of biological products and/or biomolecules. The resultant biological products and/or biolo molecules produced by these methods have utility in the treatment of human pathological conditions, diseases, symptoms or disorders.

As used herein, in the context of the use of the biological products or

biomolecules insofar as it relates to any of the other conditions recited herein below, the terms "treat," "treatment," and the like mean to relieve or alleviate at least one symptom associated with a condition, or to slow or reverse the progression or anticipated progression of a condition, such as slowing the progression of a malignancy or cancer, or increasing the clearance of an infectious organism to alleviate/reduce the symptoms caused by the infection, e.g., hepatitis caused by infection with a hepatitis virus.

By "lower" in the context of a disease marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without such disorder. As used herein, the phrases "therapeutically effective amount" and

"prophylactically effective amount" refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes or an overt symptom of pathological processes. The specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type of pathological processes mediated by avian expression, the patient's history and age, the stage of pathological processes mediated by avian expression, and the administration of other agents that inhibit pathological processes mediated by avian expression.

As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of a targeting construct and a pharmaceutically acceptable carrier. As used herein, "pharmacologically effective amount," "therapeutically effective amount" or simply "effective amount" refers to that amount of a biological product or biomolecule to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 10% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a biological product or biomolecule for the treatment of that disease or disorder is the amount necessary to effect at least a 10% reduction in that parameter.

The term "pharmaceutically acceptable carrier" refers to a carrier for

administration of a biological product or biomolecule. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For biological product or biomolecules administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. Agents included in biological product or biomolecule formulations are described further herein below.

Biological therapies use the body's immune system, either directly or indirectly, to fight cancer or to lessen the side effects that may be caused by some cancer treatments. In one sense, biological product or biomolecules can be considered in this group of therapies in that it can stimulate immune system action against a tumor, for example. However, this approach can also be considered with other such biological approaches, e.g., immune response modifying therapies such as the administration of interferons, interleukins, colony-stimulating factors, monoclonal antibodies, vaccines, gene therapy, and nonspecific immunomodulating agents are also envisioned as anti-cancer therapies to be combined with the biological product or biomolecule.

Efficacy of treatment or amelioration of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of a biological product or biomolecule or pharmaceutical composition thereof, "effective against" a cancer indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease load, reduction in tumor mass or cell numbers, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of cancer.

A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given biological product or biomolecule drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.

Infections are broadly classified as bacterial, viral, fungal, or parasitic based on the category of infectious organism or agent involved. Other less common types of infection are also known in the art, including, e.g., infections involving rickettsiae, mycoplasmas, and agents causing scrapie, bovine spongiform encephalopthy (BSE), and prion diseases (e.g., kuru and Creutzfeldt- Jacob disease). Examples of bacteria, viruses, fungi, and parasites which cause infection are well known in the art. An infection can be acute, subacute, chronic, or latent, and it can be localized or systemic. As defined herein, a "chronic infection" refers to those infections that are not cleared by the normal actions of the innate or adaptive immune responses and persist in the subject for a long duration of time, on the order of weeks, months, and years. A chronic infection may reflect latency of the infectious agent, and may be include periods in which no infectious symptoms are present, i.e., asymptomatic periods. Examples of chronic infections include, but are not limited to, HIV infection and herpesvirus infections. Furthermore, an infection can be predominantly intracellular or extracellular during at least one phase of the infectious organism's or agent's life cycle in the host.

Exemplary viruses include, but are not limited to: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III), HIV-2, LAV or HTLV-III/LAV, or HIV-III, and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses);

Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); adenovirus; Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses, i.e., Rotavirus A, Rotavirus B. Rotavirus C); Birnaviridae; Hepadnaviridae (Hepatitis A and B viruses); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, Human herpes virus 6, Human herpes virus 7, Human herpes virus 8, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Epstein-Barr virus; Rous sarcoma virus; West Nile virus; Japanese equine encephalitis, Norwalk, papilloma virus, parvovirus B19; Poxyiridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); Hepatitis D virus, Hepatitis E virus, and unclassified viruses (e.g., the etiological agents of Spongiform

encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class l=enterally transmitted; class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astro viruses).

Bacteria include both Gram negative and Gram positive bacteria. Examples of

Gram positive bacteria include, but are not limited to Pasteurella species, Staphylococci species, and Streptococcus species. Examples of Gram negative bacteria include, but are not limited to, Escherichia coli (including enterohaemorrhagic E. coli (EHEC) strain 0104), Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include but are not limited to: Helicobacter pylons, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria spp. (e.g., M. tuberculosis, M. avium, M.

intracellulare, M. kansasii, M. gordonae, M. leprae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus),

Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis,

Streptococcus (anaerobic spp.), Streptococcus pneumoniae, pathogenic Campylobacter spp., Enterococcus spp., Haemophilus influenzae {Hemophilus influenza B, and

Hemophilus influenza non-typable) , Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium spp., Erysipelothrix rhusiopathiae, Clostridium perfringens,

Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides spp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, Leptospira, Rickettsia, Actinomyces israelii, meningococcus, pertussis, pneumococcus, shigella, tetanus, Vibrio cholerae, yersinia, Pseudomonas species, Clostridia species, Salmonella typhi, Shigella dysenteriae, Yersinia pestis, Brucella species, Legionella pneumophila, Rickettsiae, Chlamydia,

Clostridium perfringens, Clostridium botulinum, Staphylococcus aureus, Pseudomonas aeruginosa, Cryptosporidium parvum, Streptococcus pneumoniae, and Bordetella pertussis.

Exemplary fungi and yeast include, but are not limited to, Cryptococcus neoformans, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanathii, Candida lusitaniae, Rhodotorula mucilaginosa, Aspergillus fumigatus, Aspergillus flavus, Blastomyces dermatitidis , Aspergillus clavatus, Cryptococcus neoformans, Chlamydia trachomatis, Coccidioides immitis, Cryptococcus laurentii, Cryptococcus albidus, Cryptococcus gattii, Nocardia spp, Histoplasma capsulatum, Pneumocystis jirovecii (or Pneumocystis carinii), Stachybotrys chartarum, and any combination thereof.

Exemplary parasites include, but are not limited to: Entamoeba histolytica;

Plasmodium species {Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax), Leishmania species (Leishmania tropica, Leishmania braziliensis, Leishmania donovani), Toxoplasmosis (Toxoplasma gondii), Trypanosoma gambiense, Trypanosoma rhodesiense (African sleeping sickness), Trypanosoma cruzi (Chagas' disease), Helminths (flat worms, round worms), Babesia microti, Babesia divergens, Giardia lamblia, and any combination thereof.

The invention further relates to the use of a biological product or biomolecule for the treatment of an infectious disease, such as hepatitis B or a chronic bacterial infection, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating such infectious diseases or disorders (e.g., antibiotics, anti-viral agents). In other embodiments, administration of a biological product or biomolecule is performed in combination with an anti-viral medicament or agent.

In other embodiments, administration of a biological product or biomolecule is performed in combination with an anti-fungal medicament or agent. An "antifungal medicament" is an agent that kills or inhibits the growth or function of infective fungi. Anti-fungal medicaments are sometimes classified by their mechanism of action. Some anti-fungal agents function as cell wall inhibitors by inhibiting glucose synthase, other antifungal agents function by destabilizing membrane integrity, and other antifungal agents function by breaking down chitin (e.g., chitinase) or immunosuppression (501 cream).

In further embodiments, administration of a biological product or biomolecule is administered in combination with an anti-parasitic medicament or agent. An

"antiparasitic medicament" refers to an agent that kills or inhibits the growth or function of infective parasites.

Delivery of Biological Products

The delivery of a biological product or biomolecule to a subject in need thereof can be achieved in a number of different ways. In vivo delivery can be performed directly by administering a composition comprising a biological product or biomolecule, e.g, antibody, etc to a subject. Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the biological product or biomolecule. These alternatives are discussed further below.

"Introducing into a cell," when referring to a biological product or biomolecule, means facilitating or effecting uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of a biological product or biomolecule can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a biological product or biomolecule may also be "introduced into a cell," wherein the cell is part of a living organism. In such an instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, biological product or biomolecules can be injected into a tissue site or administered systemically. It is also contemplated by the inventors that introduction into cells or tissues may effected ex vivo, in situ and in ovo. In the case of transplants or within the field of stem cell technologies, it is contemplated that "introduction into a cell" will embrace the introduction to cells of any lineage or state, whether presently stem cells or which are intended to produce stem cells or progenitors or precursors thereof, as well as tissues, explants, organs and even organ systems.

When the organism to be treated is a mammal such as a human, the biological product of the methods described here may be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial {e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection.

Direct delivery

In general, any method of delivering a nucleic acid molecule can be adapted for use with a biological product or biomolecule (see e.g., Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5): 139-144 and WO94/02595, which are incorporated herein by reference in their entireties). However, there are three factors that are important to consider in order to successfully deliver a biological product or biomolecule in vivo: (a) biological stability of the delivered molecule, (2) preventing non-specific effects, and (3) accumulation of the delivered molecule in the target tissue. The non-specific effects of a targeting construct can be minimized by local administration, for example by direct injection or implantation into a tissue (as a non-limiting example, a tumor) or topically administering the preparation.

Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that may otherwise be harmed by the agent or that may degrade the agent, and permits a lower total dose of the biological product or biomolecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA agent, biological product or biomolecule, is administered locally. For administering a biological product or biomolecule systemically for the treatment of a disease, the biological product or biomolecule can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the molecule by endo- and exo-nucleases (in the case of nucleic acid based targeting constructs) in vivo. Modification of a biological product or biomolecule or the pharmaceutical carrier can also permit targeting of the biological product or biomolecule composition to the target tissue and avoid undesirable off-target effects. Biological product or biomolecule molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. In like fashion, the biological product or biomolecule of the present invention may be conjugated to one or more aptamers.

In an alternative embodiment, the biological product or biomolecule can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of a targeting construct molecule (when negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of a biological product or biomolecule by the cell. Cationic lipids, dendrimers, or polymers can either be bound to a biological product or biomolecule, or induced to form a vesicle or micelle (see e.g., Kim SH., et al (2008) Journal of Controlled Release 129(2): 107-116) that encases a biological product or biomolecule. The formation of vesicles or micelles further prevents degradation of the biological product or biomolecule when administered systemically.

Methods for making and administering cationic biological product or biomolecule complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, DR., et al (2003) J. Mol. Biol 327:761-766; Verma, UN., et al (2003) Clin. Cancer Res. 9: 1291- 1300; Arnold, AS et al (2007) J. Hypertens. 25: 197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of biological product or biomolecule include DOTAP (Sorensen, DR., et al (2003), supra; Verma, UN., et al (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS., et al (2006) Nature 441 : 111-114), cardiolipin (Chien, PY., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26: 1087-1091), polyethyleneimine (Bonnet ME., et al (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly- Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, DA., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16: 1799-1804). In some embodiments, a biological product or biomolecule forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of a biological product or biomolecule and cyclodextrins can be found in U.S. Patent No. 7, 427, 605, which is herein incorporated by reference in its entirety.

Methods of direct delivery are disclosed in PCT/US2007/079203 filed September 21 , 2007 (Applicant docket number ALE-027) incorporated by reference herein in its entirety. These methods are useful in the present invention. Disclosed in

PCT/US09/38437 filed 26-Mar-2009 (Applicant docket number ALN-062) and USSN 12/591,629 25-Nov-2009 (Applicant docket number ALN-080) are further methods of delivery useful in the present invention. Each of these documents is incorporated by reference herein in its entirety.

VI. Kits and Assays

Any of the compositions described herein may be comprised in a kit. In a non- limiting example, reagents for generating targeting constructs, including iRNA agents and specifically siRNA molecules are included in a kit. The kit may further include reagents or instructions for creating or synthesizing the targeting construct. It may also include one or more buffers, such as a nuclease buffer, transcription buffer, or a hybridization buffer, compounds for preparing the DNA template or a dsRNA, and components for isolating the resultant template, dsRNA, or siRNA. Other kits of the invention may include components for making a nucleic acid array comprising iRNA agents, e.g., siRNA, and thus, may include, for example, a solid support.

The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the targeting constructs, e.g., nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. In some embodiments, labeling dyes are provided as a dried power. It is contemplated that 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 micrograms or at least or at most those amounts of dried dye are provided in kits of the invention. The dye may then be resuspended in any suitable solvent, such as DMSO.

The container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the targeting construct, e.g., nucleic acid formulations are placed, preferably, suitably allocated. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.

The kits of the present invention may also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained.

Kits may also include components that facilitate isolation of a DNA template, long dsR A, or iR A agent, e.g., siRNA. It may also include components that preserve or maintain the nucleic acids or that protect against their degradation. Such components may be R Ase-free or protect against R Ases, such as R ase inhibitors. Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution.

A kit can include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented.

In some embodiments, kits are provided for testing the effect of a targeting construct or a series of targeting construct son the production of a biological product by the cell, where the kits comprise a substrate having one or more assay surfaces suitable for culturing cells under conditions that allow production of a biological product. In some embodiments, the exterior of the substrate comprises wells, indentations, demarcations, or the like at positions corresponding to the assay surfaces. In some embodiments, the wells, indentations, demarcations, or the like retain fluid, such as cell culture media, over the assay surfaces.

In some embodiments, the assay surfaces on the substrate are sterile and are suitable for culturing host cells (e.g., avian host cells) under conditions representative of the culture conditions during large-scale (e.g., industrial scale) production of the biological product. Advantageously, kits provided herein offer a rapid, cost-effective means for testing a wide-range of agents and/or conditions on the production of a biological product, allowing the cell culture conditions to be established prior to full- scale production of the biological product.

In some embodiments, one or more assay surfaces of the substrate comprise a concentrated test agent, such as a targeting construct, such that the addition of suitable media to the assay surfaces results in a desired concentration of the targeting construct surrounding the assay surface. In some embodiments, the targeting construct may be printed or ingrained onto the assay surface, or provided in a lyophilized form, e.g., within wells, such that the effector molecules can be reconstituted upon addition of an appropriate amount of media. In some embodiments, the targeting constructs are reconstituted by plating cells onto assay surfaces of the substrate. In some embodiments, kits provided herein further comprise cell culture media suitable for culturing a cell under conditions allowing for the production of a biological product and/or biomolecule of interest. The media can be in a ready to use form or can be concentrated (e.g., as a stock solution), lyophilized, or provided in another reconstitutable form.

In further embodiments, kits provided herein further comprise one or more reagents suitable for detecting production of the biological product or biomolecule by the cell, cell culture, or tissue culture. In further embodiments, the reagent(s) are suitable for detecting a property of the cell, such as maximum cell density, cell viability, or the like, which is indicative of production of the desired biological product. In some

embodiments, the reagent(s) are suitable for detecting the biological product or a property thereof, such as the in vitro or in vivo biological activity, homogeneity, or structure of the biological product or biomolecule.

In some embodiments, one or more assay surfaces of the substrate further comprise a carrier for which facilitates uptake of targeting constructs by cells. Carriers for targeting constructs are known in the art and are described herein. For example, in some embodiments, the carrier is a lipid formulation such as LIPOFECTAMINE™

transfection reagent (Invitrogen; Carlsbad, CA) or a related formulation. Examples of such carrier formulations are described herein. In some embodiments, the reagent that facilitates targeting construct uptake comprises a charged lipid, an emulsion, a liposome, a cationic or non-cationic lipid, an anionic lipid, a transfection reagent or a penetration enhancer as described throughout the application herein. In particular embodiments, the reagent that facilitates targeting construct uptake comprises a charged lipid as described in U.S. Application Ser. No. 61/267,419, filed on December 7, 2009.

In some embodiments, one or more assay surfaces of the substrate comprise a targeting construct or series of targeting constructs and a carrier, each in concentrated form, such that plating test cells onto the assay surface(s) results in a concentration the targeting construct(s) and the carrier effective for facilitating uptake of the targeting construct (s) by the cells and modulation of the expression of one or more genes targeted by the targeting constructs. In some embodiments, the substrate further comprises a matrix which

facilitates 3-dimensional cell growth and/or production of the biological product by the cells. In further embodiments, the matrix facilitates anchorage-dependent growth of cells. Non-limiting examples of matrix materials suitable for use with various kits described herein include agar, agarose, methylcellulose, alginate hydrogel (e.g., 5% alginate + 5% collagen type I), chitosan, hydroactive hydrocolloid polymer gels, polyvinyl alcohol- hydrogel (PVA-H), polylactide-co-glycolide (PLGA), collagen vitrigel, PHEMA (poly(2- hydroxylmethacrylate)) hydrogels, PVP/PEO hydrogels, BD PURAMATRIX™ hydrogels, and copolymers of 2-methacryloyloxyethyl phophorylcholine (MPC).

In some embodiments, the substrate comprises a microarray plate, a biochip, or the like which allows for the high-throughput, automated testing of a range of test agents, conditions, and/or combinations thereof on the production of a biological product or biomolecule by cultured cells. For example, the substrate may comprise a 2-dimensional microarray plate or biochip having m columns and n rows of assay surfaces (e.g., residing within wells) which allow for the testing of m x n combinations of test agents and/or conditions (e.g., on a 24-, 96- or 384-well microarray plate). The microarray substrates are preferably designed such that all necessary positive and negative controls can be carried out in parallel with testing of the agents and/or conditions.

In further embodiments, kits are provided comprising one or more microarray substrates seeded with a set of targeting constructs designed to modulate a particular pathway, function, or property of a cell which affects the production of the biological product or biomolecule. For example, in some embodiments, the targeting constructs are directed against target genes comprising a pathway involved in the expression, folding, secretion, or post-translational modification of a recombinant protein product by the cell.

In further embodiments, kits are provided herein comprising one or more microarray substrates seeded with a set of targeting constructs designed to address a particular problem or class of problems associated with the production of an

immunogenic agent in cell-based systems. For example, in some embodiments, the targeting constructs are directed against target genes expressed by latent or endogenous viruses; or involved in cell processes, such as cell cycle progression, cell metabolism or apoptosis which inhibit or interfere production or purification of the biological product. In further embodiments, the targeting constructs are directed against target genes that mediate enzymatic degradation, aggregation, misfolding, or other processes that reduce the activity, homogeneity, stability, and/or other qualities of the biological product. In yet further embodiments, the targeting constructs are directed against target genes that affect the infectivity of exogenous or adventitious contaminating microbes. In one embodiment, the biological product or biomolecule includes a glycoprotein, and the targeting constructs are directed against target genes involved in glycosylation (e.g., fucosylation) and/or proteolytic processing of glycoproteins by the host cell. In another embodiment, the biological product is a multi-subunit recombinant protein and the targeting constructs are directed against target genes involved in the folding and/or secretion of the protein by the host cell. In another embodiment, the targeting constructs are directed against target genes involved in post-translation modification of the biological product in the cells, such as methionine oxidation, glycosylation, disulfide bond formation, pyroglutamation and/or protein deamidation. In another embodiment, the targeting constructs are directed against avian transcripts involved in any one or more of the functions outlined in Table 5.

In some embodiments, kits provided herein allow for the selection or optimization of at least one factor for enhancing production of the biological product or biomolecule. For example, the kits may allow for the selection of a targeting construct from among a series of candidate targeting constructs, or for the selection of a concentration or concentration range from a wider range of concentrations of a given targeting construct. In some embodiments, the kits allow for selection of one or more targeting constructs from a series of candidate targeting constructs directed against a common target gene. In further embodiments, the kits allow for selection of one or more targeting constructs from a series of candidate targeting constructs directed against two or more functionally related target genes or two or more target genes of a common host cell pathway.

In some embodiments, kits provided herein allow for the selection or optimization of a combination of two or more factors in the production of a biological product or biomolecule. For example, the kits may allow for the selection of a suitable targeting construct from among a series of candidate targeting constructs as well as a concentration of the targeting construct. In further embodiments, kits provided herein allow for the selection of a first targeting construct from a first series of candidate targeting constructs and a second targeting construct from a second series of candidate targeting constructs. In some embodiments, the first and/or second series of candidate targeting constructs are directed against a common target gene. In further embodiments, the first and/or second series of targeting constructs are directed against two or more functionally related target genes or two or more target genes of a common host cell pathway.

In another embodiment, a kit for enhancing production of a biological product in a cell, comprising at least a first targeting construct, a portion of which is complementary to at least a first target gene of a latent or endogenous virus; a second targeting construct, a portion of which is complementary to at least a second target gene of the cellular immune response; and, optionally, a third targeting construct, a portion of which is complementary to at least a third target gene of a cellular process. The kit can further comprise at least additional targeting construct that targets a cellular process including, but not limited to, carbon metabolism and transport, apoptosis, RNAi uptake and/or efficiency, reactive oxygen species production, cell cycle control, protein folding, pyroglutamation protein modification, deamidase, glycosylation, disulfide bond formation, protein secretion, gene amplification, viral replication, viral infection, viral particle release, control of cellular pH, and protein production.

VII. Bioprocessing

In one embodiment of the invention are methods for producing a biological product or biomolecule in an avian host cell by contacting the cell with a targeting construct (e.g., iRNA agent) capable of modulating expression of an avian transcript, wherein the modulation enhances production of the biological product or biomolecule.

According to the present invention, bioprocessing methods may be improved by targeting (using one or more of the targeting constructs of the present invention) avian genes or transcripts expressed endogenously or expressed as a result of engineering the host cells to express said targets. They may also be improved by supplementing, replacing or adding one or more avian transcripts or transcript variants or avian products. Bioprocessing methods of using targeting constructs (e.g., siRNA, miRNA, dsRNA, saRNA, shRNA, piRNA, tkRNAi, eiRNA, pdRNA, a gapmer, an antagomir, or a ribozyme, etc.) are disclosed in co-owned applications 61/223,370, filed July 6, 2009, entitled COMPOSITIONS AND METHODS FOR ENHANCING PRODUCTION OF A BIOLOGICAL PRODUCT by Maraganore et al; U.S. Provisional Patent Application No. 61/244,868 filed September 22, 2009, entitled COMPOSITIONS AND METHODS FOR ENHANCING PRODUCTION OF A BIOLOGICAL PRODUCT, by Maraganore et al; U.S. Provisional Patent Application No. 61/293,980, filed January 11 , 2010, entitled COMPOSITIONS AND METHODS FOR ENHANCING PRODUCTION OF A BIOLOGICAL PRODUCT, by Rossomando et al; U.S. Provisional Patent Application No. 61/319,589, filed March 31 , 2010, entitled CELL-BASED BIOPROCESSING by Rossomando et al; each of which is incorporated fully herein by reference. These methods may be employed in the process of targeting avian transcripts to improve bioprocessing.

As such, methods of practicing bioprocessing including enhancing bioprocessing by controlling host cell immune responses, improving host cell viability, improving post- translational processing taught in the aforementioned commonly owned applications are also applicable to the avian systems described herein. Likewise, the methods for reducing bicontamination by endogenous, exogenous, latent and adventitious viruses are also applicable here.

The phrase "genome information" as used herein and throughout the claims and specification is meant to refer to sequence information from partial or entire genome of an organism, including protein coding and non-coding regions. These sequences are present every cell originating from the same organisms. As opposed to the transcriptome sequence information, genome information comprises not only coding regions, but also, for example, intronic sequences, promoter sequences, silencer sequences and enhancer sequences. Thus, the "genome information" can refer to, for example an avian genome, a human genome, a mouse genome, a rat genome. One can use complete genome information or partial genome information to add an additional dimension to the database sequences to increase the potential targets to modify with a targeting construct. The phrase "play a role" refers to any activity of a transcript or a protein in a molecular pathway known to a skilled artisan or identified elsewhere in this specification. Such pathways an cellular activities include, but are not limited to apoptosis, cell division, glycosylation, growth rate, a cellular productivity, a peak cell density, a sustained cell viability, a rate of ammonia production or consumption, or a rate of lactate production.

A "bioreactor", as used herein, refers generally to any reaction vessel suitable for growing and maintaining host cells such that the host cells produce a biological product, and for recovering such biological product. Bioreactors described herein include cell culture systems of varying sizes, such as small culture flasks, Nunc multilayer cell factories, small high yield bioreactors (e.g., MiniPerm, INTEGRA-CELLine), spinner flasks, hollow fiber- WAVE bags (Wave Biotech, Tagelswangen, Switzerland), and industrial scale bioreactors. In some embodiments, the biological product is produced in a "large scale culture" bioreactor having a 1 L capacity or more, suitable for

pharmaceutical or industrial scale production of biological products (e.g., a volume of at least 1 L, least 2 L, at least 5 L, at least 10 L, at least 25 L, at least 50 L, at least 100 L, or more, inclusive), often including means of monitoring pH, glucose, lactate, temperature, and/or other bioprocess parameters. In one embodiment, a large scale culture is at least 1 L in volume.

In one embodiment, a large scale culture is at least 2 L in volume. In one embodiment, a large scale culture is at least 5 L in volume. In one embodiment, a large scale culture is at least 25 L in volume. In one embodiment, a large scale culture is at least 40 L in volume. In one embodiment, a large scale culture is at least 50 L in volume. In one embodiment, a large scale culture is at least 100 L in volume.

A "host cell", as used herein, is any avian cell, cell culture, cellular biomass or avian tissue, capable of being grown and maintained in cell culture under conditions allowing for production and recovery of useful quantities of a biological product or biomolecule, as defined herein. A host cell can be a hybridoma derived from the combination of any avian cell with a yeast, insect, amphibian, fish, reptile, bird, mammal or human cell. Host cells can be unmodified cells or cell lines, or cell lines which have been genetically modified (e.g., to facilitate production of a biological product). In some embodiments, the host cell is a cell line that has been modified to allow for growth under desired conditions, such as in serum-free media, in cell suspension culture, or in adherent cell culture.

In some embodiments, the host cells are suitable for growth in suspension cultures.

In some embodiments, the host cell is an attachment dependent cell which is grown and maintained in adherent culture. Examples of avian host cells useful in methods provided herein include avian cells from the superorders including both the

Palaeognathae and Neognathae. Orders of birds within the Paleaegnathae include the Struthioniformes (ostriches, emus, kiwis) and Tinamiformes (tinamous) any of whose cells may encode avian transcripts as targets of the invention. The superorder

Neognathae includes the orders Anseriformes (waterfowl), Galliformes (fowl),

In one embodiment, the avian host cells are those of the Order Anseriformes (waterfoul) and in particular those of ducks. Cells of ducks which may be used in the present invention include those from Abacot Ranger (also known as Streicher), Allier Duck (Blanc d'Allier), Ancona duck, Aylesbury Duck, Bali Duck, Black East Indian, Blue Swedish duck, Buff Orpington Duck, Call Duck, Challans, Cayuga Duck, Chara Chemballi Duck, Crested Duck, Danish Duck, Duclair, Dutch Hookbill

(kromsnaveleend), East Indie Duck, Forest Duck (Eend van Vorst), Gimbsheimer, Golden Cascade, Gressingham Duck (Wild Mallard crossed with Pekin), Huttengem Duck, Indian Runner Duck, Magpie Duck, Majorcan Duck, Muscovy duck, Orpington duck, Pekin Duck (also known as Long Island duck), Rouen Duck, Saxony Duck, Semois, Silver Appleyard Duck, Silver Bantam Duck, Termonde Duck, Venetian Duck (Germanata Veneta), Welsh Harlequin Duck, Wood Duck and the Swedish Duck.

In one embodiment the cells are from the Pekin duck. Cells of the Pekin duck can be obtained from ATCC (Mannassas, VA; number CCL-141; Anas platyrhynchus domesticus). Other duck cells which may be used in the methods of the present invention include those disclosed in US Publications 20090081251, 20100062489 and

20100226912, each of which is incorporated herein in its entirety.

Host cells can be unmodified or genetically modified (e.g., a cell from a transgenic animal). For example, CEFs from transgenic chicken eggs can have one or more genes essential for the IFN pathway, e.g. interferon receptor, STATl, etc., has been disrupted, i.e., is a "knockout." See, e.g., Sang, 12 Trends Biotech. 415 (1994); Perry et al, 2 Transgenic Res. 125 (1993); Stern, 212 Curr Top Micro. Immunol. 195-206 (1996); Shuman, 47 Experientia 897 (1991). Also, the cell can be modified to allow for growth under desired conditions, e.g., incubation at 30°C.

In some embodiments, the host cell is an attachment dependent cell which is grown and maintained in adherent culture. In some embodiments, the host cell is contained in an egg, such as a fish, amphibian, or avian egg.

"Isolating biological product from the host cell" means at least one step in separating the biological product away from host cellular material, e.g., the host cell, host cell culture medium, host cellular biomass, or host tissue. Thus, isolating biological products that are secreted into, and ultimately harvested from, the host cell culture media are encompassed in the phrase "isolated from the host cell." A useful quantity includes an amount, including an aliquot or sample, used to screen for or monitor production, including monitoring modulation of target gene expression.

The present invention provides for the production of biological product or biomolecules such as a polypeptide, a metabolite, a nutraceutical, a chemical

intermediate, a biofuel, a food additive, an antibiotic (antiviral, antibacterial, antifungal, etc), or an immunogenic agent. More specifically, a "biological product" can include any substance capable of being produced by a host cell and recovered in useful quantities, including but not limited to, polypeptides (e.g., glycoproteins, antibodies, peptide -based growth factors), carbohydrates, lipids, fatty acids, metabolites (e.g., polyketides, macrolides), and chemical intermediates. This also includes the term "biologies", a preparation, such as a drug, a vaccine, or an antitoxin, that is synthesized from living organisms or their products, and used as a diagnostic, preventive, or therapeutic agent. Thus, biological products can be used for a wide range of applications, including as biotherapeutic agents, vaccines, research or diagnostic reagents, fermented foods, food additives, nutraceuticals, biofuels, industrial enzymes (e.g., glucoamylase, lipase), industrial chemicals (e.g., lactate, fumarate, glycerol, ethanol), in the fields of biodefense, biowarfare, environmental applications and the like.

In some embodiments, the biological product is a polypeptide. The polypeptide can be a recombinant polypeptide or a polypeptide endogenous to the host cell. In some embodiments, the polypeptide is a glycoprotein and the host cell is a mammalian cell. Non-limiting examples of polypeptides that can be produced according to methods provided herein include receptors, membrane proteins, cytokines, chemokines, hormones, enzymes, growth factors, growth factor receptors, antibodies, antibody derivatives and other immune effectors, interleukins, interferons, erythropoietin, integrins, soluble major histocompatibility complex antigens, binding proteins, transcription factors, translation factors, oncoproteins or proto-oncoproteins, muscle proteins, myeloproteins, neuroactive proteins, tumor growth suppressors, structural proteins, and blood proteins (e.g., thrombin, serum albumin, Factor VII, Factor VIII, Factor IX, Factor X, Protein C, von Willebrand factor, etc.). As used herein, a polypeptide encompasses glycoproteins or other polypeptides which has undergone post-translational modification, such as deamidation, glycation, and the like.In some embodiments, the biological product is an antibody (e.g., a monoclonal antibody). Monoclonal antibodies produced in mammalian host cells contain an N-linked glycosylation site on each heavy chain. The heavy chain glycans are typically complex structures with high levels of core fucosylation. The fucose residues attached via an al,6 linkage to the innermost N-acetylglucosamine (GlacNAc) residues of the Fc region N-linked oligosaccharides are the most important carbohydrate structures for antibody activity. For example, non-fucosylated antibodies are associated with dramatically increased antibody-dependent cellular cytotoxicity (ADCC) activity. ADCC activity can also be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al, 95 PNAS 652 56 (1998).

In other embodiments, the biological product or biomolecule is an immunogenic viral, bacterial, protozoan, or recombinant protein derived from an expression vector. An example approach for producing viral-based vaccines involves the use of attenuated live virus vaccines, which are capable of replication but are not pathogenic, and, therefore, provide lasting immunity and afford greater protection against disease. The conventional methods for producing attenuated viruses involve the chance isolation of host range mutants, many of which are temperature sensitive, e.g., the virus is passaged through unnatural hosts, and progeny viruses which are immunogenic, yet not pathogenic, are selected. Efficient vaccine production requires the growth of large quantities of virus produced in high yields from a host system. Different types of virus require different growth conditions in order to obtain acceptable yields. The host in which the virus is grown is therefore of great significance. As a function of the virus type, a virus can be grown in embryonated eggs, primary tissue culture cells, or in established cell lines.

Thus, in some embodiments of the present invention, the biological product or biomolecule is a viral product, for example, naturally occurring viral strains, variants or mutants; mutagenized viruses (e.g., generated by exposure to mutagens, repeated passages and/or passage in non-permissive hosts), reassortants (in the case of segmented viral genomes), and/or genetically engineered viruses (e.g., using the "reverse genetics" techniques) having the desired phenotype. The viruses of these embodiments can be attenuated; i.e., they are infectious and can replicate in vivo, but generate low titers resulting in subclinical levels of infection that are generally non pathogenic.

Additionally, the biological product or biomolecule of the present invention can be derived from an intracellular parasite for which a biological product can be enhanced using the compositions, cells, and/or methods of the present invention, e.g., using a targeting construct. For example, alternative embodiments of the present invention provide for production of a bacterial immunogen in a eukaryotic avian cell. These bacteria include Shigella flexneri, Listeria monocytogenes, Rickettsiae tsutsugamushi, Rickettsiae rickettsiae, Mycobacterium leprae, Mycobacterium tuberculosis, Legionella pneumophila, Chlamydia ssp. Additional embodiments of the present invention provide for production of a protozoan immunogen, in a eukaryotic cell. These protozoa include Plasmodium falciparum, Tripanosoma cruzi, and Leishmania donovani.

In some embodiments, the enhancement of production of a biological product is achieved by improving viability of the cells in culture. As used herein, the term

"improving cell viability" refers to an increase in cell density (e.g., as assessed by a Trypan Blue exclusion assay) or a decrease in apoptosis (e.g., as assessed using a

TUNEL assay) of at least 10% in the presence of a targeting construct(s) compared to the cell density or apoptosis levels in the absence of such a treatment. In some embodiments, the increase in cell density or decrease in apoptosis in response to treatment with a targeting construct(s) is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or even 100% compared to untreated cells. In some embodiments, the increase in cell density in response to treatment with a targeting construct(s) is at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or higher than the cell density in the absence of the targeting construct(s).

As used herein, "immunogenic agent" refers to an agent used to stimulate the immune system of a subject, so that one or more functions of the immune system are increased and directed towards the immunogenic agent. An antigen or immunogen is intended to mean a molecule containing one or more epitopes that can stimulate a host immune system to make a secretory, humoral and/or cellular immune response specific to that antigen. Immunogenic agents can be used in the production of antibodies, both isolated polyclonal antibodies and monoclonal antibodies, using techniques known in the art. Immunogenic agents include vaccines.

As used herein, "vaccine" refers to an agent used to stimulate the immune system of a subject so that protection is provided against an antigen not recognized as a self- antigen by the subject's immune system. Immunization refers to the process of inducing a high level of antibody and/or cellular immune response in a subject,that is directed against a pathogen or antigen to which the organism has been exposed. Vaccines and immunogenic agents as used herein, refer to a subject's immune system: the anatomical features and mechanisms by which a subject produces antibodies and/or cellular immune responses against an antigenic material that invades the subject's cells or extra-cellular fluids. In the case of antibody production, the antibody so produced can belong to any of the immunological classes, such as immunoglobulins, A, D, E, G, or M. Vaccines that stimulate production of immunoglobulin A (IgA) are of interest, because IgA is the principal immunoglobulin of the secretory system in warm-blooded animals. Vaccines are likely to produce a broad range of other immune responses in addition to IgA formation, for example cellular and humoral immunity. Immune responses to antigens are well-studied and reported widely. See, e.g., Elgert, IMMUNOL. (Wiley Liss, Inc., 1996); Stites et al, BASIC & CLIN. IMMUNOL., (7th Ed., Appleton & Lange, 1991). By contrast, the phrase "immune response of the host cell" refers to the responses of unicellular host organisms to the presence of foreign bodies.

"Bioprocessing" as used herein is an exemplary process for the industrial-scale production of a biological product or biomolecule (e.g., a heterologous polypeptide) in cell culture (e.g., in a host cell), that typically includes the following steps: (a) inoculating host cells containing a transgene encoding the heterologous protein into a seed culture vessel containing cell culture medium and propagating the cells to reach a minimum threshold cross-seeding density; (b) transferring the propagated seed culture cells, or a portion thereof, to a large-scale bioreactor; (c) propagating the large-scale culture under conditions allowing for rapid growth and cell division until the cells reach a

predetermined density; (d) maintaining the culture under conditions that disfavor continued cell growth and/or host cell division and facilitate expression of the heterologous protein.

Steps (a) to (c) of the above method generally comprise a "growth" phase, whereas step (d) generally comprises a "production" phase. In some embodiments, fed batch culture or continuous cell culture conditions are tailored to enhance growth and division of the host cells in the growth phase and to disfavor cell growth and/or division and facilitate expression of the heterologous protein during the production phase. For example, in some embodiments, a heterologous protein is expressed at levels of about 1 mg/L, or about 2.5 mg/L, or about 5 mg/L, or about 1 g/L, or about 5 g/L, or about 15 g/L, or higher. The rate of cell growth and/or division can be modulated by varying culture conditions, such as temperature, pH, dissolved oxygen (d02) and the like. For example, suitable conditions for the growth phase can include a pH of between about pH 6.5 and pH 7.5, a temperature between about 30°C to 38°C, and a C02 between about 5% to 90% saturation. In some embodiments, the expression of a heterologous protein can be enhanced in the production phase by inducing a temperature shift to a lower culture temperature (e.g., from about 37°C to about 30°C), increasing the concentration of solutes in the cell culture medium, or adding a toxin (e.g., sodium butyrate) to the cell culture medium. In some embodiments, the expression of a heterologous protein can be enhanced in the production phase by inducing a temperature shift to about 28°C, e.g., to increase protein expression in the absence of cell division. A variety of additional protocols and conditions for enhancing growth and/or protein expression during the production phase are known in the art.

The host cells can be cultured in a stirred tank bioreactor system in a fed batch culture process in which the host cells and culture medium are supplied to the bioreactor initially and additional culture nutrients are fed, continuously or in discrete increments, throughout the cell culture process. The fed batch culture process can be semi- continuous, wherein periodically whole culture (including cells and medium) is removed and replaced by fresh medium. Alternatively, a simple batch culture process can be used in which all components for cell culturing (including the cells and culture medium) are supplied to the culturing vessel at the start of the process. A continuous perfusion process can also be used, in which the cells are immobilized in the culture, e.g., by filtration, encapsulation, anchoring to microcarriers, or the like, and the supernatant is continuously removed from the culturing vessel and replaced with fresh medium during the process.

In one embodiment, after the production phase the biological product or biomolecule is recovered from the cell culture medium using various methods known in the art. For example, recovering a secreted heterologous protein typically involves removal of host cells and debris from the medium, for example, by centrifugation or filtration. In some cases, particularly if the biological product is a protein is not secreted, protein recovery can also be performed by lysing the cultured host cells, e.g., by mechanical shear, osmotic shock, or enzymatic treatment, to release the contents of the cells into the homogenate. The protein can then be separated from subcellular fragments, insoluble materials, and the like by differential centrifugation, filtration, affinity chromatography, hydrophobic interaction chromatography, ion-exchange

chromatography, size exclusion chromatography, electrophoretic procedures (e.g., preparative isoelectric focusing (IEF)), ammonium sulfate precipitation, and the like. Procedures for recovering and purifying particular types of proteins are known in the art.

In some embodiments, it is desirable to adapt cells to serum free media and adapt adherent cells to cell growth in suspension. In some embodiments, cells are adapted to grow in serum- free medium. In one aspect of the invention, adaptation of cells is facilitated by increasing cell plactisity by using a targeting construct that targets genes involved in control of plasticity.

Stem Cells

In one embodiment of the invention, avian directed agents (targeting constructs) find utility in the study, research, manipulation, production and application of stem cell- based technologies. According to the present invention, the differentiation status, survival, proliferation or regeneration of a stem cell or population of cells may be altered by the administration or introduction into a cell of a targeting construct. It is also contemplated that cellular populations may be affected or altered by the administration of a transcript which comprises an avian transcript or gene. It is also contemplated that cells may be contacted with a combination of one or more targeting constructs or avian transcripts to modify the differentiation status, survival, proliferation or regeneration of the cells.

Several protein coding genes have been identified in the art which affect the differentiation status, survival, proliferation or regeneration of stem cells or their precursors (See US Publication 20100166714 and 20060246446 which discloses genes affecting cardiac stem cells each of which is incorporated herein by reference in its entirety).

In one embodiment of the invention are provided compositions comprising targeting constructs that function to regulate or modulate the expression of avian transcripts which in turn alter or modulate the protein coding genes which have been associated with differentiation status, survival, proliferation or regeneration of stem cells. For example, targeting constructs of the present invention may be used to target transcription factors known to alter the status of stem cell lineages. These targeting constructs may target the transcripton factors at the DNA, R A or protein level.

Systems and methods for selecting targeting constructs

Based on the known Duck transcriptome, we have developed methods and systems for selecting targeting constructs to affect the cells through manipulating cellular processes, for example, to improve production of biomolecules in the cells.

Accordingly, the invention provides databases and system comprising and using the Duck transcriptome sequences and optionally also an organized compilation of the Duck transcriptome outlining at least one functional aspect of each of the transcript, such as the transcripts role in a particular cellular process or pathway, and the corresponding siRNAs to allow design and selection of targets and targeting constructs for optimization of biological processes, particularly in the Duck cells.

Functional aspects of transcripts relate to their role in, for example apoptosis, cell cycle, DNA amplification (DHFR), virus gene production, e.g., in the case of viral promoters that are used to drive biomolecule production in the cells, glycosylation, carbon metabolism, prooxidant enzymes, protein folding, methionine oxidation, protein pyroglutamation, disulfide bond formation, protein secretion, cell viability, specific productivity of cell, nutrient requirements, internal cell pH. Other cellular processes are known to a skilled artisan, and can be found, for example, at Gene Ontology database available through world wide web at geneontology.org.

Analogous systems useful for exploitation of the duck transcriptome are taught in for example U.S. Provisional Patent Application No. 61/354,932, filed June 15, 2010, entitled CHINESE HAMSTER OVARY (CHO) CELL TRANSCRIPTOME, CORRESPONDING SIRNAS AND USES THEREOF, by Rossomando et al; which is incorporated fully herein by reference. These methods may be employed in the process of targeting avian transcripts to improve bioprocessing.

The terms "system", "computing device", and "computer-based system" refer to the computer hardware, associated software, and data storage devices used to analyze the information of the present invention. In one embodiment, the computer-based systems of the present invention comprises one or more central processing units (e.g., CPU, PAL, PLA, PGA), input means (e.g., keyboard, cursor control device, touch screen), output means (e.g., computer display, printer) and data storage devices (e.g., RAM, ROM, volatile and non-volatile memory devices, hard disk drives, network attached storage, optical storage devices, magnetic storage devices, solid state storage devices). As such, any convenient computer-based system can be employed in the present invention.

Further, the computing device can included an embedded system based on a combination computing hardware and associated software or firmware.

A "processor" includes any hardware and/or software combination which can perform the functions under program control. For example, any processor herein can be a programmable digital microprocessor such as available in the form of an embedded system, a programmable controller, mainframe, server or personal computer (desktop or portable). Where the processor is selectively programmable, suitable programs, software or firmware can be communicated from a remote location to the processor, or previously saved in a computer program product (such as a portable or fixed computer readable storage medium, whether magnetic, optical or solid state device based). For example, a magnetic medium or optical disk can store the program or operating instructions and can be read and transferred to each processor at its corresponding station.

"Computer readable medium" as used herein refers to any storage or transmission medium that participates in providing instructions and/or data to a computer for execution and/or processing. Examples of storage media include floppy disks, magnetic media (tape, disk), UBS, optical media (CD-ROM, DVD, Blu-Ray), solid state media, a hard disk drive, a RAM, a ROM or integrated circuit, a magneto-optical disk, or a computer readable card such as a PCMCIA card and the like, whether or not such devices are internal or external to the computer. A file containing information can be "stored" on computer readable medium, where "storing" means recording information such that it is accessible and retrievable at a later date by a computer.

With respect to computer readable media, "permanent memory" or "non-volatile memory" refers to memory that is permanently stored on a data storage medium.

Permanent memory is not erased by termination of the electrical supply to a computer or processor. A computer hard-drive, ROM, CD-ROM, floppy disk and DVD are all examples of permanent memory. Random Access Memory (RAM) is an example of non- permanent or volatile memory.

To "record" or "store" data, programming or other information on a computer readable medium refers to a process for storing information, using any convenient method. Any convenient data storage structure can be chosen, based on the means used to access the stored information.

A "memory" or "memory unit" refers to any device which can store information for subsequent retrieval by a processor, and can include magnetic or optical devices (such as a hard disk, floppy disk, CD, or DVD), or solid state memory devices (such as volatile or non-volatile RAM). A memory or memory unit can have more than one physical memory device of the same or different types (for example, a memory can have multiple memory devices such as multiple hard drives or multiple solid state memory devices or some combination of hard drives and solid state memory devices)

Accordingly, the present invention provides a system for selecting a sequence of at least one targeting construct suitable for modulating protein expression in a cell, the system comprising: a computing device, having a processor and associated memory, and a database comprising at least one cell transcriptome information, the information comprising, a sequence for each transcript of the transcriptome, and optionally, a name of the transcript, and a pathway the transcript plays a role; and at least one targeting construct information, the information comprising at least the sequence of the targeting construct and optionally target specificity of the targeting construct, wherein each targeting construct is designed to match at least one or more sequences in the at least one cell transcriptome; a computer program, stored in memory, executed by the computing device and configured to receive from a user via a user input device, parameters comprising a cell type selection, a target organism selection, a cellular pathway selection, a cross-reactivity selection, a target gene name and/or sequence selection, and optionally a method of delivery selection comprising either in vivo or in vitro delivery options; and further optionally user address information; a first module configured to check the parameters against the sequences in the database for a matching combination of the parameters and transcriptome transcript sequences; and a second module to display a selected sequence of at least one targeting construct suitable for modulating protein expression in the cell.

The computing device and associated programs stored in memory can be adapted and configured to provide a user interface, such as a graphical user interface which allows the user to input search target parameters, for example, using one or more drop down menus or structured or free form text input, and selects the appropriate parameters for finding an appropriate target in the desired cell. For example, if a user wishes to find a target for modulating carbon metabolism in an Avian cell, the user identifies the target cell as "Avian" e.g, chicken, duck, etc, and pathway as "carbon metabolism", and the server performs a search through the database that would identify, e.g., transcripts for Gluts, PTEN and LDH genes and matches them with the appropriate siRNA molecules from the siRNA database part. This output information can be presented to the user on a computer display or other output device, such as a printer.

The system can be a stand alone system or an internet-based system, wherein the computations and selection of targeting constructs is performed in same or different locations. The transcriptome information can be stored in database and accessed by computing device. As used herein, the term database includes any organization of data regardless of whether it is structured or unstructured that allows retrieval of the information requested. The database can be a flat file or set of flat files stored in memory, one or more tables stored in memory, a set of discrete data elements stored in memory. The database can also include any well known database program that allows a user to directly or indirectly (through another program) access the data. Examples of these include Microsoft Access, Oracle database, and MySQL. In an alternative embodiment the system can be a network based system.

One can also provide a system by selling software to be run by a computer, wherein the databases and algorithms matching the parameters with sequence information and other information are provided to the user. The user may then either synthesize the targeting constructs or separately order them from a third party provider.

In some embodiments, the system further comprises a storage module for storing the at least one targeting construct in a container, wherein if there are two or more targeting constructs, each targeting construct is stored in a separate container, and a robotic handling module, which upon selection of the matching combination, selects a matching container, and optionally adds to the container additives based on a user selection for in vivo or in vitro delivery, and optionally further packages the container comprising the matching targeting construct to be sent to the user address.

The storage module can be a refrigerated module linked to the system

components. The system may also be linked to a nucleic acid or other biomolecule synthesizer.

The robotic handling module can be any system that can retrieve, and optionally mix components from the storage module, and or the biomolecule synthesizer, and optionally package the container(s). The robotic handling module can comprise one or more parts functioning based upon the commands from the system. The robotic handling module may be in the same or different location as where the computations are performed.

In some embodiments, the system further comprises genome information of the cell, wherein by a user selection, the targeting constructs can be matched to target genomic sequences, comprising promoters, enhancers, introns and exons present in the genome.

VIII. Business Methods

Given the broad application of improved bioprocessing methods in avian cells, the present invention may also be used in commerce in other fields. Provided herein are improved methods of protein production (including enzymes and antibodies) which can be exploited in the marketplace to alter current distribution, storage and chemical processing methods. These methods provide alternatives to current economic paradigms around manufacture, transport and distribution chains in the areas of not only biological processes (including food production) but those of crop science, electronics and textiles.

In one embodiment is a a method of doing a business for the production of a biological product, comprising providing a service for the rapid production of a biological product from an avian host cell using one or more iR A targeting constructs of the present invention and assessing the efficacy of the biological product. In this embodiment, the biological product may be useful in the treatment of one or more infections and assessment of efficacy may be against one or more infective agents suspected of causing the infection. To be clear, the infection being targeted may be that of a human subject or may be the infection of a separate cell culture or batch or lot of cells. The resultant effective biological product may then be provided to a consumer or customer where the end result may be the treatment of an animal subject or the treatment of a culture of cells.

Further, the biological products made by the processes of the present invention or via the utilization of the targeting constructs of the present invention may be useful in the manufacture of textiles, in the chemicals industry and/or in the manufacture, cleaning or production of electronic devices or components. To this end the biological products may be enzymes that find utility in the above-mentioned processes or structural proteins likewise useful.

EXAMPLES

Example 1. Identification of Avian Transcripts (ATs)

Duck transcriptome

The duck (Anas platyrhynchos) transcriptome was obtained from Ensembl (www.ensembl.org) and was supplemented with novel transcripts and miRNAs from internal sequencing of the CCL-141 cell line. The Avian Transcripts (SEQ ID NOs 1- 18039) are listed in Lengthy Table 1 which is incorporated herein.

Certain avian genes of the present invention were identified from the Ensembl database (www.ensembl.org) and their respective RNA transcripts were extracted (SEQ ID 1-17169, having transcript names ENSAPLT00000000001 through

ENSAPLT00000017169). Other microRNA avian transcripts were idenfied via internal discovery efforts and these are also listed in Table 1 (SEQ ID NOs: 17170 through 18039). To be clear, Table 1 lists the Ensembl gene identifier of each avian transcript with the prefix, ENSAPLT while internal identifiers have been given to transcripts identified internally.

Example 2. Function of Avian Transcripts

Evaluation of the avian transcripts to determine function of each was conducted by comparative analysis to publicly available databases using the transcript sequence information. Table 5 lists the functional annotation for a subset of the transcripts listed in Table 1. The avian transcripts are identified here by SEQ ID number.

Table 5. Molecular Function

cyclic-AMP phosphodiesterase activity

7672 3',5'-cyclic-nucleotide phosphodiesterase activity;catalytic

activity ;protein binding;3',5'-cyclic-GMP phosphodiesterase activity

2982 3'-5'-exoribonuclease activity;RNA binding;polyribonucleotide

nucleotidyltransferase activity

14147 4-alpha-glucanotransferase activity ;amylo-alpha-l ,6-glucosidase

activity;catalytic activity ;protein binding;polyubiquitin

binding;polysaccharide binding;glycogen debranching enzyme activity

12859 4-aminobutyrate transaminase activity;transaminase activity;pyridoxal phosphate binding ;protein homodimerization activity ;protein binding

7182 6-pyruvoyltetrahydropterin synthase activity;protein binding;identical protein binding;protein homodimerization activity;metal ion binding

2649 7S RNA binding

14151 acetylglucosaminyltransferase activity

11300 acetylglucosaminyltransferase activity ;transferase activity;transferase activity, transferring glycosyl groups ;protein xylosyltransferase activity

3388, 4395, 10637, 10640 acid phosphatase activity

11289 acid phosphatase activity;inositol hexakisphosphate 5-kinase

activity;inositol 1, 3,4,5, 6-pentakisphosphate kinase

activity;diphosphoinositol-pentakisphosphate kinase activity

8615, 13494 acid phosphatase activity ;protein binding

2046, 2904, 11065 acid-amino acid ligase activity

16725 acid-amino acid ligase activity ;binding

8429 acid-amino acid ligase activity ;binding;protein binding;zinc ion

binding;thyroid hormone receptor binding

2992 acid-amino acid ligase activity;phosphopantetheine binding

4165, 16094 acid-amino acid ligase activity ;protein binding

13953 acid-amino acid ligase activity ;protein binding;ligase activity

4472 acid-amino acid ligase activity ;protein binding ;ubi qui tin-protein ligase activity;proline-rich region binding;protein domain specific binding;beta- 2 adrenergic receptor binding; sodium channel inhibitor activity;RNA polymerase binding;ubiquitin binding;phosphoserine

binding;phosphothreonine binding

10945 acid-amino acid ligase activity ;protein binding ;ubiquitin-protein ligase activity;R-SMAD binding;I-SMAD binding;activin binding

10322 acid-amino acid ligase activity ;protein binding ;ubiquitin-protein ligase activity ;S MAD binding;identical protein binding

11093 acid-amino acid ligase activity ;protein binding ;ubi qui tin-protein ligase activity;syntaxin binding

2489 acid-amino acid ligase activity ;ubi qui tin-protein ligase activity;metal ion binding;binding

15267 acid-amino acid ligase activity ;ubiquitin-protein ligase activity ;protein binding;transcription coactivator activity

11268, 16025 actin binding

6656 actin binding;binding 7700, 10122 actin binding;calcium ion binding;hydrolase activity, hydrolyzing O- glycosyl compounds;protein binding

1919 actin binding;oxidoreductase activity;transition metal ion binding

8712 actin binding;phospholipid binding;phosphatidylinositol binding;protein binding;clathrin binding

3709, 9624 actin binding;profilin binding

12597, 14230 actin binding;Rho GTPase binding;binding

4212 actin binding;sequence-specific DNA binding transcription factor activity;sequence-specific DNA binding;protein binding

13434 actin filament binding

5772 acyl-CoA binding

12754 acyl-CoA dehydrogenase activity;oxidoreductase activity, acting on the

CH-CH group of donors

11099 acyl-CoA oxidase activity;oxidoreductase activity, acting on the CH-CH group of donors;acyl-CoA dehydrogenase activity

13545 acyl-CoA thioesterase activity

13401 acyl-CoA thioesterase activity;protein binding

5734 acylglycerol lipase activity ;protein homodimerization activity

4643, 7450 acyltransferase activity

5746 acyltransferase activity; 1 -acylglycerol-3-phosphate O-acyltransferase activity

10248 acyltransferase activity;ATP binding;ATPase activity, coupled to

transmembrane movement of substances

14940 acyltransferase activity;carnitine O-octanoyltransferase activity

6399 acyltransferase activity;DNA binding;catalytic activity;ATP

binding;glycerol-3-phosphate O-acyltransferase activity

2280 acyltransferase activity ;glycerol-3 -phosphate O-acyltransferase activity

5656 acyltransferase activity;nucleotide binding;aminoacyl-tRNA ligase activity;ATP binding;protein binding;dihydrolipoyllysine-residue acetyltransferase activity

12923 acyltransferase activity ;protein binding

9535 acyltransferase activity;transferase activity

16614 adenosylhomocysteinase activity

8290 adenosylhomocysteinase activity ;protein binding

12422 adenyl-nucleotide exchange factor activity;protein homodimerization activity;chaperone binding;protein binding;unfolded protein binding

8271 alpha- 1 ,3-mannosylglycoprotein 2-beta-N-acetylglucosaminylrransferase activity;beta-l,3-galactosyl-0-glycosyl-glycoprotein beta-l ,3-N- acetylglucosaminyltransferase activity

598 alpha2-adrenergic receptor activity;protein homodimerization

activity;protein heterodimerization activity ;protein binding; alpha-2 A adrenergic receptor binding;epinephrine binding;G-protein coupled receptor activity

8718 alpha-L-fucosidase activity

5646 alpha-mannosidase activity;catalytic activity;carbohydrate binding;mannosidase activity;heme binding

13425 alpha-tubulin binding;gamma-tubulin binding;protein binding

9514 amidophosphoribosyltransferase activity;transferase activity;transferase activity, transferring glycosyl groups;metal ion binding;iron-sulfur cluster binding;4 iron, 4 sulfur cluster binding

10911 aminoacylase activity;catalase activity;oxidoreductase activity, acting on peroxide as acceptor;heme binding;protein homodimerization activity;NADP+ or NADPH binding;peroxidase activity;iron ion binding;oxidoreductase activity ;metal ion binding;protein binding

14316 aminoacyl-tRNA ligase activity;ATP binding

2999 aminoacyl-tRNA ligase activity;ATP binding;nucleotide

binding;tryptophan-tRNA ligase activity ;protein binding

1793 aminopeptidase activity

10689 aminopeptidase activity;manganese ion binding

14008 aminopeptidase activity;manganese ion binding ;protein

homodimerization activity;metalloaminopeptidase activity

2575 aminopeptidase activity;metalloexopeptidase activity

13584 antioxidant activity;oxidoreductase activity

8103 antioxidant activity;oxidoreductase activity;identical protein

binding;protein kinase binding;caspase inhibitor activity;protein C- terminus binding;protein binding;kinase binding

8174 ARF GTPase activator activity;zinc ion binding

6100, 15849 ARF GTPase activator activity;zinc ion binding;GTP binding

7121 ARF GTPase activator activity;zinc ion binding;phosphatidylinositol- 3,4,5-trisphosphate binding ;protein binding;inositol 1,3,4,5 tetrakisphosphate binding

4105, 5937, 6919, 10164 ARF GTPase activator activity;zinc ion binding;protein binding

2885, 13887, 14713, 15655 ARF guanyl-nucleotide exchange factor activity

1894, 2771 ARF guanyl-nucleotide exchange factor activity;binding

10669 ARF guanyl-nucleotide exchange factor activity;binding;guanyl- nucleotide exchange factor activity

12766 ARF guanyl-nucleotide exchange factor activity;protein binding

3715 ARF guanyl-nucleotide exchange factor activity;protein

binding;phosphatidylinositol-3 ,4, 5 -trisphosphate binding

7192 arginine-tRNA ligase activity;ATP binding;nucleotide

binding;aminoacyl-tRNA ligase activity

1875, 6120, 14210 aspartic-type endopeptidase activity

14965 aspartic-type endopeptidase activity;endopeptidase activity ;protein binding

12348 aspartic-type endopeptidase activity;protein binding;ATP

binding;chromatin binding

4865 aspartic-type endopeptidase activity;zinc ion binding

8700, 11725, 15101 ATP binding

11162 ATP binding;5-formyltetrahydrofolate cyclo-ligase activity

12818 ATP binding;ATPase activity 4608, 13992 ATP binding;ATPase activity, coupled to transmembrane movement of ions, phosphorylative mechanism;catalytic activity;ATPase activity;hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances

6379 ATP binding;ATPase activity, coupled to transmembrane movement of ions, phosphorylative mechanism;catalytic activity;calcium-transporting ATPase activity;calcium ion transmembrane transporter

activity;hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances;calmodulin binding;calcium ion binding;PDZ domain binding;protein C-terminus binding;protein binding;calcium-dependent ATPase activity

4952, 11254 ATP binding;ATPase activity, coupled to transmembrane movement of ions, phosphorylative mechanism;catalytic activity;calcium-transporting ATPase activity ;hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances;signal transducer

activity;manganese-transporting ATPase activity;manganese ion binding;calcium ion binding

6909, 10171, 11797, 13669 ATP binding;ATPase activity, coupled to transmembrane movement of ions, phosphorylative mechanism;catalytic activity;magnesium ion binding;phospholipid-translocating ATPase activity

8812 ATP binding;ATPase activity, coupled to transmembrane movement of ions, phosphorylative mechanism;catalytic activity;magnesium ion binding;phospholipid-translocating ATPase activity;hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances

2472 ATP binding;ATPase activity, coupled to transmembrane movement of ions, phosphorylative mechanism;catalytic activity;magnesium ion binding;phospholipid-translocating ATPase activity ;phospholipid transporter activity

7640 ATP binding;ATPase activity, coupled to transmembrane movement of ions, phosphorylative mechanism;catalytic activity;metal ion binding;copper ion binding;copper-exporting ATPase activity;metal ion transmembrane transporter activity;hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances

5291, 9238, 15455 ATP binding;ATPase activity, coupled to transmembrane movement of ions, phosphorylative mechanism;magnesium ion binding;phospholipid- translocating ATPase activity

9982 ATP binding;ATPase activity, coupled to transmembrane movement of ions, phosphorylative mechanism;magnesium ion binding;phospholipid- translocating ATPase activity;hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances

13459 ATP binding;ATPase activity, coupled to transmembrane movement of ions, phosphorylative mechanism;queuine tRNA-ribosyltransferase activity;catalytic activity;calcium-transporting ATPase activity;calcium ion transmembrane transporter activity;hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances

8784, 11941 ATP binding;ATPase activity, coupled to transmembrane movement of substances

4044, 9186 ATP binding;ATPase activity, coupled to transmembrane movement of substances;ATPase activity;nucleotide binding;nucleoside-triphosphatase activity 15752 ATP binding;ATPase activity, coupled to transmembrane movement of substances;ATPase activity;nucleotide binding;nucleoside-triphosphatase activity;oxidoreductase activity

3667 ATP binding;ATPase activity, coupled to transmembrane movement of substances;ATPase activity;nucleotide binding;nucleoside-triphosphatase activity;phosphopantetheine binding

3541 ATP binding;ATPase activity, coupled to transmembrane movement of substances;ATPase activity;nucleotide binding;nucleoside-tripliospliatase activity;protein homodimerization activity

2409 ATP binding;ATPase activity, coupled to transmembrane movement of substances;ATPase activity;nucleotide binding;nucleoside-tripliospliatase activity;protein homodimerization activity;peptide-transporting ATPase activity;substrate-specific transmembrane transporter activity

14397 ATP binding;ATPase activity, coupled to transmembrane movement of substances;ATPase activity;nucleotide binding;nucleoside-tripliospliatase activity ;transporter activity;protein kinase activity

11575 ATP binding;ATPase activity, coupled to transmembrane movement of substances;efflux transmembrane transporter activity;heme-transporting ATPase activity ;heme binding

3006, 6428, 7316, 7435 ATP binding;ATPase activity nucleotide binding;nucleoside- triphosphatase activity

9239 ATP binding;ATPase activity;nucleotide binding;nucleoside- triphosphatase activity;phospholipid transporter activity ;protein homodimerization activity;protein heterodimerization

activity;glycoprotein transporter activity;sterol-transporting ATPase activity;toxin transporter activity;cholesterol transporter activity;ADP binding

14716 ATP binding;ATPase activity;nucleotide binding;nucleoside- triphosphatase activity ;protein homodimerization activity ;protein heterodimerization activity

4888 ATP binding;inositol pentakisphosphate 2 -kinase activity

16040 ATP binding;kinase activity

4244 ATP binding;ligase activity, forming aminoacyl-tRNA and related

compounds ;nucleotide binding;protein binding;aminoacyl-tRNA editing activity

16711 ATP binding;mismatched DNA binding

11928 ATP binding;nucleic acid binding;zinc ion binding

7041, 7315, 15147 ATP binding;nucleobase, nucleoside, nucleotide kinase activity; adenylate kinase activity

8474 ATP binding;nucleobase, nucleoside, nucleotide kinase activity;cAMP- dependent protein kinase regulator activity;nucleotide

binding;nucleoside-triphosphatase activity

9309 ATP binding;nucleotide binding;nucleoside-triphosphatase activity

16082 ATP binding;nucleotide binding;nucleoside-triphosphatase activity;4 iron, 4 sulfur cluster binding

7424 ATP binding;nucleotide binding;nucleoside-triphosphatase

activity;binding;ATPase activity

3483 ATP binding;nucleotide binding;nucleoside-triphosphatase

activity;binding;protein binding;syntaxin binding; ATPase activity, coupled 15946 ATP binding;phosphotransferase activity, alcohol group as

acceptor;nucleotide binding;nucleoside-triphosphatase activity;kinase activity ;protein binding

13917 ATP binding;receptor activity;transcription cofactor activity;transcription factor binding;transcription activator activity;transcription coactivator activity ;protein binding;RNA polymerase II transcription mediator activity;ligand-dependent nuclear receptor binding;estrogen receptor binding;thyroid hormone receptor binding;peroxisome proliferator activated receptor binding;retinoic acid receptor binding; vitamin D receptor binding;DNA binding;nuclear hormone receptor

binding;chromatin binding

13841 ATP binding;small protein activating enzyme activity;catalytic activity

5519, 10032 ATP binding;small protein activating enzyme activity;catalytic

activity;ISG15 activating enzyme activity

9508 ATP binding;small protein activating enzyme activity ;transporter

activity ;lipid binding;catalytic activity ;protein binding;FAT10 activating enzyme activity

15271 ATP binding;zinc ion binding

15739 ATPase activator activity;protein binding;chaperone binding

6638, 7279 ATPase activity

15968 ATPase activity, coupled to transmembrane movement of

substances;proton-transporting ATPase activity, rotational mechanism;protein binding

13387 ATPase binding

11190 ATP-dependent peptidase activity;nucleotide binding;nucleoside- triphosphatase activity;ATP binding;serine-type endopeptidase activity;ATPase activity;single-stranded RNA binding;sequence-specific DNA binding;protein binding;mitochondrial heavy strand promoter anti- sense binding;mitochondrial light strand promoter anti-sense binding;DNA polymerase binding;G-quadruplex DNA binding;ADP binding;single-stranded DNA binding

4017 ATP-dependent peptidase activity ;protein binding

5595 beta-amyloid binding;protein binding;ATP binding

10961 beta-catenin binding;DNA binding;transcription factor

binding;transcription coactivator activity;histone acetyltransferase activity ;protein binding;acetyltransferase activity;acyltransferase activity

14897 beta-catenin binding;identical protein binding;protein binding

7602 beta-catenin binding;protein binding;armadillo repeat domain binding

9368 beta-tubulin binding;protein binding;single-stranded DNA binding

11267 beta-tubulin binding;Ran GTPase binding;Ran guanyl-nucleotide

exchange factor activity

1033, 2724, 2884, 3338, 4061 , binding

4890, 5763, 6284, 6719, 6769,

6785, 7419, 7653, 8085, 8199,

8332, 8453, 8869, 8892, 8966,

10000, 10143, 10603, 11082,

11122, 11124, 1 1234, 11792,

12039, 12931, 12978, 13282,

13978, 14398, 14817, 15469,

4648 catalytic activity;6-phosphofructo-2-kinase activity;ATP

binding;identical protein binding;protein binding

3357, 16650 catalytic activity;acetate-CoA ligase activity; AMP binding;protein binding

9292 catalytic activity;acyl carrier activity

10647 catalytic activity;ATP binding;protein binding

6403 catalytic activity;axon guidance receptor activity

14362 catalytic activity;biotin-[acetyl-CoA-carboxylase] ligase activity;biotin- [propionyl-CoA-carboxylase (ATP-hydrolyzing)] ligase activity;biotin binding;enzyme binding;biotin-protein ligase activity

3340 catalytic activity;branched-chain-amino-acid transaminase activity

13641, 13957 catalytic activity;coenzyme binding;3-beta-hydroxy-delta5-steroid dehydrogenase activity;dTDP-4-dehydrorhamnose reductase activity

14346 catalytic activity;coenzyme binding;3-beta-hydroxy-delta5-steroid dehydrogenase activity;dTDP-4-dehydrorhamnose reductase activity ;protein binding

13195 catalytic activity;coenzyme binding;3-beta-hydroxy-delta5-steroid dehydrogenase activity;dTDP-4-dehydrorhamnose reductase activity ;protein binding ;methionine adenosyltransferase regulator activity

13900 catalytic activity;coenzyme binding;3-beta-hydroxy-delta5-steroid dehydrogenase activity;UDP-glucose 4-epimerase activity;dTDP-4- dehydrorhamnose reductase activity ;protein homodimerization activity

8584 catalytic activity;coenzyme binding ;binding

14446 catalytic activity;coenzyme binding;GDP-mannose 4,6-dehydratase activity;oxidoreductase activity

16759 catalytic activity;CTP synthase activity;protein binding

3992 catalytic activity;ferric-chelate reductase activity

10738 catalytic activity;flavin adenine dinucleotide binding;oxidoreductase activity;ATP binding;alkylglycerone-phosphate synthase activity

14846 catalytic activity;glutathione transferase activity;protein

homodimerization activity;protein binding

8749 catalytic activity;glycerophosphodiester phosphodiesterase

activity;carbohydrate binding ;phosphoric diester hydrolase activity

5137 catalytic activity ;hydrolase activity

12942 catalytic activity;long-chain fatty acid-CoA ligase activity

13353 catalytic activity ;metal ion binding;4 iron, 4 sulfur cluster binding;iron- sulfur cluster binding

1974, 2236 catalytic activity;metalloendopeptidase activity;ubiquinol-cytochrome-c reductase activity;zinc ion binding;metal ion binding;protein binding

6581 catalytic activity ;molybdenum ion binding;pyridoxal phosphate binding

3693 catalytic activity;phosphatidylinositol-5-phosphate

binding;phosphatidylinositol-3-phosphate binding;phosphatidylinositol- 4-phosphate binding

12242 catalytic activity;phosphoglycolate phosphatase activity; acireductone synthase activity ;metal ion binding 6583 catalytic activity;phosphoprotein phosphatase activity ;protein tyrosine phosphatase activity;protein binding;hydrolase activity

8599, 10318, 12700, 14551 catalytic activity ;protein binding

13646 catalytic activity ;protein binding;glycerone-phosphate O-acyltransferase activity;transferase activity;acyltransferase activity

8530, 9095 catalytic activity ;protein binding;lipid binding;oxidoreductase

activity;transferase activity;transferase activity, transferring acyl groups other than amino-acyl groups;sterol binding;propanoyl-CoA C- acyltransferase activity

3125 catalytic activity ;protein binding;long-chain fatty acid-CoA ligase activity

8987 catalytic activity ;protein binding;very long-chain fatty acid-CoA ligase activity;long-chain fatty acid-CoA ligase activity

8361 catalytic activity ;protein kinase activity;ATP binding;3-oxoacyl-[acyl- carrier-protein] synthase activity

1226 catalytic activity ;protein serine/threonine phosphatase activity

714 catalytic activity;pyridoxal phosphate binding

14150 catalytic activity ;sphingosine-l -phosphate phosphatase activity

7775 catalytic activity;thiosulfate sulfurtransferase activity ;protein

binding;nucleotidyltransferase activity ;URM1 activating enzyme activity

2549, 13243 catalytic activity;transcription factor binding

12187 catalytic activity;transcription factor binding;oxidoreductase

activity ;protein binding

15815 catalytic activity;transketolase activity ;protein binding

7876 catalytic activity ;transporter activity

12683 catalytic activity;UDP -glucose 6-dehydrogenase

activity;binding;oxidoreductase activity;oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor;NAD+ or NADH binding

10388 catalytic activity;zinc ion binding;protein binding

3027, 4577, 5414, 7961, 12562 cation transmembrane transporter activity

9618 cation transmembrane transporter activity;cytochrome-c oxidase

activity;copper ion binding;heme binding;protein binding

8340 cation transmembrane transporter activity;nucleotide binding

4336 cation hloride symporter activity ;transporter activity

271 1 ceramidase activity

2562 chloride channel activity

5171 chloride channel activity ;protein binding

10207 cholesterol binding

10672 chondroitin-glucuronate 5-epimerase activity

6147 chromatin binding;catalytic activity

9701 chromatin binding;catalytic activity ;protein binding

9440 chromatin binding;DNA binding ;methylated histone residue binding 15718 chromatin binding;histone demethylase activity (H3-K36 specific)

8112 chromatin binding;methylated histone residue binding;ubiquitin-protein ligase activity;protein binding;single-stranded RNA binding

7833 chromatin binding ;protein tyrosine kinase activity;protein

binding;histone kinase activity;histone binding;histone acetyl-lysine binding;zinc ion binding;nucleotide binding;vitamin D receptor activator activity ;protein complex scaffold;vitamin D receptor binding

4882 chromatin binding;sequence-specific DNA binding transcription factor activity;calcium ion binding;protein binding;chromatin DNA binding;sequence-specific DNA binding;receptor activity

15855 cobalamin binding

4251 copper ion binding

11542 copper ion binding;copper chaperone activity;enzyme activator activity

13915 copper ion binding;electron carrier activity;low-density lipoprotein binding

4862 copper ion binding;electron carrier activity ;protein binding;enzyme binding

5585 copper ion binding;oxidoreductase activity

6534 copper ion transmembrane transporter activity

16478 cyanate hydratase activity;guanyl-nucleotide exchange factor

activity;GTP binding;GTPase binding

13317 cyanate hydratase activity;guanyl-nucleotide exchange factor

activity;GTP binding;GTPase binding;binding

13733 cyclin-dependent protein kinase regulator activity

3582 cyclin-dependent protein kinase regulator activity;protein binding;zinc ion binding

10386 cyclin-dependent protein kinase regulator activity;zinc ion

binding;ubiquitin-protein ligase activity

11211, 13932, 14532 cysteine-type endopeptidase activity

8782, 8807 cysteine-type endopeptidase activity;calcium ion binding ;protein

binding;low-density lipoprotein receptor activity

3405, 3626, 3628, 3632, 9554, cysteine-type endopeptidase activity;calcium-dependent cysteine-type 10805 endopeptidase activity

9776, 15370 cysteine-type endopeptidase activity;cysteine-type peptidase activity

13972 cysteine-type endopeptidase activity;cysteine-type peptidase

activity;peptidase activity ;protein binding

6982 cysteine-type endopeptidase activity;cysteine-type peptidase

activity ;protein binding

905, 1146, 1151 cysteine-type endopeptidase activity;nucleotide binding;nucleoside- triphosphatase activity;phosphopantetheine binding;ATP

binding;ATPase activity

8300, 15973 cysteine-type endopeptidase activity;peptidase activity

1020 cysteine-type endopeptidase activity;protein binding

9216 cysteine-type endopeptidase activity;protein binding;peptidase

activity;cysteine-type peptidase activity ;hydrolase activity

11229 cysteine-type endopeptidase activity;protein binding;zinc ion binding

3608 cysteine-type endopeptidase activity;receptor activity;binding

592, 2906 DNA binding;ATP binding;chromatin binding;nucleic acid

binding;helicase activity;protein binding

8533 DNA binding;ATP binding;hydrolase activity

4820 DNA binding;ATP binding;nucleic acid binding;helicase activity

9672 DNA binding;ATP binding;nucleic acid binding;helicase

activity;ATPase activity ;protein binding;histone binding;transcription activator activity

10457 DNA binding;ATP binding ;nucleic acid binding;helicase activity;ATP- dependent helicase activity;hydrolase activity ;nucleotide binding;ATPase activity ;protein binding

6373 DNA binding;ATP binding;nucleic acid binding;helicase activity;binding

2725 DNA binding;ATP binding;nucleic acid binding;helicase

activity;chromatin binding

13756 DNA binding;ATP binding;nucleic acid binding;helicase activity;protein binding;DNA-dependent ATPase activity;transcription regulator activity

6696 DNA binding;ATP binding;nucleic acid binding;helicase activity;protein binding;zinc ion binding;catalytic activity;chromo shadow domain binding;chromatin binding

2302 DNA binding;ATP binding;nucleic acid binding;helicase activity;protein kinase binding;transcription cofactor activity

17049 DNA binding;ATP binding;nucleic acid binding;helicase

activity;transcription elongation regulator activity;chromatin binding;protein complex binding;protein C-terminus binding ;protein N- terminus binding;protein binding;DNA-dependent ATPase activity

10782 DNA binding;ATP binding;nucleic acid binding;helicase activity;zinc ion binding;protein binding

3586 DNA binding;ATP binding;nucleotide binding;nucleoside-triphosphatase activity;ATP-dependent DNA helicase activity;DNA helicase activity ;protein binding

14869 DNA binding;ATP binding ;protein binding;nucleic acid binding;helicase activity;zinc ion binding

13354 DNA binding;ATP-dependent DNA helicase activity;double-stranded telomeric DNA binding;telomeric DNA binding;protein C-terminus binding;promoter binding;protein binding;5'-deoxyribose-5-phosphate lyase activity

14867 DNA binding;beta-catenin binding;protein kinase binding;RNA

polymerase II transcription factor activity;gamma-catenin

binding;sequence-specific DNA binding transcription factor activity;transcription factor binding;sequence-specific DNA binding;protein binding;specific transcriptional repressor activity;nuclear hormone receptor binding

3001, 11266 DNA binding;binding

2773, 5013 DNA binding;catalytic activity ;protein binding

16529 DNA binding;catalytic activity;protein binding;protein phosphatase 2A binding

16305 DNA binding;catalytic activity;zinc ion binding;hydrolase activity;DNA

5 '-adenosine monophosphate hydrolase activity;metal ion binding

11299 DNA binding;protein binding;zinc ion binding 3882 DNA binding;protein binding;zinc ion binding;calcium ion

binding;histone-lysine N-methyltransferase activity;chromatin binding

3825 DNA binding;protein binding;zinc ion binding;histone methyltransferase activity (H3-K4 specific)

13648 DNA binding;protein binding;zinc ion binding;histone-lysine N- methyltransferase activity

1099 DNA binding;protein binding;zinc ion binding;transcription activator activity;chromatin binding

17158 DNA binding;sequence-specific DNA binding transcription factor activity; ATP binding

7822 DNA binding;sequence-specific DNA binding transcription factor activity ;protein binding

3287 DNA binding;sequence-specific DNA binding transcription factor activity;protein binding ;protein C-terminus binding;transcription regulator activity

11755 DNA binding;sequence-specific DNA binding transcription factor activity ;protein binding;transcription activator activity;transcription regulator activity;sequence-specific DNA binding

9458 DNA binding;sequence-specific DNA binding transcription factor activity ;protein binding;transcription activator activity;transcription repressor activity;sequence-specific DNA binding

8692 DNA binding;sequence-specific DNA binding transcription factor activity ;protein binding ;ubi qui tin protein ligase binding

7845 DNA binding;sequence-specific DNA binding transcription factor activity ;receptor activity;protein binding;zinc ion binding;sequence- specific DNA binding;metal ion binding; steroid hormone receptor activity

5314 DNA binding;sequence-specific DNA binding transcription factor activity;RNA polymerase II transcription factor activity;sequence- specific DNA binding

2787 DNA binding;sequence-specific DNA binding transcription factor activity;sequence-specific DNA binding

7584 DNA binding;sequence-specific DNA binding transcription factor activity;sequence-specific DNA binding;transcription activator activity ;protein binding

8546 DNA binding;sequence-specific DNA binding transcription factor activity;transcription regulator activity;sequence-specific DNA binding

11107 DNA binding;sequence-specific DNA binding transcription factor activity;transcription repressor activity;general transcriptional repressor activity;sequence-specific DNA binding

2384 DNA binding;sequence-specific DNA binding transcription factor activity;zinc ion binding;transcription repressor activity;enzyme binding;sequence-specific DNA binding;nucleic acid

binding;transcription regulator activity;promoter binding

14466 DNA binding;signal transducer activity;endonuclease activity;zinc ion binding;protein binding;histone methyltransferase activity (H3- 27 specific)

11041 DNA binding;signal transducer activity ;protein binding;histone

methyltransferase activity;chromatin binding

11182 DNA binding;signal transducer activity ;protein binding;transcription corepressor activity 6086 DNA binding;site-speciflc DNA-methyltransferase (cytosine-N4- speciflc) activity

7855 DNA binding;transcription corepressor activity;bistone deacetylase binding;protein binding;sequence-specific DNA binding;sequence- specific DNA binding transcription factor activity ;protein domain specific binding;transcription repressor activity;promoter binding;retinoic acid receptor binding;retinoid X receptor binding;thyroid hormone receptor binding;histone deacetylase regulator activity

6533 DNA binding;transcription factor binding;transcription activator

activity;zinc ion binding;histone acetyltransferase activity ;protein binding;acetyltransferase activity

4524 DNA binding;transcription regulator activity

2292 DNA binding;transcription regulator activity;nucleic acid binding

5714 DNA binding;transporter activity

15577 DNA binding;transporter activity;zinc ion binding

2442, 5214, 7382, 15420, 15700 DNA binding;zinc ion binding

11043 DNA binding;zinc ion binding ;protein binding

12968 DNA binding;zinc ion binding ;protein dimerization activity

6483, 6510 DNA binding;zinc ion binding;sequence-specific DNA binding

transcription factor activity

10980 DNA binding;zinc ion binding;sequence-specific DNA binding

transcription factor activity;sequence-specific DNA binding;protein binding;transcription activator activity

15521 DNA binding;zinc ion binding;sequence-specific DNA binding

transcription factor activity;transcription factor binding

15369 DNA binding;zinc ion binding;signal transducer activity

4576 DNA-directed DNA polymerase activity;protease binding ;protein

binding;DNA binding;transferase activity;nucleotidyltransferase activity

5219 DNA-directed RNA polymerase activity

10220 DNA-directed RNA polymerase activity;nucleotide binding;catalytic activity ;protein kinase activity

4542 dolichyl-diphosphooligosaccharide-protein glycotransferase

activity ;protein binding

10324 dopamine beta-monooxygenase activity

14722 dopamine receptor binding

4423 double-stranded RNA binding;protein binding

3500 double-stranded RNA binding ;protein kinase inhibitor activity ;protein binding;protein kinase binding

10096 double-stranded RNA binding;RNA binding;adenosine deaminase activity;double-stranded RNA adenosine deaminase activity

6027 electron carrier activity

12599 electron carrier activity;iron-sulfur cluster binding;dihydroorotate

oxidase activity;dihydroorotate dehydrogenase activity

1743 electron carrier activity;iron-sulfur cluster binding;oxidoreductase

activity;oxidoreductase activity, acting on NADH or NADPH;4 iron, 4 sulfur cluster binding;NADH dehydrogenase activity;NADH dehydrogenase (ubiquinone) activity

6168, 9638 electron carrier activity;protein disulfide oxidoreductase activity

13343 electron carrier activity;protein disulfide oxidoreductase activity ;protein binding

17156 electron carrier activity;protein disulfide oxidoreductase activity ;protein binding;DNA binding;transcription activator activity

7161 endodeoxyribonuclease activity, producing 5'-phosphomonoesters

15651 endopeptidase activity;serine-type endopeptidase activity ;peptide

binding;transmembrane receptor protein tyrosine kinase activity;ATP binding

15649 endopeptidase activity;serine-type endopeptidase activity ;protein binding

11378 endopeptidase activity;threonine-type endopeptidase activity;RNA binding;NF-kappaB binding;protein binding

8257 endopeptidase inhibitor activity ;protein binding

15153 endopeptidase inhibitor activity ;protein binding; semaphorin receptor binding

9805 endopeptidase inhibitor activity;semaphorin receptor binding

14977 enzyme binding

16647 enzyme inhibitor activity;histone deacetylase binding;histone binding

17047 epoxide hydrolase activity

3146 ethanolamine kinase activity

9283 ethanolamine kinase activity;choline kinase activity;drug binding;protein homodimerization activity;cholinesterase activity

16526 eukaryotic initiation factor 4E binding;protein binding

3402 eukaryotic translation initiation factor 2alpha kinase activity

12460 exonuclease activity;nucleic acid binding

5222, 5338 exonuclease activity;nucleic acid binding;3'-5' exonuclease activity

2610, 2712, 16340 extracellular ligand-gated ion channel activity

16063 extracellular ligand-gated ion channel activity;benzodiazepine receptor activity

10558 extracellular ligand-gated ion channel activity;protein binding

14639 extracellular matrix binding

13437 extracellular matrix structural constituent ;binding

13821 extracellular matrix structural constituent;binding;protein binding

10969 extracellular matrix structural constituent ;protein binding;enzyme

binding;glycosphingolipid binding;calcium ion binding;unfolded protein binding

3443 extracellular matrix structural constituent;protein

binding;glycosphingolipid binding;calcium ion binding; signal transducer activity;unfolded protein binding

8658 extracellular matrix structural constituent;receptor binding;peptidase inhibitor activity;SMAD binding

13547 extracellular matrix structural constituent;receptor binding;structural molecule activity 6024 farnesyltranstransferase activity;geranyltranstransferase activity

9104 ferrous iron binding;DNA-Nl-methyladenine dioxygenase activity

12183 fibronectin binding;receptor activity ;protein binding;identical protein binding

14955 folic acid transporter activity;heme transporter activity

10984 formate-tetrahydrofolate ligase activity;ATP binding;catalytic

activity;protein homodimerization activity ;protein binding

12148 fructosamine-3 -kinase activity

16265 GABA-A receptor activity;ion channel activity;extracellular ligand-gated ion channel activity

13916 galactokinase activity;ATP binding;kinase activity;galactose binding

3925 galactosylceramidase activity

15435 galactosylceramide sulfotransferase activity

1227 galactosyltransferase activity

747 galactosyltransferase activity;UDP-galactose:beta-N-acetylglucosamine beta-1 ,3-galactosyltransferase activity

3533 gamma-glutamylcyclotransferase activity;protein homodimerization activity

13980 gamma-glutamyltransferase activity

12633 general RNA polymerase II transcription factor activity

15361 general RNA polymerase II transcription factor activity ;protein kinase activity ;protein N-terminus binding;RNA polymerase II carboxy- terminal domain kinase activity;DNA-dependent ATPase activity

10272 glucosamine-6-phosphate deaminase activity

16438 glucuronyl-galactosyl-proteoglycan 4-alpha-N- acetylglucosaminyltransferase activity ; alpha- 1 ,4-N- acetylgal actos aminyltrans ferase activity

14832 glutamate-ammonia ligase activity

3193 glutamate-cysteine ligase activity ;hydrolase activity, hydrolyzing O- glycosyl compounds;magnesium ion binding;glutamate

binding;coenzyme binding;ADP binding;protein heterodimerization activity

9462 glutathione peroxidase activity

16256 glutathione synthase activity;ATP binding;magnesium ion

binding;protein homodimerization activity;glutathione binding

3288 glycerol kinase activity;phosphotransferase activity, alcohol group as acceptor

11123 glycerol-3-phosphate dehydrogenase activity;calcium ion

binding;oxidoreductase activity

14417 glycerophosphodiester phosphodiesterase activity;phosphoric diester hydrolase activity

4025 glycine hydroxymemyltransferase activity;pyridoxal phosphate

binding;protein homodimerization activity

16019 glycine hydroxymemyltransferase activity;transferase activity;transferase activity, transferring nitrogenous groups ;pyridoxal phosphate binding;glycine C-acetyltransferase activity

16992 glycogen (starch) synthase activity

binding;transporter activity ;protein binding

1208 guanyl -nucleotide exchange factor activity;protein binding

5681 guanyl -nucleotide exchange factor activity;translation initiation factor activity ;protein binding

3669, 5492, 8706, 10366, 16534 heat shock protein binding

15687 heat shock protein binding;electron carrier activity;iron-sulfur cluster binding

13140 heat shock protein binding;hormone activity;Hsp70 protein

binding;proteasome binding;chaperone binding

7018 heat shock protein binding;nucleic acid binding;zinc ion binding

10671 heat shock protein binding;protein binding

14014 heat shock protein binding;protein binding;misfolded protein

binding;ATPase binding;chaperone binding;ATPase activator activity;disulfide oxidoreductase activity

1647, 6142 heat shock protein binding;unfolded protein binding

2106, 5491, 10240 heat shock protein binding;unfolded protein binding ;protein binding

14828 heat shock protein binding;unfolded protein binding;protein

binding;small GTPase regulator activity;interferon-gamma receptor binding;protein kinase binding;NF-kappaB binding;transcription factor binding

12475, 13050 hedgehog receptor activity

8095 hedgehog receptor activity;protein binding

14103 helicase activity ;protein binding;ATPase activity;nucleotide

binding;nucleoside-triphosphatase activity;ATP binding;DNA binding

16190 heme binding;growth factor activity

16812 heme binding;iron ion binding;oxidoreductase activity

12346 heme binding;oxidoreductase activity;cytochrome-b5 reductase

activity;NAD(P)H oxidase activity;oxidoreductase activity, acting on NADH or NADPH, heme protein as acceptor

8713 heme binding;oxidoreductase activity;iron ion binding;oxidoreductase activity, acting on paired donors, with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water;protein binding

2492 heme oxygenase (decyclizing) activity;protein binding;electron carrier activity

6582, 7951, 14690 heparan sulfate proteoglycan binding

15537 heparan sulfate proteoglycan binding;protein binding;peptidyl- dipeptidase inhibitor activity

7299 heparan sulfate proteoglycan binding;sequence-specific DNA binding transcription factor activity; signal transducer activity

7550 heparin binding

1646 heparin binding;receptor binding; frizzled binding

11966 hexokinase activity;ATP binding;phosphotransferase activity, alcohol group as acceptor

4889 histone acetyltransferase activity;protein binding;phosphorylase kinase regulator activity 8722 homocysteine S-methyltransferase activity

12845 hormone activity

15142 hormone activity;endothelin B receptor binding

15307 hormone activity;protein kinase activity;ATP binding

1060 hormone binding;transcription corepressor activity;protein

homodimerization activity ;thyroid hormone binding;NADP+ or NADPH binding

8506, 13937 hydrogen ion transmembrane transporter activity

9990 hydrogen ion transmembrane transporter activity; ATP ase activity

14001 hydrogen ion transmembrane transporter activity;ATPase activity;protein binding

7814 hydrogen ion transmembrane transporter activity ;protein

binding;hydrogen-exporting ATPase activity, phosphorylative mechanism

7375 hydrogen ion transporting ATP synthase activity, rotational mechanism

15262 hydrogen ion transporting ATP synthase activity, rotational

mechanism;proton-transporting ATPase activity, rotational mechanism

8325 hydrogen ion transporting ATP synthase activity, rotational

mechanism;proton-transporting ATPase activity, rotational mechanism;ATPase activity

12014 hydrogen ion transporting ATP synthase activity, rotational

mechanism;proton-transporting ATPase activity, rotational mechanism;hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances;ATP binding

2876, 3648, 8482, 9157, 15121 hydrolase activity

161 17 hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances;ATPase binding

12141 hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds

3985 hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in cyclic amides ;hydrolase activity ;hydrolase activity, acting on carbon- nitrogen (but not peptide) bonds

10057 hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides

4539, 7710, 9046, 14399 hydrolase activity, acting on ester bonds

2731, 2822, 3160, 4734, 16226, hydrolase activity, hydrolyzing O-glycosyl compounds

16914

11598 hydrolase activity, hydrolyzing O-glycosyl compounds;alpha- galactosidase activity;catalytic activity;receptor binding;protein homodimerization activity;protein binding

15353 hydrolase activity, hydrolyzing O-glycosyl compounds ;beta-mannosidase activity

17142 hydrolase activity, hydrolyzing O-glycosyl compounds;catalytic

activity;cation binding

17131 hydrolase activity, hydrolyzing O-glycosyl compounds;chitinase activity

7752 hydrolase activity, hydrolyzing O-glycosyl compounds;protein binding

3371 hydrolase activity;adenosine-diphosphatase activity

15513 hydrolase activity;diphosphoinositol-polyphosphate diphosphatase activity

9454 insulin receptor binding; fibroblast growth factor receptor binding

3647 insulin receptor binding;protein binding;protein kinase binding

5041 insulin receptor binding;receptor signaling protein activity

101 15 insulin-like growth factor binding;protein binding

11468 insulin-like growth factor binding;serine-type endopeptidase

activity ;protein binding;catalytic activity

6449 integrin binding;calcium ion binding;heparin binding;structural molecule activity

4965 intramolecular transferase activity, phosphotransferases

3194, 5819, 6317, 6477, 8610, ion channel activity

10284, 12181, 12866, 14352

16385 ion channel activity;extracellular ligand-gated ion channel activity

8292 ion channel activity;hydrolase activity

17157 ion channel activity;inward rectifier potassium channel activity;voltage- gated ion channel activity

2131 ion channel activity;inward rectifier potassium channel activity;voltage- gated ion channel activity;ATP binding;ATPase activity, coupled to transmembrane movement of substances

15137 ion channel activity;inward rectifier potassium channel activity;voltage- gated ion channel activity;PDZ domain binding;protein binding

3946 ion channel activity;inward rectifier potassium channel activity;voltage- gated ion channel activity ;protein binding;G-protein activated inward rectifier potassium channel activity

975 ion channel activity;oxidoreductase activity;voltage-gated potassium channel activity

2972 ion channel activity;P-P -bond-hydrolysis-driven protein transmembrane transporter activity

4144 ion channel activity ;protein binding;store-operated calcium channel activity

6029 ion channel activity; structural constituent of ribosome;protein

binding;store-operated calcium channel activity

2961, 2971 ion channel activity;voltage-gated potassium channel activity;potassium channel activity;protein binding;potassium channel inhibitor activity

10095, 11507, 12448, 12544, ion channel activity;voltage-gated sodium channel activity

15276

4177 ionotropic glutamate receptor activity;extracellular-glutamate-gated ion channel activity;kainate selective glutamate receptor activity

13531 ionotropic glutamate receptor activity;extracellular-glutamate-gated ion channel activity;protein binding;kainate selective glutamate receptor activity

5363, 11958, 15445, 15486 ionotropic glutamate receptor activity;extracellular-glutamate-gated ion channel activity;transporter activity

7633 ionotropic glutamate receptor activity;extracellular-glutamate-gated ion channel activity;transporter activity;adenylate cyclase inhibiting metabotropic glutamate receptor activity;kainate selective glutamate receptor activity ;protein binding

14638 ionotropic glutamate receptor activity;extracellular-glutamate-gated ion channel activity;transporter activity;protein binding 14042 iron ion binding;cysteine dioxygenase activity

7005, 12748, 15453 iron ion binding;electron carrier activity;heme binding

970 iron ion binding;electron carrier activity;oxidoreductase activity;flavin adenine dinucleotide binding; ferric-chelate reductase activity

12802 iron ion binding;oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen;L-ascorbic acid binding

10599 iron ion binding;oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen

13507 iron ion binding;oxidoreductase activity;serine-type peptidase activity

15312 iron ion transmembrane transporter activity

16258 isomerase activity;bent DNA binding;double-stranded DNA binding

13166 isomerase activity;catalytic activity;carbohydrate binding

11347 isomerase activity;protein binding

15977 kinase activity;tRNA binding

6078 kinesin binding

3438 lactoylglutathione lyase activity;metal ion binding

10086 L-amino acid transmembrane transporter activity

13471 L-glutamate transmembrane transporter activity

13357 L-glutamate transmembrane transporter activity ;protein binding

2483 ligase activity

4609 ligase activity; acid- amino acid ligase activity

5881 lipid binding;glycolipid transporter activity;glycolipid binding

12402 lipoate synthase activity;4 iron, 4 sulfur cluster binding;catalytic

activity;iron-sulfur cluster binding;thiamine diphosphokinase activity; ATP binding

16810 magnesium ion binding;adenylosuccinate synthase activity;GTP

binding;protein binding

16081 magnesium ion binding;four-way junction DNA binding;DNA

binding;damaged DNA binding;double-stranded DNA binding;single- stranded DNA binding;protein binding;ATP binding;protein C-terminus binding;ATPase activity;centromeric DNA binding;mismatched DNA binding;guanine/thymine mispair binding;dinucleotide insertion or deletion binding;single guanine insertion binding;single thymine insertion binding;dinucleotide repeat insertion binding;oxidized purine DNA binding;MutLalpha complex binding;identical protein binding;protein homodimerization activity;ADP binding ;protein kinase binding;enzyme binding

12364 magnesium ion binding;holo-[acyl-carrier-protein] synthase

activity ;protein binding

9414 magnesium ion binding;hydrolase activity, acting on acid anhydrides, in phosphorus-containing anhydrides ;manganese ion binding;hydrolase activity

10617 magnesium ion binding;inorganic diphosphatase activity

7281 magnesium ion binding;inorganic diphosphatase

activity;pyrophosphatase activity

7080, 8068, 15126 metalloendopeptidase activity;zinc ion binding;metallopeptidase activity

1736 metalloendopeptidase activity;zinc ion binding;metallopeptidase

activity ;protein binding

2772, 9584, 10792 metalloendopeptidase activity;zinc ion binding;peptidase activity

9041 metalloendopeptidase activity;zinc ion binding;protein binding;heparin binding

4431 metalloexopeptidase activity;dipeptidyl -peptidase activity;dipeptidase activity

6765, 15136 metallopeptidase activity;metalloendopeptidase activity

4697, 10231 metallopeptidase activity;zinc ion binding

6832 metallopeptidase activity;zinc ion binding;aminopeptidase activity

3668, 10813 metallopeptidase activity;zinc ion binding;binding

12977 metallopeptidase activity;zinc ion binding;peptidase activity

14725 metallopeptidase activity;zinc ion binding;protein

binding;aminopeptidase activity

13147 methylated-DNA- [protein] -cysteine S-methyltransferase

activity;catalytic activity;methyltransferase activity

15097 methylmalonate-semialdehyde dehydrogenase (acylating)

activity;oxidoreductase activity

7846 methylmalonyl-CoA epimerase activity

15767 methylmalonyl-CoA mutase activity;cobalamin binding;intramolecular transferase activity;metal ion binding;isomerase activity;catalytic activity

10154, 10944 methyltransferase activity

16511 methyltransferase activity;diphthine synthase activity

8318 methyltransferase activity;protein binding;protein methyltransferase activity

6947 methyltransferase activity;RNA binding

16695 microtubule motor activity;alpha-tubulin binding;beta-tubulin

binding;protein binding;dynactin binding

4503, 15043, 15057 microtubule motor activity; ATP binding

7024 microtubule motor activity; ATP binding; ATP ase activity

13432 microtubule motor activity;ATP binding;nucleotide binding

5610, 5668, 8521 , 16245 microtubule motor activity;ATP binding;protein binding

11817, 15847 microtubule motor activity ;protein binding

3053, 8435, 10985 molecular function

14571 Mo-molybdopterin synthase activity

6077, 16649 monooxygenase activity;iron ion binding;electron carrier activity;heme binding

1397 monooxygenase activity;iron ion binding;electron carrier activity;heme binding;protein binding;oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen

7935 monooxygenase activity;iron ion binding;electron carrier activity;heme binding;retinoic acid binding 14441 monooxygenase activity;oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, NADH or NADPH as one donor, and incorporation of one atom of oxygen;flavin adenine dinucleotide binding

7971 motor activity

5349, 14366 motor activity;ATP binding;protein binding

7486 motor activity;ATP binding; signal transducer activity;protein binding

6081 N-acetylglucosamine-6-phosphate deacetylase activity ;hydrolase

activity;hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds

15063, 17162 NAD+ ADP-ribosyltransferase activity

10725 NAD+ ADP-ribosyltransferase activity;protein binding

16153 NADH dehydrogenase (ubiquinone) activity

15900 neurotransmitter: sodium symporter activity

4048, 12250 NF-kappaB binding;protein binding

14246 nitric-oxide synthase activity

4372 Notch binding;calcium ion binding ;protein binding;growth factor

activity;peptidase inhibitor activity

13514 nuclease activity

12392 nuclease activity;ATP binding;zinc ion binding

1202, 1907, 2476, 2835, 3649, nucleic acid binding

3940, 4466, 4992, 5224, 5281 ,

5382, 5389, 5667, 5697, 6039,

6129, 6607, 6854, 7669, 7906,

8315, 9542, 12137, 12503, 12776,

13441, 13526, 13588, 13817,

14851, 15035, 15122, 15485,

15902, 15922, 16307, 17096

10401 nucleic acid binding;AF-2 domain binding;RNA polymerase II

transcription mediator activity;receptor activator activity;ligand- dependent nuclear receptor transcription coactivator activity;estrogen receptor binding;protein binding

14309 nucleic acid binding;ATP binding;ATP-dependent helicase activity

13829 nucleic acid binding;ATP binding;ATP-dependent helicase

activity;calcium ion binding

7214 nucleic acid binding;ATP binding;ATP-dependent helicase

activity;calcium ion binding;G-protein beta/gamma-subunit complex binding

13790, 14978, 17116 nucleic acid binding;ATP binding;ATP-dependent helicase

activity;helicase activity

5723 nucleic acid binding;ATP binding;ATP-dependent helicase

activity;helicase activity;DNA binding;hydrolase activity

12215 nucleic acid binding;ATP binding;ATP-dependent helicase

activity;helicase activity;nucleotide binding;nucleoside-triphosphatase activity

14420 nucleic acid binding;ATP binding;ATP-dependent helicase

activity;helicase activity;protein binding;transcription repressor activity

7309 nucleic acid binding;ATP binding;ATP-dependent helicase activity ;protein binding

13149 nucleic acid binding;binding

15621, 16095 nucleic acid binding;binding;protein binding

4545 nucleic acid binding;chromatin binding;sequence-specific DNA binding transcription factor activity;protein binding;beta-catenin binding;zinc ion binding;transcription repressor activity;histone acetyltransferase binding;histone deacetylase binding;sequence-specific DNA binding;repressing transcription factor binding

1905 nucleic acid binding;copper ion binding;primary amine oxidase

activity ;quinone binding;protein binding;eukaryotic cell surface binding

13234 nucleic acid binding;DNA binding;protein binding;transcription

repressor activity;zinc ion binding

13439 nucleic acid binding;DNA binding;sequence-specific DNA binding transcription factor activity;zinc ion binding;transcription regulator activity;sequence-specific DNA binding;metal ion binding;hormone activity

13315 nucleic acid binding;DNA binding;zinc ion binding;metal ion binding

3864 nucleic acid binding;heat shock protein binding

9583, 13453 nucleic acid binding;helicase activity;ATP binding;ATP-dependent helicase activity

7794 nucleic acid binding;helicase activity;ATP binding;ATP-dependent helicase activity;ATP-dependent RNA helicase activity

9195, 13968 nucleic acid binding;helicase activity;ATP binding;ATP-dependent helicase activity ;nucleotide binding;nucleoside-triphosphatase activity

13164 nucleic acid binding;helicase activity;ATP binding;ATP-dependent helicase activity ;protein binding

13616 nucleic acid binding;helicase activity;ATP binding;ATP-dependent helicase activity;RNA binding;protein binding

14411 nucleic acid binding;helicase activity;ATP binding;nucleotide

binding;nucleoside-triphosphatase activity

10012 nucleic acid binding;helicase activity;ATP binding;nucleotide

binding;nucleoside-triphosphatase activity;protein binding

7481 nucleic acid binding;helicase activity;ATP binding;transcription

repressor activity

9833, 16698 nucleic acid binding;helicase activity;ATP binding;zinc ion

binding;ATP-dependent helicase activity;carboxy-lyase activity;protein binding

12712 nucleic acid binding;hydrolase activity;metal ion binding

6189, 12251, 15678 nucleic acid binding;methyltransferase activity

14795 nucleic acid binding;methyltransferase activity;endopeptidase

activity;metallopeptidase activity

16472 nucleic acid binding;methyltransferase activity;metallopeptidase activity

3971 nucleic acid binding;methyltransferase activity;protein binding ;protein methyltransferase activity

10864 nucleic acid binding;NAD+ ADP-ribosyltransferase activity;protein binding

13101 nucleic acid binding;pancreatic ribonuclease activity;protein binding 8822 nucleic acid binding;peptidyl-prolyl cis-trans isomerase activity

101 11 nucleic acid binding;peptidyl-prolyl cis-trans isomerase activity;protein binding

2642, 2669, 4309, 6452, 10053, nucleic acid binding;protein binding

11384, 13445, 13461 , 13688,

13₇04, 14407, 14530, 15365,

16267

6861 nucleic acid binding;protein binding;ligand-dependent nuclear receptor transcription coactivator activity

14915 nucleic acid binding;protein binding;methyltransferase activity;mRNA

(nucleoside-2'-0-)-methyltransferase activity

3619 nucleic acid binding ;protein binding;mRNA binding

791 1 nucleic acid binding ;protein binding;RNA binding;transcription

corepressor activity;transcription factor binding

3909 nucleic acid binding;protein binding;structural molecule activity ;protein binding, bridging

15060 nucleic acid binding;protein binding;transcription activator activity ;actin monomer binding;transcription coactivator activity;actin binding

14702 nucleic acid binding;protein binding;transcription coactivator

activity ;actin binding

15585 nucleic acid binding;protein binding;zinc ion binding

10808 nucleic acid binding;protein dimerization activity

15438 nucleic acid binding;protein kinase activity;ATP binding;protein

serine/threonine kinase activity;protein binding;ribonucleoprotein binding

13650 nucleic acid binding;pyridoxal phosphate binding;RNA binding

8772, 10160 nucleic acid binding;RNA binding

2812, 3602, 4436, 10861, 15133 nucleic acid binding;RNA binding;protein binding

4235 nucleic acid binding;RNA methyltransferase activity

6030 nucleic acid binding;RNA polymerase core enzyme binding

10277 nucleic acid binding;sequence-specific DNA binding transcription factor activity;sequence-specific DNA binding

12742 nucleic acid binding;single-stranded DNA binding;RNA binding

5642 nucleic acid binding;two-component response regulator activity;DNA binding

1655, 1808, 2438, 2754, 3147, nucleic acid binding;zinc ion binding

4241, 4441, 5553, 6178, 6502,

7565, 7597, 11326, 11818, 12096,

12839, 13487, 13836, 14323,

14549, 15367, 16149, 16314

5057 nucleic acid binding;zinc ion binding;DNA binding

14440 nucleic acid binding;zinc ion binding;DNA binding;sequence-specific

DNA binding;promoter binding ;protein binding;specific transcriptional repressor activity;repressing transcription factor binding

10937 nucleic acid binding;zinc ion binding;DNA binding ;transcription

activator activity

11546 nucleic acid binding;zinc ion binding;DNA binding ;transcription

regulator activity;metal ion binding

8893 nucleotide binding;aminoacyl-tRNA ligase activity;ATP

binding;threonine-tRNA ligase activity;ligase activity, forming aminoacyl-tRNA and related compounds

5133 nucleotide binding;aminoacyl-tRNA ligase activity;ATP

binding;tyrosine-tRNA ligase activity;RNA binding;protein binding

2539 nucleotide binding;binding;protein N-terminus binding;protein

binding;chaperone binding

5507, 5510 nucleotide binding;calcitonin gene-related polypeptide receptor

activity;catalytic activity;DNA-directed RNA polymerase activity

4899 nucleotide binding;catalytic activity;calcium-transporting ATPase

activity ;protein binding;ATP binding; ATPase activity, coupled to transmembrane movement of ions, phosphorylative

mechanism;hydrolase activity;hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances;metal ion binding;S 100 alpha binding;protein C-terminus binding

14092 nucleotide binding;catalytic activity;glutamate dehydrogenase

[NAD(P)+] activity;binding;protein binding;ATP binding;GTP binding;oxidoreductase activity;oxidoreductase activity, acting on the CH-NH2 group of donors, NAD or NADP as acceptor

7216 nucleotide binding;catalytic activity ;metal ion binding

6124 nucleotide binding;damaged DNA binding;single-stranded DNA specific endodeoxyribonuclease activity ;protein binding

9532 nucleotide binding;DNA binding;DNA-directed DNA polymerase activity ;nucleic acid binding

7647, 9506, 12180 nucleotide binding;GTP binding

17001 nucleotide binding;GTP binding;GDP binding;alpha-tubulin

binding;beta-tubulin binding

9998 nucleotide binding;GTP binding;GTPase activity;protein binding;ATP binding;transcription factor binding

9955 nucleotide binding;GTPase activity;GTP binding

7118, 7931, 8142 nucleotide binding;GTPase activity; structural molecule activity;GTP binding

831 1 nucleotide binding;magnesium ion binding;5'-nucleotidase activity

10375 nucleotide binding;magnesium ion binding;calcium-transporting ATPase activity;calmodulin binding;ATP binding;ATPase activity, coupled to transmembrane movement of ions, phosphorylative

mechanism;hydrolase activity;hydrolase activity, acting on acid anhydrides ;metal ion binding;catalytic activity;calcium ion transmembrane transporter activity;hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances

16357 nucleotide binding;motor activity ;actin binding;ATP binding;protein binding

15841 nucleotide binding;motor activity;actin binding;calmodulin binding;ATP binding;protein binding

12235 nucleotide binding;mRNA binding;protein binding;nucleic acid

binding;RNA binding

16013 nucleotide binding;nucleic acid binding

3739, 12930 nucleotide binding;nucleic acid binding;protein binding

as acceptor

2913, 7614 oxidoreductase activity;aminomethyltransferase activity

11622 oxidoreductase activity;chromatin binding;transcription factor

binding;transcription repressor activity ;protein binding;histone demethylase activity (H3-dimethyl-K4 specific);hi stone demethylase activity (H3-K9 specific);histone demethylase activity (H3-K4 specific);hi stone demethylase activity;ligand-dependent nuclear receptor transcription coactivator activity;MyoD binding;androgen receptor binding; flavin adenine dinucleotide binding;sequence-specific DNA binding transcription factor activity;promoter binding

8158 oxidoreductase activity;copper ion binding;protein binding;chaperone binding; ferroxidase activity

14968 oxidoreductase activity;D-amino-acid oxidase activity

10064 oxidoreductase activity; flavin adenine dinucleotide binding;2 iron, 2 sulfur cluster binding;caspase activator activity ;protein binding

8404, 13911 oxidoreductase activity;FMN binding

3050, 6596, 13931 oxidoreductase activity;iron ion binding;oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen;L- ascorbic acid binding

15674 oxidoreductase activity;iron ion binding;oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen;L- ascorbic acid binding ;protein binding

6992 oxidoreductase activity;iron-sulfur cluster binding;zinc ion binding

4740 oxidoreductase activity;malate dehydrogenase activity;oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor;L-malate dehydrogenase activity;catalytic activity

8407 oxidoreductase activity;NAD+ or NADH binding;NADH dehydrogenase

(ubiquinone) activity

10417 oxidoreductase activity;oxidoreductase activity, acting on a sulfur group of donors, oxygen as acceptor;prenylcysteine oxidase activity

3186 oxidoreductase activity;polyamine oxidase activity

16561 oxidoreductase activity;protein binding

4132, 15048 oxidoreductase activity ;protein binding;kinase binding

13311 oxidoreductase activity;protein binding;nitric-oxide synthase

activity;FMN binding;iron ion binding;calmodulin binding;heme binding; flavin adenine dinucleotide binding;NADP+ or NADPH binding

9002 oxidoreductase activity ;protein binding; signal transducer activity

15911 oxidoreductase activity;saccharopine dehydrogenase (NAD+, L- glutamate-forming) activity

131 17 oxidoreductase activity;transporter activity

6731 oxidoreductase activity;zinc ion binding;flavin adenine dinucleotide binding;histone demethylase activity (H3-monomethyl-K4

specific);histone demethylase activity (H3-dimethyl-K4 specific)

5978 oxygen binding;heme binding

7272 p53 binding;protein binding;protein-lysine N-methyltransferase

activity;histone-lysine N-methyltransferase activity 15924 palmitoyl-CoA hydrolase activity

8096 palmitoyltransferase activity

722, 4131 pantothenate kinase activity;ATP binding

3280 pantothenate kinase activity;ATP binding;protein binding

6729, 7130, 7397, 11664, 13005, peptidase activity

15458

10820 peptidase activity;serine-type peptidase activity

2949 peptidase inhibitor activity

1449 peptidase inhibitor activity;endopeptidase inhibitor activity

7463 peptide transporter activity

13335 peptide-aspartate beta-dioxygenase activity

6653, 9475, 9654, 9655, 1 1983, peptidyl-prolyl cis-trans isomerase activity

12886

15378 peptidyl-prolyl cis-trans isomerase activity ;protein binding;heat shock protein binding

5436 peptidyl-prolyl cis-trans isomerase activity ;protein

binding;ribonucleoprotein binding

7523 peroxidase activity;antioxidant activity;oxidoreductase

activity;peroxiredoxin activity;protein binding;thioredoxin peroxidase activity

6738 peroxidase activity;protein binding;electron carrier activity;protein- disulfide reductase activity

16949 peroxisome matrix targeting signal-2 binding

5509 PH domain binding

13664 phosphatase activity

9279 phosphatase activity;phosphotransferase activity, alcohol group as acceptor;5'-nucleotidase activity

2448 phosphatase activity;protein tyrosine phosphatase activity

12082 phosphatase activity;protein tyrosine phosphatase activity;calcium- activated potassium channel activity

922 phosphatase activity;protein tyrosine phosphatase

activity;phosphatidylinositol-3 -phosphatase activity

10713 phosphatase activity;protein tyrosine phosphatase activity ;protein

binding;inositol or phosphatidylinositol phosphatase activity

13550 phosphatase activity;zinc ion binding ;protein tyrosine phosphatase activity

8234 phosphatase activity;zinc ion binding ;protein tyrosine phosphatase activity ;protein serine/threonine phosphatase activity

4837 phosphatidylcholine-sterol O-acyltransferase activity;calcium- independent phospholipase A2 activity

4646 phosphatidylinositol N-acetylglucosaminyltransferase activity

5275 phosphatidylinositol phosphate kinase activity; 1 -phosphatidylinositol-4- phosphate 5 -kinase activity

4033 phosphatidylinositol phosphate kinase activity ;protein binding; 1- phosphatidylinositol-4-phosphate 5-kinase activity

7027 phosphatidylinositol-3,5-bisphosphate binding 16971 phosphatidylinositol-4,5-bisphosphate bindmg;phosphotyrosme

binding;beta-amyloid binding;protein complex binding;protein binding;signaling adaptor activity;protein binding, bridging;clathrin binding;receptor signaling complex scaffold activity;low-density lipoprotein receptor binding

3026 phosphatidylinositol-4,5-bisphosphate binding ;protein

binding;phosphatidylinositol-5-phosphate binding;phosphatidylinositol- 3-phosphate binding;phosphatidylinositol-3,5-bisphosphate binding;phosphatidic acid binding;phosphatidylinositol-4-phosphate binding;phosphatidylinositol-3,4-bisphosphate binding

10565 phospholipase A2 activity

11121 phospholipase A2 activity;calcium ion binding

8828 phospholipase activator activity;phospholipase binding;Rho GTPase activator activity

9201 phospholipase activity ;protein binding;phospholipase Al activity

7635 phospholipase C activity;phosphatidylinositol phospholipase C

activity;calcium ion binding;phosphoric diester hydrolase activity

6774, 16321 phospholipase C activity;phosphatidylinositol phospholipase C

activity;phosphoric diester hydrolase activity

13477 phospholipase C activity;phosphatidylinositol phospholipase C

activity;phosphoric diester hydrolase activity;enzyme binding

12854 phospholipid binding

11019 phospholipid binding;protease binding;cell surface binding

4978 phosphomannomutase activity

12954, 13259 phosphopantetheine binding

9964 phosphopantetheine binding;catalytic activity

16349 phosphopantetheine binding;protein binding

5403 phosphoprotein phosphatase activity;actin binding;protein binding

15393 phosphoprotein phosphatase activity;protein binding;CTD phosphatase activity

4338, 13075 phosphoprotein phosphatase activity;protein tyrosine phosphatase activity ;receptor activity;hydrolase activity;phosphatase activity

11000 phosphoribosylamine-glycine ligase activity;catalytic

activity;hydroxymethyl-, formyl- and related transferase

activity;phosphoribosylglycinamide formyltransferase

activity;phosphoribosylformylglycinamidine cyclo-ligase activity;ATP binding

16275 phosphoric diester hydrolase activity;single-stranded DNA

binding;double-stranded DNA binding;protein binding; 3 '-tyrosyl-DNA phosphodiesterase activity

1092, 5692, 9050, 14536 phosphorus-oxygen lyase activity; adenylate cyclase activity

11430 phosphorus-oxygen lyase activity;guanylate cyclase activity

11675 phosphorus-oxygen lyase activity;guanylate cyclase activity;heme binding

16976 phosphorus-oxygen lyase activity;nucleotide binding;aminoacyl-tRNA ligase activity;ATP binding;guanylate cyclase activity 5806, 11 169, 16635 phosphotransferase activity, alcohol group as acceptor

12334 phosphotransferase activity, alcohol group as acceptor;binding

2475 phosphotransferase activity, alcohol group as acceptor;binding;protein

binding

7683 phosphotransferase activity, alcohol group as acceptor;binding;protein binding;kinase activity;phosphoprotein binding;protein serine/threonine kinase activity

3258 phosphotransferase activity, alcohol group as acceptor;inositol or phosphatidylinositol kinase activity;sequence-specific DNA binding transcription factor activity;sequence-specific DNA

binding;oxidoreductase activity;transition metal ion

binding;binding;protein binding

14523 phosphotransferase activity, alcohol group as acceptor;protein binding

7275 phosphotransferase activity, for other substituted phosphate groups

3502, 8263, 10988, 11496 polypeptide N-acetylgalactosaminylrransferase activity

2298 polypeptide N-acetylgalactosaminyltransferase activity;manganese ion

binding

13069 polyubiquitin binding;protein binding

8423 potassium channel activity

9973 P-P-bond-hydrolysis-driven protein transmembrane transporter

activity ;protein binding

5922 P-P-bond-hydrolysis-driven protein transmembrane transporter

activity ;protein binding;ribosome binding

6192 P-P-bond-hydrolysis-driven protein transmembrane transporter

activity;ribosome binding

11705 procollagen-lysine 5-dioxygenase activity ;oxidoreductase activity ;iron ion binding;oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen;L-ascorbic acid binding

3646 proline-rich region binding;protein binding

601 , 843, 1096, 1 131, 1 169, 1339, protein binding

1438, 1459, 1473 , 1632 1730,

1806, 1856 1864 , 2026, 2032,

2042 2137 2146 , 2238 2287,

2396, 2401, 2422 , 2455 2631 ,

2635 2699 2721 , 2723 2757,

2763 2830 2845 , 2860_: 2889,

2952 3086 3087 , 3159, 3251 ,

3282 3329 3367 , 3387 3421 ,

3434_: 3463, 3514 , 3532 3540,

3549, 3618, 3653 , 3684, 3710,

3713 3728, 3736 , 3813 3874,

3887 3900 3916 , 3958 3981 ,

4009_: 4030, 4077 , 4113 4157,

4200, 4263, 4300 , 4374, 4442,

4464, 4489, 4510 , 4513 4534,

4601 4642, 4665 , 4728 4761 ,

4804, 4924, 4928 , 4945 4973,

5032 5037, 5165 , 5194_: 5283,

5483 5518, 5568 , 5579, 5582,

5620, 5660 5674 , 5700, 5720, 5770, 5798, 5804 5813 5866, 5877 5896 5897, 5973 5974, 5988 5994 5997 6211 6242, 6297 6319 6388, 6443 6492, 6495 6554 6574, 6618 6622, 6639, 6773 6790 6807 6814, 6905 6917 6950 6960, 6990, 7023 7073 7136, 7137 7138, 7142 7144 7191, 7290_: 7330, 7412 7422 7429, 7509, 7528, 7536, 7630 7636, 7657 7689, 7701 7728 7758, 7762 7798, 7821 7880 7898, 7974, 7991 , 7993 8048 8063 8082 8131 , 8155 8161 8175, 8252 8293, 8335 8368 8378, 8389_: 8481 , 8487 8639 8681, 8709, 8746, 8759_: 8773 8876, 8968 9017, 9076_: 9125 9153, 9187 9208, 9254, 9275 9327, 9349, 9360, 9394_: 9448 9490 9553 9563, 9594, 9628 9670 9700_: 9740, 9758 9821 9859 9940, 9946, 9965 10020, 10028, 10108, 101 10, 10157, 10183, 10239, 10247, 10296, 10302, 10334, 10339, 10357, 10395, 10443, 10466, 10491, 10551 , 10657, 10683, 10700, 10730, 10781, 10838, 10857, 10908, 10920, 10926, 10931, 10970, 11039, 11046, 11105, 1 1119, 11128, 11173, 11278, 1 1396, 11448, 11451, 11479, 1 1499, 11508, 11525, 11544, 1 1593, 11635, 11650, 11662, 1 1682, 11727, 11796, 11800, 1 1825, 11834, 11933, 11978, 12026, 12147, 12152, 12194, 12329, 12416, 12418, 12512, 12526, 12563, 12679, 12753, 12772, 12798, 12817, 12833, 12843, 12846, 12872, 12907, 12914, 12973, 12990, 13059, 13061 , 13064, 13070, 13104, 13122, 13184, 13199, 13218, 13246, 13250, 13264, 13265, 13414, 13447, 13452, 13479, 13527, 13532, 13566, 13645, 13757, 13774, 13853, 13899, 13924, 13933, 13983, 13999, 14018, 14029, 14041, 14044, 14058, 14078, 14207, 14394, 14527, 14566,

8273 protein binding;cysteine-type endopeptidase inhibitor activity

696 protein binding;cystic fibrosis transmembrane conductance regulator binding;small GTPase regulator activity

12439 protein binding;cytoskeletal adaptor activity;dynactin binding;Rab

GTPase binding;dynein binding;proteinase activated receptor binding

8678 protein binding;cytoskeletal protein binding

3017 protein binding;delta-opioid receptor activity

8605, 9517, 11410 protein binding;DNA binding

3230 protein binding;DNA binding;sequence-specific DNA binding RNA polymerase II transcription factor activity

6679 protein binding;DNA binding;sequence-specific DNA binding

transcription factor activity

2785, 3519, 7773, 8030 protein binding;DNA binding;zinc ion binding

744 protein binding;DNA binding;zinc ion binding;methyl-CpG binding

2370 protein binding;DNA binding;zinc ion binding;transcription repressor activity;sequence-specific DNA binding

11179 protein binding;drug transmembrane transporter activity

11711 protein binding;epidermal growth factor binding

14567 protein binding;GABA receptor binding

4586, 9818, 14106 protein binding;gamma-tubulin binding

10367 protein binding;glycoprotein binding

9169 protein binding;glycoprotein binding;carbohydrate binding;beta-amyloid binding

16159 protein binding;G-protein activated inward rectifier potassium channel activity

14251 protein binding;G-protein coupled photoreceptor activity

5380 protein binding;GTP binding; ATP ase activity;ATP binding;Rab GTPase binding

12952 protein binding;GTPase inhibitor activity

4088 protein binding;guanyl-nucleotide exchange factor activity

2593 protein binding;heparin binding

12533 protein binding;histone deacetylase activity

5096 protein binding;histone-lysine N-methyltransferase activity

5157 protein binding;Hsp90 protein binding;histone binding

5966 protein binding;hydrogen ion transmembrane transporter

activity;hydrogen ion transporting ATP synthase activity, rotational mechanism

2429 protein binding;hydrolase activity;catalytic activity

6532, 9850, 14558 protein binding;identical protein binding

3460 protein binding;identical protein binding;CARD domain binding

4972 protein binding;insulin receptor substrate binding;insulin-like growth factor receptor binding;ErbB-3 class receptor binding;insulin receptor binding;protein phosphatase binding;insulin binding

10953 protein binding;integrin binding;protein homodimerization

activity ;protein heterodimerization activity 6031 protein binding;iron ion binding

5900 protein binding;kinase activity

12885 protein binding;kinesin binding

13721 protein binding ;kinetochore binding

4390 protein binding;laminin-l binding

9410 protein binding;ligand-dependent nuclear receptor transcription

coactivator activity;protein domain specific binding;transcription coactivator activity;histone acetyltransferase activity;ligand-dependent nuclear receptor binding

7887 protein binding;microtubule binding;tubulin binding;phospholipid

binding;SH3 domain binding

6106 protein binding;microtubule motor activity;ATP binding

6545, 14568 protein binding;N-acetyltransferase activity;ribosome

binding;acetyltransferase activity

12246 protein binding;neurexin binding

1242, 3861 protein binding;nitric-oxide synthase activity

16450 protein binding;Notch binding;transcription repressor activity

5183, 7516 protein binding;nucleic acid binding

5339, 9848 protein binding;nucleotide binding;ATP binding;identical protein binding

12913 protein binding;oxidoreductase activity

1569, 8859 protein binding;oxidoreductase activity, acting on a sulfur group of donors, disulfide as acceptor;flavin adenine dinucleotide binding

4409, 10251 , 10788 protein binding;oxidoreductase activity;transition metal ion binding

13333 protein binding;oxidoreductase activity;transition metal ion

binding;kinase binding;kinase activator activity

5297 protein binding;peptidase inhibitor activity ;heparin binding;collagen binding;collagen V binding

16459 protein binding;peroxidase activity;heme binding;peptidase inhibitor activity;extracellular matrix structural constituent

16312 protein binding;phosphatidylinositol 3 -kinase binding ;kines in

binding;mitogen-activated protein kinase p38 binding;JUN kinase binding

4166, 5018, 6331, 7513, 9479, protein binding;phosphatidylinositol binding

11893, 13273, 13595, 13773,

16627

16467 protein binding;phosphatidylinositol binding; 1 -phosphatidylinositol binding;protein homodimerization activity;ubiquitin protein ligase binding

13213 protein binding;phosphatidylinositol binding;microtubule motor

activity; ATP binding

5794 protein binding;phosphatidylinositol binding;nucleic acid binding;zinc ion binding

6312 protein binding;phosphatidylinositol binding;protein C-terminus

binding;low-density lipoprotein receptor binding

4143 protein binding;phosphatidylinositol binding;protein transporter activity 3849 protein binding;phosphatidylinositol binding; SH2 domain

binding;phosphatidylinositol-3-phosphate binding;phosphatidylinositol- 3,5-bisphosphate binding;phosphatidylinositol-5-phosphate binding;phosphatidylinositol-4-phosphate binding

7363 protein binding;phosphatidylinositol transporter activity

4579 protein binding;phosphatidylinositol-4,5-bisphosphate binding

1516, 12924 protein binding;phospholipase A2 activity

4148 protein binding;P-P-bond-hydrolysis-driven protein transmembrane transporter activity;unfolded protein binding

1291 protein binding;protein binding, bridging

15994 protein binding;protein complex binding ;protein kinase regulator activity

5775, 7172 protein binding;protein complex scaffold

1877, 4086 protein binding;protein C-terminus binding

3822 protein binding;protein C-terminus binding;calcium ion binding

15805 protein binding;protein C-terminus binding;enzyme binding

8775 protein binding;protein C-terminus binding;receptor binding;phosphatase binding

6167 protein binding;protein C-terminus binding;transcription regulator activity;beta-catenin binding

12171 protein binding;protein disulfide isomerase activity

7195, 15065 protein binding;protein domain specific binding

8507 protein binding;protein domain specific binding;protein C-terminus binding

3018 protein binding;protein domain specific binding;protein kinase

binding;NF-kappaB binding;heat shock protein binding

5091 protein binding;protein domain specific binding;receptor tyrosine kinase binding

6969, 10547 protein binding;protein heterodimerization activity

7504 protein binding;protein heterodimerization activity;hydrolase activity, hydrolyzing O-glycosyl compounds;identical protein binding

3879 protein binding;protein kinase activator activity

2832 protein binding;protein kinase activity;ATP binding;protein

serine/threonine kinase activity;calmodulin binding

4671 protein binding;protein kinase B binding

1043, 14877 protein binding;protein kinase binding;kinesin binding;MAP-kinase scaffold activity

11048 protein binding;protein kinase binding ;protein homodimerization

activity ;protein tyrosine kinase inhibitor activity

3318 protein binding;protein kinase binding ;protein tyrosine kinase inhibitor activity

16331 protein binding;protein kinase C binding;Rho GTPase binding;GTP- dependent protein binding

11945 protein binding;protein N-terminal glutamine amidohydrolase activity

10739 protein binding;protein phosphatase 2A binding

4446 protein binding;protein self-association;PDZ domain binding 17038 protein binding;protein serine/threonine phosphatase activity

10356 protein binding;protein serine/threonine phosphatase inhibitor activity

5093, 11380, 16685 protein binding;protein transporter activity

15949 protein binding;Rab GTPase binding

11038 protein binding;Rab guanyl-nucleotide exchange factor activity

13596 protein binding;receptor activity

16994 protein binding;receptor activity;complement receptor activity

7514, 16077 protein binding;receptor binding

9691 protein binding;ribonucleoside-diphosphate reductase activity

3949 protein binding;RNA binding

1927 protein binding;RNA binding;hydrolase activity, acting on ester bonds

3215 protein binding;RNA binding;phenylalanine-tRNA ligase activity

6231 protein binding;semaphorin receptor activity

4544 protein binding;sequence-specific DNA binding transcription factor activity

13371 protein binding;sequence-specific DNA binding transcription factor activity;sequence-specific DNA binding

4786, 13046 protein binding;sequence-specific DNA binding transcription factor activity;sequence-specific DNA binding;DNA binding ;protein dimerization activity

8159 protein binding;sequence-specific DNA binding transcription factor activity;signal transducer activity

5581 protein binding;SH2 domain binding

998 protein binding;SH3/SH2 adaptor activity

4358 protein binding;SNARE binding;syntaxin binding;calmodulin

binding;phospholipid binding

1000, 1002, 8904 protein binding;snoRNA binding

7107 protein binding ;snRNA binding

9778 protein binding;stem cell factor receptor binding

10814 protein binding;structural molecule activity

3490 protein binding;structural molecule activity ;binding

3530, 6923, 8630 protein binding;transcription coactivator activity

6229, 13995 protein binding;transcription coactivator activity;RNA polymerase II transcription factor binding;peroxisome proliferator activated receptor binding;histone deacetylase binding;phosphatidate phosphatase activity

4121 protein binding;transcription corepressor activity

12275 protein binding;transcription corepressor activity;chromatin binding

4997, 5002 protein binding;transcription corepressor activity;toxin receptor

binding;protein domain specific binding

16883 protein binding;transcription elongation regulator activity

10807 protein binding;transcription regulator activity

2410 protein binding;transcription repressor activity

3248, 11904 protein homodimerization activity

3554, 6509 protein homodimerization activity;protein binding

6169 protein homodimerization activity;protein heterodimerization

activity;identical protein binding;protein binding

10361 protein homodimerization activity;protein heterodimerization

activity;protein binding;glucuronosyltransferase

activity;acetylglucosaminylrransferase activity ;transferase activity, transferring glycosyl groups

10056 protein kinase activity;ATP binding;calcium ion binding ;protein

serine/threonine kinase activity;protein tyrosine kinase activity;protein binding

13433 protein kinase activity;ATP binding;calcium ion binding;protein

serine/threonine kinase activity;protein tyrosine kinase activity;protein binding;receptor activity

10216 protein kinase activity;ATP binding;ephrin receptor activity ;protein serine/threonine kinase activity;protein tyrosine kinase activity;axon guidance receptor activity

9893 protein kinase activity;ATP binding;heat shock protein binding;protein serine/threonine kinase activity;protein tyrosine phosphatase activity;phosphatase activity

6171 protein kinase activity;ATP binding;motor activity;protein

serine/threonine kinase activity;micro filament motor activity;calmodulin binding;actin-dependent ATPase activity;plus-end directed microfilament motor activity;ADP binding

2133 protein kinase activity;ATP binding;protein adenylyltransferase activity

13481, 13884 protein kinase activity;ATP binding;protein binding

11587 protein kinase activity;ATP binding;protein binding;phosphatidylinositol binding;protein serine/threonine kinase activity

6144 protein kinase activity;ATP binding;protein binding;phosphatidylinositol binding;protein serine/threonine kinase activity ;protein C-terminus binding

8531, 14603 protein kinase activity;ATP binding;protein binding ;protein

serine/threonine kinase activity

3378, 5870, 10323, 11317 protein kinase activity;ATP binding;protein binding;protein

serine/threonine kinase activity;protein tyrosine kinase activity

5954 protein kinase activity;ATP binding;protein binding;protein

serine/threonine kinase activity;signal transducer activity

9828 protein kinase activity;ATP binding;protein binding;zinc ion

binding;protein serine/threonine kinase activity;binding;protein kinase binding;JUN kinase kinase activity

14577 protein kinase activity;ATP binding;protein kinase inhibitor

activity;transcription factor binding;protein binding;mitogen-activated protein kinase kinase binding;ubiquitin-protein ligase regulator activity;ubiquitin protein ligase binding

2502, 3359, 3455, 4104, 4327, protein kinase activity;ATP binding;protein serine/threonine kinase 4937, 5117, 5970, 7749, 7925, activity

10013, 10202, 10329, 10809,

10846, 11002, 1 1311 , 12820,

13513, 15085, 15477, 16399, 16728, 17050

11272 protein kinase activity;ATP binding;protein serine/threonine kinase activity;calmodulin-dependent protein kinase activity

14989 protein kinase activity;ATP binding;protein serine/threonine kinase activity;catalytic activity;NF-kappaB-inducing kinase activity;protein binding;IkappaB kinase activity

15450 protein kinase activity;ATP binding;protein serine/threonine kinase activity;catalytic activity ;protein binding;protein complex binding

12312 protein kinase activity;ATP binding;protein serine/threonine kinase activity;catalytic activity ;protein C-terminus binding ;protein binding;troponin I binding

3756 protein kinase activity;ATP binding;protein serine/threonine kinase activity;enzyme binding

16182 protein kinase activity;ATP binding;protein serine/threonine kinase activity;eukaryotic translation initiation factor 2alpha kinase activity;identical protein binding;protein binding

15347 protein kinase activity;ATP binding;protein serine/threonine kinase activity;GTP binding;protein binding

6746 protein kinase activity;ATP binding;protein serine/threonine kinase activity ;heme binding

4730, 5968 protein kinase activity;ATP binding;protein serine/threonine kinase activity;magnesium ion binding

16948 protein kinase activity;ATP binding;protein serine/threonine kinase activity;magnesium ion binding;cAMP -dependent protein kinase activity

4054 protein kinase activity;ATP binding;protein serine/threonine kinase activity;magnesium ion binding;identical protein binding;protein binding

1395, 3474, 4167, 15983 protein kinase activity;ATP binding;protein serine/threonine kinase activity;magnesium ion binding ;protein binding

14402 protein kinase activity;ATP binding;protein serine/threonine kinase activity;magnesium ion binding ;protein binding ;mitogen- activated protein kinase kinase kinase binding

17167 protein kinase activity;ATP binding;protein serine/threonine kinase activity;MAP kinase activity;JlIN kinase activity;protein binding;kinase activity

2278, 6079 protein kinase activity;ATP binding;protein serine/threonine kinase activity;MAP kinase activity;protein binding

4282 protein kinase activity;ATP binding;protein serine/threonine kinase activity;MAP kinase activity;protein binding;magnesium ion binding

2958 protein kinase activity;ATP binding;protein serine/threonine kinase activity;MAP kinase activity;transcription factor binding ;protein binding;ubiquitin protein ligase binding;magnesium ion binding; SH2 domain binding

8686, 14547 protein kinase activity;ATP binding;protein serine/threonine kinase activity;MAP kinase kinase kinase activity ;magnesium ion binding;identical protein binding;protein binding

4746 protein kinase activity;ATP binding;protein serine/threonine kinase activity;MAP kinase kinase kinase activity ;protein homodimerization activity;identical protein binding;protein binding

3379, 41 12, 11003, 11753 protein kinase activity;ATP binding;small GTPase regulator

activity ;protein serine/threonine kinase activity;protein binding

13696 protein kinase activity;ATP binding;transmembrane receptor protein tyrosine kinase activity;protein serine/threonine kinase activity ;protein tyrosine kinase activity;platelet-derived growth factor receptor binding;platelet-derived growth factor beta-receptor activity;growth factor binding ;receptor binding ;protein binding;platelet-derived growth factor binding

13578 protein kinase activity;ATP binding;transmembrane receptor protein tyrosine kinase activity;protein serine/threonine kinase activity ;protein tyrosine kinase activity;protein binding

3991 protein kinase activity;protein binding;ATP binding;kinase

activity;chemorepellent activity;ephrin receptor activity ;protein serine/threonine kinase activity;protein tyrosine kinase activity

5938 protein kinase activity;protein binding;RNA polymerase II carboxy- terminal domain kinase activity;DNA-dependent ATPase activity

9493 protein kinase activity;protein serine/threonine kinase activity;protein tyrosine kinase activity;ATP binding

4416 protein kinase activity ;protein tyrosine kinase activity ;non-membrane spanning protein tyrosine kinase activity ;protein binding;ATP binding;kinase activity;protein domain specific binding;nucleotide binding;transferase activity ;protein serine/threonine kinase activity ;receptor binding;SH2 domain binding

5662, 13560 protein kinase activity ;protein tyrosine kinase activity;transmembrane receptor protein tyrosine kinase activity;protein binding;ATP binding;kinase activity;cytokine binding ;protein homodimerization activity ;protein serine/threonine kinase activity

14849 protein kinase activity;protein tyrosine kinase activity;transmembrane receptor protein tyrosine kinase activity;protein binding;ATP binding;kinase activity;protein serine/threonine kinase activity

14935 protein kinase activity;protein tyrosine kinase activity ;transmembrane receptor protein tyrosine kinase activity;receptor signaling protein tyrosine kinase activity;insulin receptor activity;insulin-like growth factor receptor binding ;protein binding;ATP binding;GTP binding;kinase activity;insulin-like growth factor I binding;insulin-like growth factor II binding;SH2 domain binding;phosphatidylinositol 3-kinase binding;insulin binding;insulin receptor substrate binding;PTB domain binding;nucleotide binding;receptor activity;transferase activity;insulin- like growth factor binding;protein serine/threonine kinase activity

2060 protein kinase activity;transmembrane receptor protein tyrosine kinase activity;protein binding;ATP binding;kinase activity;Wnt-protein binding; Wnt receptor activity ;protein serine/threonine kinase activity ;protein tyrosine kinase activity;frizzled binding ;transmembrane receptor activity

11472 protein kinase binding ;protein binding

9388 protein kinase C activity;calcium channel regulator activity;protein binding;kinase activity;metal ion binding ;protein kinase activity;ATP binding;protein serine/threonine kinase activity

6840, 14556 protein methyltransferase activity 9196 protein methyltransferase activity;protein homodimerization

activity;protein heterodimerization activity;identical protein binding;protein binding;S-adenosylmethionine-dependent

methyltransferase activity;histone-arginine N-methyltransferase activity ;protein-arginine omega-N asymmetric methyltransferase activity ;protein-arginine omega-N monomethyltransferase activity

5064 protein N-terminal asparagine amidohydrolase activity

2891 protein N-terminus binding ;protein binding

13299 protein phosphatase inhibitor activity

131 13, 14292, 16430 protein phosphatase type 2A regulator activity;binding;protein binding

8765 protein phosphatase type 2A regulator activity;protein binding

9620 protein prenyltransferase activity

14242 protein prenyltransferase activity;acetylcholine receptor regulator activity

16308 protein serine/threonine kinase activity

3566 protein serine/threonine kinase activity;ATP binding

6728 protein serine/threonine kinase activity;ATP binding;protein binding

5225, 5308 protein serine/threonine kinase activity;ATP binding;protein binding;zinc ion binding

4356 protein serine/threonine kinase activity;ATP binding;protein tyrosine kinase activity;catalytic activity

586, 15162 protein serine/threonine phosphatase activity;catalytic activity

5294 protein serine/threonine phosphatase activity;catalytic

activity;magnesium ion binding ;phosphoprotein phosphatase activity;manganese ion binding;signal transducer activity;protein binding

7709, 15572 protein serine/threonine phosphatase activity;catalytic activity ;protein binding

11979 protein serine/threonine/tyrosine kinase activity peptidase activity; serine- type peptidase activity;hydrolase activity;metal ion binding

10077, 10686 protein transporter activity;binding

351 1, 7799, 1031 1 protein transporter activity;binding;protein binding

9090 protein transporter activity;binding;protein binding;histone binding

2022 protein transporter activity;binding;protein binding;tRNA binding

10876 protein transporter activity;importin-alpha export receptor

activity;binding;protein binding

13670 protein transporter activity;P-P -bond-hydrolysis-driven protein

transmembrane transporter activity

9209 protein transporter activity;protein binding

12585 protein tyrosine kinase activator activity;SH3 domain binding;SH2 domain binding

2692, 10475, 14135 protein tyrosine phosphatase activity;phosphatase activity

2824 protein tyrosine phosphatase activity;phosphatase activity;beta-catenin binding;alpha-catenin binding;delta-catenin binding;gamma-catenin binding;protein binding;cadherin binding;alpha-actinin binding;thiolester hydrolase activity

6688 protein tyrosine phosphatase activity;phosphatase

activity;phosphatidylinositol-3 -phosphatase activity 10393 protein tyrosine phosphatase activity;phosphatase

activity;phosphotyrosine binding ;protein binding;ATPase binding

13787 protein tyrosine phosphatase activity;phosphatase activity ;protein binding

14341 protein tyrosine phosphatase activity;phosphatase activity ;protein binding;cytoskeletal protein binding;non-membrane spanning protein tyrosine phosphatase activity

10307 protein tyrosine phosphatase activity;phosphatase activity ;protein binding;protein homodimerization activity

15425 protein tyrosine phosphatase activity;phosphatase activity ;protein binding;protein kinase binding

8985 protein tyrosine phosphatase activity;phosphatase activity;protein tyrosine/serine/threonine phosphatase activity

6691 protein tyrosine phosphatase activity;phosphatase activity;protein tyrosine/serine/threonine phosphatase activity ;protein binding

14019, 15573 protein tyrosine phosphatase activity;phosphatase activity;transferase activity ;protein binding

9028 protein tyrosine phosphatase activity;phosphatase

activity ;transmembrane receptor protein tyrosine phosphatase activity;protein binding;cadherin binding

17019, 17022 protein tyrosine phosphatase activity ;protein binding

14408 protein tyrosine phosphatase activity ;protein tyrosine/serine/threonine phosphatase activity;phosphatase activity

5899 protein tyrosine phosphatase activity ;protein tyrosine/serine/threonine phosphatase activity;phosphatase activity;identical protein binding;protein binding

2560 protein tyrosine/serine/threonine phosphatase activity;phosphatase activity

15675 protein tyrosine/serine/threonine phosphatase activity;phosphatase activity;mRNA guanylyltransferase activity;DNA ligase (ATP) activity;ATP binding;protein tyrosine phosphatase activity

12055 protein tyrosine/serine/threonine phosphatase activity;phosphatase activity;phosphoprotein phosphatase activity; actin binding

15760 protein tyrosine/serine/threonine phosphatase activity;phosphatase activity ;protein binding

4385, 71 12, 10312, 121 10 protein tyrosine/serine/threonine phosphatase activity;phosphatase activity ;protein tyrosine phosphatase activity

2760 protein tyrosine/serine/threonine phosphatase activity;phosphatase activity ;protein tyrosine phosphatase activity;MAP kinase phosphatase activity;phosphoprotein phosphatase activity

3089, 13565 protein tyrosine/serine/threonine phosphatase activity;phosphatase activity ;protein tyrosine phosphatase activity ;protein binding

16365 protein tyrosine/serine/threonine phosphatase activity;phosphatase activity ;protein tyrosine phosphatase activity;protein serine/threonine phosphatase activity;phosphatidylinositol-3-phosphatase activity;PDZ domain binding;protein binding;enzyme binding;phosphatidylinositol- 3,4-bisphosphate 3-phosphatase activity;inositol-l, 3,4,5- tetrakisphosphate 3-phosphatase activity;phosphatidylinositol-3,4,5- trisphosphate 3-phosphatase activity

exchange factor activity; 1 -phosphatidylinositol binding 5524 Rho guanyl-nucleotide exchange factor activity;guanyl-nucleotide exchange factor activity;DNA binding;Ras guanyl-nucleotide exchange factor activity ;protein binding

10521 Rho guanyl-nucleotide exchange factor activity;guanyl-nucleotide exchange factor activity ;protein binding

3380 Rho guanyl-nucleotide exchange factor activity;guanyl-nucleotide exchange factor activity ;protein binding;Rac guanyl-nucleotide exchange factor activity;Rac GTPase activator activity

6037 Rho guanyl-nucleotide exchange factor activity;guanyl-nucleotide exchange factor activity ;protein binding ;recep tor signaling protein activity

3593 Rho guanyl-nucleotide exchange factor activity;guanyl-nucleotide exchange factor activity ;protein binding ;recep tor signaling protein activity;phospholipid binding;ephrin receptor binding

7618 Rho guanyl-nucleotide exchange factor activity;guanyl-nucleotide exchange factor activity ;protein domain specific binding ;protein binding

9633 Rho guanyl-nucleotide exchange factor activity;guanyl-nucleotide exchange factor activity ;protein homodimerization activity;Rab guanyl- nucleotide exchange factor activity;Rac guanyl-nucleotide exchange factor activity;Rab GTPase binding;protein serine/threonine kinase activator activity;protein binding

9520, 10259 Rho guanyl-nucleotide exchange factor activity;guanyl-nucleotide exchange factor activity ;protein serine/threonine kinase activity;GTPase activator activity

10847 Rho guanyl-nucleotide exchange factor activity;guanyl-nucleotide exchange factor activity;signal transducer activity;protein binding;Rho GTPase binding

2659 Rho guanyl-nucleotide exchange factor activity;protein binding

15657 Rho guanyl-nucleotide exchange factor activity;protein binding;nucleic acid binding;methyltransferase activity;G-protein-coupled receptor binding

6708 Rho guanyl-nucleotide exchange factor activity;protein kinase

activity;ATP binding;protein serine/threonine kinase activity

6735 Rho guanyl-nucleotide exchange factor activity;Rho GTPase

binding;guanyl-nucleotide exchange factor activity;GTP binding;GTPase binding

11175 Rho guanyl-nucleotide exchange factor activity;signal transducer

activity ;protein binding

12228 Rho guanyl-nucleotide exchange factor activity;zinc ion binding;protein binding

16423 ribonuclease activity;ribonuclease P activity ;protein binding

10374 ribonuclease activity; structural constituent of ribosome

5673 ribonucleoside-diphosphate reductase activity;oxidoreductase

activity;transition metal ion binding

5942 ribulose-phosphate 3-epimerase activity;catalytic activity ;protein binding

3068, 7746, 9362, 13674, 17102 RNA binding

16871 RNA binding;ATP binding;transferase activity;double-stranded RNA binding;DNA binding;protein binding;transcription regulator activity

6571 RNA binding;DNA binding;hydrolase activity, acting on ester bonds 7257 RNA binding;endoribonuclease activity;5'-3' exonuclease activity;protein binding

11302, 16060 RNA binding;neurorransmitter:sodium symporter activity

2428, 11255 RNA binding;nucleic acid binding

3187, 4116, 4581, 13656, 14435, RNA binding;protein binding

15391

7072, 9630, 13828 RNA binding;pseudouridine synthase activity

4857 RNA binding;pseudouridine synthase activity;protein binding

16269, 16354 RNA binding;pseudouridine synthase activity;protein binding;DNA binding;ligand-dependent nuclear receptor transcription coactivator activity

13450 RNA binding;ribonuclease activity

4071, 13211 RNA binding;ribonuclease activity;guanyl-nucleotide exchange factor activity ;protein binding

10833 RNA binding;ribonuclease activity;nucleotide binding;nucleoside- triphosphatase activity ;nucleic acid binding;zinc ion binding

8140 RNA binding;ribonuclease III activity;double-stranded RNA

binding;protein binding

6666 RNA binding;RNA methyl transferase activity;methylrransferase activity

14682 RNA binding;S-adenosylmethionine-dependent methyltransferase activity;methyltransferase activity

727, 3504, 6048 RNA binding;structural constituent of ribosome

16610, 16923 RNA binding;translation initiation factor activity ;protein binding

1968 RNA polymerase binding

4676 RNA polymerase II transcription factor activity;ATP binding;catalytic activity ;protein binding

4023 RNA polymerase II transcription factor activity;catalytic activity ;beta- amyloid binding;protein binding

7810 RNA polymerase II transcription factor activity;DNA binding;protein N- terminus binding;transcription coactivator activity;protein

binding;thyroid hormone receptor binding;vitamin D receptor binding

2541, 2715, 6725 RNA polymerase II transcription factor activity ;protein binding

8475 RNA polymerase II transcription mediator activity

1238, 6690 RNA polymerase II transcription mediator activity;protein binding

10569 RNA polymerase II transcription mediator activity ;receptor

activity;transcription cofactor activity;transcription activator activity;transcription coactivator activity ;thyroid hormone receptor binding

14255 Roundabout binding

6580 rRNA (adenine-N6,N6-)-dimethyltransferase activity;rRNA

methyltransferase activity ;protein binding

9891 rRNA (adenine-N6,N6-)-dimethyltransferase activity;rRNA

methyltransferase activity;rRNA (adenine) methyltransferase activity

5873 rRNA N-glycosylase activity;protein binding

8683 scavenger receptor activity 4129 scavenger receptor activity;copper ion binding;oxidoreductase activity, acting on the CH-NH2 group of donors, oxygen as acceptor

9934 scavenger receptor activity;polysaccharide binding;catalytic

activity ;nucleic acid binding;hydrolase activity;metal ion binding

8384 scavenger receptor activity ;protein binding;ferritin receptor activity

11248 scavenger receptor activity;serine-type endopeptidase activity;catalytic activity

1197 scavenger receptor activity;serine-type endopeptidase activity;catalytic activity;peptidase activity

2928, 2953 selenium binding

10346 semaphorin receptor activity

3657 sequence-specific DNA binding

6043, 7840, 9705, 15073 sequence-specific DNA binding transcription factor activity

5876 sequence-specific DNA binding transcription factor activity;DNA

binding;protein domain specific binding;transcription factor binding;protein binding

13577 sequence-specific DNA binding transcription factor activity;DNA

binding;sequence-specific DNA binding

16745 sequence-specific DNA binding transcription factor activity;iron ion binding;nucleotide binding;nucleoside-triphosphatase

activity;microtubule motor activity;ATP binding;ATPase activity

2056 sequence-specific DNA binding transcription factor activity;nucleoside transmembrane transporter activity

5152, 5160, 8464 sequence-specific DNA binding transcription factor activity;protein binding

1256 sequence-specific DNA binding transcription factor activity ;protein binding;beta-catenin binding;type I transforming growth factor beta receptor binding;promoter binding ;ubiquitin protein ligase binding;transforming growth factor beta receptor, inhibitory cytoplasmic mediator activity;I-SMAD binding;activin binding;collagen binding

15058 sequence-specific DNA binding transcription factor activity ;protein binding;DNA binding;transcription activator activity

13442 sequence-specific DNA binding transcription factor activity ;protein binding;zinc ion binding;transcription activator activity;promoter binding;transcription repressor activity ;protein self-association

600, 4878, 6968, 8741, 9427, sequence-specific DNA binding transcription factor activity;sequence- 111 16, 11671, 13036, 13379, specific DNA binding

14628

6249 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;binding;DNA binding;transcription repressor activity

5304 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;DNA binding

935, 13766 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;DNA binding;protein dimerization activity

13365 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;DNA binding;protein dimerization

activity ;promoter binding;cAMP response element binding 11840 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;DNA binding;protein dimerization activity ;protein binding;transcription repressor activity

12806 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;DNA binding;protein dimerization

activity;transcription repressor activity;identical protein binding;protein binding

15195 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;DNA binding;transcription factor binding ;protein binding;enzyme binding;transcription activator activity

14258, 17069 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;DNA binding;transcription regulator activity

2695 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;DNA binding;transcription regulator activity;transcription repressor activity;chromatin binding

12102 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;nucleic acid binding;zinc ion binding;chromatin binding;transcription repressor activity ;protein binding

15729 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;nucleic acid binding;zinc ion binding;protein binding

3970, 3975, 6747 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;nucleotide binding;protein binding

10810 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;promoter binding;transcription activator activity

10575, 14596, 16154 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;protein binding

9662 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;protein binding;DNA binding;protein heterodimerization activity;sequence-specific enhancer binding RNA polymerase II transcription factor activity

11774 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;protein kinase binding;transcription activator activity;protein binding;DNA binding

7088, 11782 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;sphingosine N-acyltransferase activity

14089 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;transcription activator activity

9019, 14169 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;transcription activator activity;transcription regulator activity;protein binding

11583 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;transcription regulator activity;DNA binding;transcription repressor activity

9081 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;transcription repressor activity ;protein binding;DNA binding

7999, 12842 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;zinc ion binding 11402 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;zinc ion binding;DNA binding ;protein binding

5119 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;zinc ion binding;DNA binding ;protein dimerization activity ;protein binding

1357, 12017 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;zinc ion binding ;protein binding

8163 sequence-specific DNA binding transcription factor activity;sequence- specific DNA binding;zinc ion binding ;protein binding;chromatin binding

12996 sequence-specific DNA binding transcription factor activity;steroid hormone receptor activity;zinc ion binding;sequence-specific DNA binding

12593, 14704 sequence-specific DNA binding transcription factor activity;steroid hormone receptor activity;zinc ion binding;sequence-specific DNA binding;metal ion binding;protein binding;transcription regulator activity;DNA binding;transcription activator activity ;protein heterodimerization activity

6076 sequence-specific DNA binding transcription factor activity;steroid hormone receptor activity;zinc ion binding;sequence-specific DNA binding;protein binding;DNA binding

11742 sequence-specific DNA binding transcription factor activity;steroid hormone receptor activity;zinc ion binding;sequence-specific DNA binding;transcription activator activity;RNA polymerase II core promoter proximal region sequence-specific DNA binding transcription factor activity involved in positive regulation of transcription

7546 sequence-specific DNA binding transcription factor activity;transcription activator activity ;protein binding;DNA binding;sequence-specific DNA binding;promoter binding

3829 sequence-specific DNA binding transcription factor activity;transcription initiation factor activity;DNA binding;NF-kappaB binding

12306, 13533, 13684, 13919, sequence-specific DNA binding transcription factor activity;zinc ion 15206, 15893, 16255, 16746 binding

11686 sequence-specific DNA binding transcription factor activity;zinc ion binding;DNA binding;nucleotide binding;nucleoside-triphosphatase activity

5771 sequence-specific DNA binding transcription factor activity;zinc ion binding;nucleic acid binding;protein binding;ATP binding

9061, 11690 sequence-specific DNA binding transcription factor activity;zinc ion binding;palmitoyltransferase activity

7705, 17029 sequence-specific DNA binding transcription factor activity;zinc ion binding;protein binding

8695 sequence-specific DNA binding transcription factor activity;zinc ion binding;sequence-specific DNA binding

12655 sequence-specific DNA binding transcription factor activity;zinc ion binding;sequence-specific DNA binding;DNA binding

9997, 10319 sequence-specific DNA binding transcription factor activity;zinc ion binding;sequence-specific DNA binding;DNA binding;protein binding

5182 signal transducer activity ;actin binding ;protein domain specific binding

9160 signal transducer activity;GTPase regulator activity;protein

binding;GTPase activator activity;receptor signaling protein activity

4916, 9289 signal transducer activity;guanyl nucleotide binding

11491 signal transducer activity;guanyl nucleotide binding;GTP binding

9950 signal transducer activity;guanyl nucleotide binding;GTP binding ;protein binding

3703 signal transducer activity;guanyl nucleotide binding;protein binding

9416 signal transducer activity ;protein binding

6107, 6513 signal transducer activity ;protein binding;frizzled binding;protease binding;beta-catenin binding;receptor binding;protein heterodimerization activity

16050 signal transducer activity ;protein binding;guanyl nucleotide binding

9497, 12792 signal transducer activity ;protein binding;identical protein binding

11461 signal transducer activity ;protein binding;phosphatidylinositol binding

4286 signal transducer activity ;protein binding;WW domain binding

12425, 15289 signal transducer activity ;protein kinase activity;ATP binding;protein serine/threonine kinase activity

11022 signal transducer activity ;protein kinase activity;ATP binding ;protein serine/threonine kinase activity;G-protein coupled receptor kinase activity

8254 signal transducer activity ;protein kinase activity;ATP binding;protein serine/threonine kinase activity;protein binding

9905 signal transducer activity ;recep tor activity;G-protein coupled receptor activity

933 signal transducer activity ;recep tor activity;G-protein coupled receptor activity;cannabinoid receptor activity ;protein binding

3295 signal transducer activity ;recep tor activity;transmembrane receptor activity;G-protein coupled receptor activity;hydrogen ion transmembrane transporter activity;protein binding; Wnt receptor activity; Wnt-protein binding

15288 signal transducer activity ;recep tor activity ;transmembrane receptor activity;G-protein coupled receptor activity;Wnt-protein binding;protein binding;PDZ domain binding

8948 signal transducer activity ;recep tor binding

11329 signal transducer activity;transcription regulator activity

10262 signal transducer activity;transcription regulator activity;transcription coactivator activity;protein N-terminus binding;histone acetyltransferase activity ;protein binding;ligand-dependent nuclear receptor binding;nuclear hormone receptor binding;chromatin binding

4109 signal transducer activity;two-component response regulator

activity;3',5'-cyclic-nucleotide phosphodiesterase activity;catalytic activity

14746 signal transducer activity;two-component sensor activity;ion channel activity

13692 signal transducer activity;two-component sensor activity;ion channel activity ;nucleotide binding 7756 signal transducer activity;two-component sensor activity;ion channel activity ;protein binding

13795 single-stranded DNA binding

3202, 5752, 9798, 12150, 12579, small conjugating protein ligase activity

13852, 15128, 15366, 16525

1290 small conjugating protein ligase activity;DNA binding ;protein

binding;transcription regulator activity

6821 small conjugating protein ligase activity;oxidoreductase activity;catalytic activity

7407, 14881 small conjugating protein ligase activity ;protein binding

5760 small conjugating protein ligase activity;ubiquitin-protein ligase

activity;ISG15 ligase activity

9246, 11363 small conjugating protein ligase activity;ubiquitin-protein ligase

activity ;protein binding

15078, 15081 SNAP receptor activity

17014 SNAP receptor activity;protein binding

6280 SNAP receptor activity;protein binding;SNARE binding;calcium channel inhibitor activity

10859 sodiunxdicarboxylate symporter activity;aminopeptidase activity;L- glutamate transmembrane transporter activity

7816 sodiunxdicarboxylate symporter activity ;protein binding;amino acid binding;glutamate binding;high-affmity glutamate transmembrane transporter activity

10030 sodiunxdicarboxylate symporter activity ;transporter activity;chloride channel activity;L-threonine transmembrane transporter activity;L-serine transmembrane transporter activity;L-proline transmembrane transporter activity;L-cystine transmembrane transporter activity;L-alanine transmembrane transporter activity;L-hydroxyproline transmembrane transporter activity;neutral amino acid transmembrane transporter activity

5965, 13497 sodiunxhydrogen antiporter activity;solute:hydrogen antiporter activity

4428, 4849 sphingomyelin phosphodiesterase activity

9302, 13676 sphingosine N-acyltransferase activity

8354 sterol binding;DNA binding;binding

3477 sterol esterase activity;lipase activity

13239 structural constituent of cytoskeleton;protein C-terminus

binding;identical protein binding;protein binding

8938 structural constituent of nuclear pore

2281, 4254, 4451, 4722, 5635, structural constituent of ribosome

5815, 5851, 6975, 8337, 8813,

8886, 11957, 12278, 13634,

14633, 14758, 15039, 15252,

15705, 16946, 16962, 16988,

17149

3702 structural constituent of ribosome;caspase activator activity;translation activator activity

877, 2478, 3442, 3921, 3979, structural constituent of ribosome;protein binding

5607, 8321, 9503, 9889, 1 1180,

12077, 12401, 15868, 17118 2430 structural constituent of ribosome;protein N-terminus binding

7339, 16595 structural constituent of ribosome;RNA binding;protein binding

7783, 12247 structural constituent of ribosome;rRNA binding

6918, 14327 structural molecule activity

9083 structural molecule activity;beta-catenin binding;gamma-catenin

binding;protein binding;vinculin binding;cadherin binding;actin filament binding

16397 structural molecule activity;metal ion binding;iron-sulfur cluster binding

6101 structural molecule activity ;protein binding

2977 structural molecule activity ;protein binding;binding

7326 succinate dehydrogenase activity;protein binding;succinate

dehydrogenase (ubiquinone) activity;electron carrier

activity;oxidoreductase activity;oxidoreductase activity, acting on the CH-CH group of donors;flavin adenine dinucleotide binding

13529 sugar binding;glutamine-fructose-6-phosphate transaminase

(isomerizing) activity;transporter activity

10279 suganhydrogen symporter activity

3539 suganhydrogen symporter activity;phosphatase activity ;protein binding

4536, 14979 suganhydrogen symporter activity;protein binding

6460 sulfate adenylyltransferase (ATP) activity;ATP binding;kinase

activity;transferase activity, transferring phosphorus -containing groups;adenylylsulfate kinase activity

643, 1371, 1876, 2510, 8309, sulfotransferase activity

8669, 15449, 16866, 17092

10967 sulfotransferase activity; flavonol 3-sulfotransferase activity

15223 sulfotransferase activity;N-acetylgalactosamine 4-sulfate 6-0- sulfotransferase activity;3'-phosphoadenosine 5'-phosphosulfate binding

7159 sulfotransferase activity;protein binding; [heparan sulfate] -glucosamine 3- sulfotransferase 1 activity

1073 sulfotransferase activity;protein binding;chondroitin 4-sulfotransferase activity

2714 sulfuric ester hydrolase activity;catalytic activity

10385 sulfuric ester hydrolase activity;catalytic activity;arylsulfatase activity

3795 syntaxin binding ;neurotransmitter transporter activity

13691 telomeric DNA binding;protein binding

2899 thiamine diphosphokinase activity;ATP binding

8467 thiol oxidase activity

9206 thiol oxidase activity;protein binding

1719 thiosulfate sulfurtransferase activity

10439 threonine-type endopeptidase activity;endopeptidase activity

10252 threonine-type endopeptidase activity;endopeptidase

activity;lipopolysaccharide binding

4357 threonine-type endopeptidase activity;endopeptidase activity ;protein binding

6212 thymidylate kinase activity;ATP binding;cytidylate kinase activity;UMP kinase activity

16346 thymidylate synthase activity

9355, 13909 toxin binding

8497, 14876 transaminase activity;pyridoxal phosphate binding

4223 transcription activator activity

860 transcription activator activity;DNA binding ;protein complex

binding;protein binding

12604 transcription activator activity ;protein binding

14448 transcription activator activity;transcription coactivator

activity;chromatin binding;protein binding;ligand-dependent nuclear receptor transcription coactivator activity;enzyme binding;thyroid hormone receptor binding

13626 transcription coactivator activity;histone acetyltransferase activity

4962 transcription coactivator activity;ligand-dependent nuclear receptor transcription coactivator activity

10345 transcription coactivator activity ;protein binding

15872 transcription coactivator activity;protein binding;androgen receptor binding;ribonucleoprotein binding

16762 transcription coactivator activity;protein binding;transcription regulator activity

9910 transcription coactivator activity ;protein kinase binding;protein

binding;peptide antigen binding

9429 transcription coactivator activity;TBP-class protein binding

16329 transcription cofactor activity;histone acetyltransferase activity;zinc ion binding;protein binding;transcription coactivator activity;transcription factor binding;p53 binding;acetyltransferase activity ;MyoD binding;DNA binding;chromatin binding

12670 transcription cofactor activity;transcription coactivator activity;histone acetyltransferase activity;protein binding;N-acetyltransferase activity;transcription factor binding;acetyltransferase activity;histone deacetylase binding;protein kinase binding;cyclin-dependent protein kinase inhibitor activity;lysine N-acetyltransferase activity

16327 transcription corepressor activity;p53 binding ;transcription repressor activity ;protein binding;protein-lysine N-methyltransferase activity;histone-lysine N-methyltransferase activity

6446 transcription corepressor activity ;protein N-terminus binding;protein binding

15028 transcription corepressor activity;sequence-specific DNA binding transcription factor activity;protein C-terminus binding;transcription activator activity;protein N-terminus binding;transcription coactivator activity ;protein binding

13769 transcription factor binding

15493 transcription factor binding;protein binding

8255, 9844, 13068 transcription regulator activity

3341 transcription regulator activity;DNA binding

3243 transcription regulator activity;DNA binding;histone deacetylase binding;transcription repressor activity

7374 transcription regulator activity;DNA binding;protein binding 17021 transcription regulator activity;DNA binding;protein

binding;transcription repressor activity

6730, 13188 transcription regulator activity ;protein binding

11444 transcription regulator activity;protein binding;calcium ion binding

4020 transcription regulator activity;protein binding;DNA binding;sequence- specific DNA binding transcription factor activity;chromatin binding

8320 transcription regulator activity;protein binding;sequence-specific DNA binding;sequence-specific DNA binding transcription factor activity;protein dimerization activity

12204 transcription regulator activity;sequence-specific DNA binding

transcription factor activity;protein binding

3331 transcription regulator activity;transcription coactivator activity;histone acetyltransferase activity

3336 transcription termination factor activity;transcription corepressor

activity ;protein binding

3536 transferase activity, transferring acyl groups other than amino-acyl groups ;catalytic activity; acetyl -Co A C-acyltransferase activity

15196 transferase activity, transferring acyl groups other than amino-acyl groups ;catalytic activity ;protein binding

15562 transferase activity, transferring acyl groups, acyl groups converted into alkyl on transfer

9250, 16283 transferase activity, transferring alkyl or aryl (other than methyl) groups

13628, 13640 transferase activity, transferring alkyl or aryl (other than methyl)

groups;poly(ADP-ribose) glycohydrolase activity

2995, 4411, 6268, 7155, 16493 transferase activity, transferring glycosyl groups

14526 transferase activity, transferring glycosyl groups;UDP-xylosyltransferase activity

2107, 4669, 9297 transferase activity, transferring hexosyl groups

5083 transferase activity, transferring hexosyl

groups;acetylgalactosaminyltransferase activity

5944, 8156 transferase activity, transferring hexosyl groups;alpha-l,3- mannosyltransferase activity

6201 transferase activity, transferring hexosyl groups;glucuronosyl-N- acetylgalactosaminyl-proteoglycan 4-beta-N- acetylgal actos aminyltrans ferase activity

14938 transferase activity, transferring hexosyl groups;glucuronosyltransferase activity;peptidoglycan glycosyltransferase

activity;acetylgalactosaminyltransferase activity;glucuronosyl-N- acetylgalactosaminyl-proteoglycan 4-beta-N- acetylgal actos aminyltrans ferase

activity;glucuronylgalactosylproteoglycan 4-beta-N- acetylgal actos aminyltrans ferase activity

771 1, 16017 transferase activity, transferring nitrogenous groups;pyridoxal phosphate binding

10249 transferase activity, transferring nitrogenous groups;pyridoxal phosphate binding;L-alanine:2-oxoglutarate aminotransferase activity

6230 transferase activity, transferring nitrogenous groups;pyridoxal phosphate binding;serine C-palmitoyltransferase activity ;protein binding 3172 transferase activity, transferring phosphorus -containing groups

1466 transferase activity, transferring phosphorus -containing groups ;dolichol kinase activity

2852 transferase activity;dynein binding

11600 transferase activity;transferase activity, transferring nitrogenous

groups ;pyridoxal phosphate binding;serine C-palmitoyltransferase activity

9949, 12115 trans-hexaprenyltranstransferase activity ;protein heterodimerization activity;trans-octaprenyltranstransferase activity

8941 translation elongation factor activity;signal transducer activity;protein binding

10458, 14734 translation initiation factor activity;actin binding;Rho GTPase

binding;binding

1201, 2671 translation initiation factor activity;protein binding

7312, 9722 translation release factor activity;translation release factor activity, codon specific

3174 transmembrane receptor activity

8014, 11762, 13430 transmembrane receptor activity

14943 transmembrane receptor activity ;binding;protein binding

4013 transmembrane receptor activity ;protein binding

1622, 14310 transmembrane receptor activity ;protein kinase binding;protein

binding; Wnt-protein binding

11078 transmembrane receptor protein serine/threonine kinase

activity;transforming growth factor beta receptor activity ;protein kinase activity;ATP binding;protein serine/threonine kinase activity

5730 transmembrane receptor protein serine/threonine kinase

activity;transforming growth factor beta receptor activity ;protein kinase activity;ATP binding;protein serine/threonine kinase activity; structural constituent of ribosome;activin receptor activity, type I

839, 121 1, 3930, 4386, 4610, transporter activity

461 1, 6744, 6847, 7157, 9596,

12804, 13125, 14113, 15210,

16440

7715 transporter activity;G-protein coupled receptor activity;protein binding

4717 transporter activity;lipid binding;sugar:hydrogen symporter

activity;calcium ion binding

12261 transporter activity; sugar binding;copper ion binding

6661 triglyceride lipase activity

15772 tRNA (guanine-N7-)-methyltransferase activity;methyltransferase activity

5837 tRNA binding;phenylalanine-tRNA ligase activity;ATP binding

6253 tRNA binding;protein homodimerization activity;cell surface binding

2078, 7933 tRNA dihydrouridine synthase activity; flavin adenine dinucleotide binding

8659 tropomyosin binding

10310 tubulin binding;protein binding

961 1, 15664 tubulin-tyrosine ligase activity 7678 two-component response regulator activity;3',5'-cyclic-nucleotide

phosphodiesterase activity;catalytic activity

11999 type 5 melanocortin receptor binding;type 4 melanocortin receptor binding;type 3 melanocortin receptor binding;adrenocorticotropin hormone receptor binding;type 1 melanocortin receptor binding

1372, 7293, 13262 ubiquinol-cytochrome-c reductase activity

5150 ubiquitin binding

1991, 10293 ubiquitin protein ligase binding

8732 ubiquitin protein ligase binding;cyclin binding;ubiquitin-protein ligase activity ;protein binding

9698, 10964 ubiquitin protein ligase binding;protein binding

15248 ubiquitin protein ligase binding;protein kinase binding;protein

homodimerization activity;protein binding

8768, 14755, 15726 ubiquitin thiolesterase activity

5844 ubiquitin thiolesterase activity;binding

8113 ubiquitin thiolesterase activity;calcium ion binding

15528 ubiquitin thiolesterase activity;calcium ion binding;calcium-dependent phospholipid binding;binding

7386 ubiquitin thiolesterase activity;cysteine-type endopeptidase activity;G- protein-coupled receptor binding;zinc ion binding;protein binding

1141 ubiquitin thiolesterase activity;endopeptidase inhibitor

activity;proteasome binding;protein binding

6324 ubiquitin thiolesterase activity;protein binding

5546 ubiquitin thiolesterase activity;ubiquitin-specific protease

activity;cysteine-type endopeptidase activity ;protein binding

3058 ubiquitin thiolesterase activity;ubiquitin-specific protease activity;protein binding

2558 ubiquitin thiolesterase activity;zinc ion binding

9150 ubiquitin thiolesterase activity;zinc ion binding;cysteine-type

endopeptidase activity;G-protein-coupled receptor binding

3444 ubiquitin thiolesterase activity;zinc ion binding;histone binding

3572, 11758 ubiquitin thiolesterase activity;zinc ion binding;protein binding

5009 ubiquitin thiolesterase activity;zinc ion binding;ubiquitin-specific

protease activity;cysteine-type endopeptidase activity;transcription coactivator activity;ubiquitin binding;histone binding

9257, 13967 ubiquitin-protein ligase activity ;protein binding

2315 ubiquitin-protein ligase activity;protein binding;protein C-terminus binding;zinc ion binding;metal ion binding

8717 ubiquitin-protein ligase activity ;protein binding; specific transcriptional repressor activity;transcription activator activity;beta-catenin binding;protein phosphorylated amino acid binding

10121 ubiquitin-protein ligase activity;protein complex binding;protein binding

14084, 17009 ubiquitin-protein ligase activity ;protein homodimerization activity

13797 ubiquitin-protein ligase activity;ubiquitin conjugating enzyme

binding;enzyme binding

6137 ubiquitin-protein ligase activity;zinc ion binding

9224 zinc ion binding;DNA binding;sequence-specific DNA binding

transcription factor activity;sequence-specific DNA binding;transcription repressor activity ;protein binding

9093 zinc ion binding;DNA binding;transcription corepressor activity

17105 zinc ion binding;histone demethylase activity (H3-K9 specific) ;histone demethylase activity (H3-K27 specific);iron ion binding;oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors ;histone demethylase activity (H4- K20 specific);methylated histone residue binding;histone demethylase activity (H3-K36 specific)

1684 zinc ion binding;histone-lysine N-methyltransferase activity

10812 zinc ion binding;hydrogen ion transporting ATP synthase activity, rotational mechanism;proton-transporting ATPase activity, rotational mechanism

129, 533 zinc ion binding;hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides;NAD+ binding;NAD+ ADP- ribosyltransferase activity ;protein binding

5306 zinc ion binding;hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides;NAD+ binding;protein binding

9657 zinc ion binding;hydrolase activity;catalytic activity

14086 zinc ion binding;identical protein binding;protein binding;specific

transcriptional repressor activity

3936, 4072 zinc ion binding;metal ion binding ;protein binding

13981 zinc ion binding;methylated histone residue binding

8358 zinc ion binding;monooxygenase activity;electron carrier

activity;oxidoreductase activity

4010 zinc ion binding;nucleic acid binding

12274 zinc ion binding;nucleic acid binding transcription factor activity ;protein binding

10268 zinc ion binding;nucleic acid binding;DNA binding

101 14 zinc ion binding;nucleic acid binding;methyltransferase activity

7699 zinc ion binding;nucleotide binding ;protein binding

677 zinc ion binding;oxidoreductase activity

4169, 8999, 9439, 9544 zinc ion binding;oxidoreductase activity

5050 zinc ion binding;oxidoreductase activity;L-iditol 2-dehydrogenase activity;NAD+ or NADH binding

11094 zinc ion binding;p53 binding ;protein binding;protein-lysine N- methyltransferase activity;histone methyltransferase activity (H3-K36 specific);RNA polymerase II core binding

2018, 4249, 4763, 5212, 7662, zinc ion binding;protein binding

7670, 9912, 11033, 12446, 12713,

13438, 15491, 15492, 15634,

16713

16945 zinc ion binding;protein binding;CD40 receptor binding;ubiquitin-protein ligase activity ;sphingolipid binding

4437, 13498 zinc ion binding;protein binding;DNA binding;sequence-specific DNA binding transcription factor activity 14457 zinc ion binding;protein binding;DNA binding;transcription corepressor activity

9755 zinc ion binding;protein binding;DNA binding;transcription repressor activity;chromatin binding

14958 zinc ion binding;protein binding;electron carrier activity

9453 zinc ion binding;protein binding;GTP binding;ubiquitin-protein ligase activity;GTPase activity;GDP binding

8484 zinc ion binding;protein binding;K63 -linked polyubiquitin binding

6441 zinc ion binding;protein binding;methylated histone residue

binding;histone methyltransferase activity;chromatin binding

10950 zinc ion binding;protein binding;R-SMAD binding;co-SMAD binding

6506 zinc ion binding;protein binding;sequence-specific DNA binding;RNA polymerase II core promoter proximal region sequence-specific DNA binding;RNA polymerase II core promoter proximal region sequence- specific DNA binding transcription factor activity involved in positive regulation of transcription

1121 zinc ion binding;protein binding;signal transducer activity;ubiquitin- protein ligase activity

3015 zinc ion binding;protein binding;transcription corepressor activity

9992 zinc ion binding;protein binding;transcription regulator activity

12784 zinc ion binding;protein binding ;ubiquitin-protein ligase activity

3858 zinc ion binding;protein binding ;ubiquitin-protein ligase

activity;chromatin binding;p53 binding;transcription coactivator activity;estrogen response element binding;histone acetyl-lysine binding;sequence-specific DNA binding;ligand-dependent nuclear receptor binding;transcription repressor activity ;protein kinase activity

5397 zinc ion binding;protein binding ;ubiquitin-protein ligase activity;metal ion binding

14047 zinc ion binding;protein binding ;ubiquitin-protein ligase activity ;protein kinase binding;histone deacetylase binding;protein N-terminus binding;transcription repressor activity;mitogen-activated protein kinase kinase kinase binding;protein kinase B binding

9073 zinc ion binding;protein kinase C binding;protein kinase binding;protein binding;SH2 domain binding

8050 zinc ion binding;receptor activity;binding;oxidoreductase

activity;electron carrier activity;iron-sulfur cluster binding

13759 zinc ion binding;retinoic acid-responsive element binding

11088 zinc ion binding;transcription activator activity;translation initiation factor activity ;protein binding

5874 zinc ion binding;transcription coactivator activity

4789 zinc ion binding;transcription regulator activity;ligand-dependent nuclear receptor binding

15759 zinc ion binding;transcription regulator activity;translation initiation factor activity ;protein binding;transcription factor binding;thyroid hormone receptor binding;DNA binding

889, 13830 zinc ion binding;transcription repressor activity;protein binding

16358 zinc ion binding;transcription repressor activity;transcription corepressor activity;transcription activator activity;transcription factor binding 15384 zinc ion binding;ubiquitin thiolesterase activity ;protein binding

13337 zinc ion binding;ubiquitin thiolesterase activity;ubiquitin-specific protease activity;proline-rich region binding;protein kinase binding;protein binding

11313, 16942 zinc ion binding;ubiquitin-protein ligase activity

13160 zinc ion binding;ubiquitin-protein ligase activity;protein kinase

binding;PDZ domain binding;protein binding;kinase binding;chaperone binding

From the table, it is clear that various cellular processes can be affected by targeting the transcripts listed. In one embodiment, targeting constructs are designed to modulate the expression of one or more of the avian transcripts listed in Table 5. Where similar functions have been annotated, it is expected that one or more targeting

constructs, or a cocktail of targeting constructs may be designed to alter partial or entire signaling pathways associated with that particular function.

Example 3. Targeting Constructs: siRNA Design

Using the Avian transcripts identified in Example 1 , oligonucleotide design was carried out to identify siR As targeting the transcripts of Table 1. All sequences were obtained as described above using both the ENSEMBL datasets as well as the internal microRNA sequencing results.

All siRNA duplexes were designed with 100% identity to their respective transcripts with a total of 1 ,744,220 strands (872,1 10 duplexes) having SEQ ID NO 18040 (the first sense strand) through SEQ ID NO 1762259 (the last antisense strand). These are presented in in the sequence listing submitted with the present application and incorporated herein in its entirety.

siRNA Design and Specificity Prediction

Putative siRNA duplex designs were created in a sliding 19mer window across each avian transcript. The specificity of the 19mer oligonucleotide set was predicted from each sequence. The Avian Transcripts were used in a comprehensive search against the Anas platyrhynchos transcriptome (from the ENSEMBL dataset as well as novel microRNA transcripts identified via sequencing) using the BLASTN algorithm and a perl script was used to parse the alignment and generate a scorescore based on the position and number of mismatches between the siRNA and any potential 'off-target' transcript. The off-target score is weighted to emphasize differences in the 'seed' region of siRNAs, in positions 2-9 from the 5' end of the molecule. The off-target score is calculated as follows: mismatches between the oligo and the transcript are given penalties. A mismatch in the seed region in positions 2-9 of the oligo is given a penalty of 2.8;

mismatches in the putative cleavage sites 10 and 11 are given a penalty of 1.2, and all other mismatches a penalty of 1. The off-target score for each oligo-transcript pair is then calculated by summing the mismatch penalties. The lowest off-target score from all the oligo-transcript pairs is then determined and used in subsequent sorting of oligos. Both siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderate specific. In picking which oligonucleotides to synthesize, these scores can be sorted from high to low by the off-target score of the antisense strand and the best (lowest off-target score) oligonucleotide pairs may be synthesized for further study.

Example 4. Oligonucleotide synthesis

Source of reagents

Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Oligonucleotide Synthesis.

All oligonucleotides are synthesized on an AKTAoligopilot synthesizer.

Commercially available controlled pore glass solid support (dT-CPG, 500A, Prime Synthesis) and RNA phosphoramidites with standard protecting groups, 5'-0- dimethoxytrityl N6-benzoyl-2'-i-butyldimethylsilyl-adenosine-3'-0-N,N'-diisopropyl-2- cyanoethylphosphoramidite, 5'-0-dimethoxytrityl-N4-acetyl-2'-i-butyldimethylsilyl- cytidine-3 '-0-N,N'-diisopropyl-2-cyanoethylphosphoramidite, 5 '-0-dimethoxytrityl-N2- -isobutryl-2'-i-butyldimethylsilyl-guanosine-3'-0-N,N'-diisopropyl-2- cyanoethylphosphoramidite, and 5'-0-dimethoxytrityl-2'-i-butyldimethylsilyl-uridine-3'- 0-N,N'-diisopropyl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies) were used for the oligonucleotide synthesis. The 2'-F phosphoramidites, 5'-0- dimethoxytrityl-N4-acetyl-2'-fluro-cytidine-3'-0-N,N'-diisopropyl-2-cyanoethyl- phosphoramidite and 5 '-0-dimethoxytrityl-2'-fluro-uridine-3 '-0-N,N'-diisopropyl-2- cyanoethyl-phosphoramidite are purchased from (Promega). All phosphoramidites are used at a concentration of 0.2M in acetonitrile (CH₃CN) except for guanosine which is used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recycling time of 16 minutes is used. The activator is 5-ethyl thiotetrazole (0.75M, American International Chemicals); for the PO-oxidation iodine/water/pyridine is used and for the PS-oxidation PADS (2%) in 2,6-lutidine/ACN (1 : 1 v/v) is used.

3 '-ligand conjugated strands are synthesized using solid support containing the corresponding ligand. For example, the introduction of cholesterol unit in the sequence is performed from a hydroxyprolinol-cholesterol phosphoramidite. Cholesterol is tethered to /raTO-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain a hydroxyprolinol- cholesterol moiety. 5 '-end Cy-3 and Cy-5.5 (fiuorophore) labeled iRNAs are synthesized from the corresponding Quasar-570 (Cy-3) phosphoramidite are purchased from

Biosearch Technologies. Conjugation of ligands to 5'-end and or internal position is achieved by using appropriately protected ligand-phosphoramidite building block. An extended 15 min coupling of 0.1 M solution of phosphoramidite in anhydrous CH₃CN in the presence of 5-(ethylthio)-lH-tetrazole activator to a solid-support-bound

oligonucleotide. Oxidation of the internucleotide phosphite to the phosphate is carried out using standard iodine -water as reported (1) or by treatment with te/ -butyl

hydroperoxide/acetonitrile/water (10: 87: 3) with 10 min oxidation wait time conjugated oligonucleotide. Phosphorothioate is introduced by the oxidation of phosphite to phosphorothioate by using a sulfur transfer reagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucage reagent. The cholesterol phosphoramidite is synthesized in house and used at a concentration of 0.1 M in dichloro methane. Coupling time for the cholesterol phosphoramidite is 16 minutes.

Deprotection I (Nucleobase Deprotection) After completion of synthesis, the support is transferred to a 100 mL glass bottle (VWR). The oligonucleotide is cleaved from the support with simultaneous deprotection of base and phosphate groups with 80 mL of a mixture of ethanolic ammonia [ammonia: ethanol (3: 1)] for 6.5 h at 55°C. The bottle is cooled briefly on ice and then the ethanolic ammonia mixture is filtered into a new 250-mL bottle. The CPG is washed with 2 x 40 mL portions of ethanol/water (1 : 1 v/v). The volume of the mixture is then reduced to ~ 30 mL by roto-vap. The mixture is then frozen on dry ice and dried under vacuum on a speed vac.

Deprotection II (Removal of 2'-TBDMS group)

The dried residue is resuspended in 26 mL of triethylamine, triethylamine trihydro fluoride (TEA^»3HF) or pyridine-HF and DMSO (3:4:6) and heated at 60°C for 90 minutes to remove the ieri-butyldimethylsilyl (TBDMS) groups at the 2' position. The reaction is then quenched with 50 mL of 20 mM sodium acetate and the pH is adjusted to 6.5. Oligonucleotide is stored in a freezer until purification.

Analysis

The oligonucleotides are analyzed by high-performance liquid chromatography (HPLC) prior to purification and selection of buffer and column depends on nature of the sequence and or conjugated ligand.

HPLC Purification

The ligand-conjugated oligonucleotides are purified by reverse-phase preparative

HPLC. The unconjugated oligonucleotides are purified by anion-exchange HPLC on a TSK gel column packed in house. The buffers are 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractions containing full-length oligonucleotides are pooled, desalted, and lyophilized. Approximately 0.15 OD of desalted oligonucleotidess are diluted in water to 150 μL· and then pipetted into special vials for CGE and LC/MS analysis.

Compounds are then analyzed by LC-ESMS and CGE.

iRNA preparation For the general preparation of iR A agents, specifically siR As, equimolar amounts of sense and antisense strand are heated in IxPBS at 95°C for 5 min and slowly cooled to room temperature. Integrity of the duplex is confirmed by HPLC analysis.

Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 6. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5'-3'-phosphodiester bonds unless otherwise noted.

Table 6: Abbreviations of nucleotide monomers

Example 5: Synthesis of iRNA Sequences

Sequences are synthesized on a MerMade 192 synthesizer at Ι μηιοΐ scale.

For all the sequences in the list, 'endolight' chemistry may be applied as detailed below.

· All pyrimidines (cytosine and uridine) in the sense strand contain 2'-0-

Methyl bases (2' O-Methyl C and 2'-0-Methyl U)

• In the antisense strand, pyrimidines adjacent to (towards 5' position) ribo A nucleoside are replaced with their corresponding 2-O-Methyl nucleosides

· A two base dTsdT extension at 3' end of both sense and antisense

sequences are introduced

• The sequence file is converted to a text file to make it compatible for loading in the MerMade 192 synthesis software Synthesis, Cleavage and deprotection:

The synthesis of sequences uses solid supported oligonucleotide synthesis using phosphoramidite chemistry.

The synthesis of the above sequences are performed at lum scale in 96 well plates. The amidite solutions are prepared at 0.1 M concentration and ethyl thio tetrazole (0.6M in Acetonitrile) is used as activator.

The synthesized sequences are cleaved and deprotected in 96 well plates, using methylamine in the first step and fluoride reagent in the second step. The crude sequences are precipitated using acetone: ethanol (80:20) mix and the pellet re-suspended in 0.02M sodium acetate buffer. Samples from each sequence are analyzed by LC-MS to confirm the identity, UV for quantification and a selected set of samples by IEX chromatography to determine purity.

Purification and desalting:

Avian transcript siRNA sequences are purified on AKTA explorer purification system using Source 15Q column. A column temperature of 65C is maintained during purification. Sample injection and collection is performed in 96 well (1.8mL -deep well) plates. A single peak corresponding to the full length sequence is collected in the eluent. The purified sequences are desalted on a Sephadex G25 column using AKTA purifier. The desalted sequences are analyzed for concentration (by UV measurement at A260) and purity (by ion exchange HPLC). The single strands are then submitted for annealing.

Example 6. Expression Analysis in Avian Cells

Cells and Growth Conditions:

Duck cells (ATCC, Mannassas, VA; ATCC number CCL-141 ; Anas

platyrhynchus domesticus(duck, Pekin)) were plated into 24 well plates the night before infection at a cell density of 100k cells per well in Eagle's Minimum Essential Medium supplemented with fetal bovine serum to a final concentration of 10%. The following day the cells in half the wells were infected with B/Brisbane influenza virus at an MOI of 0.5 for 1 hr, and the media changed to DMEM supplemented with trypsin overnight (~16- 18hrs). The cells were expected to adhere, so any floating cells in the media were spun down.

RNA Preparation:

Qiagen RNeasy was used to isolate RNA from the infected and uninfected cells according to the manufacturer's directions. Each separate RNA sample for microRNA and transcriptome analysis was pooled from 4 wells of the 24well plate.

MicroRNA Sequencing:

The microRNA sequences from infected and uninfected CCL-141 cells were determined by LC Sciences, LLC (2575 West Bellfort St., Houston, TX 77054). In brief, total RNA was processed to generate a short sequence cDNA library that was then deep- sequenced with a next-gen Illumina Ilg DNA sequencing machine. The resulting sequences were clustered in microRNA based on similarity to known micro RNAs as well as by ab-initio, structure -based methods. Novel microRNA target transcript sequences are reported in Table 1.

Expression Analysis:

The relative expression levels of transcripts in infected versus uninfected CCL- 141 cells were determined by deep-sequencing of RNA transcripts. Paired end 50 base reads from a total of four RNA samples (two infected, two uninfected) were sequenced using an Illumina Ilg. The average number of reads per thousand base window of each transcript was determined after normalizing for the total number of reads obtained for the four samples.

Relative changes in expression levels for each transcript were determined by the ratio of average number of reads per thousand base window between infected and uninfected cells (See Table 7). Table 7 is list of the differentially expressed duck genes from cells infected with the B/Brisbane flu virus as measured by normalized counts of high-throughput sequencing reads. Transcripts with at least a two fold increase in expression levels in the infected versus uninfected cells are listed. Whereas, transcripts expressed in infected cells but not in uninfected cells are listed in Table 8. Fold Diff. stands for Fold differentially expressed.

Table 7: Differential Expression Target Target Transcript Fold Target Target Transcript Fold SEQ ID Diff. SEQ ID Diff.

1356 ENSAPLT00000001356 2129 14176 ENSAPLT00000014176 4

7416 ENSAPLT00000007416 1460 14494 ENSAPLTOOOOOO 14494 4

16167 ENSAPLT00000016167 1117 12488 ENSAPLTOOOOOO 12488 4

7867 ENSAPLT00000007867 1103 8196 ENSAPLT00000008196 4

16416 ENSAPLT00000016416 961 2796 ENSAPLT00000002796 4

4306 ENSAPLT00000004306 790 10791 ENSAPLTOOOOOO 10791 4

11350 ENSAPLT00000011350 726 3869 ENSAPLT00000003869 4

14810 ENSAPLT00000014810 623 5715 ENSAPLT00000005715 4

14535 ENSAPLT00000014535 572 14553 ENSAPLT00000014553 4

6241 ENSAPLT00000006241 549 5530 ENSAPLT00000005530 4

16707 ENSAPLTOOOOOO 16707 458 13884 ENSAPLT00000013884 4

9044 ENSAPLT00000009044 427 7905 ENSAPLT00000007905 4

16544 ENSAPLT00000016544 422 5661 ENSAPLT00000005661 4

9567 ENS APLT00000009567 416 11853 ENSAPLT00000011853 4

7080 ENSAPLT00000007080 407 3754 ENSAPLT00000003754 4

11595 ENSAPLT00000011595 400 8556 ENSAPLT00000008556 4

15248 ENSAPLTOOOOOO 15248 395 12213 ENSAPLTOOOOOO 12213 4

13397 ENSAPLT00000013397 361 8610 ENSAPLT00000008610 4

12882 ENSAPLT00000012882 359 1958 ENS APLTOOOOOOO 1958 4

2053 ENSAPLT00000002053 350 1169 ENSAPLT00000001169 4

1611 ENSAPLT00000001611 343 4296 ENSAPLT00000004296 4

16054 ENSAPLTOOOOOO 16054 333 2309 ENSAPLT00000002309 4

9771 ENSAPLT00000009771 323 12883 ENSAPLT00000012883 4

1602 ENS APLTOOOOOOO 1602 314 4500 ENSAPLT00000004500 4

8502 ENSAPLT00000008502 280 4882 ENSAPLT00000004882 4

3024 ENSAPLT00000003024 275 8812 ENSAPLT00000008812 4

2193 ENSAPLT00000002193 258 14867 ENSAPLTOOOOOO 14867 4

6212 ENS APLT00000006212 231 2922 ENSAPLT00000002922 4

10833 ENSAPLT00000010833 216 16144 ENSAPLT00000016144 4

13295 ENSAPLTOOOOOO 13295 203 17103 ENSAPLT00000017103 4

13172 ENSAPLT00000013172 201 7525 ENSAPLT00000007525 4

12297 ENSAPLTOOOOOO 12297 193 2477 ENSAPLT00000002477 4

10261 ENSAPLT00000010261 188 3611 ENSAPLT00000003611 4

3137 ENSAPLT00000003137 178 16805 ENSAPLTOOOOOO 16805 4

16969 ENSAPLTOOOOOO 16969 157 4631 ENSAPLT00000004631 4

13037 ENSAPLT00000013037 151 5790 ENSAPLT00000005790 4

2381 ENSAPLT00000002381 149 10558 ENSAPLT00000010558 4

15510 ENSAPLT00000015510 145 14934 ENSAPLTOOOOOO 14934 4 15770 ENSAPLT00000015770 144 8008 ENSAPLT00000008008 4

2352 ENSAPLT00000002352 141 12603 ENSAPLTOOOOOO 12603 4

10244 ENSAPLTOOOOOO 10244 137 16154 ENSAPLT00000016154 4

882 ENSAPLT00000000882 135 7936 ENSAPLT00000007936 4

14453 ENSAPLT00000014453 134 8656 ENSAPLT00000008656 4

4188 ENSAPLT00000004188 134 17073 ENSAPLTOOOOOO 17073 4

5288 ENSAPLT00000005288 132 11735 ENSAPLT00000011735 4

7802 ENSAPLT00000007802 130 11491 ENS APLTOOOOOO 11491 4

16189 ENSAPLT00000016189 121 17082 ENSAPLTOOOOOO 17082 4

13762 ENSAPLT00000013762 121 13495 ENSAPLTOOOOOO 13495 4

9077 ENSAPLT00000009077 118 3753 ENSAPLT00000003753 4

7157 ENSAPLT00000007157 117 12265 ENSAPLT00000012265 4

9570 ENS APLT00000009570 112 11645 ENSAPLTOOOOOO 11645 4

11330 ENSAPLT00000011330 111 15874 ENSAPLT00000015874 4

12824 ENSAPLTOOOOOO 12824 108 11582 ENSAPLT00000011582 4

9829 ENSAPLT00000009829 108 11538 ENSAPLT00000011538 4

7705 ENSAPLT00000007705 107 2737 ENSAPLT00000002737 4

15846 ENSAPLT00000015846 105 10385 ENSAPLTOOOOOO 10385 4

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3998 ENSAPLT00000003998 5 4530 ENSAPLT00000004530 2

16056 ENSAPLT00000016056 5 15037 ENSAPLT00000015037 2

11700 ENSAPLTOOOOOO 11700 5 12723 ENSAPLTOOOOOO 12723 2

13709 ENSAPLT00000013709 5 8646 ENSAPLT00000008646 2

12762 ENSAPLTOOOOOO 12762 5 10521 ENSAPLT00000010521 2

655 ENSAPLT00000000655 5 6897 ENSAPLT00000006897 2 8964 ENSAPLT00000008964 5 12235 ENSAPLT00000012235 2

12585 ENSAPLTOOOOOO 12585 5 16522 ENSAPLT00000016522 2

8399 ENSAPLT00000008399 5 1639 ENSAPLTOOOOOOO 1639 2

3414 ENSAPLT00000003414 5 9082 ENSAPLT00000009082 2

11702 ENSAPLTOOOOOO 11702 5 10496 ENSAPLTOOOOOO 10496 2

1338 ENSAPLT00000001338 5 12113 ENSAPLT000000121 13 2

4323 ENSAPLT00000004323 5 8236 ENSAPLT00000008236 2

13879 ENSAPLT00000013879 5 3517 ENSAPLT00000003517 2

14155 ENSAPLT00000014155 5 16404 ENSAPLTOOOOOO 16404 2

4301 ENSAPLT00000004301 5 5888 ENSAPLT00000005888 2

12086 ENSAPLTOOOOOO 12086 5 11488 ENSAPLTOOOOOO 11488 2

16294 ENSAPLTOOOOOO 16294 5 6412 ENS APLT00000006412 2

12786 ENSAPLT00000012786 5 9651 ENSAPLT00000009651 2

3328 ENSAPLT00000003328 5 11573 ENSAPLT00000011573 2

5743 ENSAPLT00000005743 5 12903 ENSAPLTOOOOOO 12903 2

6231 ENSAPLT00000006231 5 7452 ENSAPLT00000007452 2

13894 ENSAPLT00000013894 5 5534 ENSAPLT00000005534 2

12784 ENSAPLTOOOOOO 12784 5 13704 ENSAPLT00000013704 2

10514 ENSAPLTOOOOOO 10514 5 3667 ENSAPLT00000003667 2

932 ENSAPLT00000000932 5 2997 ENSAPLT00000002997 2

6126 ENSAPLT00000006126 5 3296 ENSAPLT00000003296 2

16259 ENSAPLT00000016259 5 6491 ENS APLT00000006491 2

5343 ENSAPLT00000005343 5 9280 ENSAPLT00000009280 2

8095 ENSAPLT00000008095 5 14534 ENSAPLTOOOOOO 14534 2

3339 ENSAPLT00000003339 5 14851 ENSAPLT00000014851 2

4863 ENSAPLT00000004863 5 16289 ENSAPLT00000016289 2

2795 ENSAPLT00000002795 5 9315 ENSAPLT00000009315 2

12440 ENSAPLTOOOOOO 12440 5 2096 ENSAPLT00000002096 2

7521 ENSAPLT00000007521 5 12819 ENSAPLTOOOOOO 12819 2

4559 ENSAPLT00000004559 5 17115 ENSAPLT000000171 15 2

12063 ENSAPLTOOOOOO 12063 5 71 13 ENSAPLT000000071 13 2

10114 ENSAPLT000000101 14 5 12563 ENSAPLTOOOOOO 12563 2

15211 ENSAPLTOOOOOO 15211 5 14338 ENSAPLT00000014338 2

10301 ENSAPLT00000010301 5 12500 ENSAPLTOOOOOO 12500 2

10477 ENSAPLTOOOOOO 10477 5 8294 ENSAPLT00000008294 2

7368 ENSAPLT00000007368 5 5732 ENSAPLT00000005732 2

10064 ENSAPLTOOOOOO 10064 5 4978 ENSAPLT00000004978 2

5175 ENSAPLT00000005175 5 6076 ENSAPLT00000006076 2

13300 ENSAPLT00000013300 5 15035 ENSAPLT00000015035 2 12685 ENSAPLT00000012685 5 13351 ENSAPLT00000013351 2

9027 ENSAPLT00000009027 5 11762 ENSAPLT00000011762 2

13573 ENSAPLT00000013573 5 4282 ENSAPLT00000004282 2

2887 ENSAPLT00000002887 5 3276 ENSAPLT00000003276 2

10937 ENS APLT00000010937 5 6837 ENSAPLT00000006837 2

8288 ENSAPLT00000008288 5 3400 ENSAPLT00000003400 2

634 ENSAPLT00000000634 5 3654 ENSAPLT00000003654 2

8660 ENSAPLT00000008660 5 8958 ENSAPLT00000008958 2

8733 ENSAPLT00000008733 5 13154 ENSAPLT00000013154 2

11643 ENSAPLT00000011643 5 2080 ENSAPLT00000002080 2

3185 ENSAPLT00000003185 5 16662 ENSAPLTOOOOOO 16662 2

11413 ENSAPLT00000011413 5 16963 ENSAPLTOOOOOO 16963 2

6672 ENSAPLT00000006672 5 4584 ENS APLT00000004584 2

7211 ENSAPLT00000007211 5 7672 ENSAPLT00000007672 2

742 ENSAPLT00000000742 5 9238 ENSAPLT00000009238 2

4649 ENSAPLT00000004649 5 9866 ENSAPLT00000009866 2

6000 ENSAPLT00000006000 5 14359 ENSAPLTOOOOOO 14359 2

2875 ENSAPLT00000002875 5 10387 ENSAPLTOOOOOO 10387 2

17158 ENSAPLT00000017158 5 5027 ENSAPLT00000005027 2

16651 ENSAPLT00000016651 5 11984 ENSAPLTOOOOOO 11984 2

6033 ENSAPLT00000006033 5 6097 ENSAPLT00000006097 2

10980 ENSAPLT00000010980 5 15491 ENSAPLTOOOOOO 15491 2

4697 ENSAPLT00000004697 5 15717 ENSAPLT00000015717 2

7367 ENSAPLT00000007367 5 5023 ENSAPLT00000005023 2

7432 ENSAPLT00000007432 5 4871 ENSAPLT00000004871 2

13725 ENSAPLT00000013725 5 2912 ENS APLT00000002912 2

9356 ENSAPLT00000009356 5 16300 ENSAPLT00000016300 2

17019 ENSAPLT00000017019 5 2546 ENSAPLT00000002546 2

16843 ENSAPLTOOOOOO 16843 5 15026 ENSAPLTOOOOOO 15026 2

4994 ENSAPLT00000004994 5 4425 ENSAPLT00000004425 2

1899 ENSAPLTOOOOOOO 1899 5 11511 ENSAPLT00000011511 2

6677 ENSAPLT00000006677 5 1068 ENSAPLTOOOOOOO 1068 2

6254 ENSAPLT00000006254 5 9997 ENSAPLT00000009997 2

12228 ENSAPLTOOOOOO 12228 5 5237 ENSAPLT00000005237 2

5281 ENSAPLT00000005281 5 11500 ENSAPLT00000011500 2

12691 ENSAPLTOOOOOO 12691 5 11741 ENSAPLTOOOOOO 11741 2

15307 ENSAPLT00000015307 5 13929 ENSAPLTOOOOOO 13929 2

13003 ENSAPLTOOOOOO 13003 5 6425 ENSAPLT00000006425 2

6028 ENSAPLT00000006028 5 3302 ENSAPLT00000003302 2 10780 ENS APLTOOOOOO 10780 5 11046 ENS APLTOOOOOO 11046 2

2187 ENSAPLT00000002187 5 15637 ENSAPLT00000015637 2

12201 ENSAPLT00000012201 5 13784 ENSAPLT00000013784 2

1687 ENSAPLT00000001687 5 11263 ENSAPLT00000011263 2

7538 ENSAPLT00000007538 4 16554 ENSAPLTOOOOOO 16554 2

16558 ENSAPLT00000016558 4 12855 ENSAPLT00000012855 2

11146 ENSAPLT00000011146 4 13186 ENSAPLT00000013186 2

6652 ENSAPLT00000006652 4 10071 ENSAPLTOOOOOO 10071 2

9200 ENSAPLT00000009200 4 14753 ENSAPLT00000014753 2

13816 ENSAPLT00000013816 4 8558 ENSAPLT00000008558 2

10158 ENSAPLT00000010158 4 16446 ENSAPLTOOOOOO 16446 2

12625 ENSAPLT00000012625 4 16333 ENSAPLT00000016333 2

15010 ENSAPLT00000015010 4 9129 ENS APLT00000009129 2

14750 ENS APLTOOOOOO 14750 4 14436 ENSAPLTOOOOOO 14436 2

3489 ENSAPLT00000003489 4 14012 ENSAPLTOOOOOO 14012 2

9875 ENSAPLT00000009875 4 5552 ENSAPLT00000005552 2

8663 ENSAPLT00000008663 4 6135 ENSAPLT00000006135 2

13801 ENSAPLT00000013801 4 15477 ENSAPLTOOOOOO 15477 2

2933 ENSAPLT00000002933 4 1086 ENSAPLTOOOOOOO 1086 2

6008 ENSAPLT00000006008 4 11753 ENSAPLT00000011753 2

16831 ENSAPLT00000016831 4 4574 ENS APLT00000004574 2

2371 ENSAPLT00000002371 4 3860 ENSAPLT00000003860 2

6483 ENSAPLT00000006483 4 11285 ENSAPLT00000011285 2

6791 ENSAPLT00000006791 4 11 130 ENSAPLT00000011130 2

3253 ENSAPLT00000003253 4 2923 ENSAPLT00000002923 2

1025 ENSAPLTOOOOOOO 1025 4 3377 ENSAPLT00000003377 2

11858 ENSAPLT00000011858 4 9017 ENSAPLT00000009017 2

4100 ENS APLT00000004100 4 2200 ENSAPLT00000002200 2

16729 ENSAPLTOOOOOO 16729 4 9397 ENSAPLT00000009397 2

13773 ENSAPLT00000013773 4 694 ENSAPLT00000000694 2

2037 ENSAPLT00000002037 4 8720 ENSAPLT00000008720 2

14901 ENSAPLTOOOOOO 14901 4 13520 ENSAPLT00000013520 2

13178 ENSAPLT00000013178 4 3098 ENSAPLT00000003098 2

9268 ENSAPLT00000009268 4 13347 ENSAPLT00000013347 2

4847 ENSAPLT00000004847 4 6845 ENSAPLT00000006845 2

811 ENSAPLT00000000811 4 11924 ENS APLTOOOOOO 11924 2

6130 ENSAPLT00000006130 4 1945 ENSAPLTOOOOOOO 1945 2

15650 ENSAPLT00000015650 4 8423 ENSAPLT00000008423 2

2181 ENSAPLT00000002181 4 2466 ENSAPLT00000002466 2 3284 ENSAPLT00000003284 4 6875 ENSAPLT00000006875 2

16128 ENSAPLT00000016128 4 5832 ENSAPLT00000005832 2

9975 ENSAPLT00000009975 4 9001 ENS APLT00000009001 2

8139 ENSAPLT00000008139 4 10688 ENSAPLTOOOOOO 10688 2

10875 ENSAPLT00000010875 4 14955 ENSAPLT00000014955 2

1766 ENSAPLT00000001766 4 8065 ENSAPLT00000008065 2

4639 ENSAPLT00000004639 4 8765 ENSAPLT00000008765 2

15260 ENSAPLT00000015260 4 8071 ENSAPLT00000008071 2

14739 ENSAPLTOOOOOO 14739 4 16242 ENSAPLTOOOOOO 16242 2

1144 ENSAPLT00000001144 4 16351 ENSAPLTOOOOOO 16351 2

9399 ENSAPLT00000009399 4 14313 ENSAPLTOOOOOO 14313 2

13268 ENSAPLT00000013268 4 10493 ENSAPLTOOOOOO 10493 2

5249 ENSAPLT00000005249 4 1369 ENS APLTOOOOOOO 1369 2

14709 ENSAPLTOOOOOO 14709 4 7327 ENSAPLT00000007327 2

9573 ENSAPLT00000009573 4 6733 ENSAPLT00000006733 2

16360 ENSAPLTOOOOOO 16360 4 6079 ENSAPLT00000006079 2

1448 ENS APLT00000001448 4 14237 ENSAPLT00000014237 2

8127 ENSAPLT00000008127 4 2328 ENSAPLT00000002328 2

2385 ENSAPLT00000002385 4 12325 ENSAPLT00000012325 2

5256 ENSAPLT00000005256 4 4327 ENSAPLT00000004327 2

15969 ENSAPLTOOOOOO 15969 4 3068 ENSAPLT00000003068 2

2101 ENSAPLT00000002101 4 12084 ENSAPLTOOOOOO 12084 2

10562 ENSAPLTOOOOOO 10562 4 11802 ENSAPLTOOOOOO 11802 2

12439 ENSAPLTOOOOOO 12439 4 9886 ENSAPLT00000009886 2

8187 ENSAPLT00000008187 4 4594 ENSAPLT00000004594 2

11418 ENSAPLT00000011418 4 11844 ENSAPLTOOOOOO 11844 2

7779 ENSAPLT00000007779 4 3814 ENSAPLT00000003814 2

2030 ENSAPLT00000002030 4 6016 ENSAPLT00000006016 2

9996 ENSAPLT00000009996 4 6039 ENSAPLT00000006039 2

7895 ENSAPLT00000007895 4 5830 ENSAPLT00000005830 2

6060 ENSAPLT00000006060 4 3480 ENSAPLT00000003480 2

9449 ENSAPLT00000009449 4 8164 ENSAPLT00000008164 2

9079 ENSAPLT00000009079 4 5996 ENSAPLT00000005996 2

6091 ENS APLT00000006091 4 9252 ENSAPLT00000009252 2

822 ENSAPLT00000000822 4 8494 ENSAPLT00000008494 2

170 ENS APLTOOOOOOOO 170 4 1554 ENSAPLT00000001554 2

2990 ENSAPLT00000002990 4 16945 ENSAPLTOOOOOO 16945 2

12618 ENSAPLTOOOOOO 12618 4 3549 ENSAPLT00000003549 2

16893 ENSAPLTOOOOOO 16893 4 5836 ENSAPLT00000005836 2 3513 ENSAPLT00000003513 4 11052 ENSAPLTOOOOOO 11052 2

6564 ENS APLT00000006564 4 4567 ENS APLT00000004567 2

10535 ENSAPLT00000010535 4 4059 ENSAPLT00000004059 2

15784 ENSAPLT00000015784 4 15814 ENSAPLT00000015814 2

11044 ENS APLTOOOOOO 11044 4 8324 ENSAPLT00000008324 2

12227 ENSAPLT00000012227 4 12842 ENSAPLTOOOOOO 12842 2

8046 ENSAPLT00000008046 4 9910 ENS APLT00000009910 2

7346 ENSAPLT00000007346 4 14455 ENSAPLT00000014455 2

516 ENSAPLT00000000516 4 10467 ENSAPLTOOOOOO 10467 2

7303 ENSAPLT00000007303 4 7976 ENSAPLT00000007976 2

11127 ENSAPLT00000011127 4 3593 ENSAPLT00000003593 2

16409 ENS APLTOOOOOO 16409 4 8679 ENSAPLT00000008679 2

2215 ENSAPLT00000002215 4 9093 ENSAPLT00000009093 2

5267 ENSAPLT00000005267 4 10440 ENSAPLTOOOOOO 10440 2

16850 ENSAPLT00000016850 4 7896 ENSAPLT00000007896 2

2314 ENSAPLT00000002314 4 8709 ENSAPLT00000008709 2

14178 ENSAPLT00000014178 4 7584 ENSAPLT00000007584 2

7761 ENSAPLT00000007761 4 10589 ENSAPLTOOOOOO 10589 2

8375 ENSAPLT00000008375 4 16194 ENSAPLT00000016194 2

10356 ENSAPLT00000010356 4 11492 ENS APLTOOOOOO 11492 2

3955 ENSAPLT00000003955 4 16385 ENSAPLTOOOOOO 16385 2

4479 ENSAPLT00000004479 4 12033 ENSAPLT00000012033 2

3281 ENSAPLT00000003281 4 3855 ENSAPLT00000003855 2

2969 ENSAPLT00000002969 4 10729 ENSAPLTOOOOOO 10729 2

14706 ENSAPLT00000014706 4 3930 ENSAPLT00000003930 2

12259 ENS APLTOOOOOO 12259 4 15471 ENSAPLTOOOOOO 15471 2

3970 ENSAPLT00000003970 4 10375 ENSAPLTOOOOOO 10375 2

11873 ENSAPLT00000011873 4 1211 ENSAPLT00000001211 2

10972 ENSAPLT00000010972 4 6771 ENSAPLT00000006771 2

6952 ENSAPLT00000006952 4 6978 ENSAPLT00000006978 2

7726 ENSAPLT00000007726 4 12051 ENSAPLTOOOOOO 12051 2

15063 ENSAPLTOOOOOO 15063 4 7487 ENSAPLT00000007487 2

14832 ENSAPLT00000014832 4 12056 ENSAPLTOOOOOO 12056 2

11527 ENSAPLT00000011527 4 16684 ENSAPLTOOOOOO 16684 2

6671 ENSAPLT00000006671 4 7309 ENSAPLT00000007309 2

3133 ENSAPLT00000003133 4 1922 ENS APLTOOOOOOO 1922 2

16960 ENSAPLTOOOOOO 16960 4 2538 ENSAPLT00000002538 2

17093 ENSAPLTOOOOOO 17093 4 2705 ENSAPLT00000002705 2

10324 ENSAPLTOOOOOO 10324 4 1412 ENSAPLT00000001412 2 857 ENSAPLT00000000857 4 4621 ENSAPLT00000004621 2

8915 ENSAPLT00000008915 4 10849 ENSAPLTOOOOOO 10849 2

6233 ENSAPLT00000006233 4 9692 ENSAPLT00000009692 2

7565 ENSAPLT00000007565 4 14815 ENSAPLTOOOOOO 14815 2

14235 ENSAPLT00000014235 4 13996 ENSAPLTOOOOOO 13996 2

15152 ENSAPLT00000015152 4 4459 ENSAPLT00000004459 2

3657 ENSAPLT00000003657 4 3369 ENSAPLT00000003369 2

13018 ENSAPLTOOOOOO 13018 4 5524 ENSAPLT00000005524 2

16998 ENSAPLTOOOOOO 16998 4 5676 ENSAPLT00000005676 2

14859 ENSAPLTOOOOOO 14859 4 16163 ENSAPLT00000016163 2

3433 ENSAPLT00000003433 4 5998 ENSAPLT00000005998 2

11080 ENSAPLTOOOOOO 11080 4 1154 ENSAPLT00000001154 2

9300 ENSAPLT00000009300 4 15247 ENSAPLTOOOOOO 15247 2

12088 ENSAPLTOOOOOO 12088 4 12403 ENSAPLTOOOOOO 12403 2

8038 ENSAPLT00000008038 4 15055 ENSAPLT00000015055 2

4997 ENSAPLT00000004997 4 4218 ENSAPLT00000004218 2

6583 ENSAPLT00000006583 4 10465 ENSAPLTOOOOOO 10465 2

4242 ENSAPLT00000004242 4 9069 ENSAPLT00000009069 2

10713 ENSAPLTOOOOOO 10713 4 4502 ENSAPLT00000004502 2

9375 ENSAPLT00000009375 4 15755 ENSAPLT00000015755 2

15281 ENSAPLT00000015281 4 11009 ENSAPLTOOOOOO 11009 2

16733 ENSAPLT00000016733 4 13094 ENSAPLTOOOOOO 13094 2

9914 ENS APLT00000009914 4 2928 ENSAPLT00000002928 2

3574 ENSAPLT00000003574 4 4068 ENSAPLT00000004068 2

2339 ENSAPLT00000002339 4 8844 ENSAPLT00000008844 2

11649 ENSAPLTOOOOOO 11649 4 14427 ENSAPLTOOOOOO 14427 2

3425 ENSAPLT00000003425 4 15473 ENSAPLTOOOOOO 15473 2

4084 ENSAPLT00000004084 4 12958 ENSAPLT00000012958 2

5699 ENSAPLT00000005699 4 6266 ENSAPLT00000006266 2

15648 ENSAPLT00000015648 4 3719 ENSAPLT00000003719 2

16449 ENSAPLTOOOOOO 16449 4 16136 ENSAPLT00000016136 2

15000 ENSAPLTOOOOOO 15000 4 12896 ENSAPLTOOOOOO 12896 2

17089 ENSAPLTOOOOOO 17089 4 16041 ENSAPLTOOOOOO 16041 2

991 ENS APLT00000000991 4 7225 ENSAPLT00000007225 2

15036 ENSAPLT00000015036 4 12915 ENSAPLTOOOOOO 12915 2

14490 ENSAPLTOOOOOO 14490 4 6193 ENS APLT00000006193 2

10217 ENSAPLTOOOOOO 10217 4 569 ENS APLT00000000569 2

15003 ENSAPLTOOOOOO 15003 4 13044 ENSAPLTOOOOOO 13044 2

15042 ENSAPLTOOOOOO 15042 4 5219 ENSAPLT00000005219 2 13972 ENSAPLT00000013972 4 11458 ENSAPLT00000011458 2

9086 ENSAPLT00000009086 4 8923 ENSAPLT00000008923 2

11490 ENS APLT00000011490 4 7868 ENSAPLT00000007868 2

2507 ENSAPLT00000002507 4 11703 ENSAPLTOOOOOO 11703 2

2953 ENSAPLT00000002953 4 16010 ENSAPLTOOOOOO 16010 2

2547 ENSAPLT00000002547 4 3852 ENSAPLT00000003852 2

7490 ENSAPLT00000007490 4 9302 ENSAPLT00000009302 2

4911 ENSAPLT00000004911 4 15645 ENSAPLT00000015645 2

11861 ENSAPLT00000011861 4 16633 ENSAPLT00000016633 2

3795 ENSAPLT00000003795 4 11221 ENSAPLTOOOOOO 11221 2

11601 ENSAPLT00000011601 4 16862 ENSAPLT00000016862 2

2624 ENSAPLT00000002624 4 10066 ENSAPLTOOOOOO 10066 2

4024 ENSAPLT00000004024 4 6922 ENSAPLT00000006922 2

6827 ENSAPLT00000006827 4 8272 ENSAPLT00000008272 2

15153 ENSAPLT00000015153 4 2895 ENSAPLT00000002895 2

7002 ENSAPLT00000007002 4 14329 ENSAPLTOOOOOO 14329 2

2736 ENSAPLT00000002736 4 8274 ENSAPLT00000008274 2

5595 ENSAPLT00000005595 4 3826 ENSAPLT00000003826 2

15945 ENSAPLT00000015945 4 1358 ENSAPLT00000001358 2

8041 ENS APLT00000008041 4 11515 ENSAPLT00000011515 2

11409 ENS APLT00000011409 4 13743 ENSAPLT00000013743 2

11295 ENSAPLT00000011295 4 1021 ENSAPLTOOOOOOO 1021 2

3427 ENSAPLT00000003427 4 12913 ENSAPLTOOOOOO 12913 2

8792 ENSAPLT00000008792 4 9929 ENSAPLT00000009929 2

16542 ENSAPLT00000016542 4 5216 ENSAPLT00000005216 2

250 ENSAPLT00000000250 4 10569 ENSAPLTOOOOOO 10569 2

679 ENSAPLT00000000679 4 4917 ENSAPLT00000004917 2

13925 ENSAPLTOOOOOO 13925 4 7141 ENSAPLT00000007141 2

17162 ENSAPLT00000017162 4 2293 ENSAPLT00000002293 2

8585 ENSAPLT00000008585 4 15696 ENSAPLT00000015696 2

12137 ENSAPLT00000012137 4 10923 ENSAPLTOOOOOO 10923 2

10575 ENSAPLTOOOOOO 10575 4 9381 ENS APLT00000009381 2

6655 ENSAPLT00000006655 4 16235 ENSAPLT00000016235 2

3879 ENSAPLT00000003879 4 9041 ENS APLT00000009041 2

13597 ENSAPLT00000013597 4 15936 ENSAPLT00000015936 2

1364 ENS APLTOOOOOOO 1364 4 16413 ENSAPLTOOOOOO 16413 2

1477 ENSAPLTOOOOOOO 1477 4 5705 ENSAPLT00000005705 2

7112 ENSAPLT000000071 12 4 10805 ENSAPLTOOOOOO 10805 2

1419 ENSAPLT00000001419 4 4041 ENS APLT00000004041 2 1111 ENSAPLT000000011 11 4 10914 ENSAPLTOOOOOO 10914 2

5749 ENSAPLT00000005749 4 11859 ENSAPLT00000011859 2

12493 ENSAPLTOOOOOO 12493 4 14926 ENSAPLTOOOOOO 14926 2

13016 ENSAPLTOOOOOO 13016 4 626 ENSAPLT00000000626 2

9934 ENSAPLT00000009934 4 5572 ENSAPLT00000005572 2

1016 ENSAPLT00000001016 4 5360 ENSAPLT00000005360 2

3390 ENSAPLT00000003390 4 17084 ENSAPLTOOOOOO 17084 2

6963 ENSAPLT00000006963 4 16844 ENSAPLTOOOOOO 16844 2

17017 ENSAPLTOOOOOO 17017 4 1647 ENS APLTOOOOOOO 1647 2

2044 ENSAPLT00000002044 4 4454 ENSAPLT00000004454 2

4042 ENSAPLT00000004042 4 8772 ENSAPLT00000008772 2

9495 ENSAPLT00000009495 4 1853 ENSAPLT00000001853 2

12257 ENSAPLT00000012257 4 7397 ENSAPLT00000007397 2

2579 ENSAPLT00000002579 4 16732 ENSAPLT00000016732 2

910 ENS APLT00000000910 4 3475 ENSAPLT00000003475 2

3563 ENSAPLT00000003563 4 5007 ENSAPLT00000005007 2

15743 ENSAPLT00000015743 4 9722 ENSAPLT00000009722 2

3494 ENSAPLT00000003494 4 4106 ENS APLT00000004106 2

16325 ENSAPLT00000016325 4 3705 ENSAPLT00000003705 2

9589 ENS APLT00000009589 4 6855 ENSAPLT00000006855 2

17083 ENSAPLTOOOOOO 17083 4 5300 ENSAPLT00000005300 2

13222 ENSAPLTOOOOOO 13222 4 8405 ENSAPLT00000008405 2

2235 ENSAPLT00000002235 4 7273 ENSAPLT00000007273 2

2278 ENSAPLT00000002278 4 10533 ENSAPLT00000010533 2

2752 ENSAPLT00000002752 4 4448 ENSAPLT00000004448 2

1313 ENSAPLT00000001313 4 6406 ENSAPLT00000006406 2

14914 ENSAPLTOOOOOO 14914 4 12379 ENSAPLTOOOOOO 12379 2

804 ENSAPLT00000000804 4 7543 ENSAPLT00000007543 2

11133 ENSAPLT00000011133 4 6263 ENSAPLT00000006263 2

5370 ENSAPLT00000005370 4 1605 ENS APLTOOOOOOO 1605 2

16777 ENSAPLT00000016777 4 14677 ENSAPLTOOOOOO 14677 2

6637 ENSAPLT00000006637 4 9158 ENSAPLT00000009158 2

5682 ENSAPLT00000005682 4 4287 ENSAPLT00000004287 2

6435 ENSAPLT00000006435 4 5560 ENSAPLT00000005560 2

11621 ENSAPLTOOOOOO 11621 4 6334 ENSAPLT00000006334 2

10746 ENSAPLTOOOOOO 10746 4 3183 ENSAPLT00000003183 2

13715 ENSAPLT00000013715 4 10156 ENSAPLT00000010156 2

3077 ENSAPLT00000003077 4 1060 ENSAPLTOOOOOOO 1060 2

7848 ENSAPLT00000007848 4 1458 ENS APLTOOOOOOO 1458 2 12152 ENSAPLT00000012152 4 4927 ENSAPLT00000004927 2

13307 ENSAPLT00000013307 4 10034 ENSAPLT00000010034 2

716 ENSAPLT00000000716 4 5018 ENSAPLT00000005018 2

9985 ENSAPLT00000009985 4 8590 ENSAPLT00000008590 2

11738 ENSAPLT00000011738 4 11 195 ENSAPLT00000011195 2

17143 ENSAPLT00000017143 4 12495 ENS APLT00000012495 2

9591 ENSAPLT00000009591 4 15664 ENSAPLT00000015664 2

2802 ENSAPLT00000002802 4 6397 ENSAPLT00000006397 2

4257 ENSAPLT00000004257 4 16791 ENSAPLTOOOOOO 16791 2

8254 ENSAPLT00000008254 4 9756 ENSAPLT00000009756 2

2584 ENSAPLT00000002584 4 7612 ENSAPLT00000007612 2

1713 ENSAPLT00000001713 4 135 ENSAPLT00000000135 2

5036 ENSAPLT00000005036 4 16098 ENSAPLTOOOOOO 16098 2

4016 ENSAPLT00000004016 4 13331 ENSAPLT00000013331 2

2325 ENSAPLT00000002325 4 15140 ENSAPLT00000015140 2

12652 ENSAPLT00000012652 4 3422 ENSAPLT00000003422 2

14500 ENS APLT00000014500 4 12194 ENSAPLT00000012194 2

14956 ENS APLT00000014956 4 2324 ENSAPLT00000002324 2

6961 ENSAPLT00000006961 4 15103 ENSAPLT00000015103 2

5981 ENSAPLT00000005981 4

From the table, it is evident that certain transcripts are expressed at much higher level in the CCL- 1 cells upon viral infection. It will be advantageous to target these in order to optimize the production of biological products. Preferred targets are those that are upregulated by at least 20, 50, 100, 200, 500, 1000 or 2000 fold.

Table 8: Infected Only Transcripts

16110 ENSAPLT00000016110 1431 1 ENS APLTOOOOOO 1431 1

537 ENSAPLT00000000537 9689 ENSAPLT00000009689

11168 ENSAPLT00000011 168 5348 ENSAPLT00000005348

6118 ENSAPLT00000006118 1212 ENSAPLT00000001212

728 ENSAPLT00000000728 10709 ENS APLTOOOOOO 10709

11916 ENSAPLT00000011916 9485 ENSAPLT00000009485

5578 ENSAPLT00000005578 7234 ENSAPLT00000007234

13134 ENSAPLT00000013134 6605 ENSAPLT00000006605

895 ENSAPLT00000000895 16353 ENSAPLT00000016353

1990 ENS APLT00000001990 12442 ENS APLTOOOOOO 12442

2321 ENSAPLT00000002321 11868 ENS APLTOOOOOO 1 1868

16266 ENS APLTOOOOOO 16266 9110 ENSAPLT00000009110

9566 ENSAPLT00000009566 11560 ENSAPLT0000001 1560

6604 ENSAPLT00000006604 9853 ENS APLT00000009853

2464 ENSAPLT00000002464 14368 ENSAPLT00000014368

13970 ENSAPLT00000013970 13891 ENSAPLT00000013891

2038 ENSAPLT00000002038 4914 ENS APLT00000004914

5608 ENS APLT00000005608 1451 ENSAPLT00000001451

2609 ENSAPLT00000002609 16377 ENSAPLT00000016377

404 ENSAPLT00000000404 15644 ENS APLTOOOOOO 15644

220 ENSAPLT00000000220 1072 ENS APLTOOOOOOO 1072

1863 ENSAPLT00000001863 5461 ENS APLT00000005461

11996 ENSAPLT00000011996 13398 ENSAPLT00000013398

5709 ENS APLT00000005709 14419 ENSAPLT00000014419

13960 ENSAPLT00000013960 3297 ENSAPLT00000003297

10540 ENS APLTOOOOOO 10540 15309 ENSAPLT00000015309

2300 ENSAPLT00000002300 2829 ENSAPLT00000002829

16878 ENS APLTOOOOOO 16878 2222 ENSAPLT00000002222

12991 ENS APLTOOOOOO 12991 8705 ENSAPLT00000008705

4154 ENS APLT00000004154 1798 ENS APLTOOOOOOO 1798

15445 ENSAPLT00000015445 1570 ENSAPLTOOOOOOO 1570

11958 ENSAPLT00000011958 8921 ENSAPLT00000008921

1249 ENS APLT00000001249 10842 ENS APLTOOOOOO 10842

1965 ENS APLT00000001965 7995 ENSAPLT00000007995

2683 ENSAPLT00000002683 15145 ENSAPLT00000015145

6050 ENSAPLT00000006050 15303 ENSAPLT00000015303

2333 ENSAPLT00000002333 8032 ENSAPLT00000008032

3021 ENSAPLT00000003021 1201 1 ENS APLTOOOOOO 1201 1

2610 ENS APLT00000002610 4954 ENSAPLT00000004954

651 ENSAPLT00000000651 12631 ENS APLTOOOOOO 12631

4726 ENSAPLT00000004726 1160 ENSAPLTOOOOOOO 1160 3588 ENSAPLT00000003588 807 ENSAPLT00000000807

15890 ENSAPLTOOOOOO 15890 14257 ENSAPLTOOOOOO 14257

16108 ENSAPLT00000016108 2954 ENSAPLT00000002954

10289 ENSAPLTOOOOOO 10289 5735 ENSAPLT00000005735

606 ENSAPLT00000000606 5340 ENSAPLT00000005340

4988 ENSAPLT00000004988 11577 ENSAPLT0000001 1577

2911 ENSAPLT00000002911 16140 ENSAPLT00000016140

14204 ENSAPLTOOOOOO 14204 5230 ENSAPLT00000005230

2305 ENSAPLT00000002305 11419 ENSAPLT0000001 1419

223 ENSAPLT00000000223 6618 ENS APLT00000006618

2639 ENSAPLT00000002639 16334 ENSAPLT00000016334

3840 ENS APLT00000003840 14072 ENSAPLTOOOOOO 14072

9555 ENSAPLT00000009555 8359 ENSAPLT00000008359

2118 ENSAPLT00000002118 14401 ENSAPLTOOOOOO 14401

1283 ENSAPLT00000001283 5149 ENS APLT00000005149

896 ENSAPLT00000000896 10946 ENSAPLTOOOOOO 10946

11554 ENSAPLT00000011554 1436 ENS APLTOOOOOOO 1436

2523 ENSAPLT00000002523 4939 ENSAPLT00000004939

10132 ENSAPLT00000010132 3350 ENSAPLT00000003350

476 ENSAPLT00000000476 9560 ENSAPLT00000009560

16602 ENSAPLTOOOOOO 16602 15629 ENSAPLTOOOOOO 15629

78 ENSAPLT00000000078 3301 ENSAPLT00000003301

15995 ENSAPLT00000015995 15952 ENSAPLT00000015952

464 ENSAPLT00000000464 5247 ENSAPLT00000005247

5982 ENSAPLT00000005982 1056 ENS APLTOOOOOOO 1056

9437 ENSAPLT00000009437 16826 ENSAPLTOOOOOO 16826

9 ENSAPLT00000000009 7387 ENSAPLT00000007387

15658 ENSAPLT00000015658 11134 ENSAPLT0000001 1134

14324 ENSAPLTOOOOOO 14324 14839 ENSAPLT00000014839

16227 ENSAPLTOOOOOO 16227 2722 ENSAPLT00000002722

125 ENS APLTOOOOOOOO 125 12260 ENSAPLTOOOOOO 12260

2918 ENS APLT00000002918 12706 ENSAPLTOOOOOO 12706

1601 ENSAPLT00000001601 16776 ENSAPLT00000016776

972 ENSAPLT00000000972 5785 ENSAPLT00000005785

2290 ENSAPLT00000002290 13356 ENSAPLT00000013356

3461 ENSAPLT00000003461 16939 ENSAPLTOOOOOO 16939

3656 ENSAPLT00000003656 2696 ENSAPLT00000002696

7871 ENSAPLT00000007871 1950 ENSAPLT00000001950

816 ENS APLT00000000816 8543 ENSAPLT00000008543

14465 ENSAPLTOOOOOO 14465 3499 ENSAPLT00000003499

14552 ENSAPLT00000014552 12360 ENSAPLT00000012360 9763 ENSAPLT00000009763 5708 ENSAPLT00000005708

13091 ENSAPLT00000013091 4144 ENS APLT00000004144

4468 ENSAPLT00000004468 14438 ENS APLTOOOOOO 14438

9051 ENS APLT00000009051 11153 ENSAPLT0000001 1153

100 ENS APLT00000000100 8202 ENSAPLT00000008202

2069 ENSAPLT00000002069 9245 ENSAPLT00000009245

2940 ENSAPLT00000002940 13272 ENS APLTOOOOOO 13272

451 ENS APLT00000000451 12396 ENS APLTOOOOOO 12396

2210 ENS APLT00000002210 4687 ENSAPLT00000004687

1386 ENSAPLT00000001386 5695 ENSAPLT00000005695

415 ENS APLT00000000415 8566 ENSAPLT00000008566

16653 ENSAPLT00000016653 1933 ENSAPLT00000001933

10 ENS APLT00000000010 7068 ENSAPLT00000007068

13051 ENSAPLT00000013051 11502 ENS APLTOOOOOO 1 1502

1424 ENS APLT00000001424 14957 ENS APLTOOOOOO 14957

1359 ENSAPLT00000001359 16598 ENS APLTOOOOOO 16598

11670 ENSAPLT00000011670 4120 ENS APLT00000004120

10873 ENS APLTOOOOOO 10873 8731 ENSAPLT00000008731

2545 ENSAPLT00000002545 3022 ENSAPLT00000003022

13614 ENSAPLT00000013614 3843 ENSAPLT00000003843

14357 ENSAPLT00000014357 10608 ENS APLTOOOOOO 10608

5229 ENSAPLT00000005229 10106 ENSAPLT00000010106

891 ENS APLT00000000891 1145 ENSAPLT00000001145

676 ENSAPLT00000000676 13200 ENS APLTOOOOOO 13200

16724 ENS APLTOOOOOO 16724 675 ENSAPLT00000000675

397 ENSAPLT00000000397 15523 ENSAPLT00000015523

6269 ENSAPLT00000006269 2197 ENS APLT00000002197

11089 ENSAPLT00000011089 4738 ENS APLT00000004738

1548 ENS APLT00000001548 5576 ENSAPLT00000005576

5748 ENS APLT00000005748 11159 ENSAPLT0000001 1159

9164 ENS APLT00000009164 16193 ENSAPLT00000016193

5066 ENSAPLT00000005066 16129 ENSAPLT00000016129

2567 ENSAPLT00000002567 8504 ENSAPLT00000008504

1195 ENS APLT00000001 195 4361 ENSAPLT00000004361

4297 ENSAPLT00000004297 1376 ENSAPLT00000001376

15178 ENSAPLT00000015178 5792 ENSAPLT00000005792

7934 ENSAPLT00000007934 10912 ENSAPLT00000010912

5076 ENSAPLT00000005076 13510 ENSAPLT00000013510

9016 ENS APLT00000009016 11414 ENSAPLT0000001 1414

1751 ENSAPLT00000001751 919 ENS APLT00000000919

12616 ENSAPLT00000012616 8434 ENSAPLT00000008434 12176 ENSAPLT00000012176 12221 ENSAPLTOOOOOO 12221

1393 ENSAPLT00000001393 13722 ENSAPLT00000013722

4209 ENSAPLT00000004209 14189 ENSAPLT00000014189

14913 ENSAPLTOOOOOO 14913 12932 ENSAPLT00000012932

15426 ENSAPLT00000015426 4821 ENS APLT00000004821

2726 ENSAPLT00000002726 1529 ENS APLTOOOOOOO 1529

2769 ENSAPLT00000002769 14430 ENSAPLT00000014430

4774 ENSAPLT00000004774 6766 ENSAPLT00000006766

3635 ENSAPLT00000003635 13063 ENSAPLTOOOOOO 13063

13468 ENSAPLT00000013468 10550 ENSAPLT00000010550

8232 ENSAPLT00000008232 10674 ENSAPLTOOOOOO 10674

11081 ENSAPLTOOOOOO 11081 6673 ENSAPLT00000006673

13084 ENSAPLT00000013084 16097 ENSAPLTOOOOOO 16097

17043 ENSAPLTOOOOOO 17043 12796 ENSAPLTOOOOOO 12796

2978 ENSAPLT00000002978 4125 ENS APLT00000004125

420 ENSAPLT00000000420 10651 ENSAPLTOOOOOO 10651

15614 ENSAPLT00000015614 15980 ENSAPLTOOOOOO 15980

10076 ENSAPLTOOOOOO 10076 9879 ENSAPLT00000009879

3941 ENSAPLT00000003941 9641 ENS APLT00000009641

6327 ENSAPLT00000006327 2288 ENSAPLT00000002288

1282 ENS APLTOOOOOOO 1282 10140 ENSAPLT00000010140

6445 ENSAPLT00000006445 12540 ENSAPLTOOOOOO 12540

12328 ENSAPLT00000012328 3103 ENSAPLT00000003103

232 ENSAPLT00000000232 8782 ENSAPLT00000008782

14451 ENSAPLTOOOOOO 14451 13009 ENSAPLTOOOOOO 13009

7938 ENSAPLT00000007938 11791 ENS APLTOOOOOO 1 1791

15428 ENSAPLT00000015428 14929 ENSAPLTOOOOOO 14929

2797 ENSAPLT00000002797 10888 ENSAPLT00000010888

543 ENSAPLT00000000543 13660 ENSAPLTOOOOOO 13660

1024 ENS APLTOOOOOOO 1024 8547 ENSAPLT00000008547

10051 ENSAPLTOOOOOO 10051 16009 ENSAPLTOOOOOO 16009

7451 ENSAPLT00000007451 11855 ENS APLTOOOOOO 1 1855

1155 ENSAPLT00000001 155 15548 ENSAPLT00000015548

685 ENSAPLT00000000685 15326 ENSAPLT00000015326

4227 ENSAPLT00000004227 8123 ENSAPLT00000008123

7980 ENSAPLT00000007980 7668 ENSAPLT00000007668

4508 ENSAPLT00000004508 4063 ENSAPLT00000004063

5577 ENSAPLT00000005577 5840 ENSAPLT00000005840

2084 ENSAPLT00000002084 11768 ENS APLTOOOOOO 1 1768

4196 ENS APLT00000004196 14393 ENSAPLTOOOOOO 14393

6004 ENSAPLT00000006004 8053 ENS APLT00000008053 2081 ENS APLT00000002081 16987 ENS APLTOOOOOO 16987

11985 ENSAPLT00000011985 11891 ENS APLTOOOOOO 1 1891

12199 ENSAPLT00000012199 12710 ENSAPLT00000012710

448 ENSAPLT00000000448 12419 ENSAPLT00000012419

8988 ENSAPLT00000008988 9363 ENSAPLT00000009363

11541 ENSAPLT00000011541 1577 ENSAPLT00000001577

6972 ENSAPLT00000006972 854 ENSAPLT00000000854

1314 ENSAPLT00000001314 5543 ENSAPLT00000005543

14542 ENS APLTOOOOOO 14542 743 ENSAPLT00000000743

3161 ENSAPLT00000003161 1698 ENS APLTOOOOOOO 1698

15625 ENSAPLT00000015625 8410 ENS APLT00000008410

3842 ENS APLT00000003842 9783 ENSAPLT00000009783

3781 ENSAPLT00000003781 5265 ENSAPLT00000005265

270 ENSAPLT00000000270 13600 ENS APLTOOOOOO 13600

1671 ENSAPLT00000001671 6228 ENSAPLT00000006228

12032 ENS APLTOOOOOO 12032 6616 ENS APLT00000006616

354 ENSAPLT00000000354 5833 ENSAPLT00000005833

690 ENSAPLT00000000690 15151 ENSAPLT00000015151

13261 ENSAPLT00000013261 16539 ENSAPLT00000016539

11424 ENSAPLT00000011424 16900 ENS APLTOOOOOO 16900

12543 ENSAPLT00000012543 3523 ENSAPLT00000003523

495 ENSAPLT00000000495 8811 ENSAPLT0000000881 1

16908 ENS APLTOOOOOO 16908 10654 ENSAPLT00000010654

1973 ENS APLT00000001973 6792 ENSAPLT00000006792

8833 ENSAPLT00000008833 16221 ENS APLTOOOOOO 16221

10526 ENS APLTOOOOOO 10526 15157 ENSAPLT00000015157

9981 ENS APLT00000009981 5081 ENSAPLT00000005081

16093 ENS APLTOOOOOO 16093 16002 ENS APLTOOOOOO 16002

11192 ENSAPLT00000011 192 9741 ENS APLT00000009741

887 ENSAPLT00000000887 982 ENSAPLT00000000982

1807 ENS APLT00000001807 14217 ENSAPLT00000014217

1703 ENS APLT00000001703 2202 ENSAPLT00000002202

371 ENSAPLT00000000371 3065 ENSAPLT00000003065

15159 ENSAPLT00000015159 14796 ENS APLTOOOOOO 14796

8840 ENSAPLT00000008840 4947 ENSAPLT00000004947

923 ENSAPLT00000000923 3978 ENSAPLT00000003978

213 ENS APLT00000000213 8260 ENSAPLT00000008260

2870 ENSAPLT00000002870 7972 ENSAPLT00000007972

14077 ENS APLTOOOOOO 14077 1946 ENS APLTOOOOOOO 1946

1353 ENSAPLT00000001353 12343 ENS APLTOOOOOO 12343

1999 ENS APLT00000001999 7277 ENSAPLT00000007277 14115 ENSAPLT00000014115 4163 ENS APLT00000004163

14060 ENS APLT00000014060 2150 ENSAPLT00000002150

4142 ENS APLT00000004142 8036 ENS APLT00000008036

12432 ENS APLT00000012432 8541 ENS APLT00000008541

1445 ENS APLTOOOOOOO 1445 11641 ENS APLT0000001 1641

3661 ENS APLT00000003661 6298 ENSAPLT00000006298

15683 ENSAPLT00000015683 1884 ENS APLTOOOOOOO 1884

3768 ENS APLT00000003768 5468 ENSAPLT00000005468

9739 ENSAPLT00000009739 16564 ENSAPLT00000016564

1084 ENS APLTOOOOOOO 1084 5814 ENSAPLT00000005814

7968 ENSAPLT00000007968 8470 ENSAPLT00000008470

1825 ENSAPLT00000001825 11379 ENSAPLT0000001 1379

4924 ENSAPLT00000004924 1164 ENSAPLT00000001164

12841 ENSAPLT00000012841 5526 ENSAPLT00000005526

11730 ENSAPLT00000011730 575 ENSAPLT00000000575

14479 ENS APLT00000014479 8667 ENSAPLT00000008667

2094 ENSAPLT00000002094 8951 ENSAPLT00000008951

6209 ENSAPLT00000006209 3370 ENSAPLT00000003370

4176 ENS APLT00000004176 1563 ENSAPLT00000001563

4520 ENSAPLT00000004520 15232 ENSAPLT00000015232

621 ENS APLT00000000621 2261 ENS APLT00000002261

39 ENSAPLT00000000039 11750 ENSAPLT0000001 1750

11760 ENSAPLT00000011760 10016 ENSAPLT00000010016

3093 ENSAPLT00000003093 5310 ENSAPLT00000005310

15399 ENSAPLT00000015399 656 ENS APLT00000000656

16196 ENSAPLT00000016196 5963 ENSAPLT00000005963

15703 ENSAPLT00000015703 7030 ENSAPLT00000007030

7586 ENSAPLT00000007586 773 ENSAPLT00000000773

14463 ENS APLT00000014463 16929 ENS APLTOOOOOO 16929

10825 ENSAPLT00000010825 1406 ENS APLTOOOOOOO 1406

15489 ENSAPLT00000015489 6797 ENSAPLT00000006797

16593 ENSAPLT00000016593 15175 ENSAPLT00000015175

5087 ENSAPLT00000005087 16992 ENS APLTOOOOOO 16992

6148 ENS APLT00000006148 6742 ENSAPLT00000006742

13165 ENSAPLT00000013165 1847 ENS APLTOOOOOOO 1847

2208 ENSAPLT00000002208 11656 ENS APLTOOOOOO 1 1656

6800 ENSAPLT00000006800 13329 ENSAPLT00000013329

9173 ENS APLT00000009173 4915 ENS APLT00000004915

11091 ENS APLT00000011091 10104 ENSAPLT00000010104

8374 ENSAPLT00000008374 8396 ENSAPLT00000008396

1213 ENSAPLT00000001213 17054 ENS APLTOOOOOO 17054 2211 ENSAPLT00000002211 6279 ENSAPLT00000006279

6240 ENSAPLT00000006240 12871 ENS APLTOOOOOO 12871

1294 ENS APLT00000001294 15304 ENSAPLT00000015304

15907 ENSAPLT00000015907 15139 ENSAPLT00000015139

3545 ENS APLT00000003545 11366 ENSAPLT0000001 1366

15149 ENSAPLT00000015149 5006 ENSAPLT00000005006

8176 ENS APLT00000008176 3528 ENSAPLT00000003528

12545 ENSAPLT00000012545 1327 ENSAPLT00000001327

10073 ENS APLT00000010073 16578 ENSAPLT00000016578

11167 ENSAPLT00000011 167 4398 ENSAPLT00000004398

9134 ENS APLT00000009134 7081 ENSAPLT00000007081

115 ENSAPLT00000000115 10340 ENS APLTOOOOOO 10340

2929 ENSAPLT00000002929 14575 ENSAPLT00000014575

10390 ENS APLT00000010390 1488 ENSAPLT00000001488

16372 ENSAPLT00000016372 4618 ENS APLT00000004618

16362 ENSAPLT00000016362 1480 ENS APLTOOOOOOO 1480

6252 ENSAPLT00000006252 3990 ENSAPLT00000003990

430 ENSAPLT00000000430 8618 ENSAPLT00000008618

7103 ENS APLT00000007103 11754 ENSAPLT0000001 1754

9546 ENSAPLT00000009546 15574 ENSAPLT00000015574

1299 ENS APLT00000001299 16789 ENS APLTOOOOOO 16789

17069 ENS APLT00000017069 1486 ENSAPLT00000001486

16070 ENS APLT00000016070 9159 ENSAPLT00000009159

10359 ENSAPLT00000010359 8516 ENSAPLT00000008516

306 ENSAPLT00000000306 8256 ENSAPLT00000008256

11829 ENSAPLT00000011829 3265 ENSAPLT00000003265

1663 ENS APLT00000001663 8210 ENS APLT00000008210

273 ENSAPLT00000000273 7606 ENSAPLT00000007606

6251 ENSAPLT00000006251 14414 ENSAPLT00000014414

13399 ENSAPLT00000013399 5448 ENSAPLT00000005448

1746 ENS APLT00000001746 3899 ENSAPLT00000003899

349 ENSAPLT00000000349 15580 ENSAPLT00000015580

1633 ENSAPLT00000001633 4119 ENSAPLT00000004119

10906 ENS APLT00000010906 10933 ENSAPLT00000010933

4321 ENS APLT00000004321 2855 ENSAPLT00000002855

1389 ENSAPLT00000001389 11519 ENSAPLT0000001 1519

16496 ENS APLT00000016496 6482 ENSAPLT00000006482

11373 ENSAPLT00000011373 12553 ENSAPLT00000012553

12945 ENS APLTOOOOOO 12945 6721 ENSAPLT00000006721

8476 ENSAPLT00000008476 6799 ENSAPLT00000006799

3145 ENSAPLT00000003145 8461 ENS APLT00000008461 5574 ENSAPLT00000005574 3767 ENSAPLT00000003767

15144 ENSAPLT00000015144 2976 ENSAPLT00000002976

1972 ENS APLT00000001972 4813 ENS APLT00000004813

3204 ENSAPLT00000003204 6938 ENS APLT00000006938

1040 ENS APLT00000001040 3349 ENSAPLT00000003349

721 ENS APLT00000000721 8689 ENSAPLT00000008689

13028 ENSAPLT00000013028 6200 ENSAPLT00000006200

1597 ENS APLT00000001597 16670 ENS APLTOOOOOO 16670

2289 ENSAPLT00000002289 15295 ENS APLTOOOOOO 15295

329 ENSAPLT00000000329 1826 ENSAPLTOOOOOOO 1826

720 ENSAPLT00000000720 9111 ENSAPLT0000000911 1

9526 ENSAPLT00000009526 8992 ENSAPLT00000008992

12136 ENSAPLT00000012136 3983 ENSAPLT00000003983

16386 ENSAPLT00000016386 6211 ENSAPLT0000000621 1

11246 ENSAPLT00000011246 16078 ENS APLTOOOOOO 16078

1690 ENS APLT00000001690 14228 ENSAPLT00000014228

200 ENSAPLT00000000200 10163 ENSAPLT00000010163

729 ENSAPLT00000000729 13534 ENSAPLT00000013534

1066 ENS APLT00000001066 15335 ENSAPLT00000015335

10884 ENS APLT00000010884 10610 ENSAPLT00000010610

2991 ENS APLT00000002991 4765 ENSAPLT00000004765

7645 ENSAPLT00000007645 627 ENSAPLT00000000627

1843 ENS APLT00000001843 10239 ENSAPLT00000010239

372 ENSAPLT00000000372 13414 ENSAPLT00000013414

522 ENSAPLT00000000522 9184 ENS APLT00000009184

4692 ENSAPLT00000004692 16198 ENSAPLT00000016198

1998 ENS APLT00000001998 11454 ENSAPLT0000001 1454

13404 ENSAPLT00000013404 10177 ENSAPLT00000010177

13926 ENSAPLT00000013926 986 ENSAPLT00000000986

1764 ENS APLT00000001764 11536 ENSAPLT0000001 1536

9638 ENSAPLT00000009638 3932 ENSAPLT00000003932

12053 ENSAPLT00000012053 11204 ENS APLTOOOOOO 1 1204

16632 ENSAPLT00000016632 15573 ENSAPLT00000015573

7472 ENSAPLT00000007472 13946 ENS APLTOOOOOO 13946

4604 ENSAPLT00000004604 3934 ENSAPLT00000003934

776 ENSAPLT00000000776 7306 ENSAPLT00000007306

12594 ENS APLTOOOOOO 12594 15013 ENSAPLT00000015013

1727 ENS APLTOOOOOOO 1727 12370 ENSAPLT00000012370

14256 ENS APLTOOOOOO 14256 14252 ENSAPLT00000014252

1494 ENS APLTOOOOOOO 1494 14763 ENS APLTOOOOOO 14763

8248 ENSAPLT00000008248 11972 ENS APLTOOOOOO 1 1972 8528 ENSAPLT00000008528 4795 ENSAPLT00000004795

94 ENSAPLT00000000094 4565 ENSAPLT00000004565

16165 ENSAPLTOOOOOOl 6165 12310 ENSAPLTOOOOOOl 2310

4562 ENSAPLT00000004562 5188 ENSAPLT00000005188

8807 ENSAPLT00000008807 1993 ENS APLTOOOOOOO 1993

3153 ENSAPLT00000003153 6377 ENSAPLT00000006377

830 ENSAPLT00000000830 12188 ENSAPLT00000012188

1308 ENS APLT00000001308 12718 ENSAPLTOOOOOOl 2718

9847 ENSAPLT00000009847 11364 ENSAPLTOOOOOOl 1364

15154 ENSAPLTOOOOOOl 5154 13290 ENS APLTOOOOOO 13290

8560 ENSAPLT00000008560 13888 ENSAPLTOOOOOOl 3888

14903 ENS APLTOOOOOO 14903 9906 ENSAPLT00000009906

12569 ENSAPLT00000012569 7074 ENSAPLT00000007074

1334 ENSAPLT00000001334 2706 ENSAPLT00000002706

14107 ENSAPLT00000014107 13631 ENSAPLTOOOOOOl 3631

1377 ENSAPLT00000001377 2461 ENS APLT00000002461

1132 ENS APLT00000001 132 11235 ENSAPLTOOOOOOl 1235

1 ENS APLT00000000001 732 ENSAPLT00000000732

10877 ENSAPLTOOOOOOl 0877 5884 ENSAPLT00000005884

8574 ENSAPLT00000008574 15016 ENSAPLTOOOOOOl 5016

3054 ENSAPLT00000003054 16370 ENSAPLTOOOOOOl 6370

3704 ENS APLT00000003704 10007 ENS APLTOOOOOO 10007

15947 ENSAPLT00000015947 16626 ENSAPLTOOOOOOl 6626

184 ENS APLTOOOOOOOO 184 758 ENS APLT00000000758

9904 ENSAPLT00000009904 7826 ENSAPLT00000007826

3192 ENSAPLT00000003192 2205 ENSAPLT00000002205

10727 ENS APLTOOOOOO 10727 4205 ENSAPLT00000004205

4335 ENSAPLT00000004335 5878 ENSAPLT00000005878

4226 ENSAPLT00000004226 15740 ENSAPLTOOOOOOl 5740

7571 ENSAPLT00000007571 7564 ENSAPLT00000007564

9244 ENSAPLT00000009244 13903 ENSAPLTOOOOOOl 3903

12456 ENS APLTOOOOOO 12456 4506 ENSAPLT00000004506

15310 ENSAPLTOOOOOOl 5310 2851 ENSAPLT00000002851

6724 ENSAPLT00000006724 5515 ENSAPLT00000005515

8983 ENSAPLT00000008983 702 ENSAPLT00000000702

274 ENSAPLT00000000274 6461 ENS APLT00000006461

13415 ENSAPLT00000013415 10490 ENS APLTOOOOOO 10490

4728 ENSAPLT00000004728 7444 ENSAPLT00000007444

2854 ENSAPLT00000002854 7824 ENSAPLT00000007824

2434 ENSAPLT00000002434 1925 ENSAPLT00000001925

2245 ENSAPLT00000002245 12009 ENS APLTOOOOOO 12009 5336 ENSAPLT00000005336 1265 ENSAPLT00000001265

10125 ENSAPLT00000010125 15812 ENSAPLT00000015812

1565 ENSAPLT00000001565 3655 ENSAPLT00000003655

16046 ENS APLT00000016046 6123 ENS APLT00000006123

8083 ENSAPLT00000008083 3392 ENSAPLT00000003392

16407 ENS APLT00000016407 9092 ENSAPLT00000009092

16976 ENS APLT00000016976 17138 ENSAPLT00000017138

3573 ENSAPLT00000003573 1423 ENSAPLT00000001423

4834 ENSAPLT00000004834 14932 ENSAPLT00000014932

2110 ENSAPLT00000002110 2620 ENSAPLT00000002620

14936 ENS APLT00000014936 12904 ENS APLTOOOOOO 12904

254 ENSAPLT00000000254 2792 ENSAPLT00000002792

14187 ENSAPLT00000014187 7844 ENSAPLT00000007844

10196 ENSAPLT00000010196 8001 ENS APLT00000008001

10069 ENS APLT00000010069 831 ENSAPLT00000000831

797 ENSAPLT00000000797 16142 ENSAPLT00000016142

870 ENSAPLT00000000870 16675 ENSAPLT00000016675

9278 ENSAPLT00000009278 9478 ENSAPLT00000009478

15045 ENSAPLT00000015045 3874 ENSAPLT00000003874

12933 ENSAPLT00000012933 12085 ENSAPLT00000012085

14406 ENS APLT00000014406 10398 ENS APLTOOOOOO 10398

7978 ENSAPLT00000007978 940 ENSAPLT00000000940

16669 ENS APLT00000016669 3417 ENSAPLT00000003417

268 ENSAPLT00000000268 13548 ENSAPLT00000013548

15350 ENSAPLT00000015350 15578 ENSAPLT00000015578

11974 ENSAPLT00000011974 12515 ENSAPLT00000012515

10848 ENS APLTOOOOOO 10848 12291 ENS APLTOOOOOO 12291

8501 ENSAPLT00000008501 6967 ENSAPLT00000006967

8825 ENSAPLT00000008825 3084 ENSAPLT00000003084

6328 ENSAPLT00000006328 14838 ENSAPLT00000014838

6141 ENSAPLT00000006141 4233 ENS APLT00000004233

12094 ENS APLTOOOOOO 12094 2301 ENS APLT00000002301

4519 ENS APLT00000004519 3057 ENSAPLT00000003057

5368 ENSAPLT00000005368 5915 ENSAPLT00000005915

5424 ENSAPLT00000005424 3323 ENSAPLT00000003323

10488 ENS APLTOOOOOO 10488 7654 ENSAPLT00000007654

11884 ENSAPLT00000011884 13712 ENSAPLT00000013712

8400 ENSAPLT00000008400 10127 ENSAPLT00000010127

8044 ENSAPLT00000008044 5250 ENSAPLT00000005250

7304 ENSAPLT00000007304 16888 ENSAPLT00000016888

6787 ENSAPLT00000006787 8442 ENSAPLT00000008442 3844 ENS APLT00000003844 585 ENSAPLT00000000585

14787 ENS APLTOOOOOO 14787 3509 ENSAPLT00000003509

9445 ENSAPLT00000009445 3993 ENSAPLT00000003993

2804 ENSAPLT00000002804 15355 ENSAPLT00000015355

2249 ENSAPLT00000002249 10977 ENSAPLT00000010977

4566 ENSAPLT00000004566 5653 ENSAPLT00000005653

15025 ENSAPLT00000015025 4705 ENSAPLT00000004705

1776 ENS APLTOOOOOOO 1776 10314 ENSAPLT00000010314

14545 ENS APLTOOOOOO 14545 10854 ENSAPLT00000010854

2774 ENSAPLT00000002774 8415 ENS APLT00000008415

5024 ENSAPLT00000005024 10941 ENS APLTOOOOOO 10941

1220 ENS APLTOOOOOOO 1220 13889 ENSAPLT00000013889

13601 ENSAPLT00000013601 16612 ENSAPLT00000016612

12767 ENSAPLT00000012767 13617 ENSAPLT00000013617

596 ENSAPLT00000000596 7449 ENSAPLT00000007449

9115 ENSAPLT00000009115 11151 ENSAPLT0000001 1151

5359 ENSAPLT00000005359 8148 ENSAPLT00000008148

13056 ENSAPLT00000013056 9473 ENSAPLT00000009473

6063 ENSAPLT00000006063 12038 ENSAPLT00000012038

16026 ENS APLTOOOOOO 16026 1576 ENSAPLT00000001576

2049 ENSAPLT00000002049 1828 ENSAPLTOOOOOOO 1828

2961 ENS APLT00000002961 13938 ENSAPLT00000013938

2435 ENSAPLT00000002435 6176 ENSAPLT00000006176

14090 ENS APLTOOOOOO 14090 5596 ENSAPLT00000005596

9905 ENSAPLT00000009905 15166 ENSAPLT00000015166

579 ENSAPLT00000000579 6225 ENSAPLT00000006225

14314 ENSAPLT00000014314 11389 ENSAPLT0000001 1389

1672 ENS APLTOOOOOOO 1672 12371 ENSAPLT00000012371

16790 ENS APLTOOOOOO 16790 12916 ENSAPLT00000012916

1054 ENS APLTOOOOOOO 1054 4124 ENS APLT00000004124

7579 ENSAPLT00000007579 3028 ENSAPLT00000003028

7458 ENSAPLT00000007458 12709 ENS APLTOOOOOO 12709

9860 ENSAPLT00000009860 3562 ENSAPLT00000003562

4998 ENSAPLT00000004998 9006 ENSAPLT00000009006

11250 ENSAPLT00000011250 5346 ENSAPLT00000005346

12277 ENS APLTOOOOOO 12277 6408 ENSAPLT00000006408

14390 ENS APLTOOOOOO 14390 5108 ENSAPLT00000005108

1033 ENSAPLT00000001033 11936 ENS APLTOOOOOO 1 1936

1382 ENSAPLT00000001382 1700 ENSAPLTOOOOOOO 1700

787 ENSAPLT00000000787 9072 ENSAPLT00000009072

8450 ENSAPLT00000008450 571 ENSAPLT00000000571 5598 ENS APLT00000005598 753 ENS APLT00000000753

12592 ENS APLTOOOOOO 12592 15143 ENSAPLT00000015143

1269 ENS APLTOOOOOOO 1269 1831 ENSAPLT00000001831

2621 ENSAPLT00000002621 3521 ENSAPLT00000003521

1381 ENSAPLT00000001381 10006 ENS APLTOOOOOO 10006

3256 ENSAPLT00000003256 14206 ENS APLTOOOOOO 14206

14619 ENSAPLT00000014619 12707 ENS APLTOOOOOO 12707

3321 ENSAPLT00000003321 16121 ENSAPLT00000016121

9189 ENS APLT00000009189 1385 ENSAPLT00000001385

16465 ENS APLTOOOOOO 16465 1036 ENS APLTOOOOOOO 1036

15533 ENSAPLT00000015533 1403 ENS APLTOOOOOOO 1403

9165 ENS APLT00000009165 3965 ENSAPLT00000003965

3803 ENS APLT00000003803 14445 ENS APLTOOOOOO 14445

11446 ENSAPLT00000011446 3069 ENSAPLT00000003069

15011 ENSAPLT00000015011 11604 ENS APLTOOOOOO 1 1604

1869 ENS APLTOOOOOOO 1869 942 ENSAPLT00000000942

8857 ENSAPLT00000008857 9664 ENSAPLT00000009664

235 ENSAPLT00000000235 2694 ENSAPLT00000002694

4875 ENSAPLT00000004875 12098 ENS APLTOOOOOO 12098

6132 ENS APLT00000006132 3262 ENSAPLT00000003262

4474 ENSAPLT00000004474 11870 ENS APLTOOOOOO 1 1870

4538 ENSAPLT00000004538 8694 ENSAPLT00000008694

2821 ENSAPLT00000002821 8144 ENS APLT00000008144

15264 ENSAPLT00000015264 8348 ENSAPLT00000008348

10191 ENSAPLT00000010191 10260 ENS APLTOOOOOO 10260

1350 ENSAPLT00000001350 4216 ENS APLT00000004216

1603 ENS APLTOOOOOOO 1603 8251 ENSAPLT00000008251

945 ENSAPLT00000000945 15588 ENSAPLT00000015588

3159 ENSAPLT00000003159 12267 ENS APLTOOOOOO 12267

15183 ENSAPLT00000015183 5406 ENSAPLT00000005406

8149 ENS APLT00000008149 16846 ENS APLTOOOOOO 16846

988 ENSAPLT00000000988 1095 ENS APLTOOOOOOO 1095

6615 ENS APLT00000006615 14966 ENS APLTOOOOOO 14966

13609 ENS APLTOOOOOO 13609 8767 ENSAPLT00000008767

16036 ENSAPLT00000016036 8245 ENSAPLT00000008245

3140 ENSAPLT00000003140 1329 ENS APLTOOOOOOO 1329

1296 ENS APLTOOOOOOO 1296 8942 ENSAPLT00000008942

12799 ENS APLTOOOOOO 12799 10910 ENSAPLT00000010910

12289 ENS APLTOOOOOO 12289 14447 ENS APLTOOOOOO 14447

10971 ENS APLTOOOOOO 10971 8675 ENSAPLT00000008675

15554 ENSAPLT00000015554 6013 ENS APLT00000006013 12428 ENS APLT00000012428 5151 ENSAPLT00000005151

13029 ENSAPLT00000013029 6745 ENSAPLT00000006745

It has surprisingly been discovered that certain transcripts are expressed in infected avian cells but not in uninfected cells. These avian transcripts represent an opportunity to modulate the overall biological product production in cells upon infection by modulating the transcripts which will be expressed upon infection. As such any one or more of these transcripts represents a unique target for the alteration of the avian cellular phenotype to improve the methods disclosed herein.

Example 7: Screening Targeting Constructs

A subset of avian transcript targets was selected for screening. Table 9 lists the siRNA duplex IDs, target (including transcript name), and the synthesized sense and antisense sequence IDs for the targeting constructs designed against the subset of nine avian target genes. These genes are bcl2 (B-cell CLL/lymphoma 2), Dicer 1 (ribonuclease type III), eIF2AK2 (eukaryotic translation initiation factor 2-alpha kinase 2), IFNAR( interferon (alpha, beta and omega) receptor 1), IFNB (interferon beta), IRF7 (interferon regulatory factor 7), MAVS (mitochondrial antiviral signaling protein), MXl (myxovirus resistance 1, interferon-inducible protein p78) and TLR3 (toll- like receptor 3). Endolight chemistry was applied as described herein.

Table 9: Targeting Constructs

AD-49257.1 BCL2 ENSAPLT00000007572 755552 755553

AD-49263.1 BCL2 ENSAPLT00000007572 755554 755555

AD-49268.1 BCL2 ENSAPLT00000007572 755556 755557

AD-49273.1 BCL2 ENSAPLT00000007572 755558 755559

AD-49234.1 BCL2 ENSAPLT00000007572 755560 755561

AD-49240.1 BCL2 ENSAPLT00000007572 755562 755563

AD-49246.1 BCL2 ENSAPLT00000007572 755564 755565

AD-49252.1 BCL2 ENSAPLT00000007572 755566 755567

AD-49258.1 BCL2 ENSAPLT00000007572 755568 755569

AD-49264.1 BCL2 ENSAPLT00000007572 755570 755571

AD-49417.1 DICERl ENSAPLT00000012965 1288360 1288361

AD-49423.1 DICERl ENSAPLT00000012965 1288362 1288363

AD-37986.2 DICERl ENSAPLT00000012965 1288366 1288367

AD-37986.1 DICERl ENSAPLT00000012965 1288366 1288367

AD-49432.1 DICERl ENSAPLT00000012965 1288368 1288369

AD-49438.1 DICERl ENSAPLT00000012965 1288370 1288371

AD-49444.1 DICERl ENSAPLT00000012965 1288372 1288373

AD-49450.1 DICERl ENSAPLT00000012965 1288374 1288375

AD-49456.1 DICERl ENSAPLT00000012965 1288376 1288377

AD-49418.1 DICERl ENSAPLT00000012965 1288378 1288379

AD-49424.1 DICERl ENSAPLT00000012965 1288380 1288381

AD-49428.1 DICERl ENSAPLT00000012965 1288382 1288383

AD-49433.1 DICERl ENSAPLT00000012965 1288384 1288385

AD-49439.1 DICERl ENSAPLT00000012965 1288386 1288387

AD-49445.1 DICERl ENSAPLT00000012965 1288388 1288389

AD-49451.1 DICERl ENSAPLT00000012965 1288390 1288391

AD-49457.1 DICERl ENSAPLT00000012965 1288392 1288393

AD-49419.1 DICERl ENSAPLT00000012965 1288394 1288395

AD-49425.1 DICERl ENSAPLT00000012965 1288396 1288397

AD-49429.1 DICERl ENSAPLT00000012965 1288398 1288399

AD-49434.1 DICERl ENSAPLT00000012965 1288400 1288401

AD-49440.1 DICERl ENSAPLT00000012965 1288402 1288403

AD-49446.1 DICERl ENSAPLT00000012965 1288404 1288405

AD-49452.1 DICERl ENSAPLT00000012965 1288406 1288407

AD-49458.1 DICERl ENSAPLT00000012965 1288408 1288409

AD-49420.1 DICERl ENSAPLT00000012965 1288410 1288411

AD-49426.1 DICERl ENSAPLT00000012965 1288412 1288413

AD-49430.1 DICERl ENSAPLT00000012965 1288414 1288415

AD-49435.1 DICERl ENSAPLT00000012965 1288416 1288417 AD-49441.1 DICERl ENSAPLT00000012965 1288418 1288419

AD-49447.1 DICERl ENSAPLT00000012965 1288420 1288421

AD-49453.1 DICERl ENSAPLT00000012965 1288422 1288423

AD-49459.1 DICERl ENSAPLT00000012965 1288424 1288425

AD-49421.1 DICERl ENSAPLT00000012965 1288426 1288427

AD-49431.1 DICERl ENSAPLT00000012965 1288428 1288429

AD-49436.1 DICERl ENSAPLT00000012965 1288430 1288431

AD-49448.1 DICERl ENSAPLT00000012965 1288432 1288433

AD-49454.1 DICERl ENSAPLT00000012965 1288434 1288435

AD-49460.1 DICERl ENSAPLT00000012965 1288436 1288437

AD-49422.1 DICERl ENSAPLT00000012965 1288438 1288439

AD-38003.2 DICERl ENSAPLT00000012965 1288442 1288443

AD-38003.1 DICERl ENSAPLT00000012965 1288442 1288443

AD-38009.1 DICERl ENSAPLT00000012965 1288444 1288445

AD-49437.1 DICERl ENSAPLT00000012965 1288446 1288447

AD-49443.1 DICERl ENSAPLT00000012965 1288448 1288449

AD-49449.1 DICERl ENSAPLT00000012965 1288450 1288451

AD-49455.1 DICERl ENSAPLT00000012965 1288452 1288453

AD-49269.1 EIF2A 2 ENSAPLT00000002924 296860 296861

AD-49274.1 EIF2A 2 ENSAPLT00000002924 296862 296863

AD-49235.1 EIF2A 2 ENSAPLT00000002924 296864 296865

AD-49241.1 EIF2A 2 ENSAPLT00000002924 296866 296867

AD-49247.1 EIF2A 2 ENSAPLT00000002924 296868 296869

AD-49253.1 EIF2A 2 ENSAPLT00000002924 296870 296871

AD-49259.1 EIF2A 2 ENSAPLT00000002924 296872 296873

AD-49265.1 EIF2A 2 ENSAPLT00000002924 296874 296875

AD-49270.1 EIF2A 2 ENSAPLT00000002924 296876 296877

AD-49275.1 EIF2A 2 ENSAPLT00000002924 296878 296879

AD-49236.1 EIF2A 2 ENSAPLT00000002924 296880 296881

AD-49242.1 EIF2A 2 ENSAPLT00000002924 296882 296883

AD-49248.1 EIF2A 2 ENSAPLT00000002924 296884 296885

AD-49254.1 EIF2A 2 ENSAPLT00000002924 296886 296887

AD-49260.1 EIF2A 2 ENSAPLT00000002924 296888 296889

AD-49266.1 EIF2A 2 ENSAPLT00000002924 296890 296891

AD-49271.1 EIF2A 2 ENSAPLT00000002924 296892 296893

AD-49276.1 EIF2A 2 ENSAPLT00000002924 296894 296895

AD-49237.1 EIF2A 2 ENSAPLT00000002924 296896 296897

AD-49249.1 EIF2A 2 ENSAPLT00000002924 296898 296899

AD-49255.1 EIF2A 2 ENSAPLT00000002924 296900 296901 AD-49461.1 IFNAR1 ENSAPLT00000011786 1171716 1171717

AD-49467.1 IFNAR1 ENSAPLT00000011786 1171718 1171719

AD-49473.1 IFNAR1 ENSAPLT00000011786 1171720 1171721

AD-49479.1 IFNAR1 ENSAPLT00000011786 1171722 1171723

AD-49485.1 IFNAR1 ENSAPLT00000011786 1171724 1171725

AD-49491.1 IFNAR1 ENSAPLT00000011786 1171726 1171727

AD-49497.1 IFNAR1 ENSAPLT00000011786 1171728 1171729

AD-49503.1 IFNAR1 ENSAPLT00000011786 1171730 1171731

AD-49462.1 IFNAR1 ENSAPLT00000011786 1171732 1171733

AD-49468.1 IFNAR1 ENSAPLT00000011786 1171734 1171735

AD-49474.1 IFNAR1 ENSAPLT00000011786 1171736 1171737

AD-49480.1 IFNAR1 ENSAPLT00000011786 1171738 1171739

AD-49486.1 IFNAR1 ENSAPLT00000011786 1171740 1171741

AD-49492.1 IFNAR1 ENSAPLT00000011786 1171742 1171743

AD-49498.1 IFNAR1 ENSAPLT00000011786 1171744 1171745

AD-49504.1 IFNAR1 ENSAPLT00000011786 1171746 1171747

AD-49463.1 IFNAR1 ENSAPLT00000011786 1171748 1171749

AD-49469.1 IFNAR1 ENSAPLT00000011786 1171750 1171751

AD-49475.1 IFNAR1 ENSAPLT00000011786 1171752 1171753

AD-49481.1 IFNAR1 ENSAPLT00000011786 1171754 1171755

AD-49487.1 IFNAR1 ENSAPLT00000011786 1171756 1171757

AD-49493.1 IFNAR1 ENSAPLT00000011786 1171758 1171759

AD-49499.1 IFNAR1 ENSAPLT00000011786 1171760 1171761

AD-49505.1 IFNAR1 ENSAPLT00000011786 1171762 1171763

AD-49464.1 IFNAR1 ENSAPLT00000011786 1171764 1171765

AD-49470.1 IFNAR1 ENSAPLT00000011786 1171766 1171767

AD-49476.1 IFNAR1 ENSAPLT00000011786 1171768 1171769

AD-49482.1 IFNAR1 ENSAPLT00000011786 1171770 1171771

AD-49488.1 IFNAR1 ENSAPLT00000011786 1171772 1171773

AD-49494.1 IFNAR1 ENSAPLT00000011786 1171774 1171775

AD-49500.1 IFNAR1 ENSAPLT00000011786 1171776 1171777

AD-49506.1 IFNAR1 ENSAPLT00000011786 1171778 1171779

AD-49465.1 IFNAR1 ENSAPLT00000011786 1171780 1171781

AD-49471.1 IFNAR1 ENSAPLT00000011786 1171782 1171783

AD-49477.1 IFNAR1 ENSAPLT00000011786 1171784 1171785

AD-49483.1 IFNAR1 ENSAPLT00000011786 1171786 1171787

AD-49495.1 IFNAR1 ENSAPLT00000011786 1171788 1171789

AD-49501.1 IFNAR1 ENSAPLT00000011786 1171790 1171791

AD-49466.1 IFNAR1 ENSAPLT00000011786 1171792 1171793 AD-49472.1 IFNAR1 ENSAPLT00000011786 1171794 1171795

AD-49484.1 IFNAR1 ENSAPLT00000011786 1171796 1171797

AD-49490.1 IFNAR1 ENSAPLT00000011786 1171798 1171799

AD-49496.1 IFNAR1 ENSAPLT00000011786 1171800 1171801

AD-49502.1 IFNAR1 ENSAPLT00000011786 1171802 1171803

AD-49726.1 IFNB ENSAPLT00000001356 143514 143515

AD-49732.1 IFNB ENSAPLT00000001356 143516 143517

AD-49738.1 IFNB ENSAPLT00000001356 143518 143519

AD-49744.1 IFNB ENSAPLT00000001356 143520 143521

AD-49750.1 IFNB ENSAPLT00000001356 143522 143523

AD-49755.1 IFNB ENSAPLT00000001356 143524 143525

AD-49760.1 IFNB ENSAPLT00000001356 143526 143527

AD-49765.1 IFNB ENSAPLT00000001356 143528 143529

AD-49727.1 IFNB ENSAPLT00000001356 143530 143531

AD-49733.1 IFNB ENSAPLT00000001356 143532 143533

AD-49739.1 IFNB ENSAPLT00000001356 143534 143535

AD-49745.1 IFNB ENSAPLT00000001356 143536 143537

AD-49751.1 IFNB ENSAPLT00000001356 143538 143539

AD-49756.1 IFNB ENSAPLT00000001356 143540 143541

AD-49761.1 IFNB ENSAPLT00000001356 143542 143543

AD-49766.1 IFNB ENSAPLT00000001356 143544 143545

AD-49728.1 IFNB ENSAPLT00000001356 143546 143547

AD-49734.1 IFNB ENSAPLT00000001356 143548 143549

AD-49740.1 IFNB ENSAPLT00000001356 143550 143551

AD-49746.1 IFNB ENSAPLT00000001356 143552 143553

AD-49752.1 IFNB ENSAPLT00000001356 143554 143555

AD-49757.1 IFNB ENSAPLT00000001356 143556 143557

AD-49762.1 IFNB ENSAPLT00000001356 143558 143559

AD-49767.1 IFNB ENSAPLT00000001356 143560 143561

AD-49729.1 IFNB ENSAPLT00000001356 143562 143563

AD-49735.1 IFNB ENSAPLT00000001356 143564 143565

AD-49741.1 IFNB ENSAPLT00000001356 143566 143567

AD-49747.1 IFNB ENSAPLT00000001356 143568 143569

AD-49753.1 IFNB ENSAPLT00000001356 143570 143571

AD-49758.1 IFNB ENSAPLT00000001356 143572 143573

AD-49763.1 IFNB ENSAPLT00000001356 143574 143575

AD-49768.1 IFNB ENSAPLT00000001356 143576 143577

AD-49730.1 IFNB ENSAPLT00000001356 143578 143579

AD-49736.1 IFNB ENSAPLT00000001356 143580 143581 AD-49742.1 IFNB ENSAPLT00000001356 143582 143583

AD-49748.1 IFNB ENSAPLT00000001356 143584 143585

AD-49754.1 IFNB ENSAPLT00000001356 143586 143587

AD-49759.1 IFNB ENSAPLT00000001356 143588 143589

AD-49764.1 IFNB ENSAPLT00000001356 143590 143591

AD-49769.1 IFNB ENSAPLT00000001356 143592 143593

AD-49731.1 IFNB ENSAPLT00000001356 143594 143595

AD-49737.1 IFNB ENSAPLT00000001356 143596 143597

AD-49743.1 IFNB ENSAPLT00000001356 143598 143599

AD-49749.1 IFNB ENSAPLT00000001356 143600 143601

AD-49818.1 IRF7 ENSAPLT00000013271 1318832 1318833

AD-49824.1 IRF7 ENSAPLT00000013271 1318834 1318835

AD-49830.1 IRF7 ENSAPLT00000013271 1318836 1318837

AD-49836.1 IRF7 ENSAPLT00000013271 1318838 1318839

AD-49842.1 IRF7 ENSAPLT00000013271 1318840 1318841

AD-49847.1 IRF7 ENSAPLT00000013271 1318842 1318843

AD-49852.1 IRF7 ENSAPLT00000013271 1318844 1318845

AD-49857.1 IRF7 ENSAPLT00000013271 1318846 1318847

AD-49819.1 IRF7 ENSAPLT00000013271 1318848 1318849

AD-49825.1 IRF7 ENSAPLT00000013271 1318850 1318851

AD-49831.1 IRF7 ENSAPLT00000013271 1318852 1318853

AD-49837.1 IRF7 ENSAPLT00000013271 1318854 1318855

AD-49843.1 IRF7 ENSAPLT00000013271 1318856 1318857

AD-49848.1 IRF7 ENSAPLT00000013271 1318858 1318859

AD-49853.1 IRF7 ENSAPLT00000013271 1318860 1318861

AD-49858.1 IRF7 ENSAPLT00000013271 1318862 1318863

AD-49820.1 IRF7 ENSAPLT00000013271 1318864 1318865

AD-49826.1 IRF7 ENSAPLT00000013271 1318866 1318867

AD-49832.1 IRF7 ENSAPLT00000013271 1318868 1318869

AD-49838.1 IRF7 ENSAPLT00000013271 1318870 1318871

AD-49844.1 IRF7 ENSAPLT00000013271 1318872 1318873

AD-49849.1 IRF7 ENSAPLT00000013271 1318874 1318875

AD-49854.1 IRF7 ENSAPLT00000013271 1318876 1318877

AD-49859.1 IRF7 ENSAPLT00000013271 1318878 1318879

AD-49821.1 IRF7 ENSAPLT00000013271 1318880 1318881

AD-49827.1 IRF7 ENSAPLT00000013271 1318882 1318883

AD-49833.1 IRF7 ENSAPLT00000013271 1318884 1318885

AD-49839.1 IRF7 ENSAPLT00000013271 1318886 1318887

AD-49845.1 IRF7 ENSAPLT00000013271 1318888 1318889 AD-49850.1 IRF7 ENSAPLT00000013271 1318890 1318891

AD-49855.1 IRF7 ENSAPLT00000013271 1318892 1318893

AD-49860.1 IRF7 ENSAPLT00000013271 1318894 1318895

AD-49822.1 IRF7 ENSAPLT00000013271 1318896 1318897

AD-49828.1 IRF7 ENSAPLT00000013271 1318898 1318899

AD-49834.1 IRF7 ENSAPLT00000013271 1318900 1318901

AD-49840.1 IRF7 ENSAPLT00000013271 1318902 1318903

AD-49846.1 IRF7 ENSAPLT00000013271 1318904 1318905

AD-49851.1 IRF7 ENSAPLT00000013271 1318906 1318907

AD-49856.1 IRF7 ENSAPLT00000013271 1318908 1318909

AD-49861.1 IRF7 ENSAPLT00000013271 1318910 1318911

AD-49823.1 IRF7 ENSAPLT00000013271 1318912 1318913

AD-49829.1 IRF7 ENSAPLT00000013271 1318914 1318915

AD-49835.1 IRF7 ENSAPLT00000013271 1318916 1318917

AD-49841.1 IRF7 ENSAPLT00000013271 1318918 1318919

AD-49815.1 MAVS ENSAPLT00000003383 341904 341905

AD-49775.1 MAVS ENSAPLT00000003383 341906 341907

AD-49781.1 MAVS ENSAPLT00000003383 341908 341909

AD-49793.1 MAVS ENSAPLT00000003383 341910 341911

AD-49799.1 MAVS ENSAPLT00000003383 341912 341913

AD-49805.1 MAVS ENSAPLT00000003383 341914 341915

AD-49811.1 MAVS ENSAPLT00000003383 341916 341917

AD-49776.1 MAVS ENSAPLT00000003383 341918 341919

AD-49782.1 MAVS ENSAPLT00000003383 341920 341921

AD-49788.1 MAVS ENSAPLT00000003383 341922 341923

AD-49794.1 MAVS ENSAPLT00000003383 341924 341925

AD-49800.1 MAVS ENSAPLT00000003383 341926 341927

AD-49812.1 MAVS ENSAPLT00000003383 341928 341929

AD-49817.1 MAVS ENSAPLT00000003383 341930 341931

AD-49777.1 MAVS ENSAPLT00000003383 341932 341933

AD-49783.1 MAVS ENSAPLT00000003383 341934 341935

AD-49789.1 MAVS ENSAPLT00000003383 341936 341937

AD-49795.1 MAVS ENSAPLT00000003383 341938 341939

AD-49801.1 MAVS ENSAPLT00000003383 341940 341941

AD-49862.1 MX1 ENSAPLTOOOOOO 16707 1658300 1658301

AD-49868.1 MX1 ENSAPLTOOOOOO 16707 1658302 1658303

AD-49874.1 MX1 ENSAPLTOOOOOO 16707 1658304 1658305

AD-49880.1 MX1 ENSAPLTOOOOOO 16707 1658306 1658307

AD-49886.1 MX1 ENSAPLTOOOOOO 16707 1658308 1658309 AD-49892.1 MX1 ENSAPLTOOOOOO 16707 1658310 1658311

AD-49898.1 MX1 ENSAPLTOOOOOO 16707 1658312 1658313

AD-49904.1 MX1 ENSAPLTOOOOOO 16707 1658314 1658315

AD-49863.1 MX1 ENSAPLTOOOOOO 16707 1658316 1658317

AD-49869.1 MX1 ENSAPLTOOOOOO 16707 1658318 1658319

AD-49875.1 MX1 ENSAPLTOOOOOO 16707 1658320 1658321

AD-49881.1 MX1 ENSAPLTOOOOOO 16707 1658322 1658323

AD-49887.1 MX1 ENSAPLTOOOOOO 16707 1658324 1658325

AD-49893.1 MX1 ENSAPLTOOOOOO 16707 1658326 1658327

AD-49899.1 MX1 ENSAPLTOOOOOO 16707 1658328 1658329

AD-49905.1 MX1 ENSAPLTOOOOOO 16707 1658330 1658331

AD-49864.1 MX1 ENSAPLTOOOOOO 16707 1658332 1658333

AD-49870.1 MX1 ENSAPLTOOOOOO 16707 1658334 1658335

AD-49876.1 MX1 ENSAPLTOOOOOO 16707 1658336 1658337

AD-49882.1 MX1 ENSAPLTOOOOOO 16707 1658338 1658339

AD-49888.1 MX1 ENSAPLTOOOOOO 16707 1658340 1658341

AD-49894.1 MX1 ENSAPLTOOOOOO 16707 1658342 1658343

AD-49900.1 MX1 ENSAPLTOOOOOO 16707 1658344 1658345

AD-49906.1 MX1 ENSAPLTOOOOOO 16707 1658346 1658347

AD-49865.1 MX1 ENSAPLTOOOOOO 16707 1658348 1658349

AD-49871.1 MX1 ENSAPLTOOOOOO 16707 1658350 1658351

AD-49877.1 MX1 ENSAPLTOOOOOO 16707 1658352 1658353

AD-49883.1 MX1 ENSAPLTOOOOOO 16707 1658354 1658355

AD-49895.1 MX1 ENSAPLTOOOOOO 16707 1658356 1658357

AD-49901.1 MX1 ENSAPLTOOOOOO 16707 1658358 1658359

AD-49907.1 MX1 ENSAPLTOOOOOO 16707 1658360 1658361

AD-49866.1 MX1 ENSAPLTOOOOOO 16707 1658362 1658363

AD-49872.1 MX1 ENSAPLTOOOOOO 16707 1658364 1658365

AD-49878.1 MX1 ENSAPLTOOOOOO 16707 1658366 1658367

AD-49884.1 MX1 ENSAPLTOOOOOO 16707 1658368 1658369

AD-49890.1 MX1 ENSAPLTOOOOOO 16707 1658370 1658371

AD-49896.1 MX1 ENSAPLTOOOOOO 16707 1658372 1658373

AD-49902.1 MX1 ENSAPLTOOOOOO 16707 1658374 1658375

AD-49908.1 MX1 ENSAPLTOOOOOO 16707 1658376 1658377

AD-49867.1 MX1 ENSAPLTOOOOOO 16707 1658378 1658379

AD-49873.1 MX1 ENSAPLTOOOOOO 16707 1658380 1658381

AD-49879.1 MX1 ENSAPLTOOOOOO 16707 1658382 1658383

AD-49885.1 MX1 ENSAPLTOOOOOO 16707 1658384 1658385

AD-49891.1 MX1 ENSAPLTOOOOOO 16707 1658386 1658387 AD-49897.1 MX1 ENSAPLT00000016707 1658388 1658389

AD-49903.1 MX1 ENSAPLT00000016707 1658390 1658391

AD-49909.1 TLR3 ENSAPLT00000009346 930222 930223

AD-49921.1 TLR3 ENSAPLT00000009346 930224 930225

AD-49927.1 TLR3 ENSAPLT00000009346 930226 930227

AD-49933.1 TLR3 ENSAPLT00000009346 930228 930229

AD-49938.1 TLR3 ENSAPLT00000009346 930230 930231

AD-49943.1 TLR3 ENSAPLT00000009346 930232 930233

AD-49948.1 TLR3 ENSAPLT00000009346 930234 930235

AD-49910.1 TLR3 ENSAPLT00000009346 930236 930237

AD-49916.1 TLR3 ENSAPLT00000009346 930238 930239

AD-49922.1 TLR3 ENSAPLT00000009346 930240 930241

AD-49928.1 TLR3 ENSAPLT00000009346 930242 930243

AD-49934.1 TLR3 ENSAPLT00000009346 930244 930245

AD-49939.1 TLR3 ENSAPLT00000009346 930246 930247

AD-49944.1 TLR3 ENSAPLT00000009346 930248 930249

AD-49949.1 TLR3 ENSAPLT00000009346 930250 930251

AD-49917.1 TLR3 ENSAPLT00000009346 930252 930253

AD-49923.1 TLR3 ENSAPLT00000009346 930254 930255

AD-49929.1 TLR3 ENSAPLT00000009346 930256 930257

AD-49935.1 TLR3 ENSAPLT00000009346 930258 930259

AD-49940.1 TLR3 ENSAPLT00000009346 930260 930261

AD-49945.1 TLR3 ENSAPLT00000009346 930262 930263

AD-49950.1 TLR3 ENSAPLT00000009346 930264 930265

AD-49912.1 TLR3 ENSAPLT00000009346 930266 930267

AD-49918.1 TLR3 ENSAPLT00000009346 930268 930269

AD-49924.1 TLR3 ENSAPLT00000009346 930270 930271

AD-49930.1 TLR3 ENSAPLT00000009346 930272 930273

AD-49936.1 TLR3 ENSAPLT00000009346 930274 930275

AD-49941.1 TLR3 ENSAPLT00000009346 930276 930277

AD-49946.1 TLR3 ENSAPLT00000009346 930278 930279

AD-49951.1 TLR3 ENSAPLT00000009346 930280 930281

AD-49913.1 TLR3 ENSAPLT00000009346 930282 930283

AD-49919.1 TLR3 ENSAPLT00000009346 930284 930285

AD-49925.1 TLR3 ENSAPLT00000009346 930286 930287

AD-49931.1 TLR3 ENSAPLT00000009346 930288 930289

AD-49937.1 TLR3 ENSAPLT00000009346 930290 930291

AD-49942.1 TLR3 ENSAPLT00000009346 930292 930293

AD-49947.1 TLR3 ENSAPLT00000009346 930294 930295 AD-49952.1 TLR3 ENSAPLT00000009346 930296 930297

AD-49914.1 TLR3 ENSAPLT00000009346 930298 930299

AD-49920.1 TLR3 ENSAPLT00000009346 930300 930301

AD-49926.1 TLR3 ENSAPLT00000009346 930302 930303

AD-49932.1 TLR3 ENSAPLT00000009346 930304 930305

EXAMPLE 8: Synthesis of cationic lipids

Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid- lipid particles of the invention may be prepared by known organic synthesis techniques. Synthesis of Formula A

In one embodiment, nucleic acid-lipid particles of the invention are formulated using a cationic lipid of formula A:

where l and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[l ,3]-dioxolane). In general, the lipid of formula A above may be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.

Lipid A, where Ri and R₂ are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R₃ and R4 are independently lower alkyl or R₃ and R4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.

Scheme 2

R₂

BrMg— + R₂-CN O

Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1.

Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta- 6,9,28,31 -tetraen- 19-yl 4-(dimethylamino)butanoate) is as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N- dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.6 lg) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) is stirred at room temperature overnight. The solution is washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions are dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue is passed down a silica gel column (20 g) using a 1- 5% methanol/dichloro methane elution gradient. Fractions containing the purified product are combined and the solvent removed, yielding a colorless oil (0.54 g).

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] is performed using the following scheme 3:

Synthesis of 515:

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous

THF in a two neck RBF (1L), is added a solution of 514 (lOg, 0.04926mol) in 70 mL of THF slowly at 0 0C under nitrogen atmosphere. After complete addition, reaction mixture is warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction is monitored by TLC. After completion of reaction (by TLC) the mixture is cooled to 0 0C and quenched with careful addition of saturated Na2S04 solution.

Reaction mixture is stirred for 4 h at room temperature and filtered off. Residue is washed well with THF. The filtrate and washings are mixed and diluted with 400 mL dioxane and 26 mL cone. HC1 and stirred for 20 minutes at room temperature. The volatilities are stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400MHz): δ= 9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

Synthesis of 516:

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, is added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0 0C under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture is allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture is washed successively with IN HC1 solution (1 x 100 mL) and saturated NaHC03 solution (1 x 50 mL). The organic layer is then dried over anhyd. Na2S04 and the solvent is evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 1 lg (89%). IH-NMR (CDC13, 400MHz): δ = 7.36-7.27(m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60(m, 2H), 2.30-2.25(m, 2H). LC-MS [M+H] -232.3 (96.94%).

Synthesis of 517A and 517B:

The cyclopentene 516 (5 g, 0.02164 mol) is dissolved in a solution of 220 mL acetone and water (10: 1) in a single neck 500 mL RBF and to it is added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of Os04 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (~ 3 h), the mixture is quenched with addition of solid Na2S03 and resulting mixture is stirred for 1.5 h at room temperature. Reaction mixture is diluted with DCM (300 mL) and washed with water (2 x 100 mL) followed by saturated NaHC03 (1 x 50 mL) solution, water (1 x 30 mL) and finally with brine (lx 50 mL). Organic phase is dried over an.Na2S04 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of

diastereomers, which are separated by prep HPLC. Yield: - 6 g crude

517A - Peak-1 (white solid), 5.13 g (96%). IH-NMR (DMSO, 400MHz): δ= 7.39-7.3 l(m, 5H), 5.04(s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47(d, 2H), 3.94-3.93(m, 2H), 2.71(s, 3H), 1.72- 1.67(m, 4H). LC-MS - [M+H]-266.3, [M+NH4 +]-283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518:

Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) is obtained as a colorless oil. IH-NMR (CDC13, 400MHz): δ= 7.35-7.33(m, 4H), 7.30-7.27(m, 1H), 5.37-5.27(m, 8H), 5.12(s, 2H), 4.75(m,lH), 4.58-4.57(m,2H), 2.78-2.74(m,7H), 2.06-2.00(m,8H), 1.96-1.91(m, 2H), 1.62(m, 4H), 1.48(m, 2H), 1.37-1.25(br m, 36H), 0.87(m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519:

A solution of compound 518 (1 eq) in hexane (15 mL) is added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture is heated at 40°C over 0.5 h then cooled again on an ice bath. The mixture is carefully hydrolyzed with saturated aqueous Na2S04 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. Formulations prepared by either the standard or extrusion- free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid- nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA, e.g., siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA- binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the "free" dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 1 10 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.

Example 9. MAVS targeting siRNA

A series of siRNA was designed to target the 3' UTR of the target gene, MAVS (mitochondrial antiviral signaling protein; SEQ ID NO: 3383; ENSAPLT00000003383). These siRNA are shown in Table 10 below. Identifed in the name of the siRNA duplex is the start and stop site of the sense strand.

Table 10. siRNA duplexes targeting MAVS 3 'UTR

Duplex Sense Sequence (5'-3') Sense Antisense Sequence (5'-3') Antisens SEQ e SEQ

ID ID

176313

MAVS

CUUUGUAUUUCCACUUGA UUCAAGUGGAAAUACAAA

3UTR 714 0 1763173

A G

-732

176313

MAVS

CCCCAAACCCAAUUAAAU AAUUUAAUUGGGUUUGG

3UTR 214 1 1763174

U GG

-232

176313

MAVS

UUAAGGAAUUCUGUCUGU CACAGACAGAAUUCCUUA

3UTR 529 2 1763175

G A

-547

176313

MAVS

AAAUUUGGGUCCCGAAUU AAAUUCGGGACCCAAAUU

3UTR 125 3 1763176

U U

-143

176313

MAVS

CAGUGAAACAUUUUGCUC UGAGCAAAAUGUUUCACU

3UTR 507 4 1763177

A G

-525

176313

MAVS

GCUUUUAACACUCGGAUU AAAUCCGAGUGUUAAAAG

3UTR 582 5 1763178

U C

-600

176313

MAVS

GUCUGAUGGGGCUGAUUU AAAAUCAGCCCCAUCAGA

3UTR 298 6 1763179

U C

-316

176313

MAVS

CCCAGUGAAACAUUUUGC AGCAAAAUGUUUCACUGG

3UTR 505 7 1763180

U G

-523

176313

MAVS

UGCUCAGCCUUAAGGAAU AAUUCCUUAAGGCUGAGC

3UTR 520 8 1763181

U A

-538

176313

MAVS

CCACGGAAGCCGCAGCUU AAAGCUGCGGCUUCCGUG

3UTR 568 9 1763182

U G

-586

176314

MAVS

ACCCCAAACCCAAUUAAA AUUUAAUUGGGUUUGGG

3UTR 213 0 1763183

U GU

-231

176314

MAVS

AACCCCAAACCCAAUUAA UUUAAUUGGGUUUGGGG

3UTR 212 1 1763184

A UU

-230

MAVS GCUUCCAAACAAGCUCAA UUUGAGCUUGUUUGGAA 1763185 3UTR 195 A 176314 GC -213 2

176314

MAVS

GAAACAAACAGCGGGAUU AAAUCCCGCUGUUUGUUU

3UTR 277 3 1763186

U C

-295

176314

MAVS

CUUCCAAACAAGCUCAAA GUUUGAGCUUGUUUGGA

3UTR 196 4 1763187

C AG

-214

176314

MAVS

GCGGAAAAUCUUCCCAAA AUUUGGGAAGAUUUUCCG

3UTR 621 5 1763188

U C

-639

176314

MAVS

CUCAAAAAGCCGCGGUGA UUCACCGCGGCUUUUUGA

3UTR 429 6 1763189

A G

-447

176314

MAVS

GUGCCCAGUGAAACAUUU AAAAUGUUUCACUGGGCA

3UTR 502 7 1763190

U C

-520

176314

MAVS

CAAAGUGCCUUCCGAAAA UUUUUCGGAAGGCACUUU

3UTR 248 8 1763191

A G

-266

176314

MAVS

UGCACAAAGUGCCUUCCG UCGGAAGGCACUUUGUGC

3UTR 244 9 1763192

A A

-262

176315

MAVS

CCUGAGGAGCAGCCUUAU AAUAAGGCUGCUCCUCAG

3UTR 104 0 1763193

U G

-122

176315

MAVS

CGGAAAAUCUUCCCAAAU CAUUUGGGAAGAUUUUCC

3UTR 622 1 1763194

G G

-640

176315

MAVS

GGCUCUUAGUGGGAUUCU AAGAAUCCCACUAAGAGC

3UTR 656 2 1763195

U C

-674

176315

MAVS

UCUCAUUCACUUUGUAUU AAAUACAAAGUGAAUGA

3UTR 705 3 1763196

U GA

-723

176315

MAVS

UCACUUUGUAUUUCCACU AAGUGGAAAUACAAAGU

3UTR 71 1 4 1763197

U GA

-729 176315

MAVS

UCGGAUUUCCUCUGAAUU AAAUUCAGAGGAAAUCCG

3UTR 593 5 1763198

U A

-61 1

176315

MAVS

CCCCAAGCAGGGAGAAGA CUCUUCUCCCUGCUUGGG

3UTR 463 6 1763199

G G

-481

176315

MAVS

UUUGCUCAGCCUUAAGGA UUCCUUAAGGCUGAGCAA

3UTR 518 7 1763200

A A

-536

176315

MAVS

CACGGAAGCCGCAGCUUU AAAAGCUGCGGCUUCCGU

3UTR 569 8 1763201

U G

-587

176315

MAVS

AUGUCUGAAACCCUGAGG UCCUCAGGGUUUCAGACA

3UTR 93- 9 1763202

A U

111

176316

MAVS

CAAAUUUGGGUCCCGAAU AAUUCGGGACCCAAAUUU

3UTR 124 0 1763203

U G

-142

176316

MAVS

CCAGCAGUGCCCAGUGAA UUUCACUGGGCACUGCUG

3UTR 496 1 1763204

A G

-514

176316

MAVS

GUCUCAUUCACUUUGUAU AAUACAAAGUGAAUGAG

3UTR 704 2 1763205

U AC

-722

176316

MAVS

GCUCUUAGUGGGAUUCUU GAAGAAUCCCACUAAGAG

3UTR 657 3 1763206

C C

-675

176316

MAVS

GCACAAAGUGCCUUCCGA UUCGGAAGGCACUUUGUG

3UTR 245 4 1763207

A C

-263

176316

MAVS

UGAUUUUGGGGCUGGACG ACGUCCAGCCCCAAAAUC

3UTR 310 5 1763208

U A

-328

176316

MAVS

GGCAGAGGGCUUUCAGAA GUUCUGAAAGCCCUCUGC

3UTR 362 6 1763209

C C

-380

176316

MAVS

GGAGCAGCCUUAUUCCAA UUUGGAAUAAGGCUGCUC

3UTR 109 7 1763210

A C

-127 176316

MAVS

UGAUGGGGCUGAUUUUGG CCCAUC

3UTR 301 8 CCCAAAAUCAGC 1763211

G A

-319

176316

MAVS

ACUCGGAUUUCCUCUGAA AUUCAGAGGAAAUCCGAG

3UTR 591 9 1763212

U U

-609

176317

MAVS

GGAAACAAACAGCGGGAU

3UTR 276 0 AAUCCCGCUGUUUGUUUC 1763213

U c

-294

176317

MAVS

AAUUAAAUUCCGCAGCCA UUGGCUGCGGAAUUUAAU

3UTR 224 1 1763214

A U

-242

176317

MAVS

UUCACUUUGUAUUUCCAC AAGUG

3UTR 710 2 AGUGGAAAUACA 1763215

U AA

-728

LENGTHY TABLE

The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site. An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Claims

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of an avian

transcript, wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to an avian transcript listed in Table 1, which antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense sequences selected from SEQ ID NO 18040 through SEQ ID NO 1762259.

2. The dsRNA of claim 1 , wherein said dsRNA comprises at least one modified nucleotide.

3. The dsRNA of claim 2, wherein at least one of said modified nucleotides is chosen from the group consisting of: a 2'-0-methyl modified nucleotide, a nucleotide comprising a 5'- phosphate group, a locked nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'- deoxy-modified nucleotide, an abasic nucleotide, 2'-amino-modified nucleotide, 2'-alkyl- modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

4. The dsRNA of claim 1, wherein the region of complementarity is at least 17 nucleotides in length.

5. The dsRNA of claim 1, wherein the region of complementarity is between 19 and 21 nucleotides in length.

6. The dsRNA of claim 5, wherein the region of complementarity is 19 nucleotides in

length.

7. The dsRNA of claim 1 , wherein each strand is no more than 30 nucleotides in length.

8. The dsRNA of claim 1, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide.

9. The dsRNA of claim 1, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

10. The dsRNA of claim 1, further comprising a ligand.

11. The dsRNA of claim 10, wherein the ligand is conjugated to the 3 ' end of the sense

strand of the dsRNA.

12. A cell containing the dsRNA of claim 1.

13. A composition comprising the dsRNA of claim 1.

14. The composition of claim 13, further comprising a lipid formulation.

15. The composition of claim 14, wherein the lipid formulation is a SNALP, or XTC- containing formulation.

16. A method of inhibiting avian transcript expression in a cell, the method comprising:

(a) introducing into the cell the dsRNA of claim 1; and

(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the RNA of said avian transcript, thereby inhibiting expression of the avian transcript in the cell.

17. The method of claim 16, wherein the expression of the avian transcript is inhibited by at least 30%.

18. A vector encoding at least one strand of a dsRNA, wherein said dsRNA comprises a region of complementarity to at least a part of an avian transcript of Table 1, wherein said dsRNA is 30 base pairs or less in length, and wherein said dsRNA targets the avian transcript for cleavage.

19. The vector of claim 18, wherein the region of complementarity is at least 15 nucleotides in length.

20. The vector of claim 18, wherein the region of complementarity is 19 to 21 nucleotides in length.

21. A cell comprising the vector of claim 18.

22. A method of altering the path of lineage of an avian cell comprising,

(a) contacting said avian cell with a targeting construct and allowing said first cell to undergo cellular division to produce daughter cells,

(c) determining the RNA population signature of the daughter cells produced in (a),

(d) comparing the RNA population signature of the avian cell with the daughter cells wherein a difference in the RNA population signatures between the avian cell and the daughter cells indicates an alteration in the path of lineage of the avian cell.

23. The method of claim 22, further comprising confirming the lineal alteration of the avian cells by determining the cell type of the daughter cells.

24. The method of claim 23, wherein the cell type of the daughter cells is determined by measuring cell type specific markers.

25. The method of claim 22, wherein the avian cell is an avian gamete.

26. The method of claim 22, wherein the avian cell is an avian somatic cell.

27. The method of claim 22, wherein the avian cell is an avian stem cell.

28. The method of claim 27, wherein the stem cell is selected from the group consisting of adult, embryonic, pluripotent, and induced pluripotent.

29. The method of claim 26, wherein the somatic cell is a cell selected from the group

consisting of endodermal-derived, ectodermal-derived, and mesodermal-derived.

30. The method of claim 22, wherein the targeting construct is encoded in a vector.

31. A method of altering the level of an avian transcript in an avian cell or cell culture

comprising contacting said cell or culture with a targeting construct.

32. The method of claim 31 , wherein the targeting construct is selected from the group

consisting of iRNA agents, antisense molecules, ribozymes, aptamers, small molecules, antibodies, peptides, proteins, enzymes or fragments thereof, and vitamins.

33. A method of altering the expression of a gene comprising contacting an avian cell with a synthetic isolated targeting construct which interacts with chromatin modifying proteins or protein complexes.

34. A method of altering the epigenetic signature of an avian cell comprising contacting the cell with an artificial nucleosome protein.

35. A method of altering the methylation status or pattern of a chromosome locus comprising contacting an avian cell containing said chromosome locus with a targeting construct.

36. A method of altering the methylation status or pattern of a nucleosome protein in an avian cell comprising contacting said cell containing said nucleosome protein with an targeting construct comprising an avian transcript structural feature.

37. The method of claim 36, wherein nucleosome protein is a histone.

38. A method of altering avian cellular protein trafficking comprising contacting the avian cell with one or more targeting constructs.

39. A synthetic isolated RNA molecule comprising a structural feature of an avian transcript, wherein the avian transcript is selected from the group consisting of avian transcripts of Table 1.

40. The synthetic isolated RNA molecule of claim 40, wherein said structural feature is at least 50 nucleotides in length.

41. The synthetic isolated RNA molecule of claim 41 , wherein said structural feature is from about 200 to about 500 nucleotides in length.

385

RECTIFIED (RULE 91) - ISA/US

42. The synthetic isolated RNA molecule of claim 42, wherein said structural feature is from about 200 to about 300 nucleotides in length.

43. The synthetic isolated RNA molecule of claim 41, wherein said structural feature is from about 50 to about 100 nucleotides in length.

44. A system for selecting a sequence of at least one targeting construct suitable for

modulating protein expression in an avian cell, the system comprising:

(a) a computer system including at least one processor and associated memory, the memory storing at least one computer program for controlling the operation of the computer system;

(b) a database, connected to the computer system, comprising avian transcriptome information, the information comprising, a plurality of transcript sequences of the transcriptome, and optionally, a name of the transcript, and a pathway the transcript plays a role; and targeting construct information, the information comprising at least the sequence of the targeting construct and optionally target specificity of the targeting construct, wherein each targeting construct is designed to match at least one or more sequences in the avian transcriptome;

(c) a user interface program module executed by the computer system and configured to receive user parameters comprising at least one of, a cell type selection, a target organism selection, a cellular pathway selection, a cross-reactivity selection, an amount of transcript selection, a target gene name and/or sequence selection, and optionally a method of delivery selection comprising either in vivo or in vitro delivery options; and further optionally user address information;

(d) a first module executed by the computer system and configured to check the parameters against the sequences in the database for a matching combination of the parameters and transcriptome transcript sequences; and

(e) a second module executed by the computer system and configured to display a selected sequence of at least one targeting construct suitable for modulating protein expression in the cell.

45. The system of claim 44 further comprising a storage module for storing the at least one targeting construct in a container, wherein if there are two or more targeting constructs, each targeting construct is stored in a separate container, and a robotic handling module,

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RECTIFIED (RULE 91) - ISA/US which upon selection of the matching combination, selects a matching container, and optionally adds to the container additives based on a user selection for in vivo or in vitro delivery, and optionally further packages the container comprising the matching targeting construct to be sent to the user address.

46. The system of claim 44, wherein the avian transcriptome sequence information consists essentially of a plurality of avian transcripts listed in Table 1.

47. The system of claim 44, wherein the targeting construct is selected from the group

consisting of siR A, miRNA, dsRNA, saRNA, shRNA, piR A, tkRNAi, eiRNA, pdR A, a gapmer, an antagomir, a ribozyme and any combination thereof.

48. The system of claim 44, wherein the targeting construct is selected from the group

consisting of an siRNA, a formulated siRNA, an siRNA mixture, and any combination thereof.

49. The system of claim 44, wherein the targeting construct comprises an antisense strand comprising at least 16 contiguous nucleotides of the nucleotide sequence selected from SEQ ID NO 18040 through SEQ ID NO 1763215.

50. A method for improving an avian cell line, the method comprising modulating at least one protein translated from an avian transcript selected from Table 1.

51. A method for improving an avian cell line, the method comprising modulating at least two avian transcripts using a targeting construct, wherein a first transcript affects a first avian cell culture phenotype and a second transcript affects a second, different avian cell culture phenotype, wherein said first and second avian cell culture phenotypes are independently selected from the group consisting of a cell growth rate, a cellular productivity, a peak cell density, a sustained cell viability, a rate of ammonia production or consumption, and rate of lactate production or consumption.

52. The method of claim 51 , further comprising modulating a third avian transcript affecting a third avian cell culture phenotype different from the first and second cell culture phenotypes.

53. An engineered avian cell line with an improved cellular productivity, improved cell

growth rate, or improved cell viability, comprising a population of engineered avian cells, each of which comprises at least one targeting construct modulating one or more avian transcripts selected from Table 1.

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54. The engineered avian cell line of claim 54, wherein the targeting construct is selected from the group consisting of siR A, miRNA, dsRNA, saR A, shR A, piRNA, tkRNAi, eiRNA, pdRNA, a gapmer, an antagomir, a ribozyme and any combination thereof.

55. A method for producing a biological product in a large scale avian host cell culture, comprising:

(a) contacting an avian host cell in a large scale avian host cell culture with at least a first targeting construct, a portion of which is complementary to at least one avian transcript of the avian host cell,

(b) maintaining the avian host cell culture for a time sufficient to modulate expression of the at least one first target gene, wherein the modulation of expression improves production of a biological product in the avian host cell;

(c) isolating the biological product from the host cell;

wherein the large scale avian host cell culture is at least 1 liter in size, and wherein the avian host cell is contacted with at least a first targeting construct by addition of the targeting construct to a culture medium of the large scale avian host cell culture such that the target gene expression is transiently inhibited.

56. The method of claim 55, wherein the avian host cell in the large scale avian host cell culture is contacted with a plurality of targeting constructs, wherein the plurality of targeting constructs modulate expression of at least one avian transcript, at least two avian transcripts or a plurality of avian transcripts.

57. The method of claim 55, wherein the targeting construct, comprises a double-stranded ribonucleic acid (dsRNA), wherein said dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least part of an avian transcript of Table 1, and wherein said region of complementarity is 10-30 nucleotides in length.

58. The method of claim 55, wherein the contacting step is performed by continuous infusion of the targeting construct into the culture medium used for maintaining the avian host cell culture to produce the biological product.

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59. The method of claim 55, wherein the modulation of expression is inhibition of expression, and wherein the inhibition is a partial inhibition.

60. The method of claim 59, wherein the partial inhibition is no greater than a percent

inhibition selected from the group consisting of: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and 85%.

61. The method of claim 55, wherein the contacting step is repeated at least once.

62. The method of claim 55, wherein the contacting step is repeated multiple times at a

frequency selected from the group consisting of: 6 hr, 12 hr, 24 hr, 36 hr, 48 hr, 72 hr, 84 hr, 96 hr, and 108 hr.

63. The method of claim 55 , wherein the modulation of expression is inhibition of expression and wherein the contacting step is repeated multiple times, or continuously infused, to maintain an average percent inhibition of at least 50% for the target gene(s) throughout the production of the biological product.

64. The method of claim 63, wherein the average percent inhibition is selected from the

group consisting of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%.

65. The method of claim 55, wherein the targeting construct is contacted at a concentration of less than 100 nM.

66. The method of claim 66, wherein the targeting construct is contacted at a concentration of less than 20 nM.

67. The method of any of claim 55, wherein said contacting an avian host cell in a large scale avian host cell culture with a targeting construct is done at least 6 hr, at least 12 hr, at least 18 hr, at least 36 hr, at least 48 hr, at least 60 hr, at least 72 hr, at least 96 hr, or at least 120 hr, or at least 1 week, before isolation of the biological product or prior to harvesting the supernatant.

68. The method of claim 55, wherein the targeting construct is composition formulated in a lipid formulation.

69. The method of claim 55, wherein the targeting construct is an siR A.

70. The method of claim 69, wherein the targeting construct is chemically modified.

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71. The method of claim 55, further comprising monitoring at least one measurable parameter selected from the group consisting of cell density, medium pH, oxygen levels, glucose levels, lactic acid levels, temperature, and protein production.

72. The method of claim 56, wherein each of the plurality of different targeting constructs is added simultaneously or at different times.

73. The method of claim 56, wherein each of the plurality of different targeting constructs is added at the same or different concentrations.

74. The method of claim 56, wherein the plurality of different targeting constructs is added at the same or different frequencies.

75. The method of claim 55, further comprising contacting the cell with a second agent.

76. The method of claim 75, wherein the second agent is selected from the group consisting of: an antibody, a growth factor, an apoptosis inhibitor, a kinase inhibitor, a phosphatase inhibitor, a protease inhibitor, and a histone demethylating agent.

77. The method of claim 55, wherein the biological product is a polypeptide.

78. The method of claim 55, wherein the biological product is a metabolite.

79. The method of claim 55, wherein the biological product is a nutraceutical.

80. The method of claim 55, wherein the avian cell is contacted with the targeting construct at a phase of cell growth selected from the group consisting of: stationary phase, early log phase, mid-log phase, late-log phase, lag phase, and death phase.

81. The method of claim 56, wherein the at least first targeting construct, or at least one of the plurality of targeting constructs, is 17-28 nucleotides in length.

82. The method of claim 56, wherein the at least first targeting construct, or at least one of the plurality of targeting constructs, comprises at least one modified nucleotide.

83. The method of claim 56, wherein the avian cell is a duck cell from the species Anas

platyrhynchus domesticus; Pekin duck.

84. The method of claim 55, wherein the cell further comprises a genetic construct encoding the biological product.

85. The method of claim 55, wherein the cell further comprises a genetic construct encoding a viral receptor.

86. The method of claim 55 , wherein the target gene encodes a protein that affects protein glycosylation.

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87. The method of claim 55, wherein the target gene encodes the biological product.

88. The method of claim 56 or 57, wherein the at least first targeting construct, or at least one of the plurality of targetmg constructs, is added at a concentration selected from the group consisting of 0.1 nM, 0.5 nM, 0.75 nM, InM, 2 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 75 nM, and 100 nM.

89. The method of claim 56, wherein the at least first targeting construct, or at least one of the plurality of targeting constructs, is added at an amount of 50 molecules per cell, 100 molecules/cell, 200 molecules/cell, 300 molecules/cell, 400 molecules/cell, 500 molecules/ cell, 600 molecules/cell, 700 molecules/ cell, 800 molecules/cell, 900 molecules/cell, 1000 molecules/cell, 2000 molecules/cell, or 5000 molecules/cell.

90. The method of claim 56, wherein the at least first targeting construct, or at least one of the plurality of targeting constructs, is added at a concentration selected from the group consisting of: 0.01 fmol/10⁶ cells, 0.1 fmol/10⁶ cells, 0.5 fmol/10⁶ cells, 0.75 fmol/10⁶ cells, 1 fmol/10⁶ cells, 2 fmol/10⁶ cells, 5 fmol/10⁶ cells, 10 fmol/10⁶ cells, 20 fmol/10⁶ cells, 30 fmol/10⁶ cells, 40 fmol/10⁶ cells, 50 fmol/10⁶ cells, 60 fmol/10⁶ cells, 100 fmol/10⁶ cells, 200 fmol/10⁶ cells, 300 fmol/10⁶ cells, 400 fmol/10⁶ cells, 500 fmol/10⁶ cells, 700 fmol/10⁶ cells, 800 fmol/10⁶ cells, 900 fmol/10⁶ cells, and 1 pmol/10⁶ cells.

91. The method of claim 56, wherein the at least first targeting construct, or at least one of the plurality of targeting constructs, is selected from the group consisting of siR A, miR A, dsR A, saRNA, shR A, piRNA, tkR Ai, eiRNA, pdRNA, a gapmer, an antagomir, a ribozyme, and any combination thereof.

92. The method of claim 55, wherein the method further comprises contacting the avian cell with at least one additional targeting construct, or agent, that modulates a cellular process selected from the group consisting of: carbon metabolism and transport, apoptosis, RNAi uptake and/or efficiency, reactive oxygen species production, control of cell cycle, protein folding, protein pyroglutamation, protein deamidation, protein glycosylation, disulfide bond formation, protein secretion, gene amplification, viral replication, viral infection, viral particle release, control of cellular pH, and protein production.

93. A kit for enhancing production of a biological product by a cultured avian host cell, comprising:

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RECTIFIED (RULE 91) - ISA/US (a) a substrate comprising one or more assay surfaces suitable for culturing the avian host cell under conditions in which the biological product is produced;

(b) one or more targeting constructs, wherein at least a portion of each targeting construct is complementary to a target gene; and

(c) a reagent for detecting the biological product or production thereof by the avian cell, wherein the one or more targeting constructs is an siRNA comprising an antisense strand that comprises at least 16 contiguous nucleotides of the nucleotide sequence selected from the group consisting of SEQ ID NO 18040 through SEQ ID NO 1763215.

94. The kit of claim 93, wherein the one or more assay surfaces further comprises a matrix for supporting the growth and maintenance of avian host cells.

95. The kit of claim 93, wherein the one or more targeting constructs are deposited on the substrate.

96. The kit of claim 93, further comprising a carrier for promoting uptake of the targeting constructs by the avian host cell.

97. The kit of claim 93, further comprising cell culture media suitable for culturing the avian host cell.

98. The kit of claim 93, further comprising instructions for culturing an avian host cell in the presence of one or more targeting constructs and assaying the cell for production of the biological product.

99. A kit for optimizing production of a biological product by cultured avian cells,

comprising:

(a) a microarray substrate comprising a plurality of assay surfaces, the assay surfaces being suitable for culturing the cells under conditions in which the biological product is produced;

(c) a reagent for detecting the effect of the one or more targeting constructs on production of the biological product wherein the one or more targeting constructs is an siRNA comprising an antisense strand that comprises at least 16 contiguous nucleotides of the nucleotide sequence selected from the group consisting of SEQ ID NO 18040 through SEQ ID NO 1763215.

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100. The kit of claim 99, wherein the substrate is a multi-well plate or biochip.

101. The kit of claim 99, wherein the substrate is a two-dimensional microarray plate or

biochip.

102. The kit of claim 99, wherein the one or more targeting constructs are deposited on the assay surfaces of the substrate.

103. The kit of claim 99, wherein a plurality of different targeting constructs are deposited on assay surfaces across a first dimension of the microarray.

104. The kit of claim 103, wherein the plurality of targeting constructs are each

complementary to a different target gene.

105. The method of claim 68, wherein the lipid formulation comprises a lipid having the

following formula:

wherein:

Ri and R₂ are each independently for each occurrence optionally substituted Cio-Cso alkyl, optionally substituted Cio-C3o alkoxy, optionally substituted Cio-C3o alkenyl, optionally substituted Cio-C o alkenyloxy, optionally substituted Cio-C3o alkynyl, optionally substituted C10-C30 alkynyloxy, or optionally substituted C₁₀-C3o acyl; represents a connection between L₂ and Li which is:

(1) a single bond between one atom of L₂ and one atom of L_ls wherein

Li is C(R_X), 0, S or (Q);

L₂ is -CR₅R₆-, -0-, -S-, -N(Q)-, =C(R₅)-, -C(0)N(Q)-, -C(0)0-, -N(Q)C(0)-, -OC(O)-, or -C(O)-;

(2) a double bond between one atom of L₂ and one atom of Li; wherein

Li is C;

L₂ is -CR₅= -N(Q)= -N-, -0-N=, -N(Q)-N=, or -C(0)N(Q)-N=;

(3) a single bond between a first atom of L₂ and a first atom of L_1? and a single bond between a second atom of L₂ and the first atom of Li, wherein

Li is C;

L₂ has the formula

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wherein

X is the first atom of L2, Y is the second atom of L₂, represents a single bond to the first atom of Li, and X and Y are each, independently, selected from the group consisting of -0-, -S-, alkylene, -N(Q)-, -C(O)-, -O(CO)-, -OC(0)N(Q)-, -N(Q)C(0)0-

-C(0)0, -OC(0)0-, -OS(0)(Q₂)0-, and -OP(0)(Q₂)0-;

and Z₄ are each, independently, -0-, -S-, -CH₂-, -CHR⁵-, or -CR⁵R⁵-;

Z₂ is CH or N;

Z₃ is CH or N;

or Z₂ and Z₃, taken together, are a single C atom;

Ai and A₂ are each, independently, -0-, -S-, -CH₂-, -CHR⁵-, or -CR⁵R⁵-;

each Z is N, C(R₅), or C(R );

k is 0, 1, or 2;

each m, independently, is 0 to 5;

each n, independently, is 0 to 5;

where m and n taken together result in a 3, 4, 5, 6, 7 or 8 member ring;

(4) a single bond between a first atom of L₂ and a first atom of L_ls and a single bond between the first atom of L₂ and a second atom of L_ls wherein

(A) Li has the formula:

^ / wherein

X is the first atom of L_{l s} Y is the second atom of L_{l s} represents a single bond to the first atom of L₂, and X and Y are each, independently, selected from the group consisting of -0-, -S-, alkylene, -N(Q)-, -C(0)-, -O(CO)-, -OC(0)N(Q)-, -N(Q)C(0)0- -C(0)0, -0C(0)0-, -OS(0)(Q₂)0-, and -OP(0)(Q₂)0-;

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RECTIFIED (RULE 91) - ISA/US T₂ is CH or N;

or Ti and T₂ taken together are C=C;

L₂ is CR₅; or

(B) Li has the formula:

wherein

X is the first atom of L_1? Y is the second atom of L_1; represents a single bond to the first atom of L₂, and X and Y are each, independently, selected from the group consisting of -0-, -S-, alkylene, -N(Q)-, -C(0)-, -O(CO)-, -OC(0)N(Q)-, -N(Q)C(0)0-,

-C(0)0, -0C(0)0-, -OS(0)(Q₂)0-, and -OP(0)(Q₂)0-;

Ti is -CR₅R₅-, -N(Q)-, -0-, or -S-;

T₂ is -CR5R5-, -N(Q)-, -0-, or -S-;

L₂ is CR₅ or ;

R has the formula:

wherein

each of Yi, Y₂, Y₃, and Y₄, independently, is alkyl, cycloalkyl, aryl, aralkyl, or alkynyl; or any two of Y_1? Y₂, and Y3 are taken together with the N atom to which they are attached to form a 3- to 8- member heterocycle; or

395

RECTIFIED (RULE 91) - ISA/US Y₁₍ Y₂, and Y are all be taken together with the N atom to which they are attached to form a bicyclic 5- to 12- member heterocycle;

each R_n, independently, is H, halo, cyano, hydroxy, amino, alkyl, alkoxy, cycloalkyl, aryl, heteroaryl, or heterocyclyl;

L₃ is a bond, -N(Q)-, -0-, -S-, -(CRjRe , -C(O)-, or a combination of any two of these; L₄ is a bond, -N(Q)-, -0-, -S-, -(CR₅R₆)_a-, -C(0)-, or a combination of any two of these; L₅ is a bond, -N(Q)-, -0-, -S-, -(CR5R₆)_a-, -C(0)-, or a combination of any two of these; each occurrence of R₅ and R₆ is, independently, H, halo, cyano, hydroxy, amino, alkyl, alkoxy, cycloalkyl, aryl, heteroaryl, or heterocyclyl; or two R5 groups on adjacent carbon atoms are taken together to form a double bond between their respective carbon atoms; or two R₅ groups on adjacent carbon atoms and two R^ groups on the same adjacent carbon atoms are taken together to form a triple bond between their respective carbon atoms; each a, independently, is 0, 1, 2, or 3;

wherein

an R₅ or R₆ substituent from any of L₃, L₄, or L₅ is optionally taken with an R₅ or R₆ substituent from any of L₃, L₄, or L5 to form a 3- to 8- member cycloalkyl, heterocyclyl, aryl, or heteroaryl group; and

any one of Y_ls Y₂, or Y₃, is optionally taken together with an R₅ or R6 group from any of L₃, L₄, and L₅, and atoms to which they are attached, to form a 3- to 8- member heterocyclyl group;

each Q, independently, is H, alkyl, acyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl or heterocyclyl; and

each Q₂, independently, is 0, S, N(Q)(Q), alkyl or alkoxy.

106. A method of doing a business for the production of a biological product, comprising

(i) providing a service for the rapid production of a biological product from an avian host cell using one or more iRNA targeting constructs; and

(ii) assessing the efficacy of the biological product.

107. The method of claim 106, wherein the biological product is useful in the treatment of an infection.

108. The method of claim 107, wherein the assessment of efficacy is against one or more infective agents suspected of causing said infection.

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109. The method of claim 108 further comprising providing the effective biological product to a consumer or customer.

110. The method of claim 106, wherein the biological product is useful in the manufacture of textiles.

111. The method of claim 106, wherein the biological product is useful in the chemicals industry.

112. The method of claim 106, wherein the biological product is useful in the manufacture, cleaning or production of electronic devices or components.

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