COMPOSITIONS AND METHODS FOR PRODUCING MODIFIED

GLYCOPROTEINS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 61/510,437, filed July 21 , 2011, and U.S. Provisional Application No. 61/617,322, filed March 29, 2012, each of the foregoing applications is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Glycosylation is the enzymatic process that attaches glycans to proteins, lipids, or other organic molecules. Glycosylation is a form of co-translational or post- translational modification. Glycans serve a variety of structural and functional roles in membrane and secreted

[0003] N-glycans are covalently attached to protein at asparagine residues (Asn; particularly an Asp which occurs in the sequence Asn-Xaa-Ser/Thr/Cys, where Xaa represents any amino acid) by an N-glycosidic bond. Following attachment of the saccharide moiety, further modifications may occur in vivo. Typically these modifications occur via an ordered sequence of enzymatic reactions, known as a cascade.

[0004] Ail N-glycans share a common core sugar sequence, Manal-6(Manal- 3)Man 1 ~4GlcN Αοβ 1 -4GlcN Αοβ 1 - [ Asn-X-Ser/Thr/Cys] , and are classified into three types: (1) oligomannose, in which only mannose residues are attached to the core; (2) complex, in which "antennae" initiated by N-acetylgiucosaminyltransferases (GlcNAcTs) are attached to the core; and (3) hybrid, in which only mannose residues are attached to the Manal-6 arm of the core and one or two antennae are on the Manal-3 arm.

[0005] Complex N-glycans often also comprise galactose, fucose, and terminal sialic acid residues. In humans, nucleotide sugar precursors (e.g., UDP-N-acetylgiucosamine, UDP-

N-acetylgalactosamine, CMP-N-acetylneuraminic acid, UDP-galactose, GDP-fucose, etc.) are generally synthesized in the cytosol and transported into the Golgi, where they are attached to the core oligosaccharide by glycosyltransferases. See, e.g., Sommers and Hirschberg, 1981 J.

Ceil Biol. 91(2): A406-A406; Sommers and Hirschberg 1982 J. Biol. Chem.257( 18): 811-817;

Perez and Hirschberg 1987 Methods in Enzymoiogy 138: 709-715. Glycosyl transfer reactions typically yield a side product which is a nucleoside diphosphate or monophosphate, While monophosphates can be directly exported in exchange for nucleoside triphosphate sugars by an antiport mechanism, diphosphonucleosides (e.g., GDP) have to be cleaved by phosphatases (e,g. , GDPase) to yield nucleoside monophosphates and inorganic phosphate prior to being exported.

[ 0006] Giycosyltransferases and mannosidases are located in the inner (luminal) surface of the ER and Golgi appara tus, which provides a catalytic surface for sequential processing of glycoproteins as they proceed through the ER and Golgi network. As a glycoprotein proceeds from synthesis in the ER to full maturation in the late Golgi or trans Golgi Network (TON), it is sequentially exposed to different glycosidases, mannosidases and giycosyltransferases such that a specific N-glycan structure may be synthesized.

[0007] The modification of serine or threonine residues on proteins by addition of a GalNAc residue results in an O-linked oligosaccharide or O-glycan, O-glycan biosynthesis is initiated by the addition of the monosaccharide GalNAc (from UDP-GalNAc) to serine and threonine residues catalyzed by a polypeptide GalNAc transferase (GalNAcT). Many O-glycans are extended into long chains with variable termini that may be similar to the termini of N- glycans. However, O-glycans are less branched than most N-glycans and are commonly biantennary structures.

[0008] Many therapeutic proteins are glycosylated, including, for example, interferons (IFNs), erythropoietin (EPO), tissue plasminogen activator (tPA), antithrombin, granulocyte-macrophage colony stimulating factor (GM-CSF), and therapeutic monoclonal antibodies. The structures of N-glycans can affect therapeutic efficacies of glycoproteins in many ways, such as pharmacokinetics (e.g., half life), physical stability, protein folding, solubility, susceptibility to proteases, trafficking, transport, compartmentaiization, secretion, recognition by other proteins or factors, or immunogeniciry. See, e.g., Jenkins et al. (1996) Nature Biotechnololy, 14:975-981; Ghaderi et al. (2010) Nature Biotechnololy, 28:863-867.

[0009] For many antibodies, the N-glycosylation status of the Fc region of the antibody heavy chain (H-chain) plays an important role in antibody dependent dell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). The structure and extent of heterogeneity of these N-glycans are among the considerations in selecting a protein expression platform for a therapeutic antibody.

[0010] Accordingly, there is a need for improved methods for making glycoproteins, and for therapeutic glycoproteins that contain glycans that provide improved properties.

SUMMARY OF THE INVENTION

[0011 ] The in vention generally relates to compositions and methods for producing glycoproteins that have alter glycan structure and improved properties. The glycoproteins are produced by modifying the glycosylation pathways in a host cell using an RNA effector molecule, such as an siR A. Glycan-modified proteins produced using the methods described herein have improved properties, such as improved effector activity, improved pharmacokinetic properties, reduced immunogenicity in humans and the like.

BRIEF DESCRIPTION OF THE FIGURES

[0012 ] Figure 1 shows examples of GO, G l, and G2 N-glycans. In this figure, the glycans are fucosylated,

[0013] Figure 2 provides a schematic illustration of the glycosylation pathway in CHO cells.

[0014] Figure 3 provides a schematic illustration of fucosylation pathway in CHO ceils.

[0015] Figure 4 shows the growth curve and ceil viability of an exemplar}' 3L bioreactor. siRNA. dosing days are indicated by the blue arrows.

[0016] Figure 5 shows the qPCR results that confirmed both Fut8 and GMD silencing over the time course of the bioreactor. Arrows indicate siRNA dosing days, RNAi- treated sample mRNA. levels were normalized to the mRNA levels in the untreated control bioreactor.

[0017] Figure 6 shows the detection of the conversion of fucosylated glycans to their a-fucQsyiated forms by CE-LIF. RNAi-treated antibody samples are indicated in light blue and the untreated control antibody samples in dark blue. The structures of the indicated glycoforms are represented below the graph. Each antibody sample was analyzed four times by CE-LIF.

[0018] Figure 7 shows the detection of the conversion of fucosylated glycans to their a-fucosylated forms by LC-MS. RNAi-treated antibody samples are indicated in light blue and the untreated control antibody samples in dark blue. The structures of the indicated glycoforms are represented below the graph. Each antibody sample was analyzed three times by LC-MS.

[0019] Figure 8 shows that RNAi-treated CD20 antibody has improved FcyRJIIa binding affinity and capacity. An approximate two-fold improvement in both affinity and binding capacity was observed with the a-fucosylated CD20 antibody (square) compared to the non-RNAi treated control (triangle) and to rituximab (inverted triangle; Mabthera). The CD20 control antibody and rituximab (a commercially available anti-CD20 antibody) had similar FcyRIHa characteristics. Figure to the right of the curve is a schematic representation of the EI.JSA with expected results. Labels are indicated in figure.

[0020] Figure 9 shows that RNAi-treated CD20 antibody has improved ADCC activity. An approximate three-fold improvement in specific cell lysis was observed with the a- fueosylated CD20 antibody (square) compared to the non-RNAi treated control (triangle) and approximately a two-fold improvement over rituximab (inverted triangle; Mabthera). The CD20 control antibody and rituximab (a commercially available anti-CD20 antibody) had similar ADCC activity. Figure to the right of the curve is a schematic representation of the ADCC. Labels are indicated in figure.

DETAILED DESCRIPTION OF THE INVENTION

1. OVERVIEW

[0021] As described herein, glycoproteins that have altered glycan structures can be produced on a commercial scale by transiently reducing the expression of target genes that encode enzymes or transporters that are involved in glycosylation pathways. Transient reduction of target genes in commercial scale bioreactors can be accomplished using RNA effector molecules, such as an siRNA. Glycoproteins produced in this way have improved properties. For example, as described and exemplified herein, siRNAs were used to transiently reduce the expression of enzymes and/or transporters that are involved in the fucosylation pathway in CHO cells. Significantly higher amount of afucosyiated monoclonal antibodies were produced upon addition of siRNAs to the cell culture, and the afucosyiated antibodies showed improved ADCC activity as compared to the corresponding fucosylated antibodies,

[0022] RNA effectors, such as siRNAs, can also be used to reduce the expression levels of enzymes or transporters that are involved in the production of immunogenic epitopes on glycans. Examples of epitopes on glycans that are immunogenic in humans include, e.g., N- glycans that comprise a N-glycolylneuramiiiic acid (NeuSGc) residue, and the aGal epitope (galactose-alpha(l ,3)-gaiactose-beta(l ,4)N-acety!glucosamine-R; or Gal- f 1 ,3)-Gal-f3(l ,4)- GlcNAc-R; where R is the giycaii-protein structure). Epitopes on glycans that are immunogenic in humans are often present on glycoproteins that are produced in non-huma host ceils, such as CHO cells. Glycoproteins that lack NeuSGc or aGal epitope can reduce the likelihood of adverse immune responses (such as anaphylactic responses) when a glycoprotein is administered to a human patient, and facilitate the clinical application of many therapeutic glycoproteins. Reducing immunogenic! ty ca also preserve efficacy and potency of the glycoprotein, especially when the glycoprotein is intended for repeated (e.g., chronic) use. f 0023] The serum half life of glycoproteins is also influenced by the composition and structure of its N-giycans, and RNA effectors, such as siRNAs, can also be used to improve pharmacokinetic properties of glycoproteins, in general, maximal serum half life of a glycoprotein requires that its N-glycans terminate with a sialic acid cap (e.g., N- acetylneuraminic acid (Neu5 Ac)). RNA effectors can be used to transiently reduce the expression of sialydases, which remove the terminal sialic acid (e.g., NeuSAc) cap from giycoprotein-glycans, thereby stabilizing the caped glycan and promoting a longer serum half life. By reducing the hydrolysis of terminal sialic acid from N-linked N-glycans, therapeutic efficacies of glycoproteins can be improved.

[0024] Accordingly, in one aspect, the invention provides glycan-modified antibodies, or fusion proteins that comprise an Fc domain of an antibody, that have improved effector activities (such as ADCC).

[0025] In another aspect, the invention provides glycan-modified glycoproteins that have reduced immunogenicity in humans. [0026] In another aspect, the invention provides glyean-modified glycoproteins that have an increased serum hal f life.

[0027] A single species of RNA effector molecule can be used to reduce the expression of a single gene that encodes a protein involved in a desired giycosylation reaction. Alternatively, two or more different species of RNA. effector molecules may be used, to reduce expression of tw o or more genes that encode a proteins involved in a desired giycosylation reaction(s).

[0028] The glyean-modified glycoproteins described herein can be formulated into a pharmaceutical formulation that is suitable for in vivo administration. The invention also relates to the use of the glycoproteins described herein, or pharmaceutical compositions comprising the glycoproteins, in therapy, and to the use of the glycoproteins, or pharmaceutical compositions comprising the glycoproteins, for the manufacture of a medicament for use in therapy.

[0029] Also provided are methods for producing glyean-modified glycoproteins, by treating a large scale host cell culture with RNA. effector molecules,

2. DEFINITIONS

[0030] The term "about", as used here, refers to +/- 10% of a value.

[0031] The term "antibody" covers fully assembled immunoglobulins tha comprise two heavy chains and two light chains (e.g., human, humanized, chimeric antibodies), as well as an antigen-binding fragment of an antibody (e.g., Fab, Fab', F(ab')2, Fv, scFv, single domain antibody) that may contain natural, or introduced giycosylation site(s).

[0032] The terms "complementary," "fully complementary" and "substantially complementary" are used herein to describe the base matching between the sense strand and the antisense strand of a double-stranded RNA. (dsRN A), or between the antisense strand of a RNA effector molecule and a target sequence. A nucleotide sequence is "fully complementary" to another nucleotide sequence when there are no mismatched base pairs across the length of the shorter sequence. A nucleotide sequence is "substantially complementary" to another nucleotide sequence when there are no more than 20% of the mismatched base pairs a cross the length of the shorter sequence (e.g., no more than 5, 4, 3, 2, or 1 mismatched base pair(s) upon hybridization for a duplex up to 30 base pairs). 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, a 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, can yet be referred to as "fully complementary."

[0033] The term "Fc domain" refers to the Fc region of an antibody, i.e., the antibody fragment that comprises the heavy-chain constant region 2 (C _H 2) and the heavy-chain constant region (C _R 3) of an IgA, IgD, and IgG, and heavy-chain constant regions 2, 3, and 4 (CH2-CH3-CH4) of IgE and IgM. Typically, the Fc region of an antibody comprises a dimer of two CH2-CH3 (or CH2-CH3-CR4) chains. The Fc region does not include the variable regions of the heavy and light chains, the heavy-chain constant region 1 (Cul), and the light-chain constant region 1 (Q_.l) of the immunoglobulin. An Fc domain may further include the hinge region at the heavy-chain constant region, The term "Fc domain" also compasses fragments of the Fc region of an antibody, such as the CH ₂ domain, or the C _H 3 domain.

|0034] The term "fusion polypeptide" refers to a single polypeptide in which the amino acid sequence is derived from at least two different naturally occurring proteins or polypeptide chains.

[0035] The term "glycoform" of a protein refers to a protein comprising a particular glycan structure or structures. It is recognized that a glycoprotein having more than one glycosylation site can have the same glycan species attached to each glycosylation site, or can have different glycan species attached to different glycosylation sites, in this manner, different patterns of glycan attachment yield different glycoforms of a glycoprotein.

[0036] A "GO glycoform" refers to a glycoform in which the N-glycan has the core structure of GlcNAc ₂ Man ₃ Glc Ac ₂ , wherein no terminal galactose (Gal) residue is attached to either the two mannose arms. A "Gl glycoform" or "GT glycoform" refers to a glycoform in which the N-glycan has the core structure of GlcNAc ₂ Man ₃ GlcNAc ₂ , wherein one terminal galactose (Gal) residue is attached to one of the mannose arms. A "G2 glycoform" refers to a glycoform in which the N-glycan has the core stnicture of GlcNAc ₂ Man ₃ GlcNAc ₂ , wherein one terminal galactose (Gal) residue is attached to each of two mannose arms. Exampl es of GO, Gl (G! '), and G2 N-glycans are shown in Figure I .

[0037] The "imrnunogeniciiy" of a molecule refers to the ability of the molecule to induce a response by the immune system. The immune response may be a cell or antibody- mediated response. Irnmunogenieity may be determined by use of any suitable method known in the art, e.g. in vivo or in vitro irnmunogenieity test that quantifying the presence of antigen- specific antibodies or T-ceils. The term "reduced irnmunogenieity", when referring to composition, means that the composition gives rise to a measurably lower immune response than a reference composition, as determined under comparable conditions. Reduced

irnmunogenieity can be demonstrated by showing, e.g., decreased amount of an immunogenic epitope (e.g., an a -Gal epitope) that is present in the composition; decreased binding affinity for an antibody; or lower antibody titers induced by the composition; as compared to a reference composition, determined under comparable conditions. The reference composition may be a commercially available product (such as Rituximab, Cetuximab, Trastuzumab, Abatacept etc.),

[0038 j A "large scale culture" refers to a culture that is at least about a 10 liter in size, (e.g., a volume of at least about 10L, least about 20L, least about SOL, least about 40L, at least about SOL, least about 60 L, least about 70L, least about SOL, least about 90L, at least about 100L, least about 150L, least about 2QQL, at least about 250L, least about 300L, least about 400L, at least about 500L, least about 600L, least about TOOL, least about 8001.. least about 900L, at least about 1000 L, at least about 2000 L, at least about 3000 L, at least about 4000 L, at least about 5000 L, at least about 6000 L, at least about 10,000 L, at least about 15,000 L, at least about 20,000 L, at least about 25,000 L, at least about 30,000 L, at least about 35,000 L, at least about 40,000 L, at least about 45,000 L, at least about 50,000 L, at least about 55,000 L, at least about 60,000 L, at least about 65,000 L, at least about 70,000 L, at least about 75,000 L, at least about 80,000 L, at least about 85,000 L, at least about 90,000 L, at least about 95,000 L, at least about 100,000 L, etc.).

[0039] The expression of the target gene in a host cell is "transiently" reduced by an RNA effector molecule when the RNA effector molecule reduces the expression level of the target gene for a defined period of time (e.g., at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, etc), but the reduction in the expression level is not permanent, in other words, the RNA effector, or a nucleic acid constmct encoding the RNA effector, does not integrate into the genome of the host cell.

3. GLYCAN-MODIFIED PROTEINS

[0040] In one aspect, the invention provides glycan-modified proteins that have improved effector activity, improved pharmacokinetic properties, or reduced immunogeniciry in humans. "Glycan-modified" or "glycan modification" refer to a change in the glycan structure of a glycoprotein produced by a host cell in the presence of an RNA effector molecule that transiently reduces the expression of a target gene that encodes an enzyme or a transporter protein that is involved in a glycosylation pathway, as compared the glycan structure of the glycoprotein produced by the host cell under substantially the same conditions but in the absence of the RNA effector.

A. Glycan-Modifled Proteins That Have Improved Effector Activity

[0041] In one aspect, the invention provides glycan-modified antibodies, or fusion proteins that comprise the Fc domain of an antibody (Fc-fusion proteins), that have improved effector activity. For example, the invention provides a composition comprising an antibody or a fusion protein that comprises the Fc domain of an antibody, wherein the composition is characterized by: (a) at least about 70% of the antibody molecules, or the fusion protein molecules, comprise a complex N-glycan; and (b) about 20% to about 100% of the N-giycans are afucosyl glycans. Preferably, about 25%» to about 100%», about 30%» to about 100%» , about 35% to about 100% , about 40% to about 100%, about 45% to about 100%, or about 50% to about 100%) of the N-glycans are afucosyl glycans.

[0042] In certain embodiment, the composition comprises an antibody. The antibodies described herein are preferably monoclonal antibodies. The antibodies can be monospecific, or polyspecific (e.g., bispecific). The antibodies can be from any species, but preferably are humanized, human, or chimeric, ail of which are well-known in the art.

[0043] In other embodiments, composition comprises a fusion protein that comprises the Fc domain of an antibody. For example, the Fc domain may be fused with an enzyme, a toxin, a ligand (e.g., a growth factor), a cytokine (e.g., a chemokine), etc. The Fc fusion proteins may optionally comprise a linker. [0044] Preferably, the N-glycan is linked to the Fc domain of the antibody or the refusion protein. Naturally-occurring antibody molecules have conserved N-linked glycosyiaiion at the Fc region of each of the two heavy chains. For example, an IgG mol ecule contains an N- 1 inked gl can covalently attached at the conserved Asn297 (Kabat numbering) in each of the CH2 domains in the Fc region. The glycans found in the Fc region of serum IgGs are mostly biaiitennary complex glycans. Variations of IgG glycosvlation patterns include attachment of terminal sialic acid, a third GicNAc arm (bisecting GlcNAc), terminal galactosylation, and a- l ,6-linked core fucosylation. The N-glycans can contain zero (GO), one (Gl and Gl '), or two (G2) galactoses (see Figure 1 for examples of GO, Gl, and G2 glycans). The exact pattern of glycosvlation depends on the stractural properties of IgG subcomponents, in particular, C _H 2 and CH3 domains (see, e.g., Lund et al. (2000) Eur, J. Biochem., 267:7246-7257).

[0045] In addition, about 15%-20% polyclonal IgG molecules bear N-linked glycans in the variable regions of the light and/or heavy chains. There N-linked glycans are attached at the Asn residue of the consensus motif Asn-Xaa-Ser/Thr/Cys, where Xaa represents any amino acid). The glycans in the Fab region of serum IgGs are mostly biantennary oligosaccharides that are extensively galactosylated, fucosylated, and substantially sialylated. See, e.g., Gary Walsh (ed.), Post-translational Modification of Protein Biopharmaceuticals, Wiley- CH, 1st edition (2009), Chapter 4, Antibodies. For example, Cetuximab (Erbitux®) contains an N-glycan at Asn 99 of the VH region. N-glycosylation at the Fab region can influence antigen binding affinity of the antibody.

[0046] The compositions described herein may comprise a mixture of glycosylated and aglycosylated antibodies, or Fc-fusion proteins. Preferably, at least about 50%, and preferably, at least about 70% of antibody molecules, or Fc-fusion proteins, comprise a complex N-glycan. For example, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the antibody molecules, or Fc-fusion proteins, comprise a complex N-glycan. Preferably, the N-glycan is linked to the Fc domain of the antibody or the Fc-fusion protein.

[0047] The Fc domain of the antibodies or Fc-fu sion proteins described herein can be a naturally occurring Fc region of an antibody, which may optionally include the hinge region. Alternatively, the Fc domain can a truncated form of a naturally occurring Fc region (e.g., CH2 domain only); a fusion form that comprising a naturally occurring Fc region fused with a heterologous sequence; a mutated form of a naturally occurring Fc domain (e.g., a Fc region having amino acid substitutions), addition(s), or deietion(s)); or a combination thereof. For example, Fc variants T256A, K290A, S298A, E333A, K334A, A339T have been described as having enhanced ADCC activity as compared to naturally occurring Fc sequences (see, e.g., Shields et al. (2001) J. Biol, Chem,, 276:6591-6604), Additional mutation variants of Fc regions are disclosed in U.S. Patent Application No. 2004/0228856.

[0048] The Fc domain can be derived from the Fc region of IgG, IgA, IgD, IgE and IgM (including various isotypes, such as, IgGl, lgG2, IgG3, IgG4, JgAl, JgA2 etc). Preferably, the Fc domain is derived from IgG 1.

[0049] As described herein, reducing or inhibiting N-glycan fucosylation of antibodies, or Fc-fusion proteins, can enhance the antibody dependent cell-mediated cytotoxicity (ADCC). ADCC typically involves the activation of natural killer (NK) cells and is dependent on the recognition of antibody-coated cells by Fc receptors, most commonly CD 16 (FcyRJil)) on the surface of the NK cell. Binding of the Fc domain to Fc receptors on the NK cells is affected by the glycosylation state of the Fc domain. In addition, the type of the N-glycan at the Fc domain also affects ADCC activity. Therefore, for an antibody composition, or a Fc-fusion protein composition, increasing the relative amount of afucosyl N-glycans can increase the binding affinity for an FcyRIII, or ADCC activity, of the composition.

10050] The N-glyeosylated antibodies, or Fc-fusion proteins, as described herein, may comprise a mixture of fucosylated and afucosylated molecules. For example, from about 20% to about 100%, preferably, from about 40°/» to about 80% of the glycans are afucosyl glycans. For example, from about 20% to about 100%, from about 25% to about 100%, from about 30% to about 100%, from about 35% to about 100%, from about 40% to about 100%, from about 45% to about 100%), or from about 20% to about 100% of the N-glycans are afucosyl glycans. It is not required that all, or substantially all, of the N-glycosylated antibodies, or Fc- fusion proteins are afucosylated. In fact, as shown in Example 1 , significant enhancement in ADCC activity of a monoclonal antibody that binds to CD20 was achieve when about 48% of the N-glycans were afucosylated. f 0051] The antibody composition, or Fc-fusion protein composition described herein may comprise a mixture of GO, Gl, Gl 'and G2 glycoforms (e.g., at the Fc-lmked N-glycan). For example, from about 0% to about 70%, from about 0%s to about 65%, from about 0% to about 60%, from about 0% to about 55%, from about 0% to about 50%, from about 0% to about 45%, from about 0% to about 40%), from about 0% to about 35%, from about 0%s to about 30%», of the N-glycans may be GO glycans. Alternatively or in addition, from about 0% to about 70%, from about 0%s to about 65%», from about 0% to about 60%, from about 0% to about 55%», from about 0% to about 50%, from about 0% to about 45%, from about 0% to about 40%, from about 0% to about 35%, from about 0% to about 30%, of the N-glycans may be Gl and Gl ' glycans. Alternatively or in addition, from about 0% to about 70%, from about 0% to about 65%, from about 0% to about 60%), from about 0% to about 55%, from about Q%> to about 50°/», from about 0%) to about 45%», from about 0% to about 40%, from about 0%» to about 35%, from about 0% to about 30%, of the N-glycans may be G2 glycans, The N-glycans of the antibody composition, or Fc-fusion protem composition may also be any combination of the GO, Gl, Gl ', G2 profiles described above.

[0052] Preferably, from about 20% to about 100%, such as from about 25% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 20% to about 95%, from about 20% to about 90%, from about 20% to about 85%, from about 20% to about 80°/», from about 25% to about 95%, from about 30% to about 90%, from about 35% to about 85%), or from about 40% to about 80%, of the GO N-glycans are afucosyl GO glycans.

[0053] Alternatively or in addition, from about 0% to about 100%, such as from about 1 % to about 90%, from about 1% to about 80%, from about 1 % to about 70%, from about 1%» to about 60%», from about 1% to about 55%, from about 1%» to about 50%, from about 1% to about 45%, from about 1% to about 40%, from about 35%, of the Gl and Gl ' N-glycans are afucosyl Gl and Gl ' glycans.

[0054] Alternatively or in addition, from about 0% to about 100%, such as from about 1% to about 90%, from about 1% to about 80%, from about 1% to about 70%, from about 1 % to about 60%, from about 1% to about 55%, from about 1% to about 50%, from about 1% to about 45%, from about 1%) to about 40%), from about 35%, of the G2 N-glycans are afucosyl G2 glycans.

[0055] The afucosyl N-glycans of the antibody composition, or Fc-fusion protein composition may also be any combination of the GO, Gl, Gl ', G2 profiles described above. [0056] The glycan structure of the antibodies or Fc-fusion proteins described herein can be determined using art-known methods for analyzing glycan structures of glycoproteins, as described below,

[0057] Antibodies or Fc-fusion proteins having improved ADCC activity are useful for treating or preventing various diseases including cancers, inflammatory diseases, immune diseases such as autoimmune diseases, allergies and the like, circulator organ diseases (e.g., arteriosclerosis) and viral or bacterial infections. Preferably, the antibody or Fc-fusion protein binds to a cell-surface molecule, or a cell-surface associated molecule, such as a transmembrane receptor. If desired, the antibody or Fc-fusion protein may bind to a soluble molecule that can associate with the cell surface, for example, with a cell-surface molecule, such as a ligand that binds to a transmembrane receptor.

[0058] Examples of the antibody which recognizes a tumor-related antigen include anti-GD2 antibody (Ohta et al., Anticancer Res., 13, 331-336, 1993), anti~GD3 antibody (Qhta et al,, Cancer Immunol. Immunother., 36, 260-266, 1993), anti-GM2 antibody (Nakamura et al., Cancer Res,, 54, 1511-1516, 1994), anti-HER2 antibody (Carter et al, Proc. Natl. Acad, Sci. USA, 89, 4285-4289, 1992), anti-CD52 antibody (Carter et al, Proc. Natl. Acad. Sci. USA, 89, 4285-4289, 1992), anti-MAGE antibody (Jungbluth et al., British J, Cancer, 83, 493-497, 2000), anti-HM124 antibody (Ono et al., Molecular Immunol, 36, 387-395, 1999), anti-parathyroid hormone-related protein (PTHrP) antibody (Ogata et al, Cancer, 88, 2909-2911 , 2000), anti- basic fibroblast growth factor antibody and anti-FGF8 antibody (Matsuzaki et al., Proc. Natl. Acad, Sci, USA, 86, 9911-9915, 1989), anti-basic fibroblast growth factor receptor antibody and anti-FGFS receptor antibody (Kuo et al,, J. Biol. Chem., 265, 16455-16463, 1990), anti-insulinlike growth factor antibody (Yao et al., J. Neurosci. Res,, 40, 647-659, 1995), anti-insulin-like growth factor receptor antibody (Yao et al., J. Neurosci. Res,, 40, 647-659, 1995), anti-PMSA antibody (Murphy et al., J. Urology, 160, 2396-2401 , 1998), anti-vascular endothelial cell growth factor antibody (Presta et al, Cancer Res., 57, 4593-4599, 1997), anti-vascular endothelial cell growth factor receptor antibody ( anno et al., Oncogene, 19, 2138-2146, 2000) and the like.

[0059] Examples of the antibody which recognizes an allergy- or inflammation- related antigen include anti-interleukin 6 antibody (Abrams et al, Immunol. Rev., 121 , 5-24, 1992), anti-interleukin 6 receptor antibody (Sato et al., Molecular Immunol., 31, 371-381, 1994), anti-interleukin 5 antibody (Abrams et al, Immunol. Rev., 127, 5-24, 1992), anti-interleukin 5 receptor antibody and anti-interleukin 4 antibody (Biord et al., Cytokine, 3, 562-567, 1991 ), anti-tumor necrosis factor antibody (Tempest et al., Hybridoma, 13, 183-190, 1994), anti-tumor necrosis factor receptor antibody (Amrani et al., Molecular Pharmacol, 58, 237-245, 2000), anti-CCR4 antibody (Campbell et al., Nature, 400, 776-780, 1999), anti-chemokine antibody (Peri et al, J. Immuno. Meth., 174, 249-257, 1994), anti-chemokine receptor antibody (Wu et al., J. Exp. Med., 186, 1373-1381 , 1997) and the like. Examples of the antibody which recognizes a circulatory organ disease-related antigen include anti-GpIIb/IIIa antibody (Co et al, J. Immunol., 152, 2968-2976, 1994), anti-platelet-derived growth factor antibody (Ferns et al., Science, 253, 1 129-1132, 1991), anti-platelet-derived growth factor receptor antibody (Shulman et al., J. Biol. Chem., 272, 17400-17404, 1997) and anti-blood coagulation factor antibody (Peter et al, Circulation, 101, 1158-1 164, 2000) and the like.

[0060] Examples of the antibody which recognizes a viral or bacterial infection- related antigen include anti-gpl 20 antibody (Tugarinov et al,, Structure, 8, 385-395, 2000), anti- CD4 antibody (Schulze-Koops et al, J. Rheumatology, 25, 2065-2076, 1998), anti-CCR4 antibody and anti-Vero toxin antibody ( amali et al,, J. Clin, Microbiol, 3, 396-399, 1999) and the like.

[0061 ] Several therapeutic antibodies and Fc fusion proteins are commercially available, such as antibodies that bind to VEGF (e.g., Bevacizumab (Avastin)), EGFR (e.g., Cetuximab (Erbitux®)), HER2 (e.g., Trastuzumah (Herceptin®)), and CD20 (e.g., Rituxirnab (Rituxan®)), and Fc-fusion proteins that bind to TNFa (e.g., Etanecept (Enbrel), which comprises the receptor-binding domain of a TNF receptor (p75)), CD 2 (e.g., Alefacept

(Amevive), which contains the CD2-binding domain of LFA-3), or B7 (Abatacept (Orencia®), which comprises the B7-binding domain of CTLA4).

[0062] All of the antibodies above can be modified according to the invention to improve ADCC activities.

(1), Glycan-modified anti-CD20 antibodies

[0063] In one aspect, the invention provides a composition comprising an antibody that binds to CD20, wherein the composition is characterized by: (a) at least about 70% of the antibody molecules comprise a complex N-glycan (e.g., linked to the Fc domain of the antibody); and (b) about 40% to about 100% of the N-glycans are afucosylated.

[0064] CD20 is a transmembrane protein with a molecular weight of approximately 35 kD located on pre-B and mature B lymphocytes (Valentine et al. (1989) J. Biol. Chem.

264(19): 11282-1 1287; and Einfield et al. (1988) EMBO J. 7(3):311-717). CD20 is found on the surface of greater than 90% of B cells from peripheral blood or lymphoid organs and is expressed during early pre-B ceil development and remains until plasma cell differentiation. Although CD20 is expressed on normal B ceils, this surface antigen is usually expressed at ver high levels on neoplastic B cells. More than 90% of B-cell lymphomas and chronic lymphocytic leukemias, and about 50% of pre-B-cell acute lymphoblastic leukemias express this surface antigen. CD20 is not found on hematopoietic stem cells, pro-B cells, normal plasma cells, or other normal tissue (Tedder et al. (1985) i, Immunol. 135(2):973~979).

[0065] CD20 is expressed by malignant cells of B-cell origin, including B-cell lymphoma and chronic lymphocytic leukemia (CLL). CD20 is not expressed by malignancies of pre-B-ce ls, such as acute lymphoblastic leukemia. CD20 is therefore a good target for therapy of B-cell lymphoma, CLL, and other diseases in which B-cells are involved in the disease etiology. Other B-cell disorders include autoimmune diseases in which auto-antibodies are produced during the differentiation of B-cells into plasma cells.

[0066] In addition, CD20 has also been targeted by radioimmunotherapeutic agents to treat B-cell related diseases. One treatment consists of anti-CD20 antibodies prepared in the form of radionuclides for treating B-cell lymphoma (e.g., ^ljl l-labeled anti-CD20 antibody), as well as a ⁹ Sr- labeled form for the palliation of bone pain caused by prostate and breast cancer metastases (Endo, Gan To Kagaku Ryoho 1999, 26: 744-748).

[0067] Rituximab (Rituxan©) is a commercially available recombinant

mouse/human IgGl chimeric monoclonal antibody (mAb) in which variable domains of the hea vy and light chains of a murine anti-CD20 mAb are fused to the human constant regions of IgG l .

[0068] In certain embodiments, the light chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the light chain of Rituximab. Alternatively or in addition, the heavy chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the heavy chain of Rituximab.

[0069] Preferably, the light chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98°/», at least 99%, or 100% identical to the light chain of Rituximab; and the heavy chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the heavy chain of Rituximab.

[0070] The anti-CD20 antibody composition may comprise a mixture of GO, Gl, Gl ', and G2 glycoforms (e.g., at the Fc-linked N-glycan), as described above.

[0071] As described above, an antibody comprising an afucosylated N-glycan at the Fc region has an increased binding affinity for FcyRIII, as compared to a corresponding antibody (antibody having the same amino acid sequence) comprising a fucosylated N-glycan. Therefore, the antibody composition as described herein has an increased antibody-dependent cellular cytotoxicity (ADCC) activity, as compared to a composition comprising the same amount of the glycosylated antibody, but less than 40% of the N-glycans are afucosylated. Preferably, the anti- CD20 composition described herein has an increased antibody-dependent cellular cytotoxicity (ADCC) activity as compared to Rituximab under substantially the same conditions.

[0072] The anti-CD20 antibodies described herein may be used for treating Non- Hodgkins lymphoma.

(2), Glycan-modified anti-EGFR antibodies

[0073] In one aspect, the invention provides a composition comprising an antibody that binds to EGFR, wherein the composition is characterized by: (a) at least about 70% of the antibody molecules comprise a complex N-glycan (e.g, linked to the Fc domain of the antibody); and (b) about 40% to about 100% of the N-glycans are afucosylated.

[0074] EGFR (also known as ErbB-1 and HER! in humans) is the cell-surface receptor for members of the epidermal growth factor family (EGF-family) of extracellular protein ligands (Herbst, 2004, Int. j. Radiat. Oncol. Biol. Phys. 59 (2 Suppi):2! -6). Mutations affecting EGFR expression or activity can result in cancer. EGF receptors are over expressed in most epithelial malignancies including those of the colon and the rectum. The EGFR. is constitutively expressed in many normal epithelial tissues, including the skin and hair follicle.

[0075] Cetuximab (Erbitux©) is a commercially available recombinant

mouse/human IgGl chimeric monoclonal antibody (mAb) in whic variable domains of the heavy and light chains of a murine anti-EG FR mAb (kno wn as 225) are fused to the human constant regions of IgGl . Cetuximab has been approved for use in combination with radiation therapy for treating squamous ceil carcinoma of the head and neck (SCCHN) or as a single agent in patients who have had prior platinum-based therapy. Cetuximab is also indicated for treatment of metastatic colon cancer in combination with irmotecan (Camptosar) a DNA topoisomerase blocker. Cetuximab is believed to function mainly by blocking the EGF binding to EGFR, thereby starving the tumor of needed growth factor.

[0076] In certain embodiments, the light chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the light chain of Cetuximab.

Alternatively or in addition, the heavy chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the heavy chain of Cetuximab.

[0077] Preferably, the light chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the light chain of Cetuximab; and the heavy chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the heavy chain of Cetuximab.

[0078] The anti- EGFR antibody composition may comprise a mixture of GO, Gl , Gl ', and G2 glycoforms (e.g, at the Fc-linked N-glycan), as described above.

[0079] As described herein, an antibody comprising an afucosylated N-glycan at the Fc region has an increased binding affinity for FcyRJII, as compared to a corresponding antibody (antibody having the same amino acid sequence) comprising a fucosylated N-glycan. Therefore, the antibody composition as described herein has an increased antibody-dependent cellular cytotoxicity (ADCC) activity, as compared to a composition comprising the same amount of the glycosylated antibody, but less than 40% of the N-glycans are afucosylated. Preferably, the anti- EGFR composition described herein has an increased antibody-dependent cellular cytotoxicity (ADCC) activity as compared to Cetuximab under substantially the same conditions.

[0080] The anti-EGFR antibodies described herein may be used for treating colorectal cancer or head and neck squamous cell carcinoma.

(3), G!ycan-modified anti-HER2 antibodies f 0081] In one aspect, the invention provides a composition comprising an antibody that binds to HER2, wherein the composition is characterized by: (a) at least about 70% of the antibody molecules comprise a complex N-glycan (e.g, linked to the Fc domain of the antibody); and (b) about 40% to about 100% of the N-glycans are afucosylated.

[0082] HER2 (also known as ErbB-2) is a 185 kD transmembrane glycoprotein receptor that is encoded by the erbB2 gene. HER2 is related to the epidermal growth factor receptor (EGFR, or HER1 in human), and is over expressed in about 25%» to 30% of human breast cancer (Slamon et al.. Science 235: 177-182 , (1987); Siamoii et al., Science 244:707-712 (1989)). HER2 overexpression is commonly regarded as a predictor of a poor prognosis, especially in patients with primary disease that involves axillary lymph nodes (Slamon et al., (1987) and ( 1989), supra; Ravdin and Chamness, Gene 159: 19-27 (1995); and Hynes and Stern, Biochim Biophys Acta 1 198: 165-184 (1994)), and has been linked to sensitivity and/or resistance to hormone therapy and chemotherapeutic regimens, including C F

(cyclophosphamide, methotrexate, and fluomracil) and anthracyclines (Baselga et al., Oncology 1 1 (3 Suppl 1 ):43~48 (1997)).

[0083] Trastuzumab (Herceptin®) is a humanized anti~ErbB2 monoclonal antibody (a humanized version of the murine anti-ErbB2 antibody 4D5, referred to as rhuMAb HER2). Trastuzumab has been clinically active in patients with ErbB2-overexpressing metastatic breast cancers that had received extensive prior anti-cancer therapy (Baselga et al., j. Clin. Oncol. 14:737-744 (1996)).

[0084] In certain embodiments, the light chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%· identical to the light chain of Trastuzumab.

Alternatively or in addition, the heavy chain of the antibody comprises a sequence that is at least 75%>, at least 80%. at least 85%, at least 90%>, at least 95%, at least 96%, at least 97%>, at least 98%, at least 99%, or 100% identical to the hea vy chain of Trastuzumab,

[0085] Preferably, the light chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%), at least 99%, or 100% identical to the light chain of Trastuzumab; and the hea vy chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the heavy chain of Trastuzumab,

[0086] The anti~HER2 antibody composition may comprise a mixture of GO, G l, Gl ', and G2 glycoforms (e.g, at the Fc-linked N-glycan), as described above.

[0087] As described above, an antibody comprising an afucosylated N-glycan at the Fc region has an increased binding affinity for FcyRIII, as compared to a corresponding antibody (antibody having the same amino acid sequence) comprising a fucosylated N-glycan. Therefore, the antibody composition as described herein has an increased antibody-dependent cellular cytotoxicity (ADCC) activity, as compared to a composition comprising the same amount of the glycosylated antibody, but less than 40% of the N-glycans are afucosylated. Preferably, the anti- HER2 composition described herein has an increased antibody-dependent cellular cytotoxicity (ADCC) activity as compared to Trastuzumab under substantially the same conditions.

[0088] The anti-HER2 antibodies described herein may be used for treating breast cancer,

(4). Glycan-modified B7-Bmdisig Molecule

[ 0089] In one aspect, the invention provides a composition comprising a Fc-fusion protein that binds to B7, wherein the composition is characterized by: (a) (a) at least about 70% of the antibody molecules comprise a complex N-glycan (e.g., linked to the Fc domain of the fusion protein), and (b) about 40% to about 100% of the N-glycans are afucosylated.

[0090] B7 is a peripheral membrane protein found on activated antigen presenting cells (APC) that, when paired with either a CD28 or CD 1.52 (CTLA-4) surface protein on a T cell, can produce a costiraulatory signal to enhance or decrease the activity of a MHC-TCR signal between the APC and the T cell, respectively (see, e.g., Coico, et al. (2003) Immunology: A Short Course; Wiley-Liss. p. 131). Besides being present on activated APCs, B7 is also found on T-cells themselves (see, e.g., Taylor et al., J Immunol. 172 (1): 34-39), Binding of the B7 on T-cells to CTLA-4 causes inhibition of the activity of T-cells. There are two major types of B7 proteins, B7.1 and B7.2 or CD80 and CD86 respectively.

[0091] In certain embodiments, the Fc-fusion protein comprises the extracellular domain of Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4).

[0092] CTL A4 (Cytotoxic T-Lymphocyte Antigen 4) also known as CD 152 (Cluster of differentiation 152) is a protein that plays an important regulatory role in the immune system. In humans, the CTL.A4 protein is encoded by the CTLA4 gene (Dariavach P, et al,, Eur, J, Immunol. 18 (12): 1901-5). CTLA4 is a member of the immunoglobulin superfamily, which is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells, CTLA4 is similar to the T-cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 (B7) on antigen-presenting cells. CTLA4 transmits an inhibitory signal to T ceils, whereas CD28 transmits a stimulatory signal. Intracellular CTLA4 is also found in regulatory T cells and may be important to their function. T ceil activation through the T cell receptor and CD28 leads to increased expression of CTL A-4, an inhibitor} _' receptor for B7 molecules (CD80 and CD86).

[0093] In certain embodiments, the fusion protein comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%), at least 99%», or 100%) identical to Abatacept.

[0094 j Orencia® ( Abatacept) is a soluble fusion protein that consisting of the extracellular domain of human cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) linked to the modified Fc (hinge, CH2, and CH3 domains) portion of human immunoglobulin Gl ( IgGl ). Abatacept can bind to B7 and is a selective costimuiation modulator as it inhibits the

costimulation of T cells.

[0095] The Fc-fusion protein composition may comprise a mixture of GO, Gl, Gl ', and G2 glycoforms (e.g, at the Fc-linked N-glycan), as described above. [0096] As described above, an Fc-fusion protein comprising an afucosylated N- glycan at the Fc region has an increased binding affinity for FcyRJ !I, as compared to a corresponding fusion protein (fusion protein having the same amino acid sequence) comprising a fucosylated N-glycan. Therefore, the Fc-fusion protein composition as described herein has an increased antibody-dependent cellular cytotoxicity (ADCC) activity, as compared to a composition comprising the same amount of the glycosylated Fc-fusion protein, but less than 40% of the N-giycans are afucosylated. Preferably, the Fc-fusion protein composition described herein has an increased antibody-dependent cellular cytotoxicity (ADCC) activity as compared to Abatacept under substantially the same conditions.

B. Glycan-Modified Proteins That Have Reduced Immunogenicity in Human

[0097] As described herein, RNA effectors, such as siRNAs, can also be used to reduce the expression levels of enzymes or transporters that are involved in the production of immunogenic epitopes. Examples of immunogenic epitopes in humans include, e.g., glycans, such as N-giycans, that comprise a N-glycolylneuraminic acid (NeuSGc) residue, and the aGal epitope (galactose-alpha(l ,3)-galactose-beta( l,4)N-acetylglucosamine-R; or Gal-a(l,3)-Gai- P(l,4)~GlcNAe~R; where R is the glycan-protein structure).

[0098] The compositions described herein may comprise a mixture of glycosylated and aglycosylated proteins. Preferably, at least about 50%, and preferably, at least about 70% of the proteins comprise a glycan. For example, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the protein molecules comprise a glycan. Preferably, the glycan is a complex N-glycan.

(1). aGal Epitope

[0099] In another aspect, the invention provides a composition comprising a protein, wherein at least about 70% of the protein molecules comprise a glycan ( e.g., a complex N- glycan); and wherein (a), the protein is produced by a cell that is not a human, ape, or Old World monkey cell; and at least about 40%) of the glycosylated molecules do not comprise the a-Gal epitope.

[00100] Mammalian cells, such as Chinese hamster ovary (CHO) cells, and rodent myeloma derived NS/0 and SP2/0 cells, are widely used for producing therapeutic glycoproteins. However, glycoproteins produced by these cells often contain oligosaccharides terminated with a-galactose residues. The a-galactose residues are linked to the penultimate galactose residues at a hydroxy! of the third sugar carbon position, a(l-3) linkage. Human, apes, and old-world monkey cells, however, do not express functional -gaiactosyltransferase (al-3GT), which participates in the glycosylation of cell membrane glycoconjugates in nonprimate mammals, prosimians, and Ne World monkeys. The absence of a 1-3 Gal epitopes from human cells is due to silencing of the gene for the enzyme a.1 ,3 galactosyltransferase (GGTA1 gene). As a result, humans have up to 1 % of circulating antibodies (such as anti-aGal IgA, IgG, and IgM) directed against the enzymatic product of a 1 ,3 -galactosyltransferase ( Gal al-3Ga!pl-4Glc Ac), also called Gaiili antigen (Galili, U., Clark, M. R., Shohet, S. B., Buehler, J., and Macher, B. A. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1369-1373), or aGal epitope. As such, there is a potential risk for clinical application of glycoprotein that are produced by non-human, non-ape, and non-Old World monkey cells.

[00101] Accordingly, the invention provides compositions of glycoproteins in which at least about 20% of the glycosylated molecules do not comprise the a-Gal epitope. Preferably, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, of the glycosylated molecules do not comprise the a-Gal epitope.

[00102] Preferably, the compositions described herein comprises glycoproteins in which at least about 40% of the glycans do not comprise the a-Gal epitope, and the composition has reduced immunogenicity in human, as compared to a composition comprising the same amount of the glycosylated protein in which more than 40% of the glycans comprise the a-Gal epitope.

(2). N-gfycolylneuraminic acid

[00103] In another aspect, the invention provides a composition comprising a protem, wherein at least about 70% of the protein molecules comprise a glycan (e.g., a complex N- glycan); and wherein (a) the protein is produced by a non-human cell; and; (b) the glycans (e.g., complex N-glycans) of said protein molecules are characterized by a total sialic acid content that contain no more than about 20% of N-glycolylneuraminic acid (Neu5Gc). [00104] Sialic acids are 9-carbon backbone acidic sugars terminating the glycan chains of various glycoproteins and glycolipids at vertebrate cell surfaces. N-acetylneurarmnic acid (Neu5 Ac) and its hydrox lated form NeuSGc are the two major Sialic acids in mammals, with the activated form CMP-NeuSAc serving as the precursor for synthesis of CMP-Neu5Gc, catalyzed by the enzyme CMP-NeuSAc hydroxylase (CMAH), encoded by the CMAH gene. This enzyme is specifically inactivated in humans, in contrast to other mammals studied to date, including old world primates and pigs fVarki A., Proc. Natl. Acad. Sci. USA 2010; 107(Suppl. 2): 8939-8946). In addition, humans lack an alternative pathway for NeuSGc synthesis.

Accordingly, human immune system recognizes NeuSGc as foreign, resulting in a humoral response involving a polyclonal highly diverse antibody profile in all humans.

[00105] Accordingly, the invention provides compositions of glycoproteins in which the total sialic acid content of the glycans (e.g., complex N-glycans) contain no more than about 40% of NeuSGc, Preferably, the total sialic acid content of the glycans (e.g., complex N- glycans) contain no more than about 35%, no more than about 30%, no more than about 25%, no more than about 20%, no more than about 15%, or no more than about 10% of N euSGc.

[00106] Preferably, the compositions described herein comprises glycoproteins in which the total sialic acid content of the glycans (e.g., complex N-glycans) contain no more than about 20% of NeuSGc, and the composition has reduced immunogenicity in human, as compared to a composition comprising the same amount of the glycosylated protein, but the total sialic acid content of the glycans (e.g., complex N-glycans) contain more than about 20% of NeuSGc.

(3), Glycan-modified antibodies and Fc-fusion proteins that have reduced immunogenicity in humans

[00107] Any suitable glycoprotein may be modified according to the teachings of the invention to reduce the amount of immunogenic epitopes, such as the aGal and NeuSGc epitopes described above. Preferred classes of glycoproteins include, e.g., antibodies or Fc- fusion proteins. Therefore, in certain embodiments, the composition described herein comprises a protein that is an antibody or a fusion protein that comprises the Fc domain of an antibody; and wherein said N -glycan is linked to the Fc domain of the antibody molecules or the fusion protein. [00108] As described above, the antibody composition, or Fc-fusion protein composition described herein may comprise a mixture of GO, G l, Gl ', and G2 glycoforms (e.g., at the Fc-linked N-glycan). For example, from about 0% to about 70%, from about 0% to about 65%, from about 0% to about 60%, from about 0% to about 55%, from about 0% to about 50%, from about 0%s to about 45%, from about 0% to about 40%, from about 0% to about 35%», from about 0% to about 30%, of the N-glycans may be GO glycans. Alternatively or in addition, from about 0% to about 70%, from about 0% to about 65%, from about 0% to about 60%, from about 0% to about 55%, from about 0% to about 50%, from about 0% to about 45%, from about 0% to about 40%, from about 0% to about 35%, from about 0% to about 30%, of the N-glycans may be Gl and Gl ' glycans. Alternatively or in addition, from about 0% to about 70%, from about 0% to about 65%, from about 0% to about 60%, from about 0% to about 55%, from about 0% to about 50%), from about 0% to about 45%, from about 0% to about 40%>, from about 0% to about 35%, from about 0% to about 30%, of the N-glycans may be G2 glycans. The N-glycans of the antibody composition, or Fc-fusion protein composition may also be any combination of the GO, Gl, Gl ', G2 profiles described above.

[00109] In addition, an antibody or Fc-fusion protein may be modified such tha it has both improved effector activity (e.g., ADCC activity), and reduced immunogenicity in humans. For example, two or more different species of RNA effector molecules may be added to a host cell culture. At least one RNA effector targets the fucosylation pathway to increase the amount of afucosylated antibody or Fc-fusion protein that is produced by the host ceils, and at least another RNA effector targets a-galactosyltransferase or CMAH to reduce the amount of immunogenic epitopes produced by the host cells.

[00110] The gl can structure of the antibodies or Fc-fusion proteins described herein can be determined using art-known methods for analyzing glycan structures of glycoproteins, as described below.

[00111] In certain embodiments, the composition described herein comprises a protein that is an anti-EGFR antibody. Preferably, the light chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%>, at least 99%, or 100% identical to the light chain of Cetuximab; and/or the heavy chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the heavy chain of Cetuximab. Preferably, the anti-EGFR antibody composition described herein has reduced immunogenicity as compared to Cetuximab under substantially the same conditions (for example, as a result of reduced amount of aGal epitope, NeuSGc epitope, or both).

[00112] One study reported that among 76 Cetuximab -treated subjects, 25 had a hypersensitivity reaction to the drug, In most subjects who had a hypersensitivity reaction, IgE antibodies that are specific for the aGal epitope were detected. Therefore, preferably, the anti- EGFR antibody composition described herein has reduced immunogenicity in human, as compared Cetuximab to under similar conditions. In addition, it has been reported that NeuSGc is present in Cetuximab (see, e.g. Ghaderi et al, Nature Biotechnology Vol. 28, 863-867 (2010)).

[00113] In certain embodiments, the composition described herein comprises a protein that is an anti-CD20 antibody. Preferably, the light chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%», at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the light chain of Rituximab; and/or the heavy chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the heavy chain of Rituximab. Preferably, the anti-CD20 antibody composition described herein has reduced immunogenicity as compared to Rituximab under substantially the same conditions (for example, as a result of reduced amount of aGal epitope, NeuSGc epitope, or both).

100114] In certain embodiments, the composition described herein comprises a protein that is an anti-HER2 antibody. Preferably, the light chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%), at least 99%, or 100% identical to the light chain of Trastuzumab; and/or the heavy chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%), at least 90%, at least 95%, at least 96%), at least 97%», at least 98%, at least 99%), or 100% identical to the heavy chain of Trastuzumab. Preferably, the anti~HER2 antibody composition described herein has reduced immunogenicity as compared to Trastuzumab under substantially the same conditions (for example, as a result of reduced amount of aGal epitope, NeuSGc epitope, or both). [00115] In certain embodiments, the composition described herein comprises a Fc- fusion protein that binds to B7,

[00116] In certain embodiments, the Fc-fusion protein comprises the extracellular domain of Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4).

[00117] In certain embodiments, the fusion protein comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to Abatacept.

[00118] Preferably, the Fc-fusion protein composition described herein has reduced immunogenicity as compared to Abatacept under substantially the same conditions (for example, as a result of reduced amount of c/Gal epitope, NeuSGc epitope, or both). The Fc- fusion proteins described herein may be used to for treating rheumatoid arthritis.

C. Glycan-Modifled Proteins That Have Increased Serum Half Life

[00119] As described herein, RNA effectors, such as siRNAs, can also be used to improve pharmacokinetic properties of glycoproteins. In another aspect, the invention provides a composition comprising a protein, wherein one or more glycans comprise a sialic acid cap (e.g., N-acetylneuraminic acid (NeuSAc)}.

[00120] The serum half life of glycoproteins is dependent on the composition and structure of its N-glycans. In general, maximal serum half life of a glycoprotein requires that its N-glycans terminate with a sialic acid cap (e.g., N-acetylneuraminic acid (Ne 5Ac)). RNA effectors can be used to transiently reduce the expression of sialidases that remove the terminal sialic acid (e.g., NeuSAc) cap from a glycoprotein. By reducing the hydrolysis of terminal sialic acid from N-linked N-glycans, therapeutic efficacies of glycoproteins can be improved.

[00121] Preferably, the composition described herein has an increased serum half-life in human, as compared to a composition comprising the same amount of the glycosylated protein in which the N-glycans does not comprise NeuSAc.

[00122] In addition, a glycoprotein may be modified such that it has both reduced immunogenicity in humans, and increased serum half-life. For example, two or more different species of RNA effector molecules may be added to a host cell culture. At least one RNA effector targets α-galactosyl transferase or CMAH to reduce the amount of immunogenic epitopes produced by the host cells, and at least another RNA effector targets a sialidase to increase the amount of sialic acid-capped N-glycans that are produced by the host cell.

[00123] Any suitable glycoprotein may be modified accordingly to reduce the amount of immunogenic epitopes, such as the oiGal and NeuSGc epitopes described above. Preferred classes of glycoproteins include, e.g., antibodies or Fc-fusion proteins. Therefore, in certain embodiments, the composition described herein comprises a protein that is an antibody or a fusion protein that comprises the Fc domain of an antibody; and wherein said antibody or fusion protein comprises a glycan, Preferably, the glycan is an N-glycan. Preferably, the N-glycan is linked to the antibody or Fc-fusion protein at the Fc domain.

[00124] As described above, the antibody composition, or Fc-fusion protein composition described herein may comprise a mixture of GO, Gl, Gl ', and G2 glycoforms (e.g., at the Fc-linked N-glycan). For example, from about 0% to about 70%, from about 0% to about 65%, from about 0% to about 60%, from about 0% to about 55%, from about 0% to about 50%, from about 0% to about 45%, from about 0% to about 40%, from about 0% to about 35%, from about 0%» to about 30%, of the N-glycans may be GO glycans. Alternatively or in addition, from about 0% to about 70%, from about 0% to about 65%, from about 0% to about 60%, from about 0% to about 55%, from about 0% to about 50%, from about 0% to about 45%, from about 0% to about 40%, from about 0% to about 35%, from abou 0% to about 30%, of the N-glycans may be G l and Gl ' glycans. Alternatively or in addition, from about 0% to about 70%, from about 0% to about 65%, from about 0% to about 60%. from abou 0% to about 55%, from about 0% to about 50%, from about 0% to about 45%, from about 0% to about 40%, from about 0% to about 35%), from about 0% to about 30%, of the N-glycans may be G2 glycans. The N-glycans of the antibody composition, or Fc-fusion protein composition may also be any combination of the GO, Gl , Gl ', G2 profiles described above.

[00125] The glycan structure of the antibodies or Fc-fusion proteins described herein can be determined using art-known methods for analyzing glycan structures of glycoproteins, as described below.

[00126] In addition, an antibody or Fc-fusion protein may be modified such that it has any combination of improved effector activity (e.g., ADCC activity), reduced immunogenicity in humans, or increased serum half life, For example, two or more different species of RNA effector molecules may he added to a host cell culture. At least one RNA effector targets the fucosylation pathway to increase the amount of afucosylated antibody or Fc-fusion protein that is produced by the host cells, and ai least another RNA effector targets a sialidase to increase the amount of sialic acid-capped N-glycans that are produced by the host cell, or targets a- galactosyltransferase or CMAH to reduce the amount of immunogenic epitopes produced by the host cells. If desired, three or more different species of RNA effector molecules may be added to a host cell culture, at least one targets the fucosylation pathway, at least one targets a sialidase, and at least one targets cx-gaiactosyltransferase or CMAH.

[00127] In certain embodiments, the composition described herein comprises a protein that is an anti-EGFR antibody. Preferably, the light chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%), at least 98%, at least 99%, or 100% identical to the light chain of Cetuximab; and/or the heavy chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the heavy chain of Cetuximab, Preferably, the anti-EGFR antibody composition described herein has an increased serum half life as compared to Cetuximab under substantially the same conditions (for example, as a result of reduced hydrolysis of the sialic acid cap).

[00128] In certain embodiments, the composition described herein comprises a protein that is an anti-CD20 antibody. Preferably, the light chain of the antibody comprises a sequence that is at least 75%, at least 80%), at least 85%, at least 90%, at least 95%), at least 96%, at least 97%, at least 98%>, at least 99%, or 100% identical to the light chain of Rituximab; and/or the heavy chain of the antibody comprises a sequence that is at least 75%, at least 80%), at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the heavy chain of Rituximab. Preferably, the anti-CD20 antibody composition described herein has an increased serum half life as compared to Rituximab under substantially the same conditions (for example, as a result of reduced hydrolysis of the sialic acid cap).

[00129] In certain embodiments, the composition described herein comprises a protein that is an anti-HER2 antibody. Preferably, the light chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the light chain of Trastuzumab; and/or the heavy chain of the antibody comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the heavy chain of Trastuzumab, Preferably, the anti-HER2 antibody composition described herein has an increased serum half life as compared to Trastuzumab under

substantially the same conditions (for example, as a result of reduced hydrolysis of the sialic acid cap).

[00130] In certain embodiments, the composition described herein comprises an Fc- fusion protein that binds to B7. In certain embodiments, the Fc-fusion protein comprises the extracellular domain of Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4). Preferably, the fusion protein comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to Abatacept. Preferably, the Fc-fusion protein composition described herein has an increased serum half life as compared to Abatacept under substantially the same conditions (for example, as a result of reduced hydrolysis of the sialic acid cap),

4. METHODS FOR MODIFYING PROTEIN GLYCOSYLATION

[00131] In one aspect, the invention provides methods for producing giycan-modified glycoproteins, in particular on a large or commercial scale. The method comprises culturing a host ceil in a large scale cell culture in the presence of an RNA effector that targets a gene that encodes an enzyme or a transporter protein that is involved in a glycosylation pathway. The RNA effector transiently reduces the expression level of the target gene, thereby altering the glycosylation profile of a glycoprotein.

[00132] Examples of commercially available glycoproteins that can be modified using the methods described herein are listed in Table 1.

Table 1

Product Class Mode of action Indication Cell Company line

Aranesp Erythropoiesis Regulates red blood Anemia CHO Amgen

(Darbepoetin stimulating cell production

alfa) protein

Arcaiyst IL-1 Trap Binds IL-Ιβ to prevent Cryopo rin - associated CHO Regeneron (Rilonacept) the interaction to cell periodic syndromes

surface receptors

Avastin rMab Binds to the vascular Colorectal cancer CHO Genentech Product Class Mode of action Indication Cell Company line

(Bevacizumab) endothelial growth

factor (VEGF) to

inhibit angiogenesis

Avonex Interferon Binds to type I Multiple sclerosis (MS) CHO Biogen Idee

(Interferon interferon receptors to

β-l ) activate two Jak

tyrosine kinases

Cerezyme Enzyme Engineered to have Gaucher Disease CHO G enzyme (Imiglucerase) manno s e - 1 erminat ed

oligosaccharide chains

thai are recognized by

endocytic

carbohydrate receptors

on macrophages.

Catalyzes hydrolysis

of glycolipid

glucocerebroside to

glucose and ceramide

i those macrophages

which accumulate

lipids in Gaucher

disease

Elaprase Enzyme Hydrolyzes the 2- Hunter syndrome HT- Shire

(Iduronate sulfate esters of 1080

sulfatase) terminal iduronate

sulfate residues from

the

glycosaminoglycans

dermatari sulfate and

heparan sulfate in the

lysosomes of various

cell types

Enbrei Fusion protein Mimics inhibitory Rheumatoid arthritis CHO Amgen (Etanercept) effects of naturally

occurring soluble TNF

receptors to reduce

inflammatory response

Epogen Er lhropoiesis eco mbi ant human Anemia CHO Amgen, Kirin (Epoetin alfa) stimulating erythropoietin

protein interacts with

erythropoietin (EPO)

receptors to stimulate

production of red

blood cells from bone

marrow stem cells

Erbstux i-Mab Binds to the Colorectal cancer SP2/0 Imclone/BMS (Cetuximab) extracellular domain

of the epidermal

gro th factor (EGFR)

preventing activation

of EGFR to impair cell

growth and

orolif erai ion

Herceptin rMab Binds to HER2+ Breast cancer CHO Genentech (Trastuzurnab) tumor cells, blocks

downstream HER2

Intereron - t roug t e n ucton Product Class Mode of action Indication Cell Company line

l a) of cell membrane

components of the

major

histocompati bility

complex

Remi cade rMab Binds to TNF and Crohn's disease & SP2/0 C e n tocor/ John so n (Infliximab) inhibits TNF action Rheumatoid arthritis & Johnson

Rituxan rMab Binds to the cluster of Non-Hodgkins CHO Genentech (Riitsximab) differentiation 20 lymphoma (IDEC)

(CD20) which is

expressed on B-cells,

Fc portion mediates

ADCC and CDC

Soliris rMab Binds to the Paroxysmal nocturnal CHO Alexion

(Eculizumab) complement protein hemoglobinuria

C5, inhibiting terminal

complement mediated

intravascular

hemolysis

Synagis rMab Targets an epitope in Respiratory syncytial NSO Medlmmune (Pahvizumab) the A antigenic site of virus

the F protein of

respiratory syncytial

virus fRSV

TNKase Enzyme Recombinant fibrin- Acute myocardial CHO Genetech

(Tenecteplase) specific plasminogen infarction

(tPA) activator

Tysabri rMab Binds to o4 integral of Multiple sclerosis (MS) CHO Biogen Idee (Natalizumab) adhesion molecule

VLA-4 and sterically

inhibits binding of

VLA-4 to VCAM-1

Vectibix rMab Binds to EGFR Colorectal carcinoma CHO Amgen

(Panitiimumab)

Zenapax rMab Binds to the a subunit Acute organ rejection SP2/0 Hoffmann-La (Daclizumab) (p55 a, CD25, or Tac Roche

subunit) of human IL- 2 receptor expressed

on the surface of

activated lymphocytes

A. Target Proteins for Modifying Glycosylation

[00133] The biosynthesis of all eukaryotic N-glycans begins on the cytoplasmic face of the Ell membrane with the transfer of GlcNAc-P from UDP-GlcNAc to the iipid-like precursor dolichol phosphate (Dol-P) to generate dolichol pyrophosphate N-acetylglucosamine (Dol-P-P-Glc Ac). Fourteen sugars are sequentially added to Dol-P before en bloc transter of the entire glycan to an Asn-X-Ser/Thr/Cys motif in a protein tha t is being synthesized and translocated through the ER membrane. The protein-hound N-glycan is subsequently remodeled in the ER and Golgi by a complex series of reactions catalyzed by membrane -bound glycosidases and glycosyltransierases. Many of these enzymes are exquisitely sensitive to the physiological and biochemical state of the ceil in which the glycoprotein is expressed. Thus, the populations of sugars attached to each glycosylated asparagine in a mature glycoprotein will depend on the cell type in which the glycoprotein is expressed and on the physiological status of the cell, a status that may be regulated during development and differentiation and altered in disease.

[00134] Glycosylation is a nonlinear non-template driven process. Regulation of a particular glycan may be due to a number of orthogonal inputs such as precursor levels, donor levels, transferase levels etc, Figure 2 provides a schematic illustration of the glycosylation pathway in CHO cell line. Each one of the enzymes may be targeted by RNA effectors.

[00135] In one aspect, the invention provides a method for producing a composition comprising a glycoprotein, the method comprises: (a) culturing a host cell in a large scale cell culture, wherein (i) said host cell expresses the glycoprotein; and (ii) said host cell comprises a target gene that encodes a protein that is selected from die group consisting of: GDP-fucose transporter (GFT), solute carrier-35Cl (SLC35C1), solute carrier-35C2 (SLC35C2), SVIPDUI , and Ggtal; and (b) adding an effective amount of an RNA effector molecule to said large scale cell culture, wherein said RNA effector is substantially complementar to a target gene of (a), and reduces or prevents the expression of said target gene.

[00136] If desired, two or more genes may be targeted. For example, the host cell may further comprises a second target gene that encodes a protein that is selected from the group consisting of: dolichyl-diphosphooligosaccharide-protein glycosyltransferase, UDP

glycosyltransferase, UDP-Gal:pGlcNAcp 1 ,4-galactosyltransferase, UDP-galactose-ceramide gaiactosyltransferase, fucosyltransferase, protein O-fucosyltransferase, N- acetylgalactosaminyltransferase, O-GlcNAc transferase, oligosaccharyl transferase, O-linked N- acetylgrucosamine transferase, a-galactosidase, β-galactosidase, sialyltransferase, GMD dehydratase, FX epimerase, a-l,3-galactosyltransferase, mannosyl (a-l,3-)-glycoprotehi beta- 1,2-N-acetylglucosaminyltransferase (MGAT1), MGAT4B, SLC35D1, ST6GALNAC6, and glucosamine (UDP-N-acetyl)-2-epimerase; and step (b) may further comprise adding an effective amount of a second RNA. effector molecule to the large scale cell culture, wherein the RNA effector is substantially complementary to a target gene that encodes a protein that is selected from the group consisting of: dolichyi-diphosphooligosaccharide-protein glycosyltransferase, UDP glycosyltransferase, UDP-Gal: βΘΙοΝΑοβ 1 ,4-galactosyltransferase, UDP-galactose-ceraniide galactosyltransferase, fucosyltransferase, protein O-fucosyltransferase, N-acetylgalactosaminyltransferase, O-GlcNAc transferase, oligosaccharyl transferase, O-linked N-acetylgrucosamine transferase, a-galactosidase, β-galactosidase, sialyltransferase, GMD dehydratase, FX epimerase, a-l,3-galactosyltransferase, mannosyl (a-l,3-)-glycoprotehi beta- 1,2-N-acetylg!ucosaminyItransferase (MGA.T1), MGAT4B, SLC35D1 , ST6GALNAC6, and glucosamine (UDP-N-acetyl)-2-epimerase, and wherein said RNA effector reduces or prevents the expression of said second target gene.

[00137] In one aspect, the invention provides a method for producing a composition comprising a glycoprotein, the method comprises: (a) culturing a host cell in a large scale cell culture, wherein (i) said host cell expresses the glycoprotein; and (ii) said host cell comprises at least two target genes, the target genes independently encode a protein selected from the group consisting of: dolichyl-diphosphooligosaccbaride-protein glycosyltransferase, UDP

glycosyltransferase, UDP-Gal: GicNAc 1 ,4-galactosyltransferase, UDP-galactose-ceramide galactosyltransferase, fucosyltransferase, protein 0-fucosyltransfera.se, N- acetylgalactosaminyltransferase, O-GlcNAc transferase, oligosaccharyl transferase, O-linked N- acetylgrucosamine transferase, a-galactosidase, β-galactosidase, sialyltransferase, GMD dehydratase, FX epimerase, a-1 ,3-galactosyliransferase, mannosyl (a- 1 ,3 ^-glycoprotein beta- 1 ,2-N-acetylglucosaminyltransferase (MGAT1), MGAT4B, SLC35D 1, ST6GALNAC6, and glucosamine (UDP-N-acetyl)-2-epimerase; and (b) adding an effective amount of two or more RNA effector molecules to said large scale cell culture, wherein each of said RNA effector is substantially complementary to a target gene of (a), and reduces or prevents the expression of its target gene. In certain embodiments, the host cell is a rodent cell, such as a CHO cell.

[00138] Table 2 provides exemplary target genes that encode an enzyme or a transporter protein of the glycosylation pathway.

Table 2

Protein Name Gene ssine

sialidase NEU2

a-N-acet l-neura!rtinyl-2,3-beta-galactosyl- ! ,3-N- ST6GALNAC6

ac etylgalacto saminid e a -2,6- sialylfrans fe ras 6

CMP-N-acetylneuraminic acid hydroxylase CMAH α 1 , 3 ga iacto syl trans feras e ABO

GDP-mannose 4,6-dehydratase GMD

GDP-4-keto-6-deoxy-D-tnaiM!ose epimerase -reductase FX

GDP-fucose transporter GFT

Fu co sy Item sf eras e FUT (e.g., FUT1 to FUT12)

solute carrier-35Cl SLC35C1

solute carrier-35C2 St G G2

dolichyl-diphosphooligosaccharide-protein glycosyltransferase DADL RPN2, PN1, STT3A, STT3B, OST4,

DDOST

UDP glycosyltran sferase UGT (e.g., IJGTlal, UGTla2, UGTl aS, etc.)

UDP-Gali pGlcNAcP 1 ,4-galactosyliransferase B4GALT1 , B4GALT2, B4GALT3, B4GALT4,

B4GALT5, B4GALT6

UDP-galactose-ceramide galactosyltransferase UGT8A

protein O-fucosyltransferase POFUTl , POFUT2

N-acetylgalactosaminyltransferase MGATl, MGAT4B

O-GlcNAc transferase OCT

oligosaccharyl transferase STT3A, STT3B, DAD1 , DDOST

O-linked N-acetylglucosamine transferase OGT

a-gaiactosidase GLA

p-galactosidase GLB1

sialyltransferase CMAH

GDP-fucose transporter GFT

GMD dehydratase GMDS

FX epimerase FX

a- 1 ,3 -galactosyltransferase A3GALT2

a- 1 ,6-mannosyl-glycoproiein 2-beta-N- MGAT2

ac ety Igluco saminy Itransf eras e

a-l,3-!rtanaosyl-glycoprotein 2-beta-N- MGATl

acetylghicosaminyltransferase

a-l ,3-mannosyl-glycoprotein 4-beta-N- MGAT4B

acetylglucosaminyltransferase B

Slc35dl soluie carrier family 35 SLC35D1

a-N-acetylgalactosaminide aipha-2,6-si alyltransferase 6 ST6GALNAC6

mannose-P-dolichol utilization defect 1 MPDU1

N-acetyllactosaminide a- 1 ,3 -galactosyltransferase Ggtal

[00139] Preferred genes that may he targeted for modifying the glycosyiation include FUT8, GMDS, TSTA3, ABO, CMAH, MGATl, MGAT4B, SLC35D1, TSTA3, SLC35C1, SLC35C2, NEU2, ST6GALNAC6, and UGGT1 .

[00140] In certain embodiments, the RNA. effector transiently reduces the expression of its target gene. The nucleotide sequences of exemplary target genes from Chinese hamster and siRNA molecules that modulate the expression of these genes, are provided in the

Appendices.

[00141] In certain embodiments, the method further comprising harvesting said glycoprotein from said large scale culture.

(1). Modification of Fucosylation pathways

[00142] In one aspect, the invention provides a method for producing a composition comprising an afucosylated glycoprotein, comprising: (a) culturing a host cell in a large scale cell culture, wherein (i) said host ceil expresses the glycoprotein; and (ii) said host cell comprises a target gene that encodes a protein tha is selected from the group consisting of: GDP-fucose transporter (GFT), solute carrier-35Cl (SLC35C1), and solute carrier-35C2 (SLC35C2); and (h) adding an effective amount of an RNA effector molecule to said large scale cell culture, wherein said RNA effector is substantially complementary to said target gene, and reduces or prevents the expression of said target gene.

[00143] If desired, two or more genes may be targeted. For example, the host cell may further comprises a second target gene that encodes a protein that is selected from the group consisting of: GDP-mannose 4,6-dehydratase (GMD), GDP-4-keto-6-deoxy-D-mannose epimerase-reductase (FX), and Fucosyltransferase (Fut); and wherein step (b) further comprises adding an effective amount of a second R NA effector molecule to said large scale cell culture, wherein said RN A effector is substantially complementary to a target gene that encodes a protein that is selected from the group consisting of: GDP-mannose 4,6-dehydratase (GMD), GDP-4-keto-6-deoxy-D-mannose epimerase-reductase (FX), and Fucosyltransferase (Fut), and wherein said RNA effector reduces or pre vents the expression of said second target gene.

[00144] In one aspect, the invention provides a method for producing a composition comprising an afucosylated glycoprotein, the method comprises: (a) culturing a host cell in a large scale cell culture, wherein (i) said host ceil expresses the glycoprotein; and (ii) said host cell comprises at least two target genes that independently encode a protein that is selected from the group consisting of: GDP-mannose 4,6-dehydratase (GMD), GDP-4-keto-6-deoxy-D- mannose epimerase-reductase (FX), and Fucosyltransferase (Fut); and (b) adding an effective amount of two or more RNA effector molecules to said large scale ceil culture, wherein each of said RNA effector is substantially complementary to a target gene of (a), and reduces or prevents the expression of its target gene,

[00145] Preferably, at least one of the target genes encodes a fucosyltransfera.se.

[00146] In certain embodiment, the target gene is selected from the group consisting of: Fut8, GMD, and TSTA3.

[00147] Figure 3 provides a schematic illustration of fucosylation pathway in CHO cells. Each one of the enzymes may be targeted by RNA effectors. Preferably, two or more of the enzymes or transporter proteins are targeted. When two or more enzymes transporter proteins are targeted, it may be desirable that at least two enzymes or transporter proteins act sequentially along a metabolic pathway. For example, GMD, FX, GTF, and FutS (Figure 3) are considered act sequentially along a metabolic pathway. Similarly, Fucose kinase, GFPP, GTF, and FutS (Figure 3) are also considered act sequentially along a metabolic pathway. As such, any two of these four proteins may be targeted, By "sequentially," the two enzymes or transporter proteins do not have to act immediately one after another, as long as they align a metabolic pathway, such that a molecule is sequentially processed by the two enzymes or protein transporters (e.g., GMD and FutS are two enzymes act sequentially). By targeting two enzymes or transporter proteins act sequentially along a metabolic pathway, potential synergistic effect may be achieved.

[00148] Alternatively, it may be desirable that to target two or more enzymes or transporter proteins, each is involved in an alternative metabolic pathway that leads to the same product (see e.g., Figure 3, GMD in the de novo pathway, and fucose kinase in the salvage pathway). By targeting two enzymes or transporter proteins are involved in alternative metabolic pathways that lead to the same product, potential synergistic effect may be achieved.

[00149] In certain embodiments, the glycoprotein is an antibody or a fusion protein that comprises the Fc domain of an antibody.

[00150] Genes that may be targeted for modifying the fucosylation of N-glycans include, e.g., FutS (encodes Fucosyltransferase), GMD, 1ST A3, SLC35C1, SLC35C2, etc.

(2), Reduction of Immunogenic Epitopes [00151] In one aspect, the invention provides a method for producing a composition comprising a glycoprotein, the method comprises: (a) culturing a host cell in a large scale cell culture, wherein (i) said host cell expresses the glycoprotein; and (ii) said host cell comprises a target gene that encodes ABO a- 1,3 galactosyltransferase; and (h) adding an effective amount an RNA effector molecule to said large scale cell culture, wherein said RNA effector is substantially complementary to said target gene, and reduces or prevents the expression of the target gene, Preferably, said glycoprotein has reduced immunogenicity in a human when compared to the same glycoprotein produced in the absence of said RNA effector molecule.

100152] In one aspect, the invention provides a method for producing a composition comprising a glycoprotein, the method comprises: (a) culturing a host cell in a large scale cell culture, with the proviso that said host cell is not a human, ape, or Old World monkey cell; wherein (i) said host cell expresses the glycoprotein; and (ii) said host cell comprises a target gene that encodes a protein selected from the group consisting of ABO a-1 ,3

galactosyltransferase and Ggtal ; and (b) adding an effective amount an RNA effector molecule to said large scale cell culture, wherein said RNA effector is substantially complementary to said target gene, and reduces or prevents the expression of the target gene.

[00153] In one aspect, the invention provides a method for producing a composition comprising a glycoprotein, wherein said glycoprotein comprises a N-g!ycan, the method comprises: (a) culturing a host cell in a large scale ceil culture, with the proviso that said host cell is not a human cell; wherein: (i) said host cell expresses the glycoprotein; and (ii) said host cell comprises a target gene that encodes CMP-N-acetylneuraminic acid hydroxylase (CMAH); and (b) adding an effecti ve amount of an RN A effector molecule to said large scale cell culture, wherein said RNA effector is substantially complementary to said gene, and reduces or prevents the expression of said target gene.

(3). Increasing Serum Half-Life

[00154] In one aspect, the invention provides a method for producing a composition comprising a glycoprotein, wherein said glycoprotein comprises a N-glycan, the method comprises: (a) culturing a host cell in a large scale cell culture; wherein (i) said host cell expresses the glycoprotein; and (ii) said host cell comprises a target gene that encodes a sialidase; and (b) adding an RNA effector molecule to said large scale cell culture, wherein said RNA effector is substantially complementary to said gene, and reduces or prevents the expression of said target gene,

[00155] Preferably, the sialidase is NEU2 siaiidase or a-N-acetyl-neuraminyl-2,3- beta-galactosyl- 1 ,3)-N-acetylgaIactosaminide a-2,6-sialyltransferase 6 (ST6GALNAC6).

[00156] Two or more RNA effectors may be used, such as the glycan modified glycoproteins show a combination of improved effector activities (such as ADCC), reduced immunogenicity in humans, or increased serum half life. For example, one RNA effector may target fucosyitransferase to reduce the level of fucosylation, and is used in combination with another RNA effector that targets a sialidase to reduce the hydrolysis of terminal sialic acid. Alternatively, one RNA effector may target ABO a 1,3 gaiactosyltransferase or CMAH to reduce the level of immunogenic epitope, and is used in combination with another RNA effector that targets a siaiidase to reduce the hydrolysis of terminal sialic acid,

[00157] In another aspect, the invention provides a method for producing a glycoprotein, comprising: (a) culturing a host cell that expresses said glycoprotein in a large scale cell culture, wherein said host ceil expresses target genes that are necessary for the glvcosylation of said glycoprotein with two or more of N-glycolymeuraminic acid, fucose and galactose-a-1 ,3-galactose; (b) adding two or more RNA effector molecules to said large scale cell culture, wherein each of said RNA effectors are substantially complementary to said target genes, and cultivating the large scale culture for a sufficient period of time such that the expression of the target genes are reduced or inhibited in said host ceil, thereby producing a glycoprotein with reductions in two or more of N-glycolymeuraminic acid, fucose and galactose-a- 1,3 -galactose as compared to a glycoprotein expressed under the same conditions but in the absence of said two or more RNA effector molecules.

B. RNA effector molecules

[00158] RNA effector molecules that are suitable for modifying glycosylation process of a host cell has been disclosed in detail in WO 2011/005786, and is described brief below.

[00159] RNA effector molecules are ribonucleotide agents that are capable of reducing or preventing the expression of a target gene within a host cell, or ribonucleotide agents capable of forming a molecule that can reduce the expression level of a target gene within a host cell. A portion of a RNA effector molecule, wherein the portion is at least 10, at least 12, at least 15, at least 17, at least 1 8, at least 19, or at least 20 nucleotide long, is substantially complementary to the target gene. The complementary region may be the coding region, the promoter region, the 3' untranslated region (3'-UTR), and/or the 5'-UTR of the target gene, Preferably, at least 16 contiguous nucleotides of the RNA effector molecule are complementary to the target sequence (e.g., at least 17, at least 18, at least 19, or more contiguous nucleotides of the RNA effector molecule are complementary to the target sequence). The RNA effector molecules interact with RNA transcripts of target genes and mediate their selective degrada tion or otherwise prevent their translation.

[00160] RNA effector molecules can comprise a single RNA strand or more than one RNA strand. Examples of RNA effector molecules include, e.g., double stranded RNA

(dsRN A), microRN A (rmRNA), antisense RNA, promoter-directed RNA (pdRNA), Piwi- interacting RNA (piRNA), expressed interfering RNA (eiRNA), short hairpin RNA (shRNA), antagomirs, decoy RNA, DNA, plasmids and ap amers. The RN A effector molecule can be single- stranded or double-stranded. A single-stranded RN A effector molecule can have double- stranded regions and a double-stranded RNA effector can have single-stranded regions.

Preferably, the RNA effector molecules are double-stranded RNA, wherein the antisense strand comprises a sequence that is substantially complementary to the target gene.

[00161] Complementary sequences within a RNA effector molecule, e.g., within a dsRNA (a double-stranded ribonucleic acid) may be fully complementary or substantially complementary. Generally, for a duplex up to 30 base pairs, the dsRNA comprises no more than 5, 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to regulate the expression of its target gene.

[00162] In some embodiments, the RNA effector molecule comprises a single- stranded oligonucleotide that interacts with and directs the cleavage of RNA transcripts of a target gene. For example, single stranded RNA effector molecules comprise a 5' modification including one or more phosphate groups or analogs thereof to protect the effector molecule from nuclease degradation. The RNA effector molecule can be a single-stranded antisense nucleic acid having a nucleotide sequence that is complementary to a "sense" nucleic acid of a target gene, e.g., the coding strand of a double-stranded cDNA molecule or a RNA sequence, e.g., a pre-mRNA, mRNA, miRNA, or pre-miRNA. Accordingly, an antisense nucleic acid can form hydrogen bonds with a sense nucleic acid target.

[00163] 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 the coding or noncoding region of a RNA, 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 deoxyribonucleotid.es 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'-0-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 RN AseH activity is desired.

[00164] In certain embodiments, the RNA effector comprises a double-stranded ribonucleic acid (dsRNA), wherein said dsRNA (a) comprises a sense strand and an antisense strand that are substantially complementary to each other; and (b) wherein said antisense strand comprises a region of complementarity that is substantially complementary to one of the target genes, and wherein said region of complementarity is from 10 to 30 nucleotides in length.

[00165] In some embodiments, RNA effector molecule is a double-stranded oligonucleotide . Typically, the duplex region formed by the two strands is small, about 30 nucleotides or less in length. Such dsRNA is also referred to as siRNA. For example, the siRNA may be from 15 to 30 nucleotides in length, from 10 to 26 nucleotides in length, from 17 to 28 nucleotides in length, from 18 to 25 nucleotides in length, or from 19 to 24 nucleotides in length, etc.

[00166] 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 36 base pairs in length, e.g., 15 to 30 base pairs in length. For example, the duplex region may be 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, or 36 base pairs in length, or any sub-range there between, including, e.g., 15 to 30 base pairs, 15 to 26 base pairs, 15 to 23 base pairs, 15 to 22 base pairs, 15 to 21 base pairs, 15 to 20 base pairs, 15 to 19 base pairs, 15 to 18 base pairs, 15 to 17 base pairs, 18 to 30 base pairs, 18 to 26 base pairs, 18 to 23 base pairs, 18 to 22 base pairs, 18 to 21 base pairs, 18 to 20 base pairs, 19 to 30 base pairs, 19 to 26 base pairs, 19 to 23 base pairs, 19 to 22 base pairs, 19 to 21 base pairs, 19 to 20 base pairs, 20 to 30 base pairs, 20 to 26 base pairs, 20 to 25 base pairs, 20 to 24 base pairs, 20 to 23 base pairs, 20 to 22 base pairs, 20 to 21 base pairs, 21 to 30 base pairs, 21 to 26 base pairs, 21 to 25 base pairs, 21 to 24 base pairs, 21 to 23 base pairs, or 21 to 22 base pairs.

[00167] The two strands forming the duplex structure of a dsRNA 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 (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 dsRN A are formed by separate RNA strands, the two strands can be optionally covalently linked. Where the two strands are connected covalently by means other than a hairpin loop, the connecting structure is referred to as a "linker."

[00168] It is known that dsRNAs having a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA

interference. Elbashir et al, 20 EMBO 6877-88 (2001). [00169] A double-stranded oligonucleotide can include one or more single-stranded nucleotide overhangs, which are one or more unpaired nucleotide that protrudes from the terminus of a duplex structure of a double-stranded oligonucleotide, e.g., a dsRNA, A double- stranded oligonucleotide can comprise an o verhang 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. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the ucleotidefs) 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.

[00170] In one embodiment, at least one end of a dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides.

[00171] The overhang can comprise a deoxyribonucleoside or a nucleoside analog. Further, one or more of the internucloside linkages in the overhang can be replaced with a phosphorothioate. In some embodiments, the overhang comprises one or more

deoxyribonucleoside or the overhang comprises one or more d ^' T, e.g., the sequence 5'-dTdT-3' or 5'-dTdTdT-3\ In some embodiments, overhang comprises the sequence 5'-dT*dT-3, wherein * is a phosphorothioate internucleoside linkage.

[00172] An RNA effector molecule as described herein can contain one or more mismatches to the target sequence. Preferably, a RNA effector molecule as described herein contains no more than three mismatches. If the antisense strand of the RNA effector molecule contains one or more mismatches to a target sequence, it is preferable that the mismatch(s) is (are) not located in the center of the region of complementarity, but are 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 RNA effector molecule agent RNA, the antisense strand generally does not contain any mismatch within the central 13 nucleotides.

[00173] 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 Technologies (Novato, CA).

[00174] In some embodiments, the RNA effector molecule is a promoter-directed

RNA (pdRNA) which is substantially complementary to a noncoding region of an rnRNA transcript of a target gene. In one embodiment, the pdRNA is substantially complementary to the promoter region of a target gene mRNA at a site located upstream from the transcription start site, e.g., more than 100, more than 200, or more tha 1 ,000 bases upstream from the transcription start site. In another embodiment, the pdRNA is substantially complementary to the 3'-UTR. of a target gene mRNA transcript. In one embodiment, the pdRNA comprises dsRNA of 18-28 bases optionally having 3' di- or tri-nucleotide overhangs on each strand, in another embodiment, the pdRNA comprises a gapmer consisting of a single stranded polynucleotide comprising a DNA sequence which is substantially complementary _' to the promoter or the 3'-UTR of a target gene mRNA transcript, and flanking the polynucleotide sequences ( e.g., comprising the 5 terminal bases at each of the 5' and 3' ends of the gapmer) comprises one or more modified nucleotides, such as 2' MQE, 2'OMe, or Locked Nucleic Acid bases (LNA), which protect the gapmer from cellular nucleases.

[00175] pdRNA can be used to selectively increase, decrease, or otherwise modulate expression of a target gene. Without being limited to theory, it is believed that pdRNAs modulate expression of target genes by binding to endogenous antisense RN A 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 pdRNA effector molecule activates expression of the target gene, Thus, in some embodiments, pdRNAs can be used to selectively activate the expression of a target gene by inhibiting the negative regulation of target gene expression by endogenous antisense RN A. 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.

[00176] In some embodiments, the RNA effector molecule 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. An aptamer can fold into a specific staicture that directs the recognition of a targeted binding site on the non-nucleic acid ligand. Aptamers can contain any of the modifications described herein.

[00177] In some embodiments, the RNA effector molecule comprises an antagomir. A tagomirs 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 miRNA 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.

[00178] In some embodiments, antagomirs are stabilized against nucleoiytic 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 intemucleotide l inkages 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'~fiuoro, 2'-Q-m.ethyl, 2'-0-methox.yethyl (2'~0-MOE), 2'-0-ammopropyl (2'~Q~AP), 2'-()-dimethyiaminoethyi (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2'-0-D AP), 2'-0- dimethyiaminoethyloxyethyi (2'-0-DMAEOE), or 2'-0-N-methylacetamido (2'-0-NMA). In some embodiments, antagomirs include at least one 2 '-O-methyl-modified nucleotide.

[00179] In some embodiments, the UNA effector molecule is a promoter-directed

RNA (pdRNA) which is substantially complementary to a noncoding region of an mRNA transcript of a target gene. The pdRNA can be substantially complementary to the promoter region of a target gene mRNA 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 the 3'-UTR of a target gene mRN A 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 the promoter region or the 3'-UTR region of a target gene mRNA transcript. In another embodiment, the pdRN A comprises a gapmer consisting of a single stranded polynucleotide comprising a DNA sequence which is substantially complementary to 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. [00180] Expressed interfering RNA (eiRNA) can be used to selectively increase, decrease, or otherwise modulate expression of a target gene. Typically, eiRNA, the dsRNA is expressed in the first transfected cell from an expression vector. In such a vector, the sense strand and the aniisense 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 plasniids designed to transcribe one strand of the dsRNA while the other is designed to transcribe the other strand. Methods for making and using eiRNA effector molecules are known in the art. See, e.g., WO 2006/033756; U.S. Patent Pubs. No. 2005/0239728 and No. 2006/0035344.

[00181] In some embodiments, the RNA effector molecule comprises a small single- stranded Piwi-interacting RNA (piRNA effector molecule) which is substantially

complementary to a target gene, and which sel ectively binds to proteins of the Piwi or

Aubergine subclasses of Argonaute proteins, A piRNA effector molecule ca 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.

[00182] 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 microRNAs 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 mRN As. MicroRN As 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 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 com lementarity, e.g., complete com lementarity, to allo w duplex formation. For example, the regions of complementarity are at least 8 to 10 nucleotides long (e.g., 8, 9, or 10 nucleotides long). [00183] 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 miRN A binds its target with perfect or a high degree of complementarity .In further embodiments, the RNA effector molecule can include an

oligonucleotide agent which targets an endogenous miRNA or pre-miRNA. For example, the RNA effector can target an endogenous miRNA which negatively regulates expression of a target gene, such that the RNA effector alleviates mi NA-based inhibition of the target gene.

[00184] The miRNA can comprise naturally occurring nucieobases, sugars, and covalent intemucleotide (backbone) linkages, or comprise 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, an miRNA designed to bind to a specific endogenous mi RNA. 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 miRN A. Exemplary oligonucleiotde agents 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.

[00185] A miRN A or pre-miRNA can be 10 to 200 nucleotides in length, for example from 16 to 80 nucleotides in length. Mature miRN As can have a length of 16 to 30 nucleotides, such as 21 to 25 nucleotides, particularly 21, 22, 23, 24, or 25 nucleotides in length. miRNA precursors can ha ve a length of 70 to 100 nucleotides and can have a hairpin conformation. In some embodiments, miRNAs are generated in vivo from pre-miRNAs by the enzymes cDicer and Droslia. miRNAs or pre-miRNAs can be synthesized in vivo by a cell-based system or can be chemically synthesized. mi NAs can comprise modifications which impart one or more desired properties, such as superior stability, hybridization thermodynamics with a target nucleic acid, targeting to a particular tissue or cell-type, and/or cell permeability, e.g., by an

endocytosis-dependent or -independent mechanism. Modifications can also increase sequence specificity, and consequently decrease off-site targeting. [00186] Upon contact with a cell expressing the target gene, the RNA effector molecule inhibits the expression of the target gene by at least 10%, as assayed by, for example, a PGR or branched DNA (bDNA)-based method, or by a protein-based method, such as by western blot, Expression of a target gene in cell culture can be assayed by measuring target gene mRNA levels, e.g., by bDNA or TAQMAN® assay, or by measuring protein levels, e.g., by immunofluorescence analysis or quantitative immunobiot.

[00187] Optionally, an RNA effector may bechemically modified to enhance stability or other beneficial characteristics.

[00188] Oligonucleotides can be modified to prevent rapid degradation of the oligonucleotides by endo- and exo-nucleases and avoid undesirable off-target effects. The nucleic acids featured in the invention can be synthesized and/or modified by methods well established in the art, such as those described in CURRENT PROTOCOLS IN NUCLEIC ACID

CHEMISTRY (Beaucage et al., eds., John Wiley & Sons, Inc., NY). Modifications include, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation, conjugation, inverted linkages, etc.), or 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) intemucleoside linkage modifications, including modification or replacement of the phosphodiesier linkages. Specific examples of

oligonucleotide compounds useful in this invention include, but are not limited to RNAs containing modified backbones or no natural intemucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. Specific examples of oligonucleotide compoimds useful in this invention include, but are not limited to oligonucleotides containing modified or non-natural intemucleoside linkages.

Oligonucleotides having modified internucioside linkages include, among others, those that do not have a phosphorus atom in the intemucleoside linkage.

[00189] Modified intemucleoside linkages include (e.g., RNA backbones) mclude, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, animoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidat.es including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,

thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphat.es 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.

[00190] Representative patents that teach the preparation of the above phosphorus- containing linkages include, e.g., U.S. Patents No. 3,687,808; No. 4,469,863; No. 4,476,301; No. 5,023,243; No. 5,177,195; No, 5,188,897; No, 5,264,423; No, 5,276,019; No. 5,278,302; No. 5,286,717; No. 5,321, 131; No. 5,399,676; No. 5,405,939; No. 5,453,496; No. 5,455,233; No. 5,466,677; No. 5,476,925; No. 5,519,126; No. 5,536,821 ; No. 5,541,316; No. 5,550,1 1 1; No. 5,563,253; No. 5,571,799; No. 5,587,361; No. 5,625,050; No. 6,028,188; No. 6,124,445; No. 6,160,109; No. 6,169,170; No, 6,172,209; No, 6, 239,265; No. 6,277,603; No. 6,326,199; No. 6,346,614; No. 6,444,423; No. 6,531,590; No. 6,534,639; No. 6,608,035; No. 6,683,167; No. 6,858,715; No. 6,867,294; No. 6,878,805; No. 7,015,315; No. 7,041,816; No. 7,273,933; No. 7,321 ,029; and No. E39464.

[00191 ] Additionally, both the sugar and the internucleoside linkage may be modified, i.e., the backbone, of the nucleotide units are replaced with novel groups. One such oligomeric compound, a RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). See, e.g., U.S. Patents No. 5,539,082;

No. 5,714,331 ; and No, 5,719,262. Further teaching of PNA. compounds can be found, for example, in Nielsen et al., 254 Science 1497-1500 (1991).

[00192] Modified oligonucleotides can also contain one or more substituted sugar moieties. The RNA effector molecules, e.g., dsRNAs, can include one of the following at the 2' position: H (deoxyribose); OH (ribose); F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N- alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, aikenyl and alkynyl can be substituted, or unsubstituted Q to Go alkyl or C ₂ to Cio aikenyl and alkynyl, Exemplary suitable modifications include G[(CH ₂ ) _ri Q] _m C¾, 0(CH ₂ ) _ri GCH ₃ , 0(CH ₂ ) _ri NH ₂ , 0(CH ₂ ) _n CH ₃ , 0(CH ₂ ) _n 0NH ₂ , and 0(CH ₂ ) _n ON[(CH ₂ ) _n CH ₃ )] ₂ , where n and m are from .1 to 10, inclusive. In some embodiments, oligonucleotides include one of the following at the 2' position: G to C ₁₀ lower alkyl, substituted lower alkyl, aikaryl, aralkyl, O-alkaryl or O-aralkyl, SH, 8C¾, OCN, CI, Br, CN, CF ₃ , OCF ₃ , SOCH3, SO :(. ^' ! ! ;. ONO2, N0 ₂ , N ₃ , NH ₂ , heterocyeloalkyl, heteroeyeloalkaryl, aminoaikyiamino, polyalkylaniino, substituted silyl, a RNA cleaving group, a reporter group, an intercalaior, a group for improving the pharmacokinetic properties of an oligonucleotide (e.g., a RNA effector molecule), or a group for improving the pharmacodynamic properties of an oligonucleotide (e.g., a RNA effector molecule), and other substituents having similar properties, in some embodiments, the modification includes a 2'~methoxyethoxy (2 -0- CH ₂ CH ₂ OCH ₃ , also known as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et al, 78 Helv. Chim. Acta 486-504 ( 1995)), i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'~ dimethylaminooxyethoxy, i.e., a 0(CH ₂ ^' ) ₂ 0N(CH ₃ ) ₂ group, also known as 2 -DMAOE, as described in examples herein below, and 2'-dirn.ethylaminoethoxyethoxy (also known in the art as 2'-0-dimethylaminoethoxyethyl or 2 -DMAEOE), i.e., 2 ^, -0-CH ₂ -0-CH ₂ -N(CH ₂ )2.

[00193] Other modifications include 2'-methoxy (2'-OCH ₃ ), 2'-ammopropoxy

(2'-OCi¾CH ₂ CH ₂ NH ₂ ) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotide and the 5' position of 5' terminal nucleotide.

Oligonucleotides can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative patents that teach die preparation of such modified sugar structures include, but are not limited to, U.S. Patents No, 4,981,957; No. 5,118,800;

No. 5,319,080; No. 5,359,044; No. 5,393,878; No. 5,446, 137; No. 5,466,786; No. 5,514,785; No. 5,519,134; No. 5,567,81 1 ; No. 5,576,427; No. 5,591 ,722; No. 5,597,909; No. 5,610,300; No. 5,627,053; No. 5,639,873; No. 5,646,265; No. 5,658,873; No. 5,670,633; and

No. 5,700,920, certain of which are commonly owned with the instant application.

[00194] An oligonucleotide (e.g., a RNA effector molecule) can 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 cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2-(halo)adenine, 2~(alkyi)adenine, 2-(propyl)adenme, 2

(amino)adenine, 2-(aminoalkyll)adenine, 2 (aminopropyl }adenme, 2 (methylthio) N6

(isopentenyl)adenine, 6 (aikyl)adenirie, 6 (methyl)adenine, 7 (deaza)adenine, 8 (alkenyl)adenine,

8-(alkyl)adenine, 8 (aikynyl)adenine, 8 (amino)adenine, 8-(halo)adenine, 8-(hydroxyi)adenine, 8 (thioalkyl)adenine, 8-(thiol)adenine, N6-(isopenty[)adenine, N6 (methyl)adenine, N6, N6 (dimethyl)ademne, 2-(alkyl)guanine,2 (propyl)guanine, 6-(alkyl)guanine, 6 (methyl)guanine,

7 (alk l)guanine, 7 (methyl)guatiine, 7 (deaza)guanine, 8 (alkyl)guanine, 8-(alkenyI)guanine,

8 (alkynyl)guanine, 8~(ammo)guanine, 8 (halo)guanme, 8-(hydroxyl)guanine,

8 (thioalkyl)guanine, 8-(thiol)guanine, N (methyl)guanine, 2-(thio)cytosine, 3 (deaza) 5

(aza)cytosine, 3-(alkyl)cytosine, 3 (methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5 (halo)cytosine, 5 (methyl)cytosine, 5 (propynyl)cytosine, 5 (propynyl)cytosine,

5 (trifluoroniethyl)cytosine, 6-(azo)cytosine. N4 (acetyl)cytosine, 3 (3 amino-3

carboxypropyl)uracil, 2-(thio)uracil, 5 (methyl) 2 (thio)uracil, 5 (niethylaminomethyl)-2

(thio)uracil, 4-(thio)uracii, 5 (methyl) 4 (thio)uraciL 5 (methylaminomethyl)-4 (thio)uracil, 5 (methyl) 2,4 (dithio) uracil, 5 (methylammomethyl)-2,4 (dithio)uracil, 5 (2-aminopropyl)uracil, 5-(alkyl)uracii, 5-(alkyny[)uracil, 5-(al[ylamino)uracil, 5 (aminoallyl)uracil,

5 (ammoalky])uraeil, 5 (guanidiniumalkyl)uracil, 5 (l ,3-diazole-l ~alkyl)uracil,

5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5 (dimethylaminoalkyl)uracii, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5 oxyacetic acid, 5 (methoxycarbonylmethyl)-2-(thio)uracil,

5 (methoxycarbonyl-methyl)uracil, 5 (propynyl)uracil, 5 (propynyl)uracil, 5

(trifluoromethyl)uracil, 6 (azo)uracil, dihydrouracil, N3 (niethyl)uracil, 5 -uracil (i.e.,

pseudouracil), 2 (thio)pseudouracii,4 (thio)pseudouracii,2,4-(dithio)psuedouracil,5- (aikyi)pseudouracii, 5-(methyl)pseudouracil, 5-(alk}4)-2-(thio)pseudouracil, 5-(methyl)-2- (thio)pseudouracil, 5-(alkyl)-4 (thio)pseudouracil, 5-(methyl)-4 (thio)pseudouracil, 5-(alkyl)-2,4 (dithio)pseudouracil, 5-(methyl)-2,4 (dithio)pseudouracil, 1 substituted pseudouracil,

1 substituted 2(thio)-pseudouracil, 1 substituted 4 (thio)pseudouracil, 1 substituted 2,4- (dithio)pseudouracii, 1 (aminocarbonylethylenyl)-pseudouracil, 1 (aminocarbonylethylenyl)- 2(thio)-pseudouracil, 1 (aminocarbonylethylenyl)-4 (thio)pseudouracil,

1 (aminocarbon ! ethylenyl ^' )-2 ,4-(dithi o) seudouraci! , 1 (ammoa!k ! aminocarbonylethy ! eny 1)- pseudouracil, 1 (aniinoalkyla.rnino-carbonylethylenyl)-2(thio)-pseudouracil,

I (arninoaLkylaminocarbonylethylenyi)-4 (thio)pseudouracil,

1 iaminoalkylaminocarbonylethylenyl)-2,4-idithio)pseudouraciL l,3-(diaza)-2-(oxo)- phenoxazin- 1 ~yl, 1 -(aza)-2-(thio)-3-(aza)-phenoxazin- 1 -yl, 1 ,3-(diaza)~2-(oxo)~phenthiazm~ 1 -yl, l-(aza)-2-(thio)-3-(aza)-phenthiazin- l-yl, 7 -substituted l,3-(diaza)-2-(oxo)-phenoxazin- l-yl, 7-substituted 1 -(aza)~2-(thio)-3-(aza)~phenoxa.zm- 1 -yl, 7-substituted 1 ,3-(diaza)-2-(oxo)~ phenthiazin- 1 -yl, 7-substituted 1 -(aza)-2-(thio)-3-(aza)-phenthiazin- 1 -yl,

7-(aminoalkylhydroxy)- 1 ,3-(diaza)-2-(oxo)-pherioxazin- 1 -yl, 7-(aminoalkylhydroxy)- 1 -(aza)-2- (thio)-3-(aza)-phenoxazin- 1 -yi, 7-(aminoalkylhydroxy)- 1 ,3-(diaza)-2-(oxo)-phenthiazin- 1 -yi, 7~(ammoa3ky3hydroxy)~ 1 -(aza)~2-(tbio)-3-(aza)-pbenthiazin- 1 -yl, 7-(guanidmiurnalkylhydroxy)- 1 ,3-(diaza)-2-(oxo)-phenoxazin- 1 -yi, 7-(guanidiniunialkylhydroxy)- 1 -(aza)-2-(thio)-3-(aza)- phenoxazin-l-yl, 7-(guanidiniumalkyl-hydroxy)-l,3-(diaza)-2-(oxo)-phenthiazin -l-yl,

7-(guanidiniimialkylhydroxy)- l-(aza)-2-(th^ l,3,5-(triaza)-2,6- (dioxa)-naphthalene. inosine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, mtropyrazoiyl, nitrobenzknidazolyl, nitroindazolyl, amino indolyl, pyrrolopyrimidinyl, 3 -(methyl)isocarbostyrilyl,

5- (methyi)isocarbostyrilyi, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyS)~ 7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyriiyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl,

4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, napthaienyl, anthracenyi, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, dilluorotoiyl, 4-(fluoro)-6~(methy])benzimidazole, 4-(methyi)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5 nitroindoie, 3 nitropyrrole,

6- (aza)pyrimidine, 2 (amino)purine, 2,6-(diamino)purine, 5 substituted pyrimidines,

N2-substituted purines, N6-substituted purines, 06-substituted purines, substituted 1,2,4- triazoles, pyrrolo-pyrimidin-2-on-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, para-substituted- 6-phenyl-pyrroio-pyrimidin-2-oti-3-yl, ortho-s bstituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, para-(aminoalkylhydroxy)- 6- pbenyl-pyrro3.o-pyrimidin-2-on-3-yl, ortho-(aminoaLkylhydroxy)- 6-phenyl-pyrrolo-pyrimidin-2- on-3-yl, bis-ortho-(aminoalkylhydroxy)- 6-phenyi-pyrrolo-pyrimidin-2-on-3-yl,

pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-y3., 2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylated derivatives thereof. Modified nucleobases also include natural bases that comprise conjugated moieties, e.g., a ligand.

[00195] The oligonucleotides 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. The addition of locked nucleic acids to oligonucleotide molecules has been shown to increase oligonucleotide molecule stability in serum, and to reduce off-target effects. Elmen et al., 33 Nucl. Acids Res. 439-47 (2005); Mook et al, 6 Moi. Cancer Ther. 833-43 (2007); Grunwelier et al., 31 Nucl. Acids Res. 3185-93 (2003); U.S. Patents No. 6,268,490; No. 6,670,461; No. 6,794,499;

No. 6,998,484; No. 7,053,207; No. 7,084,125; and No. 7,399,845.

[00196] In certain instances, the oligonucleotides of a RNA effector molecule can be modified by a non-ligand group. A number of non-1 igand molecules have been conjugated to oligonucleotides in order to enhance the activity, cellular distribution or cellular uptake of the oligonucleotides, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo et al, 365 Biochem. Biophys. Res. Comm. 54-61 (2007)); Letsinger et a!., 86 PNAS 6553 ( 1989)); choiic acid (Manoharan et al, 1994); a tiiioether, e.g., hexyl-S-tritylthiol (Manoharan et al., 1992; Manoharan et al, 1993); a thiocholesterol (Oberhauser et al., 1992); an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., 1991; Kabanov et al., 259 FEBS Lett. 327 (1990); Svinarch.uk et al., 75 Biochimie 75 (.1993)); a phospholipid, e.g., di-hexadecyl- rac-glycerol or triethylammonium 1 ,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate

(Manoharan et al, 1995); Shea et al, 18 Nucl. Acids Res, 3777 (.1990)); a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, .1995); or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995); a paimityl moiety (Misiira et al, 1995); or an octadecylamine or hexylaniino-carbonyi-oxycholesterol moiety (Crooke et al., 1996). Representative United. States patents that teach the preparation of such RNA conjugates ha ve been listed herein. Typical conjugation protocols involve the synthesis of an oligonucleotide bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA. conjugate by HPLC typically affords the pure conjugate.

Delivery Methods of RNA Effector Molecules

100197] The delivery of RNA effector molecules to cells can be achieved in a number of different ways. Several suitable delivery methods are well known in the art. For example, the skilled person is directed to WO 20.1 1/005786, which discloses exemplar}' delivery methods can be used in this invention at pages 187-219, the teachings of which are incorporated herein by reference. For example, delivery can be performed directly by administering a composition comprising a RNA effector molecule, e.g., an siRNA, into cell culture. Alternatively, delivery can be performed indirectly by administering into the cell one or more vectors that encode and direct the expression of the RNA effector molecule.

[00198] A. reagent that facilitates RNA effector molecule uptake may be used, For example, an emulsion, a cationic lipid, a non-cationic lipid, a charged lipid, a liposome, an anionic lipid, a penetration enhancer, a transfection reagent or a modification to the RNA.

effector molecule for attachment, e.g., a ligand, a targeting moiety, a peptide, a lipophilic group, etc.

[00199] For example, RNA effector molecules can be delivered using a drug delivery system such as a nanoparticle, a dendrimer, a polymer, a liposome, or a cationic deliver} _' system. Positively charged cationic delivery systems facilitate binding of a RNA effector molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient cellular uptake. Cationic lipids, dendrimers, or polymers can either be bound to RNA effector molecules, or induced to form a vesicle, liposome, or micelle that encases the RNA effector molecule. See, e.g., Kim et al., 129 J. Contr. Release 107-16 (2008). Methods for making and using catioiiic-RNA effector molecule complexes are well within the abilities of those skilled in the art. See e.g., Sorensen et al 327 J. Mol. Biol. 761-66 (2003); Verma et a!., 9 Clin. Cancer Res. 1291-1300 (2003); Arnold et al, 25 J. Hypertens. 197-205 (2007).

[00200] In one embodiment, the reagent that facilitates RNA effector molecule uptake used herein comprises a charged lipid as described in U.S. Application Ser. No, 61/267,419, filed 7 December 2009.

[00201] The RNA effector molecules described herein can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, the RNA effector molecules can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosaiioic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, iinoleiiic acid, dicaprate, tricaprate, nionooiein, dilaurin, glyceryl 1-moiiocaprate,

1 -dodecyiazacycloheptan-2-otie, an acylcarnitine, an acylcholine, or a CI -20 alkyl ester (e.g., isopropylmyristate IPM), moiioglyceride, diglyceride, or acceptable salts thereof.

[00202] In one embodiment, the RNA effector molecules are fully encapsulated in the lipid formulation (e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle). The term "SNALP" refers to a stable nucleic acid- lipid particle: a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as a RNA effector molecule or a plasmid from which a RNA effector molecule is transcribed. SNALPs are described, e.g., in U.S. Patent Pubs. No, 2006/0240093, No, 2007/0135372; No. 2009/0291131 ; U.S. Patent Applications Ser. No. 12/343,342; No.12/424,367. The term "SPLP" refers to a nucleic aeid- 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). SPLPs include "pSPLP," which include an encapsulated condensing agent-nucleic acid complex as set forth in WO 00/03683. The particles in this embodiment typically have a mean diameter of about 50 nm to about 150 nm, or about 60 nm to about 130 nm, or about 70 nm to about 1 10 nm, or typically about 70 nm to about 90 nm, inclusive, and are substantially nontoxic. I n 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 reported in, e.g., U.S. Patents No. 5,976,567; No. 5,981,501; No. 6,534,484; No. 6,586,410; No. 6,815,432; and WO 96/40964.

[00203] The lipid to RNA ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) can be in ranges 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, inclusive,

[00204] A cationic lipid of the formulation can comprise at l east one protonatable group having a p a of from 4 to 15. The cationic lipid ca be, for example, N,N-dioleyl-N,N- dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3- dioleoyloxy)propyi)-N,N,N rimethylammonium chloride (DOTAP), N-(I- (2,3- dioleyloxy)propyl)-N,N,N- rimethylammoni m chloride (DOTMA), N,N-dimethyl-2,3- dioleyloxy propylamine (DODMA), 1 ,2-DiLinoleyloxy-N,N-dimethylaminopropaiie

(DLinDMA), l ₅ 2-Dilinolenyloxy-N,N-dirnethylaminopropane (DLenDMA), 1 ,2- Dilinoleylcarbamoyloxy-3-dimethylammopropane (DLin-C-DAP), l,2-Dilinoleyoxy-3- (dimethylamino)acetoxypropane (DLin-DAC), l,2-Dilvnoleyoxy-3-morpholinopropane (DLin- MA), 1 ,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1 ,2-Dilinoleylthio-3- dimethylaminopropane (DLin-8-D A), l-Linoleoyl-2-lmoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), l,2-Dilinoleyloxy-3-trimethylammopropane chloride salt (DLin-TMA.Cl), l,2-DilinoIeoyl-3-trimethyiaminopropane chloride salt (DLin-TAP.CI), l,2-Dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ), or 3-(N,N~Dilinoleylamino)~ 1 ,2-propanediol

(DLinAP), 3-(N,N-DioleyIamino)- 1 ,2-propanedio (DOAP), 1 ,2-Dilinoleyloxo-3-(2-N,N- dimeihylarmno)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethy3.aminomethyl-[l,3]- dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-dimethy[aminoethyl-[l,3]-dioxolane, or a mixture thereof. The cationic lipid can comprise from about 20 mol% to about 70 mol%, inclusive, or about 40 mol% to about 60 mol %, inclusive, of the total lipid present in the particle. In one embodiment, cationic lipid can be further conjugated to a ligand.

[00205] A non-cationic lipid can be an anionic lipid or a neutral lipid, such as distearoyl-phospbatidylcholine (DSPC), dioieoyiphosphatidyichoiine (DOPC), dipalmitoyl- phosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoyl- phosphatidyl glycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoyl- phosphatidylcholine (POPC), palmitoyloieoyl- phosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanol amine 4-(N-maleimidorn.ethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoy 1 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 can be from about 5 mol% to about 90 mol%, inclusive, of about 10 mol%, to about 58 moi%, inclusive, if cholesterol is included, of the total lipid present in the particle.

[00206] The lipid that inhibits aggregation of particles can 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 can be, for example, a PEG-dilauryloxypropyi (CI 2), a PEG- dimyristyloxypropyl (CI 4), a PEG-dipalmityloxypropyl (CI 6), or a PEG- distearyloxypropyl (CI 8). The lipid that prevents aggregation of particles can be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle, in one embodiment, PEG lipid can be further conjugated to a ligand.

[00207] In some embodiments, the nucleic acid-lipid particle further includes a steroid such as, cholesterol at, e.g., about 10 mol% to about 60 mol%, inclusive, or about 48 mol% of the total lipid present in the particle. [00208] In one embodiment, the lipid particle comprises a steroid, a PEG lipid and a cationic lipid of formula (I):

formula (])

wherein each Xa and Xb, for each occurrence, is independently C I -6 alkylene;

n is 0, 1, 2, 3, 4, or 5; each R is independently H,

m is 0, 1 , 2, 3 or 4; Y is absent, O, NR ^Z , or S; R ¹ is alkyl alkenyl or alkynyl; each of which is optionally substituted with one or more substituents; and R ² is H, alkyl alkenyl or alkynyl; each of which is optionally substituted each of which is optionally substituted with one or more substituents,

[00209] In one example, the lipidoid ND98-4HC1 (MW 1487) (Formula 2),

Cholesterol (Sigma-Aldrich), and PEG-Ceramide CI 6 (Avanti Polar Lipids) can be used to prepare lipid RNA effector molecule nanoparticles (e.g., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/mL; Cholesterol, 25 mg mL, PEG- Ceramide C I 6, 100 mg/mL, The ND98, Cholesterol, and PEG-Ceramide C 16 stock solutions can then be combined in, e.g., a 42:48: 10 molar ratio. The combined lipid solution can be mixed with aqueous RNA effector molecule (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35% to 45% and the final sodium acetate concentration is about 100 roM to 300 mM, inclusive. Lipid RNA effector molecule 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 urn 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 2

[00210] In some embodiments, the nucleic acid-iipid particle further includes a steroid such as, cholesterol at, e.g., about 10 mol% to about 60 mo!%, inclusive, or about 48 mol% of the total lipid present in the particle.

[00211] LNP01 formulations are described elsewhere, e.g., WO 2008/042973.

[00212] In one embodiment, the reagent that facilitates RNA effector molecule 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 RNA effector molecule 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:

Lipid R

[00213] Also contempl ated herein are various formulations of the lipids described above, such as, e.g., 8, 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:

[00214] In another embodiment, the RNA effector molecule 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 approxima tely 25 mol % - 100 moi% 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:

8 47.94 47.06 5

[00215] Additional exemplary lipid-siRNA formulations are as shown below in Table

TABLE 5: lipid-siRNA formulations

cationic lipid/non-cationic

Cationic Lipid lipid/cholesterol/PEG-lipid conjugate Process

Lipid :siRNA ratio

DLinDMA/DPPC/Cholesterol/PEG- l,2-Dilinolenyloxy-N,N- cDMA

SNALP

dimethylaminopropane (DLinDMA) (57.1/7.1/34.4/1.4)

Hpid:si NA ~ 7: l

XTC/DPPC/Cholesterol/PEG-cDMA

SNALP- 2,2-Dilit3oleyl-4-dimethylaminoethyl-

57.1/7.1/34.4/ L4

XTC [I ,3]-dioxolane (XTC) ^'

Sipid:siRNA ~ 7: 1

XTC/DSPC/Cholesterol/PEG-DMG

2 , 2 -Dilino ley 1-4 -d imetiiy lamino e thy 1-

L P05 57.5/7.5/31.5/3.5 Extrusion

[l ,3]-dioxolane (XTC) ^*

lipid:siRNA ~ 6: l

XTC/DSPC/Cholesterol/PEG-DMG

2,2-Dilinoleyl-4-dsmethylaminoetbyl-

LNP06 57.5/7.5/31.5/3.5 Extrusion f l,3 j-dioxolane (XTC)

lipid:siRNA ~ 11 : 1

XTC/DSPC/Cholesterol/PEG-DMG

2 , 2 -D i 1 inoley 1 - 4 - di m ethy lamino ethyl ^■■ In-line

L P07 60/7.5/31/1.5,

[l,3]-dioxolane (XTC) mixing lipid:siR.NA ~ 6: l

XTC/DSPC/Cholesterol/PEG-DMG

2,2-Dilinoleyl-4-dimethylaminoethyl- In-line

LNP08 60/7.5/31/1.5,

l ,3 j~dsoxolane (XTC) mixing

Hpid:siRNA— 11 : 1

XTC/DSPC/Cholesterol/PEG-DMG

2,2-Dilit3oleyl-4-dimethylaminoethyl- in-line

LNP09 50/10/38,5/1 ,5

[I ,3]-dioxolane (XTC) ^' mixing

Lipid :siRNA 10: 1

(3aR,5s,6aS)-N,N-dimethyl-2,2- di ((9Z,12Z)-octadeca-9, 12- ALN100 DSPC/CholesteroL'PEG-DMG

in-line

LNP 10 dienyl )tetrahydro-3aH- 50/10/38,5/1 ,5

mixing cyclopenta[d ] [ 1 , 3 dio xo 1 - 5 ^■ am ins Lipid :siRNA 10: 1

(ALN100) ^{" '}

(6Z,9Z,28Z,31Z)-heptatriaconta- MC-3/DSPC/Cholesterol/PEG-DMG

in-line

L P11 6,9,28,3 l-tetraen-19-yl 4- 50/10/38.5/1.5

mixing (dimeth lamino )b utanoai e (MC 3 ) Lipid :siRNA 10: 1

l,r-(2-(4-(2-((2-(bis(2- hydroxydodecyl)amino)ethyl)(2- Tech Gl /DSPC/CholestesOLTEG-DMG

In-line

LNP12 hydroxydodecyl)amino)ethyl)piperazin- 50/10/38.5/1.5

mixing l-y1)ethylazanediy1)didodecan-2-ol Lipid:siRNA 10: 1

(Tech Gl ) [00216] LNP09 formulations and XTC comprising formulations are described, e.g., in U.S. Provisional Serial No. 61/239,686, filed September 3, 2009, which is hereby incorporated by reference.

[00217] LNP1 1 formulations and MC3 comprising formulations are described, e.g., in U.S. Provisional Serial No. 61/244,834, filed September 22, 2009, which is hereby incorporated by reference.

|0021S] LNP12 formulations and TechGl comprising formulations are described, e.g., in U.S. Provisional Serial No. 61/175,770, filed May 5, 2009, which is hereby incorporated by reference.

[00219] 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-nanopartieles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, PA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodai. The total dsRNA effector molecule concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated RNA effector molecule can be incubated with a RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton- XI 00. The total RNA effector molecule 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" RNA effector molecule content (as measured by the signal in the absence of surfactant) from the total RN A effector molecule content. Percen t entrapped RNA effector molecule is typically >85%. For lipid nanoparticle 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 1 10 nm, or at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm, inclusive,

[00220] 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 by macrophages in vivo. In order to cross intact cell membranes, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

[00221] 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; and liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation. See, e.g., Wang et ai., DRUG DEL.IV.

PRINCIPLES & APPL. (John Wiley & Sons, Hoboken, J, 2005); Rosoff, 1988. Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicl e size and the aqueous volume of the liposomes,

[00222] 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 ceil 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 drags. 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.

[00223] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged polynucleotide molecules to form a stable complex. The positively charged polynucleotide/liposome complex binds to the negatively charged ceil 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 ai,, 147 Biochem. Biophys. Res. Commuii., 980-85 (1987). [00224] Liposomes which are pH-sensitive or negatively-charged, entrap polynucleotide rather than complex wit it. Because both the polynucleotide and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some polynucleotide 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, 19 J. Controlled Rel. 269-74 (1992).

[00225] 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

phosphatidylglyeerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidyl ethanolamine (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.

100226] 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 GMl, or (B) is derivatized with one or more hvdrophilic 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 sterical ly stabilized liposomes deri ves from a reduced uptake into cells of the reticuloendothelial system (RES). Alien et al, 223 FEBS Lett. 42 (1987); Wu et al., 53 Cancer Res. 3765 (1993).

[00227] Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (507 Ann. NY Acad. Sci. 64 (1987)), reported the ability of

monosialoganglioside GMl, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (85 PNAS 6949 (1988)). U.S. Patent No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester. U.S. Patent No, 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholiiie are disclosed in WO 97/13499 (Lim et al).

|0022S] Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of prepara tion thereof, are known in the art. Sunamoto et al. (53 Bull. Chem. Soc. Jpn. 2778 ( 1980)) described liposomes comprising a nonionic detergent, 2C1215G, that contains a PEG moiety. Ilium et al. (167 FEBS Lett. 79 (1984)), 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. Patent No, 4,426,330 and

No, 4,534,899). In addition, antibodies can be conjugated to a polyakylene derivatized liposome (see e.g., PCT Application US 2008/0014255). Klibanov et al. (268 FEBS Lett, 235 (1990)), 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. (1029 Biochim. Biophys. Acta 1029, (1990)}, extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of

distearoylphospbatidylethanolamine (DSPE) and PEG, Liposomes having covale tly bound PEG moieties on their external surface are described in European Patent No. 0445131 Bl and WO 90/04384 to Fisher,

[00229] Liposome compositions containing 1-20 mol% of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Patents No. 5,013,556;

No. 5,356,633) and Martin et al. (U.S. Patent No. 5,213,804; European Patent

No. 0 496813 Bl). Liposomes comprising a number of other lipid-poiymer conjugates are disclosed in WO 91/05545 and U.S. Patent No. 5,225,212 and in WO 94/20073. Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391. U.S. Patents

No, 5,540,935 and No. 5,556,948 describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces. Methods and compositions relating to liposomes comprising PEG can be found in, e.g., U.S. Patents No. 6,049,094; No. 6,224,903; No. 6,270,806; No. 6,471 ,326; No. 6,958,241 . [00230] As noted above, liposomes can optionally be prepared to contain surface groups, such as antibodies or antibody fragments, small effector molecules for interacting with cell-surface receptors, antigens, and other like compounds, and these groups can facilitate delivery of liposomes and their contents to specific cell populations. Such ligands ca be included in the liposomes by including in the liposomal lipids a lipid derivatized with the targeting molecule, or a lipid having a polar-head chemical group that can be derivatized with the targeting molecule in preformed liposomes. Alternatively, a targeting moiety can be inserted into preformed liposomes by incubating the preformed liposomes with a ligand-polymer-lipid conjugate.

[00231] Lipids can be derivatized using a variety of targeting moieties, such as ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin) and monoclonal antibodies by covalently attaching the ligand to the free distal end of a hydrophilic polymer chain, which is attached at its proximal end to a vesicle-forming lipid. There are a wide variety of techniques for attaching a selected hydrophilic polymer to a selected lipid and activating the free, unattached end of the polymer for reaction with a selected ligand, and as noted above, the hydrophilic polymer polyethyleneglycol (PEG) has been studied widely. Allen et al., 1237 Biochem. Biophys. Acta 99-108 ( 1995); Zalipsky, 4 Bioconj. Chem. 296-99 (1993); Zalipsky et al, 353 FEBS Lett. 1-74 (1994): Zalipsky et al, Bioconj. Chem. 705-08 (1995); Zalipsky, in STEALTH LIPOSOMES (Lasic & Martin, eds, CRC Press, Boca Raton, FL, 1995),

[00232] A number of liposomes comprising nucleic acids are known in the art, such as methods for encapsulating high molecular weight nucleic acids in liposomes. WO 96/40062. U.S. Patent No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes can include a dsRNA. U.S. Patent No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotid.es in liposomes.

WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene. In addition, methods for preparing a liposome composition comprising a nucleic acid can be found in, e.g., U.S. Patents No. 6,011,020; No. 6,074,667; No. 6,1 10,490; No. 6,147,204;

No. 6,271 ,206; No. 6,312,956; No. 6,465,188; No. 6,506,564; No. 6,750,016; No. 7,1 12,337.

[00233] Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can 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, 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.

[00234] Encapsulated nanoparticles can also be used for delivery of RNA effector molecules. Examples of such encapsulated nanoparticles include those created using yeast cell wall particles (YCWP). For example, glucan-encapsulated siRNA particles (GeRPs) are pay load delivery systems made up of a yeast cell wall particle (YCWP) exterior and a multilayered nanoparticle interior, wherein the multilayered nanoparticle interior has a core comprising a payload complexed with a trapping agent. Glucan-encapsulated delivery systems, such as those described in U.S. Patent Applications Ser. No. 12/260,998, filed October 29, 2008, can be used to deliver siRNA duplexes to achieve silencing in vitro and in vivo, ,

C. Cell Cultures

[00235] Methods described herein use host cells to produce glycoproteins having modified glycans. A host cell can be derived from a yeast, insect, amphibian, fish, reptile, bird, mammal or human, or can be a hybridoma 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 ceil 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.

[00236] A mammalian host cell can be advantageous where the glycoprotein is a mammalian glycoprotein, particularly if the glycoprotein is a biotberapeutic agent or is otherwise intended for administration to or consumption by humans. In some embodiments, the host ceil is a CHO cell, which is a cell line used for the expression of many recombinant proteins. Additional mammalian cell lines used commonly for the expression of recombinant proteins include 293HEK ceils, HeLa ceils, COS cells, NIH/3T3 cells, Jurkat Cells, NSO cells, and HUVEC cells.

[00237] In some embodiments, the host cell is a CHO cell derivative that has been modified genetically to facilitate production of recombinant proteins. For example, various CHO cell strains have been developed which permit stable insertion of recombinant DNA into a specific gene or expression region of the cells, amplification of the inserted DNA, and selection of cells exhibiting high le vel expression of the recombinant protein. Examples of CHO cell derivatives useful in methods provided herein include, but are not limited to, CHO-Kl cells, CHO-DUKX, CHO-DUKX Bl, CHO-DG44 ceils, CHO-ICAM-1 cells, and CHO-hlFNy cells, Methods for expressing recombinant proteins in CHO cells are known in the art and are described, e.g., in U.S. Patents No. 4,816,567 and No. 5,981,214.

[00238] Examples of human cell lines useful in methods provided herein include the cell lines 293T (embryonic kidney), 786-0 (renal), A498 (renal), A549 (alveolar basal epithelial), ACHN (renal), BT-549 (breast), BxPC-3 (pancreatic), CAKI-1 (renal), Capan-i (pancreatic), CCRF-CEM (leukemia), COLO 205 (colon), DLD-1 (colon), DMS 114 (small ceil lung), DU145 (prostate), EKVX (non-small cell lung), HCC-2998 (colon), HCT-15 (colon), HCT-1 16 (colon), HT29 (colon), S IT- 1080 (fibrosarcoma), HEK 293 (embryonic kidney), HeLa (cervical carcinoma), HepG2 (hepatocellular carcinoma), HL-60(TB) (leukemia), HOP-62 (non- small cell lung), HOP-92 (non-small cell lung), HS 578T (breast), HT-29 (colon

adenocarcinoma), IG -OV1 (ovarian), IMR32 (neuroblastoma), Jurkat (T lymphocyte), K-562 (leukemia), KM 12 (colon), KM20L2 (colon), LANS (neuroblastoma), LNCap.FGC (Caucasian prostate adenocarcmoma), LOX IMV1 (melanoma), LXFL 529 (non-small cell lung), M 14 (melanoma), M19-MEL (melanoma), MALME-3M (melanoma), MCFIOA (mammary epithelial), MCI ^' 7 (mammary), MDA-MB-453 (mammary epithelial), MDA-MB-468 (breast), MDA-MB-231 (breast), MDA-N (breast), MOLT-4 (leukemia), NCl/ADR-RES (ovarian), NCI- 1122.0 (non-small cell lung), NCI-H23 (non-small cell lung), NC1-H322M (non-small cell lung ), NCI-H460 (non-small cell lung), NCI-H522 (non-small cell lung), OVCAR-3 (ovarian), QVCAR-4 (ovarian), OVCAR-5 (ovarian), OVCAR-8 (ovarian), P388 (leukemia), P388/ADR (leukemia), PC-3 (prostate), PERC6® (El -transformed embryonal retina), RPMI-7951

(melanoma), RPMI-8226 (leukemia), RXF 393 (renal), RXF-631 (renal), Saos-2 (bone), SF-268 (CNS), SF-295 (C S), SF-539 (CNS), SHP-77 (small cell lung), SH-SY5Y (neuroblastoma), SK-BR3 (breast), SK-MEL-2 (melanoma), SK-MEL-5 (melanoma), SK-MEL-28 (melanoma), SK-OV-3 (ovarian), SN12K1 (renal), SN12C (renal), SNB-19 (CNS), SNB-75 (CNS) SNB-78 (CNS), SR (leukemia), SW-620 (colon), T-47D (breast), THP-1 (monocyte-derived

macrophages), TK-10 (renal), U87 (glioblastoma), U293 (kidney), U251 (CNS), UACC-257 (melanoma), UACC-62 (melanoma), UO-31 (renal), W138 (lung), and XF 498 (CNS). [00239] Examples of non-human primate cell lines useful in methods provided herein include the cell lines monkey kidney (CVI-76), African green monkey kidney (VERO-76), green monkey fibroblast (COS-1), and monkey kidney (CVI) cells transformed by SV40 (COS- 7). Additional mammalian cell lines are known to those of ordinary skill in the art and are catalogued at the American Type Culture Collection catalog (Manassas, VA).

100240] Examples of rodent cell lines useful in methods provided herein include the ceil lines baby hamster kidney (BHK) (e.g., BHK21, BH TK), mouse Sertoli (TM4), buffalo rat liver (BRL 3A), mouse mammary tumor (MMT), rat hepatoma (HTC), mouse myeloma (NS0), murine hybridoma (Sp2/0), mouse thymoma (EL4), Chinese Hamster Ovary (CHO) and CHO cell derivatives, murine embryonic (NIH/3T3, 3T3 Li), rat myocardial (H9c2), mouse myoblast (C2C12), and mouse kidney (miMCD-3).

[00241] In some embodiments, the host cell is a multipotent stem cell or progenitor cell. Examples of multipotent cells useful in methods provided herein include murine embryonic stem (ES-D3) cells, human umbilical vein endothelial (HuVEC) cells, human umbilical artery smooth muscle (HuASMC) cells, human differentiated stem (HKB-I1) cells, human

mesenchymal stem (hMSC) cells, and induced piuripotent stem (IPS) cells.

[00242] In some embodiments, the host cell is an insect cell, such as Sf9 cell line (derived from pupal ovarian tissue of Spodoptera frugiperda); Hi-5 (derived from Trichoplusia ni egg cell homogenates); or S2 ceils (from Drosophila melanogasier).

[00243] In some embodiments, the host cells are suitable for growth in suspension cultures. Suspension-competent host cells are generally monodisperse or grow in loose aggregates without substantial aggregation, Suspension-competent host cells include cells that are suitable for suspension culture without adaptation or manipulation (e.g., hematopoietic cells, lymphoid cells) and cells that have been made suspension-competent by modification or adaptation of attachment-dependent cel ls (e.g., epithelial cel ls, fibroblasts).

[00244] In some embodiments, the host ceil is an attachment dependent ceil which is grown and maintained in adherent culture. Examples of human adherent ceil lines useful in methods provided herein include the ceil lines human neuroblastoma (SH-SY5 Y, IMR32, and LANS), human cervical carcinoma (HeLa), human breast epithelial (MCFIOA), human embryonic kidney (293T), and human breast carcinoma (SK-BR3), [00245] In some embodiments, the host ceil 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. The host cell can be, for example, a human Namalwa Burkitt lymphoma cell (BLcl-kar-Namalwa), baby hamster kidney fibroblast (BHK), CHQ cell, Murine myeloma cell (NSO, SP2/0), hybridoma cell, human embryonic kidney cell (293 HEK), human retina-derived cell (PER.C6® ceils, U.S. Patent No. 7,550,284), insect cell line (Sf9, derived from pupal ovarian tissue of Spodoptera frugiperda or Hi-5, derived from Trichoplusia ni egg cell homogenates; see also U.S. Patent No. 7,041,500), Madin-Darby canine kidney cell ( Mi X ^' K h primary mouse brain cells or tissue, primary calf lymph cells or tissue, primary monkey kidney cells, embryonated chicken egg, primary chicken embryo fibroblast (CEF), Rhesus fetal lung cell (FRhL-2), Human fetal lung ceil (W!-38, MRC-5), African green monkey kidney epithelial cell (Vero, CV-1), Rhesus monkey kidney cell (LLC-MK2), or yeast cell. Additional mammalia ceil lines commonly used for the expression of recombinant proteins include, but are not limited to, HeLa cells, COS cells, NIH/3T3 cells, Jurkat Cells, and human umbilical vein endothelial cells (HUVEC) ceils.

[00246] 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, STAT1, 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.

[00247] The host cells may express the glycoprotein of interest endogenously, or alternatively, the host ceil may be engineered to express an exogenous glycoprotein. For example, a host cell may be transfected with one or more expression vectors that encode the glycoprotein. The nucleic acid molecule encoding the glycoprotein may be transiently introduced into the host cell, or stably integrated into the genome of the host cell. For example, in case of an antibody, one or more recombinant expression vectors encoding the light and/or heavy chains of the antibody (or an antigen-binding fragment of the antibody) may be transfected, such that the light and/or heavy chains are expressed in the host cell. If desired, the glycoprotein may be secreted into the medium in which the host cell is cultured, from which medium the glycoprotein can be recovered. [00248] Standard recombinant DNA methodologies may be used to obtain a nucleic acid that encodes a glycoprotein, incorporate the nucleic acid into an expression vector and introduce the vector into a host cell, such as those described in Sambrook, et al. (eds), Molecular Cloning; A. Laboratory Manual, Third Edition, Cold Spring Harbor, (2001); Ausubel, F. M, et al, (eds. ) Current Protocols in Molecular Biology, John Wiley & Sons (1995). A nucleic acid encoding the glycoprotein may be inserted into an expression vector or vectors such that the nucleic acids are operably linked to transcriptional and translational control sequences. The expression vector and expression control sequences are generally chosen to be compatible with the expression host cell used.

[00249] For example, to express an antibody, or an antigen-binding fragment thereof, nucleic acids encoding the light and heavy chain variable regions may be first obtained. These nucleic acids can be obtained by amplification and modification of human germline ligh t and heavy chain variable region genes using PCR. Germline DNA sequences for human heavy and light chain variable region genes are known in the art.

[00250] In addition to the nucleic acid that encodes the glycoprotein, the expression vector may additionally carry regulatory sequences that control the expression of the

glycoprotein in a host cell, such as promoters, enhancers or other expression control elements (e. g. , polyadenylation signals) that control the transcription or translation of the nucleic acid(s). Such regulatory sequences are known in the art (see, e.g., Goeddel, Gene Expression

Technology: Methods in Enzyrnology 185, Academic Press (1990)). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be

transformed, the l evel of expression of protein desired, etc. Exemplary regulator}' sequences for mammalian host cell expression include viral elements that direct high le vels of protein expression in mammalian cells, such as promoters and/or enhancers derived from

cytomegalovirus (CMV) (such as the CMV promoter/enhancer). Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e. g. , the adenovirus major late promoter

(AdMLP) ) and polyoma virus.

[00251] In addition to sequences encoding the glycoprotein and regulator} _' sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host ceils (e, g. , origins of replication) and selectable marker genes.

[00252] The expression vector(s) encoding the glycoprotein may be transfected into a host cell by standard techniques, such as electroporation, calcium-phosphate precipitation, or DEAE-dextran trans fection. If desired, viral vectors, such as retro-viral vectors, may also be used to generate stable cell lines (as a source of a continuous supply of the glycoprotein).

100253] The methods described herein can be applied to any size of cell culture flask and/or bioreactor. For example, the methods can be applied in bioreactors or cell cultures of 10 L, 30 L, 50 L, 100 L, 150 L, 200 L, 300 L, 500 L, 1000 L, 2000 L, 3000 L, 4000 L, 5000 L, 10,000 L or larger. In some embodiments, the cell culture size can range from 10 L to 5000 L, from 10 L to 10,000 L, from 10 I, to 20,000 L, from 10 I, to 50,000 L, from 40 I, to 50,000 L, from 100 L to 50,000 L, from 500 L to 50,000 L, from 1000 L to 50,000 L, from 2000 L to 50,000 L, from 3000 I, to 50,000 L, from 4000 L to 50,000 L, from 4500 L to 50,000 L, from 1000 L to 10,000 L, from 1000 L to 20,000 L, from 1000 L to 25,000 L, from 1000 L to

30,000 L, from 15 L to 2000 L, from 40 L to 1000 L, from 100 L to 500 L, from 200 L to 400 L, or any integer there between.

100254] Media components include, e.g., buffer, amino acid content, vitamin content, salt content, mineral content, serum content, carbon source content, lipid content, nucleic acid content, hormone content, trace element content, ammonia content, co-factor content, indicator content, small molecule content, hydrolysate content and enzyme modulator content.

Preferably, the growth medium is a chemically defined media such as Biowhittaker©

POWERCHO® (Lonza, Basel, Switzerland), HYCLO E PF CHO™ (Thermo Scientific, Fisher Scientific), GlBCO® CD DG44 (Invitrogen, Carlsbad, CA), Medium Ml 99 (Sigma- Aldrich), OPTTPRO™ SFM (Gibco), etc).

D. Regulation of Host Cell Gene Expression.

[00255] One or more RNA effector molecules are added to the cell culture to regulate the expression level (s) of target gene(s). If more than two or more RNA effector molecules are used, they may be provided at the same concentration, or different concentrations. The RNA effectors may be added simultaneously into the cell culture, or added at different times into the cell culture. [00256] An effective amount of an RNA effector is added to the cell culture to allow sufficient reduction of the expression of a target gene, For example, an effecti ve amoun t of an RNA effector is added to the cell culture such that the expression level of its target gene is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%.

100257] In general, a suitable dose of RNA effector molecule will be in the range of 0.001 to 200.0 milligrams per unit volume per day. For example, the RNA effector molecule may be provided in the range of 0.001 nM to 200 mM per day, generally in the range of 0.1 nM to 500 nM. For example, a dsRNA can be administered at 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 0.75 nM, 1 nM, 1.5 nM, 2 nM, 3 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 100 nM, 200 nM, 400 nM, or 500 nM per single dose. In one embodiment, the RNA effector molecule is administered a cell culture at a concentration less tha about 50nM,

[00258] The composition ca be added to the cell culture once daily, or the RNA effector molecule can be added as two, three, or more sub-doses at appropriate intervals throughout the day or delivery through a controlled release formulation. In that case, the RNA effector molecule contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation, which provides sustained release of the RN A effector mol ecule over a several-day-period.

[00259] The effect of a single dose on target gene transcript levels can be long-lasting, such that subsequent doses are administered at not more than 3-, 4-, or 5-day intervals, or at not more than 1-, 2-, 3-, or 4-week intervals.

[ 00260] The administration of the RNA effector molecule may be ceased 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 biol ogical product. Thus in one embodiment, contacting a host cell (e.g., in a large scale host cell culture) with a RNA effector molecule is complete 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. [00261] Sometimes, it may be beneficial to provide a RNA effector molecule to the host cell cultures in a way that a constant number (or at least a minimum number) of RNA effector molecules per each cell is maintained. Maintaining the levels of the RNA effector molecule as such can ensure that modulation of target gene expression is main tained even at high cell densities,

100262] The amount of a RNA effector molecule can also be administered according to the cell density, In such embodiments, the RNA effector molecule(s) is added at a

concentration of at least 0.01 fmoi/10 ⁶ ceils, at least 0.1 fmol/10 ^fi cells, at least 0.5 fmoi/10 ⁶ cells, at least 0,75 fmol/10 ⁶ cells, at least 1 fmoi/10 ⁶ ceils, at least 2 fmol/lO ⁶ cells, at least 5 fmol/10 ⁶ cells, at least 10 fmol/10 ⁶ cells, at least 20 fmol/10 ⁶ cells, at least 30 fmoi/10 ⁶ ceils, at least 40 fmoi/10 ⁶ ceils, at least 50 fmoi/10 ⁶ cells, at least 60 fmol/10 ⁶ cells, at least 100 fmol/10 ⁶ cells, at least 200 fmol/10 ⁶ cells, at least 300 fmol/10 ⁶ cells, at least 400 fmol/10 ⁶ cells, at least 500 fmol/10 ⁶ cells, at least 700 fmol/10 ⁶ cells, at least 800 fmol/10 ⁶ cells, at least 900 fmol/10 ⁶ cells, or at least 1 pmol/10 ⁶ cells, or more,

[00263] For example, the RNA effector molecule may be administered at a dose of at least 10 molecules per cell, at least 20 molecules per cell (molecules/cell), at least 30

molecules/cell, at least 40 molecules/cell, at least 50 molecules/cell, at least 60 molecules/cell, at least 70 molecules/cell, at least 80 molecules/cell, at least 90 molecules/cell at least 100 molecules/cell, at least 200 molecules/cell, at least 300 molecules/cell, at least 400

molecules/cell, at least 500 molecules/cell, at least 600 molecules/cell, at least 700

molecules/cell, at least 800 molecules/cell, at least 900 molecules/cell, at least 1000

molecules/cell, at least 2000 molecules/cell, at least 5000 molecules/cell or more, inclusive.

[00264] In some embodiments, the RNA effector molecule is administered at a dose within the range of 10-100 molecules/cell, 10-90 molecules/cell, 10-80 molecules/cell, 10-70 molecules/cell, 10-60 molecules/ceil, 10-50 molecules/cell, 10-40 molecules/cell, 10-30 molecules/cell, 10-20 molecules/cell, 90-100 molecules/ceil, 80-100 molecules/ceil, 70-100 molecules/cell, 60-100 molecules/cell, 50-100 molecules/ceil, 40-100 molecules/cell, 30-100 molecules/cell, 20-100 molecules/cell, 30-60 molecules/cell, 30-50 molecules/cell, 40-50 molecules/cell, 40-60 molecules/ceil, or any range there between. [00265] In one embodiment, the RNA effector molecule is administered as a sterile aqueous solution, In one embodiment, the RNA effector molecule is formulated in a non-lipid formulation. In another embodiment, the RNA effector molecule is formulated in a cationic or non-cationic lipid formulation. In still another embodiment, the RNA effector molecule is formulated in a cell medium suitable for culturing a host cell (e.g., a serum-free medium).

E. Purification of Glycoproteins

100266] The glycoproteins produced in accordance with the methods described herein can be harvested from host ceils, and purified using any suitable methods. For example, methods for purifying polypeptides by immune-affinity chromatography are known in the art. Ruiz-Arguello et al, J. Gen. Virol, 55:3677-3687 (2004). Suitable methods for purifying desired glycoprotein including precipitation and various types of chromatography, such as hydrophobic interaction, ion exchange, affinity, chelating and size exclusion are well-known in the art. Suitable purification schemes can be created using two or more of these or other suitable methods. If desired, the glycoprotein can include a "tag" that facilitates purification, such as an epitope tag or a HIS tag. Such tagged polypeptides can conveniently be purified, for example from conditioned media, by chelating chromatography or affinity chromatography. Optionally, the tag sequence may be cleaved post-purification.

[00267] For example, normal phase liquid chromatography can be used to separate giycans and/or glycoproteins based on polarity. Reverse-phase chromatography can be used, e.g., with derivatized sugars. Anion-exchange columns can be used to purify sialylated, phosphorylated, and sulfated sugars. Other methods include high pH anion exchange chromatography and size exclusion chromatography can be used and is based on size separation.

[00268] Affinity based methods can be selected that preferentially bind certain chemical units and glycan structures. Matrices such as m-aminophenylboronic acid,

immobilized lectins and antibodies can bind particular glycan structures. M- aminophenyiboronic acid matrices can form a temporary covalent bond with any molecule (such as a carbohydrate) that contains a 1 ,2-cis-diol group. The covalent bond can be subsequently disrupted to elute the protein of interest. Lectins are a family of carbohydrate-recognizing proteins that exhibit affinities for various monosaccharides. Lectins bind carbohydrates specifically and reversibly. Primary monosaccharides recognized by lectins include mannose/glucose, galactose/N-acetylgalactosamine, N-acetylglucosamine, fucose, and sialic acid (QProteome Glycoarray Handbook, Qiagen, September 2005, available at:

http://wolfson.huji.acil/u^ or similar references, Lectin matrices (e.g., columns or arrays) can consist of a number of lectins with varying and/or overlapping specificities to bind glycoproteins with specific glycan compositions, Some lectins commonly used to purify glycoproteins include concavaiin A (often coupled to Sepharose or agarose) and Wheat Germ. Anti-glycan antibodies can also be generated by methods known in the art and used in affinity columns to hind and purify glycoproteins.

[00269] The interaction of a lectin or antibody with a ligand, such as a glycoprotein, allows for the formation of cross-linked complexes, which are often insoluble and can be identified as precipitates (Varki et al, ed., "Protein-Glycan Interactions" in Essentials of Glycobiology available at world wide web at

or similar references, In this technique, a fixed amount of lectin or antibody (receptor) is titrated with a glycoprotein or a glycan, and at a precise ratio of ligand to receptor, a precipitate is formed (Varki et al.). Such precipitation is highly specific to the affinity constant of the ligand to the receptor (Varki et al.), Another precipitation approach takes advantage of the fact that a complex between a lectin and a glycan can be "salted" out or precipitated by ammonium sulfate (Varki et al.),

F. Analysis of Glycoproteins

(1). Analysis of the structure ¾nd composition of N-linked glycans

[00270] The glycan structure of the glycoproteins (such as antibodies or Fc-fusion proteins) described herein can be determined using art-known methods for analyzing glycan structures of glycoproteins, such as chromatography, mass spectrometry (MS), chromatography followed by MS, electrophoresis, electrophoresis followed by MS, nuclear magnetic resonance (NMR), and any combinations thereof. A preferred technique is Liquid chromatography-mass spectrometry (LC-MS, or alternatively HPLC-MS).

[00271] For example, an enzyme, such as an N-glycanase (e.g, N-glycanase F, N- glycanase-A), can be used to cleave the N-glycan moiety from a glycoprotein. Further, exoglycosidases (e.g., siaiidase, galactosida.se, hexosaminidase, fucosidase, mannosidase etc) can be used cleave terminal glycosidic bonds from the non-reducing end of glycans. Alternatively, acid hydrolysis (e.g., trifluoroacetic acid) can be used to release neutral saccharides (e.g., galactose, mannose, fucose) or amino saccharides (e.g., N-acetylghicosamine) from a glycan. The cleaved or hydrolyzed saccharides can be analyzed using chromatography spectrometry, or electrophoresis methods described above.

[00272] For example, glycan structure and composition can be analyzed by chromatography, including, e.g., liquid chromatography (LC), high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), thin layer chromatography (TLC), amide column chromatography, or combinations thereof.

[00273] Another method to analyze glycan structure and composition is mass spectrometry (MS), including, e.g., tandem MS, LC-MS, LC-MS/MS, matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation with mass spectrometry (IMS-MS), electron transfer dissociation (ETD-MS), or combinations thereof.

[00274] Another method to analyze glycan structure and composition is

electrophoresis, including, e.g., capillar} _' electrophoresis (CE), CE-MS, gel electrophoresis, agarose gel electrophoresis, acrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting using antibodies that recognize specific glycan structures, or combinations thereof. For example, the structure of an N-glycan can be determined by two dimensional sugar chain mapping (see, e.g., Anal, Biochem,, 171 , 73 (1988); Biochemical Experimentation Methods 23-Methods for Studying Glycoprotein Sugar Chains (Japan Scientific Societies Press) edited by Reiko Takahashi (1989)). Two dimensional sugar chain mapping is a method for deducing the structure of a saccharide chain by plotting the retention time or elution position of the saccharide chain by reverse phase chromatography as the X axis, and the retention time or elution position of the saccharide chain by normal phase chroma tography as the Y axis, respectively, and comparing them with such results of known sugar chains. The structure deduced by two dimensional sugar chain mapping can be confirmed by mass spectrometry.

[00275] Another method to analyze glycan structure and composition is nuclear magnetic resonance (NMR), including, e.g., one-dimensional NMR (I D-NMR), two- dimensional NMR (2D-NMR), correlation spectroscopy magnetic-angle spinning N MR (COSY- MR), total correlated spectroscopy NMR (TOCSY-NMR), heteronuclear single-quantum coherence NMR (HSQC-NMR), heteronuclear multiple quantum coherence (HMQC-N R), rotational nuclear overhauser effect spectroscopy NMR (ROESY-NMR), nuclear overhauser effect spectroscopy (NOE8Y-NMR), or combinations thereof.

[00276] Saccharide composition of a glycan can also be analyzed by fluorescence labeling. For example, acid-hydroiyzed glycans can be labeled with 2-aminopyridine and then analyzed by HPLC.

[00277] Immunological methods (e.g., antibody staining, lectin staining) may also be used to determine the structures of N-glycan. For example, lectin molecules can bind to the carbohydrate moieties of glycoproteins. Therefore, a lectin that binds to a specific N-glycan can be used to identify the presence and quantity of such glycoforrns in a composition (e.g., by determining the amount of glycan-bound lectin using a secondary antibody). Examples of lectins that can be used for identifying the glycan structure of an antibody, or a Fc-fusion protein, include, e.g., WGA (wheat-germ agglutinin derived from T. vulgaris), ConA

(cocanavalin A derived from C. ensiformis), RIC (a toxin derived from R. communis), L-PHA (leucoagglutinin derived from P. vulgaris), LCA (lentil agglutinin derived from L. culinaris), PSA (pea lectin derived from P. sativum), AAL (Aleuria auraiitia lectin), ACL (Amaranthus caudatus lectin), BPL (Bauhinia purpurea lectin), DSL (Datura stramonium lectin), DBA

(Dolichos bifloms agglutinin), EBL (elderberr balk lectin), ECL (Erythrina cristagalli lectin), EEL (Euonymus eoropaeus lecin), GNL (Galanthus nivalis lectin), GSL (Griff onia simplicifolia lectin), HP A (Helix pomatia agglutinin), HHL (Hippeastram hybrid lectin), jacalin, LTL (Lotus tetragonolobus lectin), LEL (Lycopersicon esculentum lectin), MAL (Maackia amurensis lectin), MPL (Maclura pomifera lectin), NPL (Narcissus pseudonarcissus lectin), PNA (peanut agglutinin), E-PHA (Phaseolus vulgaris erythroagglutiniii), PTL (Psopliocarpus tetragonolobus lectin), RCA (Ricinus communis agglutinin), STL (Solanum tuberosum lectin), SJA (Sophora japonica agglutinin), SBA (soybean agglutinin), UEA (Uiex europaeus agglutinin), WL (Vicia viilosa lectin) and WFA (Wisteria floribunda agglutinin).

[00278] For example, a lectin that specifically recognizes a complex N-glycan in which a fucose residue is linked to the N-acetylglucosarrrine in the reducing end of the N-glycan may be used. Exemplary lectins include, e.g., Lens culinaris lectin LCA (lentil agglutinin derived from Lens culinaris), pea lectin PSA (pea lectin derived from Pisum sativum), broad

7 bean lectin VFA (agglutinin derived from Vicia faba) and Aleuria aurantia lectin AAL (lectin derived from Aleuria aurantia).

[00279] Another method to analyze glycan structure and composition is by capillary electrophoresis (CE), which is described e.g., in Szabo et al., Electrophoresis, 2010 April; 31(8): 1389-1395. doi: 10.1002/elps.201000037,

[00280] Techniques described herein may be combined with one or more o ther technologies for the detection, analysis, and or isolation of glycans or glycoproteins. For example, any combination of NMR, mass spectrometry, liquid chromatography, 2-dimensioiial chromatography, SDS-PAGE, antibody staining, lectin staining, monosaccharide quantitation, capillary electrophoresis, fluorophore-assisted carbohydrate electrophoresis (FACE), micellar electrokinetic chromatography (MEKC), exoglycosidase or endoglycosidase treatments may be used. See, e.g., Anumula, Anal. Biochem. 350( 1): 1, 2006; Klein et al, Anal. Biochem,, 179: 162, 1989; Townsend, R.R. Carbohydrate Analysis, High Performance Liquid

Chromatography and Capillary Electrophoresis, Ed. Z. El Rassi, pp 181-209, 1995. For example, Qian et al. (Analytical Biochemistry 364 (2007) 8-18) discloses a method for determining the structures of glycans using orthogonal matrix-assisted laser

desorption/ionization hybrid quadrupole-quadrupoie time-of-fight mass spectrometry (oMALDI Qq-TOF MS) and tandem mass spectrometry (MS/MS) in combination with exoglycosidase digestion. The N-linked glycans are released by treatment with N-glycanase F, reductively aminated with anthranilic acid, and fractionated, by normal phase high-performance liquid chromatography (NP-HPLC). The Xuorescent-labeled oligosaccharide pool and fractions are then analyzed by oMALDI Qq-TOF MS and MS/MS in negative ion mode. Each fraction is further digested with an array of exoglycosidase mixtures, and subsequent MALD1 TOF MS analysis of the resulting produc ts yields informa tion about structural features of the glycan.

[00281] One exemplary saccharide composition analyzer is BioLC, manufactured by Dionex, which analyzes saccharide composition by HPAEC-PAD (high performance anion- exchange chromatography-pulsed amperometric detection).

(2). Analysis of the activity of glycoproteins

[00282] The biological activity of the glycoprotein compositions described herein may be assessed using any art known method, Such biological activities include, e.g., binding affinity or specificity of a glycoprotein (e.g., antibody, or a iigand for a receptor), bioavailability, pharmacokinetics, pharmacodynamics, etc. Additionally, therapeutic activity of a glycoprotein may be assessed (e.g., ADCC activity of an antibody, efficacy of a glycoprotein in decreasing severity or symptom of a disease or condition, or in delaying appearance of a symptom of a disease or condition).

100283] Methods of analyzing bioavailability, pharmacokinetics, and

pharmacodynamics of glycoprotein therapeutics have been described. See, e.g., Weiner et al, /. Pharm. Biomed. Anal. 15(5):571-9, 1997; Srinivas et al, /. Pharm. ScL 85(1): 1-4, 1996; and Srinivas et al, Pharm. Res. 14(7):911-6, 1997. Assays for measuring ADCC activity mediated by therapeutic antibodies are also known. See, e.g., Schnueriger A, et al., Mol. Immunol. (2011) 48( 12-13): 1512-7; Cancer immunology Immunotherapy, 36, 373 (1993); Cancer Research, 5A, 1511 (1994) , As would be understood to one of skill in the art, the particular biological activity or therapeutic activity that can be tested will vary depending on the particular glycoprotein or glycan structure.

[00284] The potential adverse activity or toxicity (e.g., propensity to cause

hypertension, immunogenicity/allergic reactions, thrombotic events, seizures, or other adverse events) of glycoprotein preparations can be analyzed by any available method. For example, immunogenic! ty of a glycoprotein composition can be assessed, e.g., by determining in vitro by immunoassay (e.g., using an antibody that binds to a recognized immunogenic epitope, such as the otGal epitope, or Neu5Gc epitope), or by in vivo administration to determine whether the composition elicits an antibody response in a subject.

5. PHARMACEUTICAL COMPOSITIONS, METHODS OF ADMINISTRATION, AND KITS

A. Pharmaceutical Compositions

[00285] In one aspect, the invention relates to pharmaceutical compositions comprising the glycoproteins (such as antibodies or Fc-fusion proteins) described herein.

[00286] The pharmaceutical compositions usually one or more pharmaceutical carrier(s) and/or excipient(s). A thorough discussion of such components is available in

Gennaro (2000) Remington: The Science and Practice of Pharmacy (20th edition). Examples of such carriers or additives include water, a pharmaceutical acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, carboxymethylcellulose sodium, polyacrylic sodium, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, gelatin, agar, di glycerin, glycerin, propylene glycol, polyethylene glycol. Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, a

pharmaceutically acceptable surfactant and the like. Formulation of the pharmaceutical composition wil l vary according to the route of administration selected.

[00287] Optionally, the glycoprotein can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with

conventional immunoglobulins. Any suitable lyoptiilization and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilization and

reconstitution can lead to varying degrees of activity loss and that use levels may have to be adjusted to compensate.

[00288] A variety of aqueous carriers can be used to formulate suitable

pharmaceutical compositions for administration, such as plain water (e.g. w.f.i.) or a buffer e.g. a phosphate buffer, a Tris buffer, a borate buffer, a succinate buffer, a histidme buffer, or a citrate buffer. Buffer salts will typically be included in the 5-20mM range.

[00289] The pharmaceutical compositions are preferably sterile, and may be sterilized by conventional sterilization techniques.

[00290] The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, and tonicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. Preferably, the pharmaceutical compositions of the invention may have a pH between 5.0 and 9.5, e.g. between 6.0 and 8.0.

[00291 ] Pharmaceutical compositions of the invention may include sodium salts (e.g. sodium chloride) to give tonicity. A concentration of 10+2 mg/mi NaCl is typical e.g. about 9 mg/ml. [00292] Pharmaceutical compositions of the invention may have an osmolarity of between 200 mOsm/kg and 400 mOsm kg, e.g. between 240-360 mOsm/kg, or between 290-310 mOsm/kg.

B. Methods of Administration

[00293] In another aspect, the invention provide a method for treating Non-Hodgkins lymphoma, comprising administering to a subject in need thereof a therapeutically effectively amount of an anti-CD20 antibody as described herein. The subject is preferably human.

100294] In another aspect, the invention provide a method for treating cancer or head and neck squamous cell carcinoma, comprising administering to a subject in need thereof a therapeutically effectively amount of an anti-EGFR antibody as described herein. The subject is preferably human.

[00295] In another aspect, the invention provide a method for treating breast cancer, comprising administering to a subject in need thereof a therapeutically effectively amount of an anti-HER2 antibody as described herein. The subject is preferably human.

[00296] In another aspect, the invention pro vide a method for treating breast cancer, comprising administering to a subject in need thereof a therapeutically effectively amount of an B7-binding Fc-fusion protein as described herein. The subject is preferably human.

[00297] The compositions described herein may be administered to a subject orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques. Administration by intravenous, intradermal, intramusclar, intramammary, intraperitoneal, intrathecal, retrobulbar,

intrapulmonary injection and or surgical implantation at a particular site is contemplated as well. In certain embodiments, injection, especially intravenous, is preferred.

[00298] The amounts of a glycoprotein in a given dosage will vary according to the size of the individual to whom the therapy is being administered as well as the characteristics of the disorder being treated. In exemplary treatments, it may be necessary to administer about 1 mg/day, about 5 mg/day, about 10 mg/day, about 20 nig/day, about 50 nig/day, about 75 mg/day, about 100 mg/day, about 150 mg/day, about 200 mg/day, about 250 mg/day, about 400 mg/day, about 500 mg/day, about 800 mg/day, about 1000 mg/day, about 1600 mg/day or about 2000 mg/day. The doses may also be administered based on weight of the patient, at a dose of 0.01 to 50 mg kg. The glycoprotein may be administered in a dose range of 0.015 to 30 mg/kg, such as in a dose of about 0.015, about 0.05, about 0.15, about 0.5, about 1.5, about 5, about 15 or about 30 mg/kg.

100299] Dosage can be by a single dose schedule or a multiple dose schedule.

Multiple doses will typically be administered at least 1 week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

[00300] Standard dose-response studies, first in animal models and then in clinical testing, can reveal optima] dosages for particular diseases and patient populations.

[00301] The glycoprotein compositions described herein may be administered in combination with a second therapeutic agent. For example, for cancer treatment, a

chemotherapeutic agent may be used as the second agent, For treatment of autoimmune diseases, non-steroidal anti-inflammatory drugs ( SAlDs), analgesiscs, glucocorticoids, disease- modifying antirheumatic drugs (DMARDs), may be used as die second agent. Examples of such therapeutic agents can be found, e.g., in WO 2008/1 6713.

C. Kits

[00302] In another aspect, the invention provides kits that comprise the glycoprotein compositions described herein packaged in a manner that facilitates their use for therapy. For example, such a kit includes a glycoprotein (e.g., an antibody or an Fc-fusion protein) as described herein, packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in the package that describes use of the composition in practicing the method. The kit can further comprise another container comprising a pharmaceutically- acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. Preferably, the composition is packaged in a unit dosage form. The kit may further include a device suitable for administering the composition according to a specific route of administration or for practicing a screening assay. Preferably, the kit contains a label that describes use of the composition. [00303] In another aspect, the invention provides kits for testing the effect of a RNA effector molecule or a series of RNA effector molecules on the production of a glycoprotein by the host cell, where the kits comprise a substrate having one or more assay surfaces suitable for cuituring cells under conditions that allow production of the glycoprotein. 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.

[00304] In some embodiments, the assay surfaces on the substrate are sterile and are suitable for cuituring host cells under conditions representative of the culture conditions during large-scale (e.g., industrial scale) production of the glycoprotein. Advantageously, kits provided herein offer a rapid, cost-effective means for testing a wide-range of agents and/or conditions on the production of the glycoprotein, allowing the cell culture conditions to be established prior to full-scale production of the glycoprotein.

[00305] In some embodiments, one or more assay surfaces of the substrate comprise a concentrated test agent, such as a RNA effector molecule, such that the addition of suitable media to the assay surfaces results in a desired concentration of the RNA effector molecule surrounding the assay surface. In some embodiments, the RNA effector molecules 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 RNA effector molecules are reconstituted by plating cells onto assay surfaces of the substrate.

[00306] In some embodiments, kits provided herein further comprise cell culture media suitable for cuituring a cell under conditions allowing for the production of the glycoprotein 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.

[00307] In further embodiments, kits provided herein further comprise one or more reagents suitable for detecting production of the glycoprotein 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 glycoprotein, In some embodiments, the reagent(s) are suitable for detecting the glycoprotein or a property thereof, such as the in vitro or in vivo biological activity,

homogeneity, or structure of the glycoprotein.

[00308] In some embodiments, one or more assay surfaces of the substrate further comprise a carrier for which facilitates uptake of RNA effector molecules by cells. Carriers for R A effector molecules are known in the art and are described herein. For example, in some embodiments, the carrier is a lipid formulation such as Lipofectamine™ transiection reagent (Inviirogen; Carlsbad, CA) or a related formulation. Examples of such carrier formulations are described herein. In some embodiments, the reagent that facilitates RNA effector molecule uptake comprises a charged lipid, an emulsion, a liposome, a cationic or non-cationic lipid, an anionic lipid, a transiection reagent or a penetration enhancer as described throughout the application herein. In particular embodiments, the reagent that facilitates RNA effector molecule uptake comprises a charged lipid as described in U.S. Application Ser.

No. 61/267,419, filed on December 7, 2009.

[00309] In some embodiments, one or more assay surfaces of the substrate comprise a RNA effector molecule or series of RNA effector molecules and a carrier, each in concentrated form, such that plating test cells onto the assay surface(s) results in a concentration the RNA effector moiecule(s) and the carrier effective for facilitating uptake of the RN A effector molecule(s) by the cells and modulation of the expression of one or more genes targeted by the RNA effector molecules.

[00310] In some embodiments, the substrate further comprises a matrix which facilitates 3 -dimensional cell growth and/or production of the glycoprotein 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 aicohol-hydrogel (PVA-H), polylactide-co- glycolide (PLGA), collagen vitrigei, PHEMA (poly(2-hydroxylmethacrylate)) hydrogels, PVP/PEQ hydrogels, BD PuraMatrix™ hydrogels, and copolymers of 2-methacryloyloxyettiyl phophorylcholine (MFC). [00311] 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 glycoprotein by cultured ceils. 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.

[00312] In some embodiments, kits provided herein allow for the selection or optimization of at least one factor for enhancing production of the biological product. For example, the kits may allow for the selection of a RNA effector molecule from among a series of candidate RN A effector molecules, or for the selection of a concentration or concentration range from a wider range of concentrations of a given RNA effector molecule. In some embodiments, the kits allow for selection of one or more RN A effector molecules from a series of candidate RNA effector molecules directed against a common target gene. In further embodiments, the kits allow for selection of one or more RNA effector molecules from a series of candidate RNA effector molecules directed against two or more functionally related target genes or two or more target genes of a common host cell glycosylation pathway.

[00313] In another aspect, the invention provides kits that comprise one or more container that independently contain one or more RNA effector molecules and one or more suitable host cells.

EXEMPLIFICATION

[00314] The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

EXAMPLE 1. GLYCAN-MODIFIED ANTI-CD20 ANTIBODY

[00315] In this Example, glycan-modified anti-CD20 antibody, based on Rituximab, was produced in a large scale culture using RNAi technology. The glycan-modified antibody showed enhanced ADCC activity as compared to Rituxamb under substantially the same conditions,

[00316] Rituximab (Rituxan©) is a chimeric anti-CD20 monoclonal antibody for treating non-Hodgkin's B-cell lymphoma (M I L ). Rituximab is recombinantly produced in CHO cells, and has a heterogeneous mixture of glycoforais.

[00317] The response rate for Rituximab in NHL patients is about 50-60% and is significantly correlated with a FcyRIiia receptor polymorphism (Cartron et a!. (2002) Blood 99:754-758). About 90% of patients homozygous for valine at position 158 respond to

Rituximab treatment, vviiereas patients hetero- or homozygous for phenylalanine at position 158 have a considerably lower response rate. It is believed that a lower affinity for FcyRIi ia F158 than for FcyRIiia VI 58 leads to lower ADCC activity. It has been shown that afucosyiated igGl has a higher affinity for FcyRIiia F158 (consequently higher ADCC activity) than the corresponding fucosylated IgGl (Shields et al. (2002) J. Biol. Chera. 277: 26733-26740). An afucosyiated Rituximab therefore would be a more potent therapeutic product regardless of the FcyRIi ia genotype.

[00318] In addition to ADCC, Rituximab also mediates tumor cell killing through CDC (Cragg and Glennie (2004) Blood 103: 2738-2743). A correlation between the galactose content of Rituximab N-glycans and CDC activity has been documented. As the number of galactose residues increases from 0 to 2 moles/mole of heavy chain, the level of CDC activity increases from 80%) to 150%» of the maximum CDC activity of the antibody having I mole galactose/moie of heavy chain (FD A, 1997; see, IDEC BLA 97-0260 at

www.fda.gov/Cder/biologics/review/ritugenl 12697).

1. Bioreactor operation an dosrag for modifying a-fucosylation

Bioreactor preparation for cell seeding & growth

[00319] Inoculate bioreactor with exponentially growing Anti-CD20 CHO cells to an initial seeding density of approximately 0.6 x 10 ⁶ cells/mL.

[00320] Sample bioreactor cultures daily for the following: 1) Growth (Viable Cell Density, Viability (Figure 4), and Total Cell Density) - lmL, 2) Metabolism (Glucose, gliitamine, glutamate, pH, and lactate)- ImL, 3) mRNA analysis fut8, GMD)- 0,5mL (Figure 5), and 4) Titer, product quality analysis (fucosylation)- 30-60 ml. after Day 6.

[00321] On day 3 and 6 feed with 5% each of CHO CD Efficient Feed media A and B, (Invitrogen, Catalog#'s Al 0234-01 and Al 0240-01, respectively). Starting daily on Day 7, add 1% RPMI amino acid solution (Sigma, Catalog# R7131). Add 1M glucose and gl tamax (invitrogen, Catalog# 35050-061 ) as necessary to maintain ~2-6g/L glucose and >0.5 mmol L glutamine.

2, Culture harvest & protein purification

[00322] Harvest culture at < 70% viability. Clarified culture supernatant is passed through a 50 ml. Protein A Sepharose (GE Biosciences) column. The resin washed with 20 mM phosphate buffer (pH 7.) and the protein eluted with 0.1 M citrate (pH 3.0), Eluted protein is dialyzed against 20 mM phosphate (pH 6.5) and 100 mM NaCl. Protein is sufficiently pure at this point for glycan analysis. When further polishing of the protein product is desired, the Protein A eluate is dialyzed against 50 mM NaAcetate (pFI 5.0) and applied to a 50 riiL SP Sepharose column (GE Biosciences) that was pre-equilibrated using the same buffer. The column is washed with 10-15 column volumes using 50 mM NaAcetate (pH 5.0) and the protein eluted with 250 mM NaCl in the same buffer. Eluted protein is dialyzed against 20 mM phosphate (pH 6.5) and 100 mM NaCl. The protein concentration is determined at UV280 using a scanning spectrophotometer ranging from OD240 to OD320, Protein samples are aliquoted and stored at -20 degree Celsius until needed.

3. Glycan characterization of CD20 monoclonal antibody

[00323] Purified CD20 monoclonal antibody from siRNA-treated or untreated cells was subjected to both LC/MS and Capillary Electrophoresis - Laser-induced Fluorescence (CE- LIF) to determine the extent of fucose knockdown. Both detection methods yielded similar results with an approximate 70% increase in a-fucosylated glycans. Using CE-LIF detection, the GO glycoform had the largest increase with approximately 60% conversion from GO with fucose to without followed by Gl and G2 (Figure 6).

[00324] Similar results were obtained using the LC-MS method to quantitate the glyeoforms (Figure 7). A 70% increase in the a-fucosylated glycans was observed. Again, a- iucosylated GO had the largest increase with approximately 60% followed by Gl at 10%, A- fueosylated G2 was not detected by this method.

4. CD20 monoclonal antibody hioassays

RNAi improves FcyRHIa binding to CD20 monoclonal antibody

[00325] A soluble HIS-tagged FcyRIIIa was utilized in a CD20 MAb binding ELISA to demonstrate RNAi targeting both fucosyltransferase and GMD-Dehydratase significantly decreases iucosylated CD20 MAb (Figure 6 & 7) and improves both maximum receptor binding and receptor affinity (Figure 8) by approximately two-fold.

RNAi improves CD20 antibody Antibody-Dependent Cellular Cytotoxicity (ADCC) activity

100326] Antibody-Dependent Cellular Cytotoxicity (ADCC) is a cell-based immunity mechanism that promotes the specific lyses of target cel ls bound with antibody ( CD20 MAb) by effector cells (NK) through binding interactions with FcyRJDa on the surface of effector cells. The target Cell line used in this study was Jeko-1 (Mantle cell lymphoma), Rituximab was used as a positive control. Freshly-isolated human PBMCs were used as effector cells at an Effector to Target (E/T) cell ratio of 25: 1 . The antibodies were tested in triplicate at 8 concentrations. The RNAi -treated CD20 antibody gave approximately 70% specific cell lysis compared to 20- 30% lysis for both the control CD20 antibody and rituximab (Figure 9).

5. Conclusion

[00327] RNAi is an effective means to modulate the glycan profile of therapeutic biologies to improve activity and decrease potential immunogemcity. Targeting key enzymes required for de novo GDP-fucose synthesis, GDP-fucose transport, and/or fucosyiation with siRNA. duplexes added directly to bioreactors gives the bioprocessing engineer highly specific control over the amount of fucose present on therapeutic monoclonal antibodies improving product quality that is currently unavailable today.

EXAMPLE 2, siRNAs THAT TARGET FUT8, GMDS AND EU2

[00328] In this Example, activities of siRNAs targeting Fut8, GMDS, and Neu2 were determined. Table 6 shows the results. Table 6

Neu2 0 . 388030164 AGG ACUUUGUG CCUGGACC 2 1594

Neu2 0 . 218618546 AGAQAGACCACCC ^' uCUCAG 1595

Neu2 0 . 307993119 AACCGAAGCACQU ^' uGGGCC 1596

Neu2 0 . 47 07506 CUGAUUGAGGUACACACCC 2 1597

Neu2 0 . 08951285 UUUAUUAA GACUC UG C AA 2 1598

Neu2 0 . 119298165 UCUCCUGUCUGGAAUAGCG 1599

Neu2 0 . 236577331 UCUGCUUGUCAUACAAAGG 2 1600

Neu2 0 . 231697907 AAGUUUACUCACUACCUGG 2 1601

Neu2 0 . 104061837 UUUACUCACUACCUGGUUG 2 1602

Neu2 0 . 487300265 UGAUAUUQGCCCACGGACA 2 1603

Neu2 0 . 183124124 UCGUAGGACAAACAAAUCU 2 1604

Neu2 0 . 109973778 AGCAUUGAGAUAGACCACC 2 1605

Neu.2 0 . 436004637 UACAAGUUUACUCACUACC 2 1606

Neu2 0 . 191171874 GAUAUUUGCCCACGGACAG 2 1607

Neu2 0 . 444654675 UGCAGGACAGGG CAAGUCG 2 1608

Neu2 0 . 160950067 AGC CG ^' uGUGAC AU ^' uAAC C C 2 1609

Neu2 0 . 376798761 AUAGUGACAGCCGACACAC 1610

Neu2 0 . 2844 64194 GGAUAAUUUAUUAAGACUC 1611

Neu2 0 . 371159296 CUAGCAUUGAGAUAGACCA 2 1612

Neu2 0 . 171466235 AAUAGCGUCUCCUUCUGCA 16 3

Neu2 0 . 492536463 UC C G C GG C C AG C GGG 16 4

[00329] The specification is most thoroughly understood in light of the teachings of the references cited within the specification. The embodiments within the specification provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan readily recognizes that many other embodiments are encompassed by the invention. All publications, patents, and sequences cited in this disclosure are incorporated by reference in their entirety. To the extent the materia! incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material The citation of any references herein is not an admission that such references are prior art to the present invention.

[00330] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following embodiments. Appendix 1 : nucleotide sequences of exemplar}' target genes from Chinese Hamster

Table 7

SEQ ID NO: 1: CH056727.1

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN G ATCTCAG GCCCATG GC

GACTTGCCCTGTCCTGCAGAAGGAGACGCTATTCCAGACAGGAGACTATGCTTACAG AATCCCTGCTCTGATCTACCTGTCAA

AGCAGAAGACCCTGCTGGCCTTTGCGGAAAAGCGTCTGACCAAGACAGATGAGCATG CAGATTTGTTTGTCCTACGAAGAGG

AAGCTACAATGCAGACACCCATCAGGTCCAGTGGCAAGCTGAGGAGGTGGTGACCCA AGCCTACCTGGAGGGCCACCGCTCC

ATGAGCCCATGTCCTTTGTATGACAAGCAGACAAGGACCCTTTTCCTTTTCTTCATC GCTGTCCGTGGGCAAATATCAGAACAC

CACCAGCTCCAGACTGGGGTTAATGTCACACGGCTATGCCACATCACCAGTACTGAC CATGGGAAGACCTGGAGCGCTGTCCA

GGACCTTACAGATACCACCATTGGCAGCACCCATCAAGATTGGGCCACATTTGGCGT GGGTCCTGGGCACTGTCTGCAGCTGC

GAAACACAGCTGGGAGCCTGCTGGTCCCTGCTTATGCCTATCGGAAACAACCCCCTA TCCATACACCTGCCCCCTCTGCCTTCT

GCTTCCTCAGCCATGACCATGGGAGCACATGGGAGCTGGGCCACTTTGTGTCCCAGA ACTCGCTGGAGTGCCAGGTGGCTGA

G GTTG G CACTG G CG CTG AG AG G GTG GTCTATCTCA ATG CTAG G AG CTG CCTG G G AG CCAG G GTCCAG G CAC AAAGTCCTAA

CAGTGGCCTGGATTTCCAGGACAACCAGGTAGTGAGTAAACTTGTAGAGCCTCCCAA AGGCTGCCATGGAAGTGTGATTGCT

TTCCCCAACCCCACCTCAAAGGCAGATGCCTTAGATGTGTGGCTGCTCTATACCCAC CCTACAGACTCCCGGAAGAGGACCAA

CCTGGGTGTGTACCTCAATCAGAAGCCACTGGACCCCACCACCTGGTCAGCTCCCAC CCTGTTGGCAACAGGCATCTGTGCCT

ACTCGGACTTGCAGAACATGGGGCACGGCCCTGATGGCTCCCCGCAATTTGGGTGTC TGTATGAGTCAAATAACTATGAAGA

GATTGTTTTCCTCATGTTCACCCTGAAGCAAGCTTTCCCAGCAGTGTTTGGTGCCCA GTGATCTTGCTGCATGCGGCCCAAAGT GCTTCTGTGCTCAAAACACCCATCTCTCTTTGCTTCCAGCATCCTCTGGACTCTTGAGTC CAGCTCTTGGGTAACTTCCTCAGGA GAGCAGAGAATTTGGTCTCTTGACTCTCTGCAGCCTTATTGTTTCAGCCTCTGGTTCTCT TTTCAGCCCAGAAATCAAAGGAGC CTG G CTTTCCTCAG CCTG TTG G CAG G G CAG GTG G G G AC AGTATATATAG AG G CTG CCATTCTG CATGTCG GTTGTCACTATG C TAGTTTAACCTGCCTGTTTCCCCATGCCTAGTGTTTGAATGAGTATTAATAAAATATCCA ACCCAGCC NNNNNNNNNNNNN NNNMNN

SEQ!D 0; 2: CH057849.1

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCGGCGGGGCCTGGG CGGCCAGAGCGGCG

CGGACCCCGGTTCTCGGCTCCCGGCGCCATGTGACGGGGTGCGGGGGCCGCGGGGGG CCGGGCGCTCCCCGGCGGAGGTG

TGACCCCACAACCCTGCCCCCCGGGCCACCTGCAGGACGCTGGCCTCTACCCNTCAG CAGACGCCGGAGAGAGATGAGTAGC

AACAAAGAGCAGCGGTCAGCAGTGTTTGTGGTCCTCTTTGCTCTCATCACCATCCTC ATCCTCTACAGCTCCAACAGTGCCAAC

GAGGTCTTCCACTATGGCTCCCTGAGGGGCCGCACGCGTCGGCCTGTCAACCTCAAG AAGTGGAGTTTCTCCAGCGCCTACTT

CCCTATCCTCGGCAACAAGACGCTGCCNNCCAGGTGCAACCAATGTGTGATCATCAC CAGCTCCAGCCACCTGCTGGGCACCA

AACTAGGCCCTGAGATTGAGCGGGCTGAGTGCACCATCCGCATGAACGATGCTCCCA CCTCTGG ACTCGGCNGACGTCGG

CAACAAAACCACCTTCCGCGTAGTGGCCCATTCCAGTGTGTTCCGTGTGCTGCGGAA GCCCCAGGAATTTGTCAACCGGACCC

CTGAAACGGTGTTCATCTTCTGGGGACCCCCAAACAAGATGCAGAAGCCACAGGGCA GCCTCCTGCGTGTCATCCAGCGGGC

GGGCCTCATGTTCCCTAACATGGAGGCCTATGCCGT CTCCCACCCGCATGCAGCAGTTTGACGACCTCTTCCGGGGTGAGA

CGGGCAAGGACAGGGAAAAGTCCCATTCCTGGTTGAGCACAGGCTGGTTTACCATGG TGATTGCGGTGGAGTTGTGTGACCA

TGTGCACGTGTATGGCATGGTCCCTCCTGA ATTGCAGCCAGCGGCCCCGCCTGCAACGCATGCCATACCACTACTATGAGC

CCAAGGGGCCTGACGAGTGCGTCACCTACATCCAGAATGAGCACAGCCGTAAGGGCA ATCACCACCGCTTCATCACCGAGAA

GAGGGTCTTCTCATCCTGGGCCCAGCTCTACGGCATCACCTTCTCCCACCCCTCCTG GACCTAGCCATCCTGTCCACCGATCTTG

GAGAGATAAAGGCAAAGTGGCTCTGGGCCGAACCATTTGACCTTGGCCATCTTCCAG CCAGTrCAGGTTGGCTGGAGTATTTC

CCAGCCAATCAAATCAGGGCCTTGATGAGGGTTTTTTTCCTTCCAGCCAGTGAGGGC TGGGGTTATCTCTTGTCCAATCAGGG

ATTTG G A ATCCTGTATG GTTTAATCG G GTGTCAG G G G GTCTTTCTTGTGTAATCAG G GTCTAAG CAAAGTCAATC AG G GTATA

GGGGGCTTTCTGAGTCAGTCTAAGACTCGGGTACTATCCTTTCCCAAAAGGCCTTGT GCCCGAACCCCAGGAATGGGCCCCAA

ATCATTTCCTCCTTAGCTGGGACAAAGAGGTCCTATCACAAGGATCTGGGGACTTGG TGTTGCCCCCACAGTTCCAGAATCAG

AGATACAGTACGGGGTAGGAACTGCCTGGAGGCCAGGCCAGAGAGTTTGTGGAGTTC TAGAAGTTGTAGGAAGGGCTGGG

AGGTGGAGGCGTGTGTCCAGGTCCTGGCTTCTTCCTGAGTGACCTTGAGCAAGCCCT CCTCTTTCTGGCGTGCTATAATGACAT

CACATTCCTGAGTGCAGACAGAGACCCTCACTCTATATTGTACTTAAGTGTAATTCC CCCATCGATCATCCCTCACTGGGGGAC

TCAGCTCCCTGGGAATGGGGAGCCTGGGGGGCCCTTATCCACCTTCTAGAAA TTAGGGTATTTTTGTGCAAGTGCCCCCAT

GGTTGGGGGATCCTGACAGAAATGGGGCAAACATGAAGCTGTTTCTCTAGCCCCTCA ATCCAGCTGCCGTTAGCCTGGCTCTA

GAAAGGCCAGGCACCTCTTCCTACCCTCCAGCAGGAGGCATTCCCGCTGCCCAAGGA GCCTTTGGGATGAGGAAGGGGTATC

CCAGCCACACCAGGGCN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN

NNNTGGGGGAGCTAGCCAGCAGTGAGCCCTGCACAGCAAGGTAATGGGGGGGGCAGT GACAGCCCTAGATCTGGTTTTGTA ATGATTTATACAGAAATAAACACACCTAAGCCC

SEQiD Q; 3: CH069319.1

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNMNNMNNN CAACCTCAAGG AAGGG ATCAAT I H i ! iCGAAATAANNNNNNNNNNNNNNNNNNNNNNNNNNNNN N ^ ^ Nhi N ^ ^ Nhi N N A AG AACCTCTG CA AG CACCAG G G AG G CCTG TTCAT N N AAGACATCGAGG ATT TAGATGGAAGGTCCGTTAAATNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^^

AACCCTCCTAACCCCTGGGACTCTGATCCCAGG N NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNN N NNTGCCTGCATGGACCTCAAGTTGGGAGACAAGCGAATGGTATTTGACCCTTGGTTAATT GGCCNN N NNNNNNNN NNNNNNNNNNNN N NGCTACATGAGCCTCCATCTGACTGGTTGGAGAGGCTGTGCAAAGCAGACCTCATTTATA TCAGCCAC ATGCACTCAGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNG GCGTTCATCCTG AG ATGGACACATGCATTATCGNNNNNNNCAAAGGTCATAAAATACTCAACACAGTGGACTGC ACCAGACCCAATGGGGGAAGG CTTCCTNNNNNNNNNNNNNNNNNNNNNN^

GGAAAATTTACTG AGGAATGGAAAGCCCAN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

N N N N N GACCCGATTGCTGCGGGACCCTGATATATACCATCATCTGTTTTGGAATCATTTTCAGAT AAAACTCCCTCTAAC

ACCACCCAACTGGAAGTCGTTCCTAATGCACTGTGATTAGTCTGGACCTGGTAAGTC CCAGGACCCCAGCCCAGAGGATGGTG

CCTGAACATTCAAGATGGGTCTCCCCTGCTTCGATAAACC1TTC GGAAACACTTTCATAGACACAGCCATAACTACCATTATTC

ATTGGCAGTGTCTTTTATTCAGAGATGGCAAACAATGCCCCCAAATCAAGAATTTGC TCTTAAATCTTGAACANN N NMNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN GTGGCTTGGTAGTATAGC

ATGTGCTTAGAGCATATGAGGCCCTGGGTTCCAACACACACACACACACACACACAC ACACACACACACACACACACCTGTAT

AACCCTAAATTCAN hi N ^ ^ Nhi N N ^ N N N ACCAAGG AAAGCCAAGAATG AG Nhi N ^N ^ N

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

GCGGCTCCCCACATN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN N NCATAAGTTCTGTGACTCTAG AGAACCCTGACTAATACAN N N N N N N NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

N ^ ^ Nhi N ^ ^ Nhi N ^N ^ Nhi N N ^ Nhi NCAAATTTGAAATACACTTTATTAAGAATAAN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNMN NTAATCTTAATTAACAATATATGGGTGAGTAGAAAATCTTAAATTTN NN NNNMNNN NNNNNNMNNNNNNiNiN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NTTCCTCCCTCATG ATG CAGTTTTN NNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNN

SEQ!D NO: 4: CH052173.1

NNNNNNNNNNNNNNNNNNNNNNTCTCCTGTGTGCCTGAGACCTNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACCCAGTAGG AACGATGTTCTTGTCTTGA

CTCCTTGGCTGGCTCCTATCATCTGGGANNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNN NTTTGCTATCAAAAAGTATGTGGTGTTCCTTAAGCTGTTCCTN NNNNNNNNNNNNNN

N CTTCATGGTGGGACACAAGGTCACCTACTATGTCTTCACTGACCGTCCAGCCGATGTGCC N NNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCGATGGCAGGATGTGT CCATGCACAGGATGGAGAT

GATCAGCCGC TCTCAGAGCGACGNTTTCTACGTGAGGTGGATTACCTGGTGTGTGCAGATGTGGACATGA AGTTCAGTGAC

CACGTGGGTGTGGAGATTCTCTCANNNNNNNNNNNNNNNCTGCACCCTGGCTTCTAC AGTAGCAGCCGAGAGGCCTTTACC

TATGAGCGCCGGCN N NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NTGGGGGGTCAGTGCTAGAAGTGTACCATCTCACCAAGGCCTGCCATGAAGCTATGA TGGAGGACAAGGCCAACGGCATTG

AG CCTGTGTG G CATG ATG AG AG CTATTTG AACAA ATACCTG CTTTACC ATAAG CC AACAAAG GTG CTGTCCCCAG AGTATGTG

TGGGACCAGAAGCTGCTGGGCTGGCCCTCCATCATGAAGAAGTTGAGATATGTGGCT GTACCCAAGAACCATCAGGCAATCA

GAAACTAATAGCTAAATTCCTATTGG AGAGGACAGG NNNN N NNNNNNNNN NNNNNNNNNN N N N N

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN CCCACCTCAG AGTCCAG CAG ACCCAGTATCACTTG GCTTACCACACACT

GAGCCCTGGAGTGGCAACCACCTTCAGCTCCNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNN

SEQID N0: 5: CH073348.1

NNNNNNNNNNNNNNNNCACTCCGCCTTCCTCTCGGCCGCACGCTCACCCGCCNNNNNNNN NNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNN NTACTTGGCAGAATTCCTGTTGGAGAAAGGGTACGAGGTCCATGGAATTGTACGGCG

ATCCAGTTCATTTAATACAGGTCGAATTGAACATTTATATAAGAATCCACAGGCTCA TATTGAAGGAAACATGAAGTTGCACTA

TGGTGACCTCACCGACAGCACCTGCCTAGTAAAAATCATCAATGAAGTCAAACCTAC AGAGATCTACAATCTTGGTGCCCAGA

GCCATGTCAAGATTTCCTTTGACTTAGCAGAGTACACTGCAGATGTTGATGGAGTTG GCACCTTGCGGCTTCTGGATGCAATT

AAGACTTGTGGCCTTATAAATTCTGTGAAGTTCTACCAGGCCTCAACTAGTGAACTG TATGGAAAAGTGCAAGAAATACCCCA

GAAAGAGACCACCCCTTTCTATCCAAGGTCGCCCTATGGAGCAGCCAAACTTTATGC CTATTGGATTGTAGTGAACTTTCGAGA

GGCTTATAATCTCTTTGCGGTGAACGGCATTCTCTTCAATCATGAGAGTCCTAGAAG AGGAGCTAATTTTGTTACTCGAAAAAT

TAGCCGGTCAGTAGCTAAGAT1TACCTTGGACAACTGGAATGTTTCAGTTTGGGAAA TCTGGACGCCAAACGAGACTGGGGC

CATGCCAAGGACTATGTCGAGGCTATGTGGCTGATGTTACAAAATGATGAACCAGAG GACTTTGTCATAGCTACTGGGGAAG

TTCATAGTGTCCGTGAATTTGTTGAGAAATCATTCATGCACATTGGAAAGACCATTG TGTGGGAAGGAAAGAATGAAAATGA

AGTGGGCAGATGTAAAGAGACCGGCAAAATTCATGTGACTGTGGATCTGAAATACTA CCGACCAACTGAAGTGGACTTCCTG

CAGGGAGACTGCTCCAAGGCGCAGCAGAAACTGAACTGGAAGCCCCGCGTTGCCTTT GACGAGCTGGTGAGGGAGATGGTG

CAAGCCGATGTGGAGCTCATGAGAACCAACCCCAACGCCTGAGCACCTCTACAAAAA TTCGCGAGACATGGACTATGGTGCA

GAGCCAGCCAACCAGAGTCCAGCCACTCCTGAGACCATCGACCATAAACCCTCGACT GCCTGTGTCGTCCCCACAGCTGGGCC

ACAGGTTTGTGGGCACCAGGACGGGGACACTCCAGAGCTAAGGCCACTTCGCTTTTG TCAAAGGCTCCTCTGAATGATTTTGG

GAAATCAAGAAGmAAAATCACATACTCATTITACTTGAAAmTGTCA AGACAACTTAAATTmGAGTCTTGAGATTGT^

TTTCTCTTTTCTTATTAAATGATCTTTCTATGAACCA

SEQ!D 0: 6: CHO73003.1

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

N NGTGCAGCAACAATGGGTGAGCCCCAGGGATCCAGGAGGATCCTAGTGACAGGGGGCTCT GGACTGGTGGGCAGA

GCTATCCAGAAGGTGGTCGCAGATGGCGCTGGCTTACCCGGAGAGGAATGGGTGTTT GTCTCCTCCAAAGATGCAGATCTGA

CGGATGCAGCACAAACCCAAGCCCTGTTCCAGAAGGTACAGCCCACCCATGTCATCC ATCTTGCTGCAATGGTAGGAGGCCTT

TTCCGGAATATCAAATACAACTTGGATTTCTGGAGGAAGAATGTGCACATCAATGAC AACGTCCTGCACTCAGCTTTCGAGGT

GGGCACTCGCAAGGTGGTCTCCTGCCTGTCCACCTGTATCTTCCCTGACAAGACCAC CTATCCTATTGATGAAA CAATG ATCCA

CAATGGTCCACCCCACAGCAGCAATTTTGGGTACTCGTATGCCAAGAGGATGATTGA CGTGCAGAACAGGGCCTACTTCCAGC

AGCATGGCTGCACCTTCACTGCTGTCATCCCTACCAATGTCTTTGGACCTCATGACA ACTTCAACATTGAAGATGGCCATGTGC

TGCCTGGCCTCATCCATAAGGTGCATCTGGCCAAGAGTAATGGTTCAGCCTTGACTG TTTGGGGTACAGGGAAACCACGGAG

GCAGTTCATCTACTCACTGGACCTAGCCCGGCTCTTCATCTGGGTCCTGCGGGAGTA CAATGAAGTTGAGCCCATCATCCTCTC

AGTGGGCGAGGAAGATGAAGTCTCCATTAAGGAGGCAGCTGAGGCTGTAGTGGAGGC CATGGACTTCTGTGGGGAAGTCAC

TTTTGATTCAACAAAGTCAGATGGGCAGTATAAGAAGACAGCCAGCAATGGCAAGCT TCGGGCCTACTTGCCTGATTTCCGTT

TCACACCCTTCAAGCAGGCTGTGAAGGAGACCTGTGCCTGGTTCACCGACAACTATG AGCAGGCCCGGAAGTGAAGCATGGG

ACAAGCGGGTGCTCAGCTGGCAATGCCCAGTCAGTAGGCTGCAGTCTCATCATTTGC TTGTCAAGAACTGAGGACAGTATCCA

GCAACCTGAGCCACATGCTGGTCTCTCTGCCAGGGGGCTTCATGCAGCCATCCAGTA GGGCCCATGTCCATCCTTGGGGAAAG

GCCAGACCAACATTTTGCITGTCTGCTTCTGCCCAACCTCCATGTGTCCCTGCTGGN CTAGAAACCAATAAAATGGATTTTCAT

GAAAAAAAAAAAAAAAAAA SEQID O: 7: CH063353.1

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN N NGACGGGCCCCTCTG AAGCGGTCCAGGATCCTGCGCA

TGGCGCTGACTGGAGGCTCTGCTGTCTCCGAGGAGTCAGAGAGCGGGAACAAGCCAT TTCTGCTCCGGGCGCTGCAGATCGC

GCTGGTCGTCTCTCTCTACTGGGTCACCTCCATCTCCATGGTATTCCTCAACAAGTA CCTGCTGGACAGCCCCTCCCTGCAGCTG

GATACCCCTATCTTTGTCACTTTCTACCAATGCCTGGTGACCTCTCTGCTGTGCAAG GGCCTCAGCACTCTGGCCACCTGCTGCC

CTGGCATGGTAGACTTCCCCACCCTGAACCTGGACCTTAAGGTGGCCCGAAGTGTGC TGCCGCTGTCAGTGGTCTTTATCGGC

ATGATAAGTTTCAATAACCTCTGCCTCAAGTACGTAGGGGTGGCCTTCTACAACGTG GGGCGCTCGCTCACCACCGTGTTCAA

TGTTCITCTGTCC ACCTGCTGCTCAAACAGACCAGTCGTCTATGCCCTGCTCACATGTGGCATCATCATTGGT GGTTTCTGG

CTGGGTATAGACCAAGAGGGAGCTGAGGGAACCNNNNNNNNNNNNGGCACCATCTTC GGGGTGCTGGCCAGCCTCTGCGT

CTCCCTC AATG CC ATCTATACCAAG AAG G TG CTCCCAG CAGTG G ACAAC AG CATCTG G CG CCTAACCTTCTATAACA ATGTCAA

TGCCTGTGTGCTCTTCTTGCCCCTGATGATAGTGCTGGGCGAGCTCCGTGCCCTCCT GGCCTTCACTCATCTGTACAGTGCCCA

CTTCTGGCTCATGATGACGCTGGGTGGCCTCTTCGGCTTTGCCATTGGCTATGTGAC AGGACTGCAGATCAAATTCACCAGTCC

CCTGACCCACAATGTATCAGGCACAGCCAAGGCCTGTGCGCAGACAGTGCTGGCCGT GCTCTA ATGAAGAGACTAAGAGC

TTCCTGTGGTGGACAAGCAACCTGATGGTGCTGGGTGGCTCCTCAGCCTATACCTGG GTCAGGGGCTGGGAGATGCAGAAGA

CCCAAGAGGACCCCAGCTCCAAAGAGGGTGAGAAGAGTGCTATTGGGGTGTGAGCTT CTTCAGGGACCTGGGACTGAACCC

AAGTGGGGCCTACACAGCACTGAAGGCTTCCCATGGAGCTAGCCAGTGTGGCCCTGA GCAATACTGTTTACATCCTCCTTGGA

ATATGATCTAAGAGGAGCCAGGGTCTTTCCTGGTAATGTCAGAAAGCTGCCAAATCT CCTGTCTGCCCCATCTTGTTTTGGGAA

AACCCTACCAGGAATGGCACCCCTACCTGCCTCCTCCTAGAGCCTGTCTACCTCCAT ATCATCTCTGGGGTTGGGACCAGCTGC

AGCCTTAAGGGGCTGGATTGATGAAGTGATGTCTTCTACACAAGGGAGATGGGTTGT GATCCCACTATAGCTAGTGAAGGGA

GGGTAACCCCACATCTCTGGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

N ^ ^ Nhi N ^N ^ Nhi Nhi N ^ Nhi Nhi N ^N N AT G G CCATTCTG CCCTCTTCTGTGTG G ATG G GT A

TGNN NN NNNi\iNN!\iNNN NNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCCCAACACCTCCATCTGCAGGCAG GAAGTGGAGTCCACTNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN

NNNhiNNhiNNN^NN N NCTGTCCATATCCTCATGCTGCAGAAGTACAGGCAAGCTCCTTTAAGCCTCATATAGGAA CACTAGC

CTCACTCATGAGGGTTTTACTCCATGACCTGTCAACCTCAAAGCC1TCAACATGAGG ACTCCAACGTAAATTTGGGGACAGAA

GCACTCAGACCATAACCCCAGCACCACACCCTCCTAACCTCAGGGTAGCTGTCATTC TCCTAGTCTCCTCTCTTGGGCCTTTAGA

ACCCCCATTTCCTTGGGGTAATGTCTGATGTTITTGTCCCTGTCATAAAAGATGGAG AGACTGTGTCCAGCCTTTGATTCCTACT

TCCTACAATCCCAGGTTCTAATGAAGTTTGTGGGGCCTGATGCCCTGAGTTGTATGT GATTTAATAATAAAAAAAATATAATAA

AAAAAAAAAAAAAAAAAAAAAA

SEQID 0: 8: CHO73401.1

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNN CTGCGTC

CAGCTAACCATGGGACAGTTTGGCTCTGGCCATCAGCCCCTTAGCCACCTTCAAGAG AAGCTCCTGTTCCTGCTGGCCCACGG

ATGGGGAGGTGGGCCCTGGACGTCGCCTTTGTGTGGAAGGCAGCTCTGACCCTGGGC CTGGTCCTCCTCTACTACTGCrrCTC

TATAGGCATCACCTTCTACAACAAATGGCTGACAAAGAGCITCCACTTTCCCCTCTT CATGACCATGCTGCACCTGGCCGTGAT

CTTCCTCTTCTCAGCCCTGTCCAGGGCGCTGGTTCAGTGCTCCAGCCACAGGGCCCG CGTGGTGCTCAGCTGGACTGATTACCT

CAGGAGAGTGGCCCCCACAGCACTGGCAACAGCACTTGATGTGGGCTTGTCCAACTG GAGCTTTCTTTACATCACTGTGTCAC

TGTACACAATGACCAAATCATCTGCCGTGCTCTTCATCCTGATCTTCTCTCTGATCT TCAAGCTGGAGGAGCTGCGTGCAGCCC

TGGTCCTGGTGGTCCTTCTCATCGCTGGGGGCCTCTTCATGTTTACCTATAAGTCCA CACAGTTCAACGTGGAGGGCTTTGCCT

TAGTGCTGGGGGCGTCGTTCATCGGTGGCATCCGCTGGACCCTTACACAGATGCTTC TGCAGAAAGCTGACCTGGGCCTTCAG

AACCCTATCGACACCATGTTCCACCTGCAGCCGCTCATGTTTCTGGGTCTCITCCCG CTCTTTGCCGTATTTGAAGGTCTCCATTT

GTCCACCTCAGAGAAAATCTTCCGCTTCCAAGATGCAGGGCTGCTCCTGCGGGTTCT TGGGAGCCTCTTCCTTGGCGGGATTCT AGCCnTGGTCTGGGCTTCTCTGAGTTCCTCCTTGTGTCCAGAACATCCAGCCTCACTCTC TCCATCGCCGGCATCTTTAAGGAA GTCTGCACCCTGCTGTTGGCGGCTCACCTGCTGGGTGATCAGATCAGCCTCCTGAACTGG CTGGGCTTTGCCCTCTGCCTCTCT GGCATCTCCCTGCATGTGGCCCTCAAGGCCCTGC^^

GCGCTGACCTCGAGCTGCTGCTCCAGAGCAGCCAGCGGGAGGAAGAAGACGATGATTATT TTGTGACCTAGGGGCAACAAT GACCAGTCCGGG AGCAGCAGAAGCAGGCCGCACTGCTGGCAACTCTTGGTGG N NNMNNN NNNNNNMNNN NNiNiNNNM NNN^NN^NNNhiNNNNNN^NNNNNNhiNNN^NN^NNNhiNNNNNNi^NNNNNNhi G G AGTCG AG GCTCCTG CTCTTTCCT GTGGTGCTTGGTCCAAAAAGTGAATGACAGGCTCACCAGGAGCTGAACTGGCAGTGAAGG GAGCCGGTGACCTCTTCTCCAC TGTCACTCTGGCTCTGAGAGGCAGATAAATTTGACXJTGTGGTAGATCTCAGGTTATTGG GTTCAGACTGCAGGGCCTCTGGAA GGAGTCACGGAGAGACTGGGCCTCATCACCTGGACCGCAGCTrCCTCAGAGAGTAGATTT ATTTATAGTTGAAGTAATGATTT CCTCCTCCACTGCCCAGTGTTGCCTGAGGCATGAGCACAAGGCAAGCCCTGGGGCCTGGC CTCTCCTCCGTTCCCTiNi NNNMN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN ATGCTCCTGTTTGTGCCCCAG ACCCCATCCTTGGGGCCACACTATGG ACCCACCN NNNN NAATAAATGGTGTTTGCTCAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAA

SEQ!D 0: 9: CH055897.1

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

N NTTTTTGTATTTTTGTTTATTATAGACATTTTTATTTTATTTTTATTTTTTGTA NNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNN NN NNNNNNNNNN NN NNNNNNN NN NNNNNNNNNN NNNNNNNNNN NNN NNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNN N NNNNNNNNN NNNNNNNNN N NNNNNN NN NNNNNNNNN NNN NNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNN N NNNNNNNNN NNNNNNNNN N NNNNNN NN NNNNNNNNN NNN NNNNNNNNNNNNNNNNNNN NN NNNNNNNNNN NN NNNNNNN NN NNNNNNNNNN NNN

SEQ!D NO: 10: CHO71860.1

NNNNNNNNNNNNNNN N NGCCCTCGCCGCCATTTCAAGACAGGGAGAGGTAGATGTCTAN NNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN

NNTAGAACCATTCTTGCCAGTAGTGGCGGCAGTAGAAGTCAATTACCCCTTGTTCTA TATCAAGAAGCTCCAGATGGCGTCTT

CCGTGGGCAACGTGGCCGACAGCACAGAACCAACGAAACGTATGCTTTCCTTCCAAG GGTTAGCTGAGTTGGCACATCGAGA

ATATCAGGCAGGAGATTTTGAGGCAGCTGAGAGACACTGCATGCAGCTCTGGAGACA AGAGCCTGACAATACTGGTGTCCTT

TTATTACTTTCATCTATACATTTCCAGTGTCGAAGGCTGGACAGATCTGCTCAGTTA GCACCTTGGCAATTAAACAGAATCCCC

TTCTAGCAGAAGCCTATTCGAATTTGGGGAATGTGTACAAGGAAAGAGGGCAGTTGC AGGAAGCGATTGAGCATTATCGACA

TGCCTTGCGGCTCAAGCCTGATTTCATTGATGGTTATATTAACCTGGCAGCAGCTTT GGTAGCAGCAGGTGACATGGAAGGAG

CAGTACAAGC TACGTC CTGC C1TCAGTACAATCCTGAT1TGTACTGTGTTCGCAGTGACCTGGGGAACCTGCTCAAAGCC C

TGGGTCGCTTGGAAGAAGCCAAGGCATGTTATTTGAAAGCAATTGAGACGCAACCAA ACTTTGCAGTAGCTTGGAGTAATCTC

GGCTGTGTTTTCAATGCACAAGGGGAGATTTGGCTGGCAATTCATCACTTTGAAAAG GCTGTCACCCTTGACCCCAATTTTCTG

GATGC rATATCAATTTAGGAAATGTCTTGAAAGAGGCACGCATTmGACAGAGCTGTGGCAGCmTm CGTGCCTTAAGT

TTGAGCCCAAATCACGCTGTGGTGCANGGCAACCTGGCTTGTGTATACTATGAGCAA GGCCTAATAGACTTGGCCATCGATAC

CTACAGGAGAN NNNN NGAACTGCAGCCACATTTCCCCGATGCTTACTGCAACCTAGCAAATGCTCTCAAAGAGAA GGGCAGC

GTTGCTGAAGCAGAAGATTGTTATAACACAGCTC TCGTCTGTGTCCTACCCATGCAGACTCTTTGAATAACCTTGCCAATATC

AAACGGGAACAGGGCAATATTGAAGAGGCAGTCCGCCTATATCGCAAAGCATTAGAA GTCTTCCCAGAGTTTGCTGCTGCAC

ATTCCAATTTAGCAAGTGTACTGCAACAACAGGGCAAGCTGCAGGAAGCACTGATGC ATTATAAGGAAGCCATAAGAATCAG

TCCTACATTTGCTGATGCTTACTCTAATATGGGAAACACTCTAAAGGAGATGCAAGA TGTTCANGGAGCTTTGCAGTGTTATAC

TCGTGCCATTCAGATTAATCCTGCCTTTGCAGATGCACACAGCAATCTGGCTTCCAT TCACAAGGATTCAGGGAATATCCCAGA

AG CAATAG CTTCTTACCG CACAG CTCTG AAACTTAAG CCTG ACTTTCCTG ATG CCTATTGTAACTTG G CTCATTG CTTG C AG ATT

GTCTGTGATTGGACAGACTATGATGAGCGAATGAAGAAATTGGTTAGTATTGTAGCT GAACAGCTAGAGAAGAATAGGCTGC

CITCTGTCCATCCTCAN AGTATGCTGTACCCTCTTTCTCATGGCTTCAGGAAGGCTATTGCAGAGAGGCACGGGAAT CTC

TGCTTGGATAAGATTAATGTCCTTCATAAACCACCATATGAACATCCAAAAGACTTG AAACTCAGTGATGGACGATTGCGTGT

NNNNTATGTGAGTTCTGACmGGGAACCACCCTACTTCTCACCTTATGCAGTCTATTC CAGGCATGCACAATCCTGATAAGTT

TGAGGTGTTCTGCTATGCCCTGAGCCCGGATGATGGTACAAACTTCCGAGTGAAGGT GATGGCAGAAGCCAATCATTTCATTG

ATCTTTCTCAGATTCCATGCAATGGAAAAGCAGCNGACCGCATCCACCAGGATGGAA TTCACATCCTTGTAAATATGAATGGG

TATACCAAGGGTGCTCGAAATGAGCTGTTTGCTCTTAGGCCAGCTCCTATTCAGGCG ATGTGGCTGGGNTACCCTGGAACTAG

TGGTGCACTGTTCATGGATTATATCATCACTGATCAGGAAACTTCCCCAGCTGAAGT TGCAGAGCAGTATTCTGAAAAACTGG

CTTACATG CCCCA NNNNNNNNNNNNNNNNNNNNNNNNN AAC ATGTTCCCTCACCTAAAG AAAA AAG CAGTCATCG ATTTT

AAATCCAATGGGCACATTTATGACAATCGGATAGTCCTGAATGGCATCGATCTCAAA GCATTTCTTGATAGTCTACCAGATGTG AAGATTGTCAAGATGAAATGTCCTGATGGAGGTGACAATGCAGACAGCAGTAACACAGCT CTTAATATGCCTGTCATTCCCAT

GAACACGATTGCAGAAGCAGTTATTGAAATGATTAACAGAGGACAGATTCAGATAAC AATTAATGGGTTCAGTATTAGCAAT

GGACTGGCAACTACACAGATCAATAATAAGGCTGCAACCGGAGAGGAGGTTCCCCGT ACCATTATTGTAACCACCCGTTCCCA

GTACGGGCTACCAGAAGATGCCATTGTGTACTGTAACTTTAATCAGTTATATAAAAT TGACCCTTCTACCCTACAGATGTGGGC

AAATATTCTGAAACGTGTACCTAATAGCGTGCTTTGGCTGTTGCGTTTTCCAGCAGT AGGAGAACCTAATATTCAACAATATGC

ACAAAATATGGGCCTTCCCCAGAACCGTATAATTTTTTCACCCGTGGCTCCTAAAGA GGAGCATGTCAGGAGAGGTCAGCTGG

CTGATGTCTGCCTGGACACTCCCTTGTGTAATGGGCACACTACAGGGATGGATGTTC TCTGGGCAGGAACACCCATGGTGACT

ATGCCAGGAGAGACTCTGGCCTCTCGGGTTGCAGCTTCTCAGCTTACTTGCCTAGGA TGTCTTGAGCTCATTGCTAAAAGCAG

ACAAGAATATGAAGACATAGCTGTGAAATTGGGAACCGATTTAGAATACCTGAAGAA AATTCGTGGCAAAGTTTGGAAACAG

AGAATATCTAGCCCTTTGTTCAACACCAAACAATACACAATGGAATTAGAGCGACTC TATCTGCAGATGTGGGAGCATTATGC

AGCTGGCAACAAACCTGACCACATGATTAAGCCTGTTGAAGTCACTGAGTCAGCCTG AATAAAGACTGCGCACAGGAGAATT

N N N N N NN N N N N N N NN NTCAACCTTCTGGGGG AAAGGGAACTAGATAACATGCTTTGTGTGTATCTGTGTAGTTCTGTGTTG

CAGACGGATGATATATAATGATAATAGAATAGCACATTCAGACTTGCTTCCTGCATG ATGGGGAGAGACAAGAAAGAAGAAA

TGCTATTCCACAAGGAATCTCTTAGAGTTnGCAGCAAACAGATGGTGCAGAGGTCTG GAAGGTCTGGTCTCCCTTGGTCTTC

CACGGGATGCTTAATGTGGAGGGGAGATAGAGATTAACCAGCCGTTTTGTGATGCCG TGGATTGATCGAGTCTTCTGATCCTT

TTTTTTT7T7TTATATTTTGGGTATTG

CTGAGGTTTTAAACTAAAATGTTGCTTCCTGTTTTAGTGTCTGAACTCTGACAGGGGACA GGGACCTTGCT

TATAGGTTTTATAAACCACTTGAGCCTATATCAGTCATTTTAGTGTTTGACCTAATG CTTGGCACTGTCAGTGCm

GATGACTTAAGAGTTTCAGCCTGTTTTACACACCCCAGCTCCCGTTAAATTCTCCTGTGA CTGCTAACAAACCCATGTCTGCTT

TCAGACATCCCAGAGTGTTTCTCTTGAACACGCCATCTGGTGCCAAATGAAAATTCT TAGGAGTGAATATTAATCATGAAGGG

CACAGTTGTGGTACTGTTGTTGATAATAATTAGTTCTTTGTCTAATTTTTACCTATT TCACCAGTGTTTAAGCCCTTGACTGCCCC

TCCTATGCTGCTTCCAAAAGTGGTAGTGTGCTAAGATTTTT^

CTTCCTATTTCTTTCAGCAGAAATGAAATCCCAGGTAAGTATATAAGTATTCAAATGTTT GGTTAGTAAATTACAGTTCTCTCCG TACCTTAAATGGTGTTCACN N N N N NN N N N N N N NN N N N N N N N NN N N N N N N N NN N N N N N N N NN N N N N N N N NN N N N N N N N NTCTGGACITAGCTTCAAACTCTATGGATTCGGCCTTGGTAGGGAATTGTATTACTACIT GGTTTAGAGTACAAGTTAGGC TTTATTTACCCTCCTGGAGCTTGAATTCTTGGTGGTTAAAAAAATGCATTTCAACATAGA GGGCTTGAGGCTTAAACAAAC CCGACATATGACTCTGTTTGTACTGCTCTACTGCACTTGGACTCT^

TATCTTTCCAAAGCACCTAGGCAGTGCACCTATTCTGTAGTTGCAGCTCTTGCCTGAATG TACCTTAGCTGACAAGTTATTGATA

ATTAAGACCTTGAATTTGTGATTCTTACTGCTATTTAAAACCTAAGCAAGGAGTCTC ATGTGTGATTCTGAAGCAGAATTACAT

TATAATGAACTGTGTACCmAGTAATGTAAATCAAGGAACACCAGGGAmC rAAmATTTTTAACTGAATAAATATCAGTT

AAAG GTTG CCAG CATG GTTACAGGTAAACTG ATTTTCAAAATTCACTG AACTACTAAATGTGGG CTTG G ATAATAG ACCTCCA

GTGCCGCTATGGCTGTGATCTGTGGCCATITTACATAGCACATCAITCCTCCTATAG GGATGAACTITTTTCCCGGCACGAAAA

GTAGCCACTCTGGTTGTAGCTTTGCTTATTGTAACAGGCTTTTATTTCCAGGTAACA TGTCTGTGGAAGACTTAATTCAGATAG

TTATAGATATTATTGGAAACTAATTTGTGTTTTTTTTCTATTATACTC GCTTTATTGAAAAAGTAAAGCATTTAAATTGTGCTCC

AGATATTCAGATGTTGTCTTGCGATCTTTAAACAATAAATGGATGATTTCCCCTTAA

SEQ ID NO: 11: CH064621.1

GATTGGTCCGCCCCGAGGGTAAACTTAAAAGCCCAGCTTAACTCGGTAAACTGAAGCAAA GAATTTCTATGACCATGAAGCTT

TTA AGTG GGGATCCGCGG CTG GTCTGTG AG CTTG CG CTTCGTCCCCTAGTCTTAGTTTTCTG G AGTATTCTTG G G ACCAAAG CA

CTGGACAACGGCTTGGCGCGGACTCCTACTATGGGCTGGCTGCACTGGGAACGTTTC ATGTGCAACCTCGACTGCCAAGAAG

AGCCTGATGCCTGCATCAGTGAGCAGCTGTTCATGCAGATGGCAGACCTTATGGTCT CTGAGGGCTGGAAGGACGCAGGTTA

TGAATACCTCTGCATAGATGACTGTTGGATGGCTCCTGAAAGGGACTCAAAGGGCCG AOTCAGGCAGATCCCCAGCGCTfTC

CTAGTG G AATCCAG CACCTAG CT AATTATGTCCACAG CAAAG G ATTG AAG CTAG G AATTTATG CAG ATGTTG G G AAT AA AACC

TGTG CAG GTTTCCCTG G G AGTTTTG G GTACTATG ATATTG ATG CCCAG ACATTTG CTG ACTG G G GTGTAG ATCTG CTAAAATTT

GATGGTTGTCACTGTGACAGTGTGACGTCCTTGGCAGATGGTTATAAGTACATGTCC TTGGCCTTGAATAGGACTGGCAGAAG

CATTGTATACTCCTGTGAGTGGCCGCTTTATATGAGACCCTTTCATAAGCCCAATTA TACAGATATCCAATATTATTGCAATTAC

TGGAGAAATTATGATGATATTTATGATTCCTGGGAAAGCATAAAGAATATCTTGGCC TGGACAACAGATAACCAGAAGGAGA

TTGTTGAAGTCGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGATTGGCA ACTTTGGCCTGAGTTGGGACCAGCA GGTAACACAGATGGCCCTCTGGGCTATCATGGCAGCTCCTCTACTCATGTCCAATGATCT CCGACAAATCAGCTCCCAAGCCAA

AGCTCTCCTTCAGAATGAAGATGTAATTGCTGTCAACCAAGACCCCTTGGGCAAGCA GGGGTACTGTTTTAGAAAGGAAAACA

AAATCGAAGTTTGGGAACGACCACTCTCAAATTTAGCCTGGGCTGTGGCTGTGAGAA ACCTGCAGGAAATTGGTGGACCTCG

TTCTTACACCATCCAGGTGGC TCCCTTGGTCAAGGAATAGCCTGCAATCCTGGTTGCATCATCACACAGCTCCTCCCTGAG AA

AGTACAGCTAGGCTTCTATGAATGGACCGTAACCTTAAAAACTCGAATAAATCCCTC AGGCACTGTTCTGCTTCGACTAGAAA

GATAAACTANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN

ΝΝΝί\ΙΝΝί\ΙΝΝΝ ΝΝΝί\ΙΝΝί\Ι^^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN N NCAAGACAGGGTTTCTCCATATAGCCCTGGCTGTCCTGG AACT

CACTCTGTAGACCAGGATGGCClTGAATTTAGNNNNNCGCCTGCCrCAGCCTCCAAA GTGCTGGGATTAAAGGCCTGCGCCA

CCAACGCCCAGCCrnTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNG CATTG GTAG CACACG CCTTT AATCCTAG CACTTAG G AG G CA GAGGCAGCCGGNNNNNNTCTGAATTTGAGGCCAGCCTGGTCTACAGAGTTCCAGGACAGC CAGGGCTATACAGAGAAACCC TGTCTCAG G G G AAAAAAA N NNNNNNNNNNNNNNNNNN N N CAG CG GTTAAG AG G ATTG ACTG CTCTTCCAG AG G TCATG A GTTCAAGTCCCAGCAACCACATGGTGGCTCACAATAATCTGTAATGAGATCTGATGCCCT CTTCTGNN NNNNNNNNNNNNN ΝΝΝί\ΙΝΝί\ΙΝΝΝ ΝΝΝί\ΙΝΝί\Ι^^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN SEQ!D NO: 12: CH053916.1

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN N NG ACTACAGCCGGG ACCGCTTCCTCAAGGATGGACAGCCATTCC

GGTACATCTCTGGAAGCATTCATTACTTCCGGATACCTCGCTTCTATTGGGAGGACC GGCTGCTGAAGATGAAGATGGCTGGG

CTGAACGCCATCCAGATGTACGTGCCCTGGAACTTTCATGAACCCCAGCCAGGACAA TATGAGTTTTCTGGGGACCGTGACGT

GGAATATTTCATTCACCTGGCTCACAAGCTGGGGCTCCTGGTGATCCTGAGGCCAGG ACCCTACATCTGTGCAGAGTGGGACA

TGGGAGGGTTACCTGCTTGGCTGCTAGAGAAAGAATCTATTGTTCTTCGATCTTCTG ACCCAGACTACCTTGCAGCTGTGGATA

AATGGCTGACAGTCCTTCTGCCCAAGATGAAGCCTCTGCTCTACCAGAATGGAGGGC CGATTATAACTGTGCAGGTTGAGAAT

GAGTACGGCTCCTACTTTGCCTGTGATTATGACTACCTGCGTTTCCTGGCACACCGC TTCCGTTACCATCTGGGCAATGACGTC

CTTCTCTTCACCACCGATGGGGCGAATGAAAACTTCCTTCGGTGTGGCACCCTGCAG GGCCT ATGCCACAGTGGATTTTGG

AGCAGTCAAGAATATCACACAGGCTTTCCTAATCCAGAGGAAATTCGAACCCAAAGG ACCTTTGATCAATTCTGAGTTCTATAC

TGGCTGGCTAGACCACTGGGGTGAACCCCATTACACAGTGAAGACTGAAATAGTGGC TGCCTCTCTCTATGACCTGCTTGCCC

GTGGGGCCAGTGTGAACTTGTACATGTTTATAGGTGGGACCAATTTTGCCTATTGGA ATGGTGCCAACATTCCCTATGCAGCA

CAGCCCACCAGCTATGACTACGACGCCCCGCTGAGTGAGGCAGGGGACCTGACTGAG AAGTATTTTGCTCTTCGAAATGTCAT

CCAGAAGTTTAAAGATGTCCCAAAAGGTCCTATCCCTCCATCAACTCCCAAGTTCGC CTATGGAAAAGTTGCTCTGAGAAAGTT

CAAGACAGTGGCAGAAGCTCTGGACGTCCTGTGTCCCAATGGCCCCGTGAGAAGCCG CTACCCGCTGACATTCATCCAAGTTA

AACAGTATTTTGGGTATGTGCTGTACCGAACAACACTTCCTCAAGAATGCAATGGCT CGATGCCCATCTTCTCCTCACCCTTCA

ATGGTGTCCGTGACCGGGCTTATGTGGCTGTGGACGGGGTCCCCCAAGGAATCCTTG AGCGAAACCGTGTGATATCCCTGGA CATACAGGGGAAAGCTGGAGCCGTTCTGGACATTCTGGTGGAGAACATGGGACGTGTGAA CTATGGCAGAGGCATAAATGA

TTTTAAGGGTTTGATTTCTAACATGACTCTTAACTTCAGTATTCTCACCAATTGGAC AATCTTTCCACTGGACACTGAGGCTGCA

GTACGCACCCATCTTGGGGCCTGGGAGGCCCGTGATGAGGGCCACCACGATGGACGC TTGACCTTTGGCTCTTCGAACTTTAC

GCTCCCCACGTTTTATGTGGGCAACTTCTCCATTCCCTCGGGCATCCCAGATCTGCC GCAGGATACCTTCATTCAGTTTCCTGGA

TGGACCAAGGGTCAGGTGTGGATCAACGGCTTTAACCTTGGCCGATACTGGCCTGCA CGGGGCCCACAGGTGACCTTGTTTG

TGCCCAGGCACATTCTGACGACGTCAGCCCCAAACAACATCACGGTGCTGGAGCTGG AACGCTCACCCTGCAGTGATGGGAC

CTCAGAGCTGTGTACCGTGGAGTTTGTCGACAGGCCGATTATTGGCAGGTCCCTGGC CATCAGCGGCCCTTTTCCAGATCGGT

CTC -XXAAGACTGGTGGCTGAACCCCCTC^

TGTCTCGATACAGTGGAAGGTGGATTTATCAGGTGTGTGTATCCTACCGGTGTCCCTACA GCAGTAGAGACATTGCCTGGCCT CCCACTCCTGTGGCCACCAGGAAGGCTGAAGGGAGTGG ACATCACAGG AACACACA NNNMNNN NN NNNMNNMNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNN

SEQiD NO; 13: CH071928.1

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN N AGCAGCCCTTCCCTGGCCAAGCAGGCGCCAAC NNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNN NN? viNNNP >iNNP viNNNP viNNP viNNNP viNNNP viNNP viNNNP viNNNNNNP viNNNP viNNP viNNNP v!NNN NNNNNNNNNNNNNNNNNNN NNP viNNNP viNNP viNNNP viNNP .iNNNP viNNNP viNNP viNNNP viNNNNNNP viNNNP viNNP viNNNP viNNN NNNNNNNNNNNNNNNNNNN NNP -iNNNP m viNNNP viNNP viNNNP viNNNP viNNP -iNNNP viNNNNNNP viNNNP viNNP viNNNP viNNN NNNNNNNNNNNNNNNNNNN NNP viNNNP w •iNNNP viNNP viNNNP viNNNP viNNP viNNNP viNNNNNNP viNNNP vi NP •iNNNP vfNNN NNNNNNNNNNNNNNNNNNN NNP viNNNP viNNP viNNNP viNNP .iNNNP viNNNP viNNP viNNNP viNNNNNNP viNNNP viNNP viNNNP viNNN NNNNNNNNNNNNNNNNNNN NNP viNNNP viNNP >iNNNP viNNP viNNNP viNNNP viNNP viNNNP viNNNNNNP viNNNP viNNP viNNNP viNNN NNNNNNNNNNNNNNNNNNN NNP viNNNP MNP vi viNNP viNNNP viNNNP viNNP viNNNP viNNNNNNP viNNNP viNNP •iNNNP viNNN NNNNNNNNNNNNNNNNNNN NNP viNNNP ■iNNP viNNNP viNNP viNNNP viNNNP viNNP viNNNP viNNNNNNP viNNNP viNNP viNNNP viNNN NNNNNNNNNNNNNNNNNNN NNP viNNNP viNNP viNNNP viNNP viNNNP viNNNP viNNP viNNNP viNNNNNNP viNNNP viNNP viNNNP viNNN NNNNNNNNNNNNNNNNNNN P viNNNP viNNP viNNNP viNNP viNNNP viNNNP viNNP viNNNP viNNNNNNP viNNNP viNNP viNNNP viNNN NNNNNNNNNNNNNNNNNNN NNP viNNNP ■iNNP viNNNP viNNP viNNNP viNNNP viNNP viNNNP viNNNNNNP viNNNP viNNP viNNNP viNNN NNNNNNNNNNNNNNNNNNN NNP •iNNNP viNNP viNNNP viNNP viNNNP viNNNP vi NP viNNNP viNNNNNNP •iNNNP viNNP viNNNP viNNN NN

SEQ !D NO: 14: CH068241.1

NNN NN NNN N G CCG GAG G G CCCG G G CG CTCG C AG G CCAGTCG CG G CTTGTCAG N N N N NTCG CCG CCG GAG CG G G G CG

AGGCTGCGGCGCTCGGTGTCGCCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNGGGTGCTGCATGGA

GAGGACCGAGGCGACGCTGAGCCGCGGCTCGTAGCGGCGGGGCGGCTGCTGGAGCTG CAACTGCCAGAGAGGATGCGCGG

AGCCCGGGCGGCGCGAGGCCGTTGAGAGCCTTCGGGCCCCAGGACGCCGGGGCCCGG GATGAGTTAGCGAGGGCCGCCGC

GGGGGCCAG7TCCTAGGGCTACAGGCCAAGGCGACGGCGCCGCCCGCCCGCCCCTTC CGTGCAGAGGCCGCCAGCTGCTTTC

CGCGCCCGCGCTCCCGGCCCCGGAGACCATGAGGTTCCGCATCTACAAACGGAAGGT GCTGATCCTGACGCTCGTGGTGGCC

GCCTGCGGCTTCGTCCTCTGGAGCAGCAATGGGCGACAAAGGAAGAACGACGCCCTT GGCCCGCCGCTGCTGGACGCGGAG

CCCGTACGGGGTGCGGGCCACCTTGCTGTGTCCGTAGGCATCCGCAGGGTCTCAAAC GAATCGGCCGCTCCTCTGGTTCCCGC

GGTCCCGCGGCCCGAGGTGGACAACCTAACGCTGCGGTACCGGTCGCTGGTGTACCA GTTGAACTTCGATCAGATGCTGAGG

AACGTCGGTGATGACGGCACCTGGCGCCCCGGGGAGCTGGTGCTGGTGGTCCAGGTG CATAACCGGCCGGAATACCTCAGG

CTGCTGCTAGACTCGCTTCGCAAAGCCCAGGGTATTCAGGAAGTCCTAGTCATCTTC AGCCATGACTTCTGGTCCGCAGAGAT

CAAC AGTCTG ATCTCCAG G GTG AACTTCTG CCCG GTTCTG CAAGTGTTCTTTCCATTCAG CATTCAG CTGTACCCG AGTG AGTT

TCCGGGTAGTGACCCCAGAGACTGCCCCAGAGACCTAAAGAAGAATGCAGCTCTGAA GTTGGGGTGCATCAATGCCGAATAC

CCTGACTCCTTTGGCCATTACAGAGAGGCCAAATTCTCGCAAACCAAACATCATTGG TGGTGGAAGCTGCACTTTGTGTGGGA

GAGAGTCAGAGTTCTTCAGGATTACACTGGCCTTATACTTTTCCTGGAAGAGGACCA CTACTTAGCCCCAGACTTTTACCATGT

CTTCAAAAAGATGTGGAAGTTGAAGCAGCAGGAGTGTC GGGTGTGATGTCCTCTCCCTAGGGACCTACACTGCCAGTCGG

AGTTTCTATGGCATTGCTGACAAGGTAGATGTGAAAACTTGGAAATCCACAGAGCAC AATATGGGGTTAGCCTTGACCCGAG

ATGCCTATCAGAAGTTGATCGAGTGCACAGACACTTTCTGTACTTATGATGATTATA ACTGGGACTGGACTCTTCAATATTTGA

CTGTATCTTGTCTTCCTAAATTCTGGAAAGTCTTAGTTCCTCAAGCTCCGAGGATTT TTCATGCTGGAGA GTGGTATGCATCA

CAAGAAAACATGTAGGCCATCCACCCAGAGTGCCCAGATCGAGTCATTCTTAAATAA TAACCAACAGTACATGTTTCCAGAAA

CTCTAGTTATCAGTGAGAAATTTTCTATGGCAGCCATTTCCCCACCTAGGAAAAATG GTGGGTGGGGAGATATTAGGGACCAT

GAACTCTGTAAAAGTTATAGAAGACTGCAGTGAAAACAAAAATCACAATTGCAAAAG AAAGATGACAGTCTTCTGTTTTTTAT

ATTTCTTTCAATGGGATATACAATTGAATAAAGGTATAGGAACTGGTTTCTGCTTTA ATACACAGATTTCTTGTAACAGGTGTC

CAAATATATAGTAATCCTATTCAGTTAGTCTGAmAAAmCAAACTGAAATTrrCATTI TGGGT TGGGmTAAAATTCAG

TCTATCTGCTTAAGGGTAATGATTTGATAATTATTGATTAGAAAAGGAAATTTTATT TAAATCGCATCTGTTAATCTTTCTATCT

AAAACTTTGTATACTTTTCCACTTTCAGAACTTATTTTAAGTACAGCAAGACTTATT TAAAACTGTCACAACAGTAAAAAGTATT

ACAAGAC ATTAGAGTATTGATGGGACAAACCCAGTGTCACTATTGAC TTTAT1TGGAGGAATTGTCTGACCGG1TTAATCAC

TCAGAAGTTCCGTTTGTTAAACGCCCTGTCAGAAGAGTCATTTCAGTATTGCTGATT CCTGTGCTATTGTGTTAAGATTTGCCTG

TGCTTTCAG(X:TTCATA( ACATGATTTATGTTGGAATGTATTTGGTTAATAAGAAAGTTTAAACACTGTTTTCACCTC AATGTA

6ΑΑΑΤΑαΑ6Τ66ΤΤΪΊ Ί1Ί ^" 1Τ1ΊΊΊΤΪΊ ΤΑ6Τ6ΟΤ6ααΑΑΑΑΤΑΑΑΑΤΑαταΑΤΤΤ^αΑΤ ΑΑΑΑΤΤαα0ΤΑΑΤαΑΤΤ1Τ6αΑ6

AATCTTCTATTTGTATCAATAAAGGCATTCTAGGAGTTTTGAGAATGAAAAA SEQ!D NO: 15: CHO51082.1

GCCGGCGCCACCGCCGCCATGGCGGAAGTCCACAGACGTCAGCATGCTCCGGTTAAAGGA GAAGCCCCCGCGAAATCCTCCA

CACACAGAGATGAGGAGGAGCTGGGGATGGCGCCGGCCGAAACGCTGACGGTGTTCC TGAAGCTGCTGGCCGCCGGCTTCT

ACGGCGTGAGCTCCTTCCTCATCGTGGTGGTCAACAAGAGCGTGCTCACCAACTACA GATTTCCGTCCTCGCTATGTGTTGGG

CTTGGCCAGATGGTGGCCACGGTAGCAGTTCTCTGGGTGGGAAAGGCACTCAG i^NNNNN NAAGTTTCCTGACTTTGACAGA

AATGTACCTCGAAAGACGTTTCCACTACCTCTACTATATTTTGGGAACCAAATCACC GGACTGTTCAGCACAAAGAAACTGAAC

TTGCCCATGTTTACAGTTCTGAGAAGGTTCTCCATTCTGTTTACAATGTTTGCTGAA GGAGCTCTACTCAAGAAGACTTTTTCTT

GGGGTATTAAGATGACTGTATTTGCAATGATTATCGGAGCCTTTGTAGCTGCCAGCT CTGACrrGGCATTTGATCTGGAAGGA

TATGTTTTTATTTTGATCAATGATGTCCTGACAGCAGCAAATGGTGCATATGTGAAA CAGAAGTTAGATTCGAAAGAGCTGGG

AAAGTATGGCCTGCTCTATTACAATGCACTGTTCATGATCCTTCCCACCATGGCCAT TGCGTATTTCACAGGAGATGCACAAAA

GAATATATTAATAACTTATATTGGAATGGTCTTTGGTGGAGATTATATTTTCACATG GACAAATTTTATTGGCCTAAATATAAGT

ATTG CTG G AAG CCTG GTGTATTCTTAC ATCACTTTC ACTG AAG AG CAG CTG AG CAAACAGTC N NNNMNNN NNNNNNMNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNN N ATGCTAAC TATGTTTGAATTAATTTATATCAGACTGGAAAATGTTCTGGGTGGGTTTTAATTTATTTT T NNNNNNNNNNNNNNNNNNN N ^ ^ Nhi CTTTTCTTTCTTTTCXrTTCTTTTC^

TTCTTCTTCTTCCTCCTCCTCCTCCTTCTTCTTCTCCTCCTCCTCTTCCTCCTTCTCNNC CTCCTCCTTCTCTTCCTCCTCCTTCTCTTC CTCCTN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN N AGGG AGGAGTGATTGCCAGAGCTG GGTGGCATGGCN N NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNhiNNhiNNN^NN^NNNhiNNNNNN^NN GTCGG ACCTT AGTTG AAAG G ACACAAATG AAAG SEQ!D O: 16: CH057755..1

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN N ^ ^ Nhi N ^ NGTGACCCCGTGGAAGGCGCCGATTGTGTGGGAAGGCACTTATGACACAGCTCN NN N hiNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

GAGGTCG ACTTCCTCTTCTGCATGGACGTGG ATCAAGTCTTTCAAN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN

NNNi\iNNi\iNNN NNNNNNi\iNNN^ N CCTACTCACAT

TCTCAACCTCANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNN N NGTGGTTCTCAACCTGTGGGTCGCGACCCCTTTGGGGG NNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

SEQID NO: 17: CHO70008..1

GCTGTCAGCCTCTGCCTGGCTCGCGCCGCCTTGCGCTTTCCCTCAGTCAGTGGCGCCGAA GGCTCCGTTAAGCAGCGGCCGCG GTTCCTGT7TCCGTTTCTTCCTCTCCCTTC AGTCG GGGAGTAG CATCCTCCAC NNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNGGCGGCGGCGACGGGGGACGGCGCCGACCGCCTCGC TCCCGCCTCGGGTTGCTG CTCTGG GAGGCCATCTANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNN NNMNNMNNN NNNMNNMNNN NNNN AG ACCCAG ATTCAAG CAATAAAAC ATTTCTCTG CAATACCATGTG GTTTTCTT

CAACATCATAATTCTATGGGGAGGAAGCATGTAGGATCCATGAAGCACAGGACATTC AAGCCTCCCGCCCGCGTCACCAGGA

AGATCTCTTTGTAAGAATAACCACAGTATTCCAGAGAGATTAGCCTGTCTGAAGCAT TATGTGTTGAAGCAAAAGAAACTTATT

TTCCTGTGTGGCTAACTAGAACCAGAGTACAATGTTTCCAATTCTTTGAGCTCCAGG AAGATAGAACAGAGTTGAAACTCTGA

AAATGCGGGCATGGACTGGTTCCTGGCGTTGGATTATGCTCATTCTTTTTGCCTGGG GGACCTTATTGTTTTATATAGGTGGTC

ATTTGGTTCGAGATAATGACCACCCTGACCATTCTAGCAGAGAACTCTCCAAGATTC TTGCAAAGCTGGAGCGCTTAAAACAA

CAAAATGAAGACTTGAGGAGAATGGCTGAGTCTCTCCGAATACCAGAAGGCCCTATT GATCAGGGGACAGCTACAGGAAGA

GTCCGTGTTTTAGAAGAACAGCTTGTTAAGGCCAAAGAACAGATTGAAAATTACAAG AAACAAGCTAGGAATGATCTGGGAA

AGGATCATGAAATCTTAAGGAGGAGGATTGAAAATGGAGCTAAAGAGCTCTGG I H i ! I CTACAAAGTGAATTGAAGAAATT

AAAGAAATTAGAAGGAAACGAACTCCAAAGACATGCAGATGAAATTCTTTTGGATTT AGGACATCATGAAAGGTCTATCATG

ACAGATCTATACTACCTCAGTCAAACAGATGGAGCAGGTGAGTGGCGGGAAAAAGAA GCCAAAGATCTGACAGAGCTGGTC

CAGCGGAGAATAACATATCTGCAGAATCCCAAGGACTGCAGCAAAGCCAGAAAGCTG GTATGTAATATCAACAAAGGCTGTG

GCTATGGATGTCAACTCCATCATGTGGTTTACTGCTTCATGATTGCTTATGGCACCC AGCGAACACTCATCn ^' GGA

ATTGGCGCTATGCTACTGGAGGATGGGAGACTGTGTTTAGACCTGTAAGTGAGACATGCA CAGACAGGTCTGGCCTCTCCAC

TGGACACTGGTCAGGTGAAGTGAAGGACAAAAATGTTCAAGTGGTCGAGCTCCCCAT TGTAGACAGCCTCCATCCTCGTCCTC

Cn ^' ACTTACCCTTGGCTGTACCAGAAGACCTTGCAGATCGACTCCTGAGAGTCCATG GTGATCCTGCAGTGTGGTGGGTATCC

CAGTTTGTCAAATACTTGATCCGTCCACAACCTTGGCTGGAAAGGGAAATAGAAGAA ACCACCAAGAAGCTTGGCTTCAAACA

TCCAGTTATTGGAGTCCATGTCAGACGCACTGACAAAGTGGGAACAGAAGCAGCCTT CCATCCCATTGAGGAATACATGGTAC

ACGTTGAAGAACATTTTCAGCTTCTCGAACGCAGAATGAAAGTGGATAAAAAAAGAG TGTATCTGGCCACTGATGACCCTTCT

TTGTTAAAGGAGGCAAAGACAAAGTACTCCAATTATGAATTTATTAGTGATAACTCT ATTTCTTGGTCAGCTGGACTACACAAC

CGATACACAG/VATTCACTTCGGGGCGTGATCCTGGATA^

CCCAGGTCTGTAGGGTTGCTTATGAAATCATGCAAACACTGCATCCTGATGCCTCTGCAA ACTTCCATTCTTTAGATGACATCT

ACTATTTTGGAGGCCAAAATGCCCACAACCAGATTGCAGTTTATCCTCACCAACCTC GAACTAAAGAGGAAATCCCCATGGAA

CCTGGAGATATCATTGGTGTGGCTGGAAACCATTGGAATGGTTACTCTAAAGGTGTC AACAGAAAACTAGGAAAAACAGGCC

TGTACCCTTCCTACAAAGTCCGAGAGAAGATAGAAACAGTCAAATACCCTACATATC CTGAAGCTGAAAAATAGAGATGGAGT

GTAAGAGATTAACAACAGAATTTAGTTCAGACCATCTCAGCCAAGCAGAAGACCCAG ACTAACATATGGTTCATTGACAGACA

TGCTCCGCACCAAGAGCAAGTGGGAACCCTCAGATGCTGCACTGGTGGAACGCCTCT TTGTGAAGGGCTGCTGTGCCCTCAA

GCCCATGCACAGTAAAATAATGTACTCACACATAACATACAAATGGATTATTTTCTA CTTTGCCCTTTAAATATTCTGTCCCCAT

GAAACAAACACTGCCACATTATGTAATTTAAGTGACACAGACGTTTTGTGTGAGACT TCAAACATGGTGCCTATATCTGAGAG

ACCTCTGTGATTTACTGAGAAGATGAGAACAGCTCCCTTCTGTGGGGAAGTTGGTGG TGGACTGGCCACTGAATTCACTGCAA

TCAACAGATTCAGAATGAGAATGGATGTTTTTCCTTTATATGGTTGTCTGGATTTTT TTTAAAGTAATTTCATCAGTTCAGTTCA

TCCACCTCATTAATAAATGAAGGAATATACCAATAAAATCAAATGAAATATTCACTG TCCATTAGGAAGTTTTATAAAACAATG

CCATGAACAAAAAATTCTTTAGTACTCAATGTTTCTGGACATTCTCTTTGATAACAA AAATAAATTTTAAAAAGG

Appendix 2: nucleotide sequences of exemplary siRNAs

Table 8.1

GGDUGUCACUAUGCQAGUU 92 A A C U A G C A U A G U G A C A A C C 93

UGG GGAC AGUAUAUAUAGA 94 U C UAU UAU C ϋ GU C C C CA 95

ACAACCAGGUAG ^' uGAGUAA 96 UUACUCACUACCUGGUUGU 97

GGCACAAAGUCCUAACAGU 98 ACUGUUAGGACUUUGUGCC 99

CAUGUCCUUUGUAUGACAA 100 U U G U C A U A C A A A G G A C A U G 101

UGAUCUACCUGUCAAAGCA 102 UGCUUUGACAGGUAGAUCA 103

AGUGUUUGAAUGAGUAUUA 104 UAAUACUCAUUCAAACACU 105

UG UCACUAUGCUAGUUUAA 106 f J U A C U A G C A U A G U G A C A 107

AC C C CA C CUC AAAGGC AGA 108 UCUGCCUUUGAGGUGGGGU 109

C CUG AUG GCUCCC C G CAAU 110 AUUGCGGGGAGCCAUCAGG 111

GAGGAAGCUACAAUGCAGA 112 UCUGCAUUGUAGCUUCCUC 113

CCUACAGACUCCCGGAAGA 114 UCUUCCGGGAGUCUGUAGG 115

CCUUAUUGUUUCAGCCUCU 116 A G A G G C U G A A A C A AU A A G G 117

Table 8.2

ACAAAGAGGUCCUAQCACA 166 U GO G A [J A G G A C C U C U U U GU 167

UAUUGUACuUAAGUGUAAU 68 AUUACACUUAAGUACAAUA 169

GACAGAAAUGGGGCAAACA 170 UGUUUGCCCCAUUUCUGUC 171

GCUGGUUUACCAUGGUGAU 172 AUCACCAUGGUAAACCAGC 173

CUAUAUUGUACUUAAGUGU 174 ACACUUAAGUAC AUAUAG 175

AGAGACCCUCACUCOAUAU 176 AUAUAGAGUGAGGGUCUCU 177

UCUAGAAACUUUAGGGUAU 178 AUACCCUAAAGUUUCUAGA 179

UGCCAUAC C AC UACUAUGA 180 UCAUAGUAGUGGUAUGGCA 181

GCCAGUUCAGGUUGGCUGG 182 CCAGCCAACCUGAACUGGC 183

C CAGGUG C AAC C AAUGUGU 184 AC CAUUGGUUGCACCUGG 185

GGAGAGAGAUGAGUAGCAA 186 UUGCUACUCAUCUCUCUCC 187

GCCCUGCACAGCAAGGUAA 188 UUACCUUGCUGUGC&GGGC 189

G C AUG C AG C AG UiJU G AC G A 190 UCGUCAAACUGCUGCAUGC 191

C AUGGUC C CUC CUGACUAU 192 AUAGUCAGGAGGGACCAUG 193

CGGGCUGAGUGCACCAUCC 194 GGAUGGUGCACUCAGCCCG 195

UCACUCUAUAUUGUACUUA 196 UAAGUACAAUAUAGAGUGA 197

CUGAGUCAGUCUAAGACUC 198 GAGUCUUAGACUGACUCAG 199

AGCAAAGUCAAUCAGGGUA 200 UACCCUGAUUGACUUUGCU 201

GGAGUUCUAGAAGUUGUAG 202 CUACAACUUCUAGAACUCC 203

GCAGCCUCCUGCGUGUCAU 204 A U G A C A C G C A G G A G G C U G C 205

GCGDCGGCCUGDCAACCUC 206 GAGGU [JGACAGGC C GAC GC 207

GGGUACU UG CUUUC C CAA 208 UUGGGAAAGGAUAGUACCC 209

CUGUAUGGUUUAAUCGGGU 210 AC C C GAUU AAC C AU AC G 211

CUAGAUCUGGUUUUGUAAU AU U A C A A A C C A G A U C U G 213

CAGAGAC C CU C ACUCUAU A 214 ϋ A U A G G U G G G G U CUC U G 215

CUCUAUAUUGUACUUAAGU 216 A C U U A A G U A C A A U A U A G A G 217

Table 8.3

Target gene SenseSeq SEQ ID No. An isSeq SEQ ID No.

Craah AAUCUUAAUUAACAAUAUA 218 UAUAUUGUUAAUUAAGATJU 219

GAATJUUGCUCUTJAAAUCUU 220 AAGAUUUAAGAGCAAAUTJC

GCAUGUGCUUAGAGCAUAU p AUAUGCUCUAAGCACAUGC

CUGUAUAACCCUAAAUUCA 224 UGAAUUUAGGGUUAUACAG 225

GUGAGUAGAAAAUCUUAAA 226 UUUAAGAUUUUC UACU C AC 227

GCGGGACCCUGAUAUAUAC 228 GUAUAUAUCAGGGUCCCGC 229

CAAUAUAUGGGUGAGUAGA 230 UCUAC UC AC C C AUAUAUUG 231

AUCrUUAAUUAACAAUAUAU 232 AUAUAUUGUUAAUUAAGAIJ 233

UCAAGAAUUUGCUCUUAAA 234 UUUAAGAGCLAAAUUCUUGA 235

UCAUAAAAUACUCAACACA 236 UGUGUUGAGUAUUUUAUGA 237

AAGCAGACCUCAUUUAUAU 238 AUAU AAAUG AGGUCUG CUU 239

GAG UAGAAAAUCUU AAAUU 240 AAUUUAAGAUUUUCUACUC 241 GGUGAGUAGAAAAUCUuAA 242 UUAAGAUUUUCUACUCACC 243

GCCAUA&CUACCAUUAUUC 244 GAAUAA ^' uGGUAGQUAUGGC 245

GGGUCUCCCCUGCUUCGAU 246 AUCGAAGCAGGGGAGACCC 247

GGUCAUAAAAUACUCAACA 248 UGUUG GUAUUUUAUGACC 249

CAUCUGUUUUGGAAUCAUU 250 AAUGAUUCCAAAACAGAUG 251

GGAGACAAGCGAAUGGUAU 252 AUACCAUUCGCUUGUCUCC 253

CAGAUAAAACUCCCUCUAA 254 UUAGAGGG GUUUUAUCUG 255

CUGGAAACACUUUCAUAGA 256 UCUAUGAAAGUGUUUCCAG 257

GUGUCUUUUAUUCAGAGAU 258 AUCUCUGAAUAAAAGACAC 259

GAAAAUDUACQGAGGAAUG 260 CAUUCCUCAGUAAAUUUUC 261

GAGACAAGCGAAUGGUAUU 262 AAUACCAUUCGC^JUGUCUC 263

CCUGUAUAACCCUAAAUUC 264 GAAUUUAGGGUUAUACAGG 265

CCAUCUGACUGGUUGGAGA 266 UCUCCAACCAGUCAGAUGG 267

AUAACUACCAUUAUUCAUU 268 AAUG AUAAUGGUAGUUAU 269

CUUAAUUAACAAUAUAUGG 270 CCAUAUAUUGUUAAUUAAG 271

CACACCQG ^' uAUAACCCUAA 272 UUAGGGUUAUACAGGUGUG 273

CCUGCUQCGAUAAACCUUU 274 AAAGGUiUAUCGAAGCAGG 275

GGACCCUGAUAUAUACCAU 276 AUGGUAUAUAUCAGGGUCC 277

AUAUAUGGGUGAGUAGAAA 278 UUUCUACUC CCC UAUAU 279

GUGCAAAGCAGACCUCAUU 280 AAUGAGGUCUGCUUUGCAC 281

AA GGUC UA AAUACUC 282 UGAGUAUUUUAUGACCUUU 283

CUUCGAUAAACCUUUCUGG 284 CCAGAAAGGUUUAUGG AG 285

UGAG ^' uAGAAAAUCUUAAAQ 286 AUUUAAGAUUUUCUACUCA 287

GAQAAAACUCCC ^' uCUAACA 288 UGUUAGAGGGAGUUUUAUC 289

AGGTJCAUAAAATJACUCAAC 290 GUUGAGUAUUUUAUGACCU 291

GUAGUAUAGCAUGUGCUUA ? 9 UAAGCACAUGCUAUACUAC 293

AGAAUUUGCUCUUAAAUCU 294 AGAUUUAAGAGCAAAUUCU 295

CAAGGAAGGGAUCAAUUUU 296 AAAAUUGAUGCCUUCCUUG 297

UGGGUGAGUAGAAAAUCUU 298 AAGAUUUUCUACUCACCCA 299

AUUUACUGAGGAAUGGAAA 300 UUUCCAUUCCUC GUAAAU 301

ACAAGCGAAUGGUAUU ^' uGA 302 UCAAAUACCAUUCGCUUGU 303

GUUCCAACAGACACACAGA 304 UGUGUGUGUGUGUTJGGAAC 305

CGUUCCUAAUGCACUGUGA 306 UCACAGUGCAUUAGGAACG 307

CCAUCUUGUAACGGUGAAU 308 AUUCACCGUUACAAGAUGG 309

CCCUGAU UAUACCAUCAU 310 AUGAUGGUAUAUAUCAGGG 311

CAUAACUACCAUUAUUCAU 312 AUGAAUAAUGGUAGUUAUG 313

AC CCUGUAUAACCCUAAA 314 UUUAGGGUUAUACAGGUGU 315

GAQGGAAGGUCCGUUAAAQ 316 AUUU ACGGACCUUCCAUG 317

Table 8.4

Abo GAACGAUGUUCUUGUCUUG 31 8 CAAGACAAGAACAUCGUUC 31 9

AUCAGAAACUAAUAGCUAA 32 0 UUAGCUAUUAGUUUCUGAU 32 1

AGAGCUAUUUGAACAAAUA 322 UAUUUGUUCAAAUAGCUCU 323

GCAUGAUGAGAGCUAUUUG 32 4 CAAAUAGCUCUCAUCAUGC 325

GGAACGAUGUUCUUGUCUU 32 6 AAGACAAGAACAUCGUUCC 327

CCAUCAUGAAGAAGUUGAG 32 8 CUCAACUUCUUCAUGAUGG 32 9

GGACAAGGCCAACGGCAUU 330 AAUGCCGUUGGCCUUGUCC 331

GAACCAUCAGGCAAUCAGA 332 UCUGAUUGCCUGAUGGUUC 333

CUAAUAGCUAAAUUCCUAU 334 AUAGGAAUUUAGCUAUUAG 335

AGGCCUGCCAUGAAGCUAU 33 6 AUAGCUUCAUGGCAGGCCU 337

CUGCUUUACCAUAAGCCAA 338 TrUGGCUUAUGGUAAAGCAG 33 9

ACACAAGGUCACCUACUAU 34 0 AUAGUAGGUGACCUUGUGU 34

CCGAGAGGCCUUUACCUAU 342 AUAG GUAAAGG C CUCUCGG 343

ACUAAUAGCUAAAUUCCUA 344 UAGGAAUUUAGCUAUUAGU 345

UCAGAAACUAAUAGCUAAA 34 6 UUUAGCU AUUAG UUU CUGA 347

GAAGAAGUUGAGAUAUGUG 34 8 C AC AUAU CUC AACUU CUUC 34 9

AGGCAAUCAGAAACUAAUA 350 UAUUAGUUUCUGAUUGCCU 351

GAAACUAAUAGCUAAAUUC 352 GAAUTJUAGCUAUTJAGUUUC 353

CCUACUATJGUCUUCACUGA 35 UCAGUGAAGACAUAGUAGG 355

GAGCUAUUUGAACAAAUAC 35 6 GUAUUUGUUCAAAUAG CUC 357

CCAUCAGGCAAUCAGAAAC 358 GUUUCUGAUUGCCUGAUGG 35 9

GGGACACAAGGUCACCUAC 3 60 GUAGGUG AC CUUGUG UC C C 3 61

GGC UGU AC C CAAGAAC C AU 3 62 AUGGUUCUUGGGUACAGCC 3 63

GAUGAGAGCUAUUUGAACA 3 64 UGUUCAAAUAGCUCUCAUC 3 6 5

CCCAAGAACCAUCAGGCAA 3 6 6 UUGCCUGAUGGUUCUUGGG 3 67

GCUAAAUUCCUAUUGGAGA 3 68 UCUCCAAUAGGAAUiroAGC 3 6 9

GUUCUUGUCUUGACUCCUU 37 0 AAGGAGUCAAGACAAGAAC 37 1

CGAUGUUCUUGUCUUGACU 372 AGUCAAGACAAGAACAUCG 373

UCAGGCAAUCAGAAACUAA 374 UUAG UUU CUGAUUGC CUGA 375

GU CAGUG CUAGAAG UGUAC 37 6 GUAC ACUUCUAG C ACUGAC 377

AGGUCACCUACUAUGUCUU 37 8 AAGACAUAGUAGGUGACCU 37 9

UAGCUAAAUUCCUAUUGGA 38 0 UCCAAUAGGAAUTJUAGCUA 38 1

AAUACCUGCUUUACCAUAA 382 UUAUGGUAAA'GCAGGUAUU 383

ACCAUCAGGCAAUCAGAAA. 38 4 UUUCUGAUUGCCUGAUGGU 385

AGUAUGUGGUGUUCCUUA 38 6 UUAAGGAA C AC C C UACU 387

UGGACAUGAAGUUCAGUGA 38 8 UC ACUGAACUU C AUG UC C A 38 9

AG UUGAGAUAUGUGGCUG U 3 90 ACAGCCACAUAUCUCAACU 3 91

GAGAGCUAUUUGAACAAAU 3 92 AUUUGUUCA ¾AUAGCUCUC 3 93

AAGCUAUGAUGGAGGACAA 3 94 TJuGUCCUCCAUCAUAGCUU 3 9 5

ACAAAUACCUGCUUUACCA 3 9 6 UGGUAAAGCAGGUAUUUGU 3 97

CUGCCAUGAAGCUAUGAUG 3 98 CAUCAUAGCUUCAUGGCAG 3 9 9

CCUGUGUGGCAUGAUGAGA 4 0 0 UCUC AUC AUGC C AC AC GG 4 0 1

C C AGC AG AC C C AGUAUC AC 4 02 GUGAUACUGGGUCUGCUGG 4 03

- I l l - UGAAC AAAUAC CUGCUUUA 404 UAAAGCAGGUAUUUGUUCA 405

UGAUGAGAGCUAUUQGAAC 406 GUUCAAAUAGCQC ^' uCAUCA 407

CCAGUAGGAACGAUGUUCU 408 AGAACAUCGUUCCUACUGG 409

UGAAGAAGUUGAGAUAUGU 410 ACAUAUCUCAACTJUCUUCA 411

GUAGGAACGAUGUUCUUGU 412 ACAAGA C UCGUUC CUAC 413

AUUCCUAUUGGAGAGGACA. 414 UGUC CUCUC CAAUAGGAAU 415

GGCAUGAUGAGAGCUAUUU 416 AAAUAGCUCUCAUCAUGCC 417

Table 8.5

GCUAAGG CCACUUCG CUUU 4 8 0 AAAG C G AAGUGG C CUU AG C 4 8 1

CUUCUGGA ^' uGCAAUQAAGA 4 82 UCUUAAUUGCAUCCAGAAG 4 83

CGGUCAGUAGCUAAGAUUU 4 84 AAAUC UUAGCUAC UGAC CG 4 85

AAUCAUGAGAGUCCUAGAA 4 8 6 UUCUAGGACUCUCAUGAUU 4 87

GGAGCUAAUUUUGUUACUC 4 8 8 GAGUAACAAAAUUAGCUCC 4 8 9

GGAUGCAAUUAAGACUUGU 4 90 ACAAGUCUUAAUUGCAUCC 4 91

GUAGCUAAGAUUUACCUUG 4 92 CAAGGUAAAUCUUAGCUAC 4 93

G C C CUAUGGAG C AG C C AAA 4 94 UUUGGCUGCUCCAUAGGGC 4 9 5

CAAUCAUGAGAG ^' uCCUAGA 4 9 6 UCUAGGAC UCUCAUGAUUG 4 97

GAUGUUACAAAAUGAUGAA 4 98 UUCAUCAUUUUGUAACAUC 4 9 9

AGUUCAUAGUGUCCGUGAA 50 0 UUCACGGACACUAUGAACU 50 1

GCGAUCCAGUUCAUUUAAU 502 AUUAAAUGAACUGGAUCGC 503

CCAAACUUUAUGCCUAUUG 504 C AU GGC AUAA GUUUGG 505

CU AC AAUCUUGGUG C C C AG 50 6 CUGGGCACCAAGAUUGUAG 507

GUUC AUAGUGUC CG UGAAU 50 8 AUUC ACGGAC AC UAUGAAC 50 9

GGUCAGQAGCUAAGAU ^' uUA 51 0 UAAAQCUUAGCUACUGACC 51 1

UGUCAC QAGACAAC ϋϋΑΆΑ 512 DUUAAG ^' uUGUCUAG ^' uGACA 513

AGAGGA'GCUAAUUUUGUUA 514 UAACAAAAmJAGCUCGUCU 515

CUGAAAUACUACCGACCAA 51 6 UUG'GUCGGUAGUAUUUCAG 517

Table 8.6

AUGAAACAAUGAUCCACAA 556 UUGUGGAUCAUUGUUUCAU 557

CAUCAAQGACAACGQCCUG 558 CAGGACGUUGUCAUUGAUG 559

GGCCUACUUGCCUGAUUUC 560 GAAA CAGGCAAGUAGGCC 561

CGGAGAGGAAUGGGUGUUU 562 AAACACCCAUUCCUCUCCG 563

GG GUAC AUGAAGUUGAG 564 CUCAACUUCAUUGUACUCC 565

GCGGGAGU CAAUGA GUU 566 AACUUCAUUGUACUCCCGC 567

GGCCAAG GU AUGGUUC 568 UGAACCAUUACUCUUGGCC 569

AGUCAGAUGGGCAGUAUAA 570 UUAUACUGCCCAUCUGACU 571

CCCCACAGCAGCAAUUUUG 572 CAAAAUUGCUGCUGUGGGG 573

GCAAUUUUGGGUACUCGUA 574 UACGAGUACCCAAAAUUGC 575

GGACCUCAUGAC CUUCA 576 UGAAGUUGUCAUGAGGUCC 577

CUACUUGCCUGAUUUCCGU 578 ACGGAAAUCAGGCAAGUAG 579

CGCAGAUGGCGCUGGCUUA 580 UAAGCCAGCGCCA.UCUGCG 581

GAUGAAACAAUGAUCCACA 582 UGUGGAUCAUUGUUUCAUC 583

UUGAUUCAACAAAGUCAGA 584 UCUGACUUUGUUGAAUCAA 585

GCUUCGGGCCUACUUGCCU 586 AGGCAAGUAGGCCCGAAGC 587

CAAGAGGAUGAUUGACGUG 588 CACGUCAAUCAUCCUCUUG 589

GGAGAACAGGGCCUACUUC 590 GAAGUAGGCCCUGUUCUGC 591

CGAGGAGGAUCCUAGUGAC 592 GUCACU GGAUCCUCCUGG 593

CAGUAGGGCCCAUGUCCAU 594 A.UGGACAUGGGCCCUACUG 595

G UGCAGCAC AACCCAAG 596 CUUGGGUUUGUGCUGCAUC 597

CGGAAGUGAAGCAUGGGAC 598 GUCCCAUGCUUCACUUCCG 599

CAGCAGCAAUUUUGGGUAC 600 GUACCCAAAAUUGCUGCUG 601

CUGGUUCACCGACAACUAU 602 AUAGUUGUCGGUGAACCAG 603

CCACGGAGGCAGUUCAUCU 604 AGAUGAACUGCCUCCGUGG 605

AGUUCAUCUACUCACUGGA 606 UCCAGUGAGUAGAUGAACU 607

GGAAGUCACUUUUGAUUCA 608 UGAAUCAAAAGUGACUUCC 609

AAACCACGGAGGCAGUUCA 610 UGAACUGCCUCCGUGGUUU 611

CAAUGAUCCACAAUGGUCC 612 GGACCAUUGUGGAUCAUUG 613

CCCUACCAAUGUCUUUGGA 614 UCCAAAGACAUUGGUAGGG 615

AUCAUUUGCUUGUCAAGAA 616 UUCUQGACAAGCAftAUGAU 617

Table 8.7

GGAAUAUGAU CUAAG AGGA 632 UC CUCUUAGAUC AUAUUC C 633

GGGACAGAAGCACUCAGAC 634 GUCUGAGUGCUUC UGU C C C 635

CCUCCAUCUCCAUGGUAUU 636 AAUACCAUGGAGAUGGAGG 637

UGUAUGUGAUUUAAUAAUA 638 UAUUATJUAAAUCACAUACA 639

GCAUGAUAAGUUUCAAUA 640 UUAUUGAAA CUUAUCAUGC 641

GGCAUGAUAAGUUUCAAUA 642 UAUUGAAACUUAUCAUGCC 643

GGGGUAAUGUCUGAUGUUU 644 AAC AUG G AC AUUAC C C C 645

GCUGGAUUGAUGAAGUGAU 646 AUCACUUCAUCAAUCCAGC 647

GCAUCAUCAUQGGUGGUUQ 648 AftACCACCAAUGADGAQGC 649

GCQCCUUUAAGCCUCAUAQ 650 AUAUGAGGCUUAAAGGAGC 651

C C CUG C C C AC AUGUAUC 652 GAUACAUUGUGGGUCAGGG 653

GCCCUGGCAUGGUAGACUU 654 AAGUCUACCAUGCCAGGGC 655

CCACAAUGUAUCAGGCACA 656 UGUGCCUGAUACAUUGUGG 657

CAGACCACUUCCUUCUAUG 658 C AUAG AAGGAAG UGGU CUG 659

GCAUCUGGCGCCUAACCUU 660 AAGG UUAGG C G C C AG AUG C 661

CGUAAAUUUGGGGACAGAA 662 UUCUGUC C C C AAUUU CG 663

C C CUGGCAUGGUAGACUU C 664 GAAG UCUAC C AUGC CAGGG 665

GGGUGAGAAGAGUGCUAUU 666 AAUAGCAGUCUUCUCACCC 667

C CUACUUC CUAC AAUC C C A 668 UGGGAUUGUAGGAAGUAGG 669

GCGC CUAAC CUUCUAUA C 670 GUUAUAGAAGGUUAGGCGC 671

GUCUUUAUCGG CAUG ΑϋΑΑ. 672 UUAUC AUG C CGAU AAG AC 673

GGCGCCUAACCUUCUAUAA 674 UUAU AG AAG GUU AGG C G C C 675

CUQC ^' uAUAACAA ^' uGUCAAQ 676 AUUGACAUUGUUAUAGAAG 677

AAUCCCAGGUUCUAAUGAA 678 UC UUAGAAC CU GGGAUU 679

CCUACCUGCUGCUCAAACA 680 UGUUUGAGCAGGAGGUAGG 681

AGGACUCCAACGUAAAUUU 682 AAAUUUA'C'GUUGGAGUCCU 683

AUG C CUGUC AAC CUC AAA 684 UUUG GGUUG C GGUC U 685

CUGGAUAC C C CUAUCUUUG 686 C AAAG AUAGGGGUAUC C AG 687

UUUUGUC C CUGUC AUAAAA 688 UUUUAUGACAGGGACAAAA 689

CUAAC C UUCU AUAAC AAUG 690 C AUUG UUAUAGAAGGUUAG 691

C CUUGGGGUAAUGUC UGAU 692 AUG AGAC UUAC C C C AAGG 693

GGCUCACUCAUGAGGGUUU 694 AAACCCUCAUGAGUGAGGC 695

AUAACAAUGUCAAUGCCUG 696 CAGGCAUUGACAUUGUUAU 697

AA C C CUAC C G GAAUGGC 698 UGCCAUUCCUGGUAGGGUU 699

CUGGCGCCUAACCUUCUAU 700 AUAGAAGGUUAGGCGCCAG 701

CAAUGUUCUUCUGU C CUAC 702 G UAGGAC AG AAGAAC AUUG 703

GG AC AAGC AAC CUGAUGG U 704 ACCAUCAGGUUGCUUGUCC 705

CUGAGUUGUAUGUGAUUUA 706 QAAAUCACAUACAACUCAG 707

UCCCACUAUAGCUAGUGAA 708 UUCACUAGCUAUAGUGGGA 709

GGGUCUUUCCUGGUAAUGU 710 AC UUAC CAGGAAAGAC C C 711

C C AUAUC AUCUCUGG GGUU 7 Ί ^~} AAC C C C AGAG UG AUAUGG 713

GCCUUAAGGGGCUGGAUUG 714 C AAUC CAGC C C CUUAAGGC 715

CCGUGUUCAAUGUUCUUCU 716 AGAAGAACAUUGAACACGG 717 Table 8.8

GACAAAGAGCUUCCACUUU 794 AAAG UGGAAGC UCUUUGUC 795

AGGCAUGAGCACAAGGCAA 796 UUGCCUUGUGCUCAUGCCU 797

GCAACAAUGACCAGUCCGG 798 CCGGACUGGUCAUUGUUGC 799

GUUGAAGUAAUGAUUUCCU 800 AGGAAAUCAUUACUUCAAC 801

ACAUCACUGUGUCACUGUA 802 UACAGUGA CACAGUGAUGU 803

GUUC CUC CUUGUGUC C AG A 804 UCUGGACACAAGGAGGAAC 805

CAGAUAAAUUUGACGUGUG 806 CACACGUCAAAUUUAUCUG 807

GUGCUCAGCUGGACUGAUU 808 AAU C AGUC C AGCUGAG C AC 809

CCGCUCUUUGCCGUAUUUG 810 C AAAUAC GG C AAAG AG C GG 811

CUAACCAUGGGACAGUUUG 812 CAAACUG ^[ JC C CAUGG QUAG 813

GGUGCUCAGCUGGACUGAU 814 AUCAGUCCAGCUGAGCACC 815

CACCUGCUGGGUGAUCAGA 816 UCUGAUC AC C GAGC AGGUG 817

Table 8.9

Table 8.10

GAAGAAAAUUCGUGGCAAA 860 UUUGCCACGAAUUUUCUUC 861

AAGUUAQUGAUAAUUAAGA 862 UCUQAAUUAUCAA ^' uAACUU 863

ACGGAUGAUAUAUAAUGAU 864 AUCAtfUAUAUAQCAUCCGU 865

UGAAGAAAUUGGUUAGUAU 866 AUACUAACCAAUrru CmJCA 867

CCCCGUACC UUAUUGUA 868 UUACAAUAAUGGUACGGGG 869

AGGAGAACCUAAUAUUCA 870 UUGAAUAUUAGGUUCUCCU 871

CAUUUAUGACAAUCGGAUA UAUCCGAUUGUC UA UG 873

CUAUUUAAAACCUAAGCAA 874 UUGCUUAGGUUUUAAAUAG 875

GAGA ^' uUAACCAGCCGUUUQ 876 AAAACGGCUGGUUAAUCUC 877

GAUUUGGCUGGCAAUUCAU 878 AUGAAUUGCCAGCCAAAUC 879

GAUGCUUACUCUAAUAUGG 880 CCAUAiJUAGAGUAAGCAUG 881

CCUAUAUCGCAAAGCAUUA 882 UAAUGCUtJUGGGAUAUAGG 883

GUACCUAAUAGCGUGCUUU AAAGCACGCUAUUAGGUAC 885

GGAAUUAGAGCGACUCUAU ^C O 886 AUAGAGUCGCUCUAAUUCC 887

^C O

ACACC GGGAUUUCUUAAU 888 AUUAAGAAAUCCCUGGUGU 889

GGDUCCCCGUACCAUUAUU 890 AAUAA ^' uGGUACGGGGAACC 891

CACUGAQGCADUAUAAGGA 892 UCCUUAUAAUGCAUCAGUG 893

GGAAGCGAUUGAGCATJLJAU 894 AUAAUGGUCAAUCGCmJCC 895

C CUGUUC UGGAUUAUAU 896 AUAUAAUCCAUGAAGAGUG 897

GAUUGAGCAUUAUCGACAU 898 AUGUCGAUAAUGCUCAAUC 899

AAACAAACAAGCCGACAUA. 900 UAUGUCGGCUUGUUUGUUU 901

CCAACGAAACGUAUGCUUU 902 AAAGCAUACGUUUCGUUGG 903

AGCAAGGCCUAA ^' uAGACUQ 904 AAGUCUAQUAGGCCUQGCU 905

GUUUGUAGUGCUCUAUAUU 906 AAUAUAGAGCACUACAAAC 907

CUGGCUUGUGUAUACUAUG 908 CAUAGUAUACACAAGCCAG 909

GAACCGAUUUAGAAUACCU 910 AGGUAUUCTJAAAUCGGTJUC 911

CCACAAGGAAUCUCUUAGA 912 UCUAAGAGAUUCCUUGUGG 913

GGCUUGGAUAAUAGACCUC 914 GAGGUCUAUUAUCCAAGCC 915

CGCCUAUAUCGCAAAGCAU 916 AUGCUUUGCGAUAUAGGCG 917

AUGUUUGCUUUCAGGUAUU 918 AAUACCUGAAAGCAAACAU 919

GCCAAGGCAUGUUAQU ^' uGA 920 UCAAAUAACAUGCCUUGGC 921

GUUGCAGACGGAUGAUAUA 922 UAUAUCAUCCGUCUGCAAC 923

CGIJUAAATJLJGUCCUGUGAC 924 GUCACAGGAGAATJUUAACG 925

Table 8.1 1 GUUUUGGGUACUAUGAUAU 336 AUAUC AUAGUAC C CAAAAC 337

CAGCUAGGCUUCUAQGAAU 338 AUUCAUAGAAGCCUAGCUG 333

AGO AC AG C ϋ AGG CU QC ϋ AU 340 AUAGAAGCCUAGCUGUACU 341

GUCCUUGGCAGAUGGUUAU 342 AUAACCAUCUGCCAAGGAC 343

C C C CGAGGGUA AACUUAAA. 944 UUUAAGUUUAC C CUCGGGG 945

GCACUGGGAACGUUUCAUG 946 CAUGAAACGUUCCCAGUGC 947

CUAGCUAAUUAUGUCCACA 948 UGUGGACAUAAUUAGCUAG 949

GGGAGUUUUGGGUACUAUG 950 C AUAGUAC C C AAAACUC C C 951

GGAACGACCACUCUCAAAU 952 AUUUGAGAGUGGUCGUUCC 953

UUUCAUAAGCCCAAUUAUA 954 UAUAAUUGGGCUUAUGAAA 955

CAGGGGUACUGUUUUAGAA 956 UUCUAAAAC AGUAC C C CUG 957

CCAGAUAUGUUAGUGAUUG 958 CAAUCACUAACAUAUCUGG 959

GCUAGGAAUUUAUGCAGAU 960 AUCUGCAUAAAUUCCUAGC 961

GCAACUUUGGCCUGAGUUG 362 CAACUCAGGCCAAAGUUGC 363

GUUAUAAGUAC AUG UC CUU 364 AAGGACAUGUACUUAUAAC 365

GGUUAUAAGUACAUGLICCU 366 AGGACAUGUACUUAUAACC 367

CCCGAGGGUAAACUUAAAA 368 UUUUAAGUUUACCCUCGGG 369

CAAUAUUAUUGCAAUUACU 370 AGUAATJUGCAAUAAUAUUG 971

AUAUCCAAUAUUAUUGCAA 972 UUGCAAUAAUAUUGGAUAU 973

AUA C AGAUAUC C AAU AUUA. 974 UAAUAUUGGAUAUCUGUAU 975

CGCCCCGAGGG U AA A C UU 976 UAAGUUUACCCUCGGGGCG 977

CUAUGAUAUUG AUGC C G G 978 CUGGGCAUCAAUAUCAUAG 979

C C C UAGUCUUAGUUUUCUG 980 CAGAAAACUAAGACUAGGG 981

UGCAGAUGUUGGGAAUAAA 982 UUUAUUC C CAACAUCUGCA 983

CUUCGACUAGAAAGAUAAA 984 UrrUAUCUtroCUAGUCGAAG 985

CGCAGGUUAUGAAUACCUC 986 GAGGUAUUCAUAACCUGCG 987

GACUCAAAGGGCCGACUUC 988 GAAGUCGGCCCUUUGAGUC 989

AUAUGUUAGUG UUG G C AA 390 UUGC C AAUC ACUA AC AU U 331

GUUGGAAUGAC C CAGAUAU 332 AUAUCUGGGUCAUUCCAAC 333

GCUUAACUCGGUAAACUGA 334 UC AG UUU AC C GAG UUAAG C 335

C CAAUUAUAC AGAUAUC C A 336 UGGAUAUCUGUAUAAUUGG 337

UAGAUCUGCUAAAAUUUGA 338 UCAAAUUUUAGCAGAUCUA 339

GGGUAAACUUAAAAGC C G A 1000 UGGGCTrUUAAGUrru A C C C 1001

GC C CAAUUAUAC AGAUAUC 1002 GAUAUCUGUAUAAUUGGGC 1003

GC C C CGAGGGUAAACUUAA 1004 UUAAGUUUACCCUCGGGGC 1005

GUACUGUUUUAGAAAGGAA 1006 UUC CUUUC UAAAAC AG UAC 1007

AAUAUU AUUG C AAUUACUG 1008 CAGUAAUUG CAAUAAUAUU 1009

GACCCAGAUAUGUUAGUGA 1010 UCACUAACAUAUCUGGGUC 1011

ACAGCUAGGCUUCUAUGAA 1012 UUCAUAGAAGCCUAGCUGU 1013

CAGCACCUAGCUAAUUAUG 1014 CAUAAtrUAGCUAGGUGCUG 1015

UAUACAGAUAUCCAAUAUU 1016 AAUAUUGGAUAUCUGUAUA 1017

GGCAGCUCCUCUACUCAUG 1018 C AUG A GUAG AGG A G CUG C C 1019

GCUUUAUAUGAGAC C CUUU 1020 AAAG G GU CUC AUAUAAAG C 1021 AAUCCUGGUUGCAUCAUCA 1022 UGAUGAUGCAACCAGGAUU 1023

UAUUAUUGCAAUUACUGGA 1024 UCCAGUAADUGCAA ^' uAAUA 1025

Table 8.12

C CUUC AUUC AGUUUC CUGG 1098 CCAGGAAACUGAAUGAAGG 1099

GCAGUACGCACCCAUCUUG 1100 CAAGAUGGGUGCGUACUGC 1101

AGGGUUQGAUDUCUAACAU 1102 AUGUQAGAAAUCAAACCCU 1103

GAAGUAUUUUGCUCUUCGA 1104 UCGAAGAGCAAAAUACUUC 1105

UCAUC C AAGUUAAAC AGU 1106 UACUGUUUAACUUGGAUGA 1107

AGGUGGAUUUAUCAGGUGU 1108 C AC CUGAUAAAUC C C CU 1109

C CG C AGG UA C CUUC AUUC 1110 GAAUGAAGGUAUCCUGCGG 1111

C AG C C AGGAC AAUAUGAG U 1112 ACUCAUAUUGUCCUGGCUG 1113

AAQGGAGGGCCGAUUAUAA 1114 UAU AAUCGGC C CUC C AUU 1115

GGCACACCGCUUCCGUUAC 1116 G UAAC GG AAG C GGUGUG C C 1117

GGCUGGAGUACGCACCCAU 1118 AUGGGUG C GUA GUG C AG C C 1119

CAAUGA CGUC CUUCUCUUC 1120 GAAGAGAAGGAGGUCAUUG 121

GCUGUACCGAACAACACUU 1122 A GUG UUGUUC G G UAC AG C 123

UAAAUGAUUUUAAGGGUUU 1124 AAAC C CUU AAAAUC AUUUA 1125

Table 8.13

Table 8.14

Target gene SenseSeq SEQ ID No. AntisSeq SEQ ID No. gat2 GAUAAUUAUUGAUUAGAAA 1154 UTJUCUAAUCAAUAAUUATJC 1155

CUUTJCAAUGGGAUAUACAA 1156 UUGUAUAUC C CAUUGAAAG 1157

AC ACUGG C CUU U CUUUU 1158 A A G UAUA GG C C AGUGU 1159

AGUAC AG C AAGACUUAUUU 1160 AAAUAAGU CUUG CUGU ACU 1161

AUUGAAUAAAGGUAUAGGA 1162 UC CUAUAC CUUUAUUC AAU 1163 CAUUCUUAAAUAAUAACCA 1164 UGGUUAUUAUUUAAGAAUG 1165

AAAUUU UAUUUAAA QC G C A 1166 UGCGAU ^' uUAAAUAAAAUUU 1167

ACCUCAAUGUAGAAAUACA 1168 UGUAUUUCUACAUUGAGGU 1169

CAGAAGUTJCCGUUUGTJUAA 1170 UUAACAAACGGAACUUCUG 1171

CCUCAAGCUCCGAGGAUUU 1172 AAAUCCUCGGAGCUUGAGG 1173

AAGACUAUUAGAGUAUUG 1174 UCAAUACUCUAAUAGUCUU 1175

C UAAAAUUC C CUAAUC U 1176 AUGAUUAGGGAAUUUUAUG 1177

CUUCAAUAUUUGACUGUAU 1178 AUACAGUCAAAUAUUGAAG 1179

AGACACDUUCQG ^' uACUUAQ 1180 AUAAGUACAGAAAGUGUCU 1181

AAACUGUCACAACAGUAAA 1182 UUUACUGUUGUGACAGUUU 1183

CUGTJGCUAUUGTJGUUAAGA 1184 UCUUAACACAAUAGCACAG 1185

UUUTJCAUAAAATJUC C CUAA 1186 UUAGGGAAUUUUAUGAAAA 1187

AGUUGAA CUUCGAUCAG U 1188 AUCUGAUCGAAGUUCAACU 1189

GACUAUUAGAGUAUUGAUG 1190 CAUCAAUACUCUAAUAGUC 1191

CCAGAAACUCUAGUUAUCA 1192 UGAUAACUAGAGUUUCUGG 1193

CUUGUAACAGGUGUCCAAA 1194 UUUGGAC C CUG UUACAAG 1195

UUGAUAAUUAUUGAUUAGA 1196 UCUAAUCAAUAAQUAUCAA 1197

CCCGGUUCUGCAAGUGUUC 1198 GAACACUUGCAGAACCGGG 1199

UGUAUUUGGUUAAUAAGAA 1200 UUCUUAUtLAAC C AAAUAC A 1201

GCCCGGUUCUGCAAGUGUU 1202 AACACUUGCAGAACCGGGC 1203

AG CUAUUAG GUAUUGAU 1204 AUCAAUACUCUAAUAGUCU 1205

CAAUUGAAUAAAGGUAUAG 1206 CUAUACCUUUAUUCAAUUG 1207

GUUGGAAUGUAUUUGGUUA 1208 QAACCAAAQACAUUCCAAC 1209

C C AC CUU GCUGUGU C CGUA 1210 UACGGACACAGCAAGGUGG 1211

UGAUAAUUAUUGAUUAGAA 1212 UTJCUAAUCAAUAAUUAUCA 1213

ACUUCUGGUCCGCAGAGAU 121 AUCUCUGCGGACXIAGAAGU 1215

GGAGAUAUUAGGG C C AUG 1216 C AUGGUC C CUAAUAUCUC C 1217

C C AGUGUC ACU UUG ACUU 1218 AAGUC AAUAGUG C ACUGG 1219

C AAG AAAAC AUGUAG G C C A 1220 UGGCCUACAUGUUUUCUUG 1221

AAAACUG UC AC AAC AGUAA 1222 UUACUGUUGUGACAGUUUU 1223

CCUAGUCAUCUUCAGCCAU 1224 AUGGCUGAAGAUGACUAGG 1225

UCCCUAATJCAUUUUGCAGA 1226 UCUGCAAAAUGAUUAGGGA 1227

GCUGAUUCCUGUGCUAUUG 1228 CAAUAGCACAGGAAUCAGC 1229

CAGUUGAACUUCGAUCAG 1230 UCUGAUCGAAGUUCAACUG 1231

GUG GGUGGGGA G AUAUUAG 1232 CUAAU UCUC C C C AC C C AC 1233

GGG AGAUAUUAG GGAC C AU 1234 AUGGUC C C UAAU AUCUC C C 1235

UAUUCAGUUAGUCUGAUUA 1236 UAAUCAGACUAACUGAAUA 1237

ACACUUUCUGUACUUAUGA 1238 QCAUAAGUACAGAAAGQG ^' u 1239

AGGCCAAAUUCUCGCAAAC 1240 GTrOUGCGAGAAUiraGGCCU 1241

GCGACAAAGGAAGAACGAC 1242 ^■ GUCGUUCUUCCUUUGUCGC 1243

C G C A C C AAC AUG AUUG 1244 CAAUGAUGUUUGGUUUGCG 1245

AAUUCAGUCUAUCUGCUUA 1246 U AG C AG AU GA CUGAAUU 1247

GAAGUUG AUCGAGUG CACA 1248 UGUG C ACU CGAUC AACUUC 1249 GGAUUACACUGGCCUUAUA 1250 OAUAAGGCCAGUGUAAUCC 1251

GAAUAAAGGUAUAGGAACU 1252 AGUUCCUAU CCUUUAUUC 1253

Table 8.15

UUUAUUGGCCUAAAUAUAA 1326 UUAUAUUUAGGCCAAUAAA 1327

AGAAGUUAGAUϋCGAAAGA 1328 UCUUUCGAAUCUAACUUCU 1329

GUGUAUUCUUACAUCACUU 1330 AAGUGAUGUAAGAAUACAC 1331

GAAGGUUCUCCAUUCUGUU 1332 AACAGAAUGGAGAACCUUC 1333

GAUCUGGAAGGAUAUGUUU 133 AA CAUAUCCUUCCAGAUC 1335

CCUAAAUAUAAGUAUUGCU 1336 AGCAAUACUUAUAUUUAGG 1337

CAGGAGAUGC CAAA GA 1338 UUCUUUUGUGCAUCUCCUG 1339

GAGAUUAUAUUUUCACAUG 1340 CAUGUGAAAAUAUAAUCUC 1341

AAUUUUAUUGGCCUAAAUA 1342 UAUUUAGGCCAAUAAAAUU 1343

CGUGCUCACCAACUACAGA 1344 UCUGUAGUUGGUGAGCACG 1345

GCCAGAGCUGGGUGGCAUG 1346 CAUGCCACCCAGCUCUGGC 1347

CUGGAAGCCUGGUGUAUUC 1348 GAAUACACCAGGCUUCCAG 1349

GGUUCUCCAUUCUGUUUAC 1350 GUAAACAGAAUGGAGAACC 1351

UAUGUUUGAAUUAAUUUAU 1352 AUAAAUUAAUUCAAACAUA 1353

Table 8.16

GGUCGACUUCCUCUUCUGC 1402 GCAGAAGAGGAAGUCGACC 1403

UGUGGGAAGGCACUUAUGA 1404 UCAUAAGUGCCUUCCCACA 1405

GACGUGGAUCAAGUCUUUC 1406 GAAAGACUUGAUCCACGUC 1407

GUGUGGGAAGGCACUUAUG 1408 CAUAAGUGCCUUCCCACAC 1409

CAUGGACGUGGAUCAAGUC 1410 GACUUGAUCCACGUCCAUG 1411

CCGAUUGUGUGGGAAGGCA 1412 UGCCUUC:CACACAAUGGG 1413

GAAGGCACUUAUGACACAG 1414 CUGUGUCAUAAGUGCCUUC 1415

UCUGCAUGGACGUGGAUCA 1416 UGAUCCACGUCCAUGCAGA 1417

GCGCCGAUUGUGUGGGAAG 1418 CUUCCCACACAAUCGGCGC 1413

ACGUGGAUCAAGUCUUUCA 1420 UGAAAGACUUGAUCCACGU 1421

UGGAAGGCGCCGAUUGUGU 1422 ACACAAUCGGCGCCUUCCA 1423

AGCACGAGGUCGACUUCCU 1424 AGGAAGUCG CCUCGUGCU 1425

GAAGGCGCCGAUUGUGUGG 1426 CCACACAAUCGGCGCCUUC 1427

CGACUUCCUCϋϋCUGCAUG 1428 CAUGCAG GAGGAAGUCG 1429

CUUCUGCAUGGACGUGGAU 1430 AUCCACGUCCAUGCAGAAG 1431

CCCACAUCCAGCACGAGGU 1432 ACCUCGUGCUGGAUGUGGG 1433

ACAUCCAGCACGAGG CGA 1434 UCGACCUCGUGCUGGAUGU 1435

AGGUCGACUUCCUCUUCUG 1436 CAGAAGAGGAAGUCGAGCU 1437

GAUUGUGUGGGAAGGCACU 1438 AGUGCCUUCCCACACAAUC 1439

ACUUCCUCUUCUGCAUGGA 1440 UCCAUGCAGAAGAGGAAGU 14 1

ACGAGGUCGACUUCCUCUU 1442 AAGAGGAAGUCGACCUCGU 1443

UCUUCUGCAUGGACGUGGA 1444 UCC CGUCC UGC G GA 1445

GUGGAAGGCGCCGAUUGUG 1446 CACAAUCGGCGCCUUCCAC 1447

CUACUCACAUUCUCAACCU 1448 AGGUUGAGAAUGUGAGUAG 1449

UGUGGGUCGCGACCCCUUU 1450 AAAGGGGUCGCGAGCCACA 1451

CCUGUGGGUCGCGACCCCU 1452 AGGGGUCGCGACCCACAGG 1453

Table 8.17

CCCAUGCACAGUAAAAUAA 1478 UUAUUUUACUGUGCAUGGG 1479

GAAAUUAGAAGGAAACGAA 1480 UUCGUUUCCUUCUAAUUUC 1481

CUUUGUAAGAAUAACCACA 1482 UGUGGUUAUUCUUACAAAG 1483

GAACUCUCCAAGAUUCUUG 1484 CAAGAAUCUUGGAGAGUUC 1485

AGAUGACAUCUACUAUUUU 1486 AAAAUAGU GAUGUCAUCU 1487

CACUCAUCUUGGAAUCUCA 1488 UGAGAUUCCAAGAUGAGUG 1489

CAGU AAAU UGUACUC 1490 UGAGUACAUUAUUUUACUG 1491

CAUCAUAAUUCUAUGGGGA 1492 UCCCCAUAGAAUUAUGAUG 1493

AGAUAGAACAGAGUUGAAA 1494 UUUCAACUCUGUUCUAUCU 1495

GUACAAUGUUUCCAAUUCU 1496 AGAAUUGGAAACAUUGUAC 1497

CCAGCGAACACUCAUCUUG 1498 CAAGAUGAGUGUUCGCUGG 1499

GGCGUUGGAUUAUGCUCAU 1500 AUGAGCAUAAUCCAACGCC 1501

GAAACAAGCUAGGAAUGAU 1502 AUCAUUCCUAGCUUGUUUC 1503

CAUGGUACACGUUGAAGAA 1504 UUCUUCAACGUGUACCAUG 1505

GAUAGAAACAGUCAAAUAC 1506 GUAUUUGACUGUUUCUAUC 1507

UCACCAACCUCGAACUAAA 1508 UUUAGUUCGAGGUUGGUGA 1509

AAUUCUUUUGGAUUUAGGA 1510 UCCUAAAUCCAAAAGAAUU 1511

AUUAGUGAUAACUCUAUUU 1512 AAAUAGAGUUAUCACUAAU 1513

GGUUCCUGUUUCCGUUUCU 151 AGAAACGGAAACAGGAACC 1515

CAGAAUUGGCGCUAUGCUA 1516 UAGCAUAGCGCCAAUUCUG 1517

AUUCACUGCAAUCAACAGA 1518 UCUGUUGAUUGCAGUGAAU 1519

CUUCAAACAUCCAGUUAUU 1520 AAUAACUGGAUGUUUGAAG 1521

CUUGCAGAUCGACUCCUGA 1522 UCAGGAGUCGAUCUGCAAG 1523

ACUCACACAUAACAUACAA 1524 U UGUAUGUUAUGUGUGAGU 1525

UUAUGAAUUUAUUAGUGAU 1526 AUCACUAAUAAAUUCAUAA 1527

CAGAAAGCUGGUAUGUAAU 1528 AUUAL ^' AUACCAGL ^' UUUCUG 1529

GAGCGCUUAAAACA CAAA 1530 UUUGUUGUUUUAAGCGCUC 1531

AAUACAUGGUACACGUUGA 1532 UCAACGUGUACCAUGUAUU 1533

UGUACUCACACAUAACAUA 1534 UAUGUU UGUGUG GUACA 1535

CAUGAAAUCUUAAGGAGGA 1536 UCCUCCUUAAGAUUUCAUG 1537

GCUGGUAUGUAAUAUCAAC 1538 GUUGAUAUUACAUACCAGC 1539

GAAACAGUCAAAUACCCUA 1540 UAGGGUAUUUGACUGUUUC 1541

ACUUCCAUUCUUUAGAUGA 1542 UCAUCUAAAGAAUGGAAGU 1543

CUCACCAACCUCGAACUAA 1544 UUAGUUCGAGGUUGGUGAG 1545

AACAUAUGGUUCAUUGACA 1546 UGUCAAUGAACCAUAUGUU 1547

AUGAAUUUAUUAGUGAUAA 1548 UUAUCACUAAUAAAUUCAU 1549

GAGAUGGAGUGUAAGAGAU 1550 AUCUCUUACACUCCAUCUC 1551

CCAUCAUGUGGUUUACUGC 1552 GCAGUAAACCACAUGAUGG 1553