WO2013013019A2

WO2013013019A2 - Lysosomal polypeptides, methods of making and using

Info

Publication number: WO2013013019A2
Application number: PCT/US2012/047356
Authority: WO
Inventors: Anthony Rossomando; Brian Bettencourt
Original assignee: Alnylam Pharmaceuticals, Inc.
Priority date: 2011-07-21
Filing date: 2012-07-19
Publication date: 2013-01-24
Also published as: WO2013013019A3

Abstract

The invention generally relates to compositions and methods for producing lysosomal proteins that have altered glycan structure, such that the protein can be delivered efficiently into the lysosomes of target cells (such as macrophages). The lysosomal proteins are produced by modifying the glycosylation pathways in a host cell using an RNA effector molecule, such as an siRNA. Glycan-modified lysosomal proteins produced using the methods described herein have improved properties, including e.g., increased specific uptake by target cells (such as macrophages).

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 61/510,437, filed July 21, 201 1 , U.S. Provisional Application No. 61/533,002, filed September 9, 201 1 , 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 INVENTIO

[0002] The lysosomal storage diseases (LSDs) are a group of inherited metabolic disorders that result from a lysosomal enzyme defect that causes accumulation of a metabolic substrate of the enzyme. For example, Gaucher' s disease is the most prevalent lysosomal storage disorder. It is caused by a recessive genetic disorder (chromosome 1 q21-q31) resulting in deficiency of glucocerebrosidase, also known as glucosylceramidase, which is a membrane- bound lysosomal enzyme that catalyzes the hydrolysis of the glycosphingolipid

glucocerebroside (glucosylceramide, GlcCer) to glucose and ceramide. Gaucher' s disease is caused by point mutations in the hGCD (human glucocerebrosidase) gene (GBA), which result in accumulation of GlcCer in the lysosomes of macrophages. The characteristic storage cells, called Gaucher cells, are found in liver, spleen and bone marrow. The associated clinical symptoms include severe hepatosplenomegaly, anemia, thrombocytopenia and skeletal deterioration.

[0003] Another well characterized lysosomal storage disorder is Fabry disease. Fabry disease is an X-linked lysosomal storage disease that is caused by deficient activity of lysosomal enzyme a-glucosidase A (GAA), Patients with classic Fabry disease typically have GAA activity of less than 1% and often demonstrate the full spectrum of symptoms, including severe pain in the extremities (acroparesthesias), hypohidrosis, corneal and lenticular changes, skin lesions (angiokeratoma), renal failure, cardiovascular disease, pulmonary failure, neurological symptoms and stroke. In atypical Fabry disease, individuals with residual enzyme activity demonstrate symptoms later in life, and the symptoms are usually limited to one or a few organs. Clinical manifestations in female carriers vary' greatly because of random X-

. i . chromosome inactivation. Although carriers commonly remain asymptomatic throughout life, many demonstrate clinical symptoms as variable and severe as those of affected males.

[0004] Enzyme replacement therapy has been used successfully to manage symptoms of Gaucher' s disease and other lysosomal storage diseases, such as Pompe disease and Fabry disease. Although enzyme replacement therapy is not a cure, such treatments can effectively manage the disorder when adminis tered on a regular basis. In the case of Gaucher' s disease, intravenous recombinant glucocerebrosidase administered to patients decreases liver and spleen size, reduces skeletal abnormalities, and reverses other manifestations.

[0005] Individuals with type I Gaucher disease can be treated with β- glucocerebrosidase prepared from placenta (Ceredase®) or recombinantly (Cerezyme©).

Mature human glucocerebrosidase is a 497 amino acid membrane-associated monomelic glycoprotein of about 67 kDa and has a pH optimum for enzymatic activity of about 5.5.

Glucocerebrosidase has five N-glycosylation amino acid consensus sequences (Asn-X-Ser/Thr), four of which are normally glycosylated (Asnl9, Asn59, Asn l46 and Asn270). Recombinantly produced glucocerebrosidase (Cerezyme®) differs from placenta glucocerebrosidase

(Ceredase®) in position 495, in which an arginine is substituted with a histidine.

[0006] Glycoproteins from either mammalian host cells (such as CHO ceils) or human placenta typically comprise complex N-glycans. Unmodified glucocerebrosidase that comprises complex N-glycans is primarily taken up by hepatocytes (rather than macrophages), and is therefore of limited therapeutic value. Removal of sialic acid on the placental enzyme by treatment with neuraminidase increases targeting to hepatocytes and decreases the amount taken up by non-parenchymal cell (e.g., macrophages). However, subsequent removal of galactose using β-galactosidase increases non-parenchymal cell uptake dramatically and this is further enhanced by removal of N-acetylglucosamine with hexosaminidase. The glycosidase treatment results in a glycan structure that has a terminal mannose residue. Since phagocytes have mannose receptors that recognize glycoproteins and glycopeptides with oligosaccharide chains that terminate in mannose residues, the carbohydrate remodeling of glucocerebrosidase improved the targeting of the enzyme to these cells (Van Pa tten et al., Post-translational Modification of Protein Biopharmaceuticals, 31 9-339, Ed. Gary Walsh, WILEY-VCH VerSag GmbH & Co., 2009). [0007] As such, to facilitate specific drug deliver}', it is necessary to enzymatically remove the N-eompiex glycans from recombinantly produced or placenta glucocerebrosidase. Both recombinantly produced and placenta glucocerebrosidase are treated with three different glvcosidases (neuraminidase, galactosidase, and β-Ν acetyl-glucosaminidase) to expose terminal mannoses, which enables targeting of phagocytic cells. A pharmaceutical preparation comprising the recombinantly produced enzyme is described in U.S. Pat. No, 5,549,892.

[0008] Recombinant a-Galactosidase A for enzyme replacement therapy has been produced in insect (sf9) cells (U.S. Pat. No. 7,0! 1 ,831 ), in human fibroblasts (U.S. Pat. No. 6,395,884), and in plant cells (U.S. Pat. No. 6,846,968).

[0009] Enzymatically modifying the giycan structure of a therapeutic protein can be costly. Accordingly, there is a need for improved methods for making therapeutic lysosomal proteins that can be delivered to lysosomes efficiently.

SUMMARY OF THE INVENTION

[0010] The invention generally relates to compositions and methods for producing lysosomal proteins that have altered giycan structure, such that the protein can be delivered efficiently into the lysosomes of target cell s (such as macrophages), The lysosomal proteins are produced by modifying the glycosylation pathways in a host cell using an RNA effector molecule, such as an siRNA. Glycan-modified lysosomal proteins produced using the methods described herein have improved properties, including e.g., increased specific uptake by target cells (such as macrophages).

[0011] In one aspect, the invention also provides methods for producing glycan- modified lysosomal proteins, 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 lysosomal protein. Preferred targets include, e.g., Mannose-P-dolichol utilization defect 1 protein (MPDU1) and mannosyl (a-1 ,3-)- glycoprotehi beta- 1 ,2-N-acetylglucosaminyltransferase (MGAT). [00 ! 2] In certain embodiment, the lysosomal protein is a human lysosomal protein. When the lysosomal protein is a human lysosomal protein, and the host cell is not a human host cell, it may be desirable to also reduce or prevent the expression of the corresponding host lysosomal protein,

[0013] Therefore, in one aspect, the invention provides a method for producing a composition comprising an exogenous lysosomal protein, the method comprises: culturing a large scale host cell culture in a medium that comprises an effecti ve amount of an RNA effector molecule; (a) wherein the host cell comprises (i) an exogenous nucleic acid that expresses the exogenous lysosomal protein, and (ii) an endogenous target gene that encodes an oxtholog of the lysosomal protein; (b) wherein the RNA effector is substantially complementary to the endogenous target gene that encodes the oxtholog, and reduces or prevents the expression of the endogenous target gene; and (c) wherem the host cell is cultured for a period of time sufficient for the production of the lysosomal protein, In certain embodiments, the host cell is a CHO cell, or CHO-derived cell. In certain embodiments, the exogenous lysosomal protein is a human lysosomal protein, and the endogenous gene encodes the hamster ortholog of the human lysosomal protein. In certain exnbodixnents, the invention provides a method for producing a composition comprising an human lysosomal protein, the method comprises: culturing a large scale host cell culture in a medium that comprises an effective amount of an RNA effector molecule; (a) wherein the host cell is a CHO cell or CHO-derived cell, and wherein the host cell comprises (i) an exogenous nucleic acid that expresses the human lysosomal protein, and (ii) an endogenous target gene that encodes the hamster ortholog of the human lysosomal protem; (b) wherein the RNA effector is substantially complementary to the endogenous target gene that encodes the oxtholog, and reduces or prevents the expression of the endogenous target gene; and (c) wherein the host ceil is cultured for a period of time sufficient for the production of the human lysosomal protein.

[0014] In certain embodiments, the exogenous lysosomal protein is human glucocerebrosidase, and the endogenous target gene encodes hamster glucocerebrosidase. In certain embodiments, the exogenous lysosomal protein is human acid a-glucosidase, and the endogenous target gene encodes hamster acid a-glucosidase. In certain embodiments, the exogenous lysosomal protein is a human trypsin inhibitor, and the endogenous target gene encodes the corresponding hamster trypsin inhibitor. In certain embodiments, the exogenous lysosomal protein is a human esterase inhibitor, and the endogenous target gene encodes the corresponding hamster esterase inhibitor. In certain embodiments, the exogenous lysosomal protein is human a-galactosidase A, and the endogenous target gene encodes hamster a- galactosidase A,

[0015] The RNA effector can be an siRNA, shRNA, or antisense RNA.

[0016] In another aspect, the invention provides a glycan-modified lysosomal protein that comprises at least one terminal mannose.

DETAILED DESCRIPTION OF THE INVENTION

1. OVERVIEW

[0017] As described herein, lysosomal 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 accompl ished using RNA effector molecules, such as an siRNA. Lysosomal glycoproteins produced in this way have improved properties.

[0018] For example, lysosomal proteins produced by mammalian host cells (such as CHO cells) typically comprise complex N-glycans that lack terminal mannose. siRNAs are used to transiently reduce the expression of enzymes or transporters that are in vol ved in the N-giycan biosynthesis in CHO cells. Increased amounts of lysosomal proteins that comprise at least one terminal mannose are produced upon addition of siRN As to the cell culture. The glycan- modified lysosomal proteins show increased specific uptake by target cells (such as

macrophages), as compared to unmodified lysosomal proteins (which are primarily taken up by hepatocytes)

[0019] Accordingly, in one aspect, the invention provides a method for producing a glycan-modified lysosomal protein by treating a large scale host cell culture with RNA effector molecules that target an enzyme or a transporter that are involved in the biosynthesis of glycans. In particular, Mannose-P-dolichol utilization defect 1 protein (MPDUl) and mannosyl (a-1,3-)- glyeoprotein beta- 1, 2.-N-acetylglucosaminy [transferase (MGAT) are targeted, alone or in combination, to increase the production of terminal-mannose-bearing lysosomal proteins.

[0020] 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 one, two or more genes that encode proteins involved in a desired giycosylation reaction(s). For example, MPDUl and MGAT may be targeted individually, or simultaneously. Additional gene(s) may also be targeted.

[0021] In another aspect, the invention provides a glycan-modified lysosomal protein that comprises at least one terminal mannose.

[0022] The glycan-modified lysosomal proteins 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 lysosomal proteins described herein, or pharmaceutical compositions comprising the lysosomal proteins, in therapy, and to the use of the lysosomal proteins, or pharmaceutical compositions comprising the lysosomal proteins, for the manufacture of a medicament for use in therapy.

2. DEFINITIONS

[0023] The term "about'^'', as used here, refers to +/- 10% of a value.

[0024] 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 (dsRNA), or between the antisense strand of a RNA effector molecule and a target sequence. A nucleotide sequence is "fully complementary" to another nucleoti de 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 across 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 comp 1 ementary ."

[ 0025] The term an "exogenous" protein refers to a protein that is not a protein expressed by the host cell's own genomic sequence. Exogenous proteins include a protein that is expressed by exogenously introduced nucleic acid construct, even though a gene that encodes the protein may also be present in the host cell's own genomic sequence. The term "exogenous" nucleic acid refers to a nucleic acid that is not naturally found in or produced by a host cell; and an "endogenous" nucleic acid refers to a nucleic acid that is naturally found in or produced by a host cell.

[0026] The term "glycoform" of a protein refers to a protein comprising a particular glycan stnicture 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 giycoforms of a glycoprotein.

[0027] The term "isolating" a protein refers to the separation of a protein molecule from its original environment found in nature (e.g., from host cells) with respect to its association with other molecules. Typically, an "isolated" protein is at least about 50% pure, at least about 55% pure, at least about 60% pure, at least about 65% pure, at least about 70% pure, at least about 75% pure, at least about 80% pure, at least about 85% pure, at least about 90% pure, at least about 95% pure, or at least about 99% pure.

[0028] 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 lOL, least about 20L, least about SOL, least about 40L, at least about SOL, least about 60L, least about 70L, least about 80L, least about 90L, at least about ! OQL, least about 150L, least about 200L, at least about 250L, least about 300L, least about 400L, at least about 500L, least about 600L, least about 700L, least about 800L, 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).

[0029] The term that an effective amount of an RNA effector "reduces" the expression of a target gene means that an effective amount of an RNA effector is added to a cell culture, such that the expression level of the target gene is reduced by at least about 5%, preferably at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%», at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%), at least about 85%, at least about 90%, at least about 95%, or at least about 99%.

[0030] The term "terminal maimose" refers to a mannose at the terminus of a branch of a glycan. A glycan can comprise a chain of residues having se veral different branch points (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.), resulting in a plurality of "branches." The terminus of each branch comprises a terminal group (e.g., mannose, galactose, N-acetylglucosamine, sialic acid, etc.). Thus, a "terminal mannose" refers to a mannose at the terminus of a single branch. A singl e glycan can comprise a plurality of terminal marmoses at the termini of a plurality of branches.

[0031] 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 construct encoding the RNA effector, does not integrate into the genome of the host cell.

3. GLYCAN-MODIFIED LYSOSOMAL PROTEINS [0032] In one aspect, the invention provides glycan-modified lysosomal proteins that comprise at least one terminal mannose. "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 (e.g., a gene that encodes an enzyme or a transporter protein that is involved in a glycosyiation 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.

[0033] In one aspect, the invention provides a composition comprising a lysosomal protein, wherein the composition is characterized by: (a) at least about 60% of the lysosomal protein molecules are glycosylated; and (b) at least about 50% of the glycosylated lysosomal protein molecules comprise a glycan that comprises: (i) at least one terminal marmose, and (ii) no more than 5 mannose residues total. For example, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the lysosomal protein molecules are glycosylated; and at least about 50%, at least about 55°/», at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, about 96%, at least about 97%, at least about 98%, or at least about 99% of the glycosylated lysosomal protein molecules comprise a glycan that comprises: (i) at least one termmal mannose, and (ii) no more than 5 mannose residues total.

[0034] The presence of terminal-mannose-bearing glycans facilitates the specific uptake of the lysosomal protein to its target cells, such as macrophages, as the Mannose receptor is uniquely found on macrophages, and is not found on monocytes. As such, macrophages bind terminal-mannose-bearing glycoproteins with specificity.

[0035] Preferably, the terminal mannose is exposed (i.e., the mannose residue is positioned such that, it is able to bind to a mannose receptor). One of skill in the art can determine the presence of an exposed terminal mannose by assessing the ability of a

mannosidase to remove the mannose, or by comparing the activity of the glycoprotein with a glycoform of the corresponding protein that lacks a terminal mannose. Alternatively, one can determine if a terminal mamiose is exposed by detecting binding of the glycoprotein to a mannose receptor,

[0036] Preferably, the glycan-modified lysosomal protein is internalized more efficiently by a target cell (e.g., a macrophage) tha that the corresponding unmodified lysosomal protein. For example, the glycan-modified lyososmal protein may be internalized more efficiently than the corresponding unmodified lysosomal protein by, e.g., at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%), about 70%), about 80%), or about 90% in a given time period. Alternatively, at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 fold as much of the glycan-modified lyososmal protein may be internalized, relative to the unmodified lysosomal protein, in a given time period. A. given time period may be, for example, 10 minutes, 1 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 10 hours, 12 hours, 24 hours, 48 hours, 72 hours etc.

[0037] It is also desirable that at least about 50% of the glycosylated lysosomal protein molecules comprise a glycan that comprises (i) at least one terminal mannose, and (ii) no more than 5 mannose residues total. It has been reported that in case of glucocerebrosidase, larger oiigomannose structures (e.g., Man₉GlcNAc₂) increased the binding of glucocerebrosidase to serum mannose-binding lectin (MBL). See, Van Patten et al., Glycobioiogy vol. 17, 467-478, (2007). MBL. is a collectin involved in the innate immune response and is present in serum at concentrations of 1-5 mg/niL. MBL eliminates intruding microorganisms by binding to carbohydrate structures on their surface, leading to complement system activation, opsonization, and phagocytosis. Therefore, an increase in MBL binding is undesirable and could affect the pharmacokinetic behavior of a lysosomal protein, It has also been reported that MBL could block the uptake of mannosylated liposomes into macrophages. See, Van Patten et al. (supra).

[0038] In certain embodiments, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%>, at least about 97%>, at least about 98%, or at least about 99%> of the lysosomal protein molecules are N-glycosylated. [0039] In certain embodiments, at least about 50%, at least about 55%, at least about 60%), at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%), at least about 99%), or 100% of the glycosylated lysosomal protein molecules comprise a glycan that is: Man₂GlcNAc₂, an₂GlcNAc₂Fuc, Ma¾GlcNAc₂, Man₃GlcNAc₂puc, Man₄GlcNAc2, Man₄Glc Ac2Fuc, Man₅Glc^'NAc₂, or Man₅GicNAc₂Fuc.

10040] In certain embodiments, at least about 50%, at least about 55%, at least about 60%), at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90°/», at least about 95%, at least about 96%, at least about 97%, at least about 98%), at least about 99%), or 100% of the glycosylated lysosomal protein molecules comprise a glycan that does not comprise a xylose or an a(l ,3)-fucose.

[0041] In one preferred embodiment, the lysosomal protein is a glucocerebrosidase,

[0042] In other embodiments, the lysosomal protein is not a glucocerebrosidase. In certain embodiments, the lysosomal protein is selected from the group consisting of glucosidase, galactocerebrosidase, galactosidase, iduronidase, hexosaminidase, mannosidase, fucosidase, arylsulfatase, V-acetylgalactosamine-6-sulfate sulfatase, acteylgalactosaminidase,

aspartylglucosaminidase, iduronate-2-sulfatase, a-glucosaminide-N-acetyltransferase, acetyl- CoA:a-glucosaminide N-acetyltransferase, β-D-giucoronidase, hyaluronidase, mannosidase, neurominidase, phosphotransferase, acid lipase, acid ceramidase, sphinogmyelinase,

thioesterase, cathepsin K, siaiidase and lipoprotein lipase.

[0043] In certain embodiments, the lysosomal protein is selected from the group consisting of: idursulfase, alglucosidase alfa, galsuliase, agalsidase β, laronidase, acid a- glucosidase, and a protease inhibitor.

[0044] In certain embodiments, the lysosomal protein is selected from the group consisting of idursulfase, alglucosidase alfa. galsuliase, agalsidase β, and laronidase.

[0045] Exemplary lysosomal proteins that may be used for treating LSDs are listed in Tables 1-4. Table 1: Examples of LSDs m glycoprotein degradation

Table 2: Examples of LSDs in glycosaminoglycan degradation - the mucopolysaccharidoses

Lysosomal Storage Disorder Defective Lysosomal Protein

Tay-Saehs β-bexosamimdase A

Sandhoff β-hexosaminidase A and B

GMl gangliosidosis β-galactosidase

Sialidosis sialidase

Farber acid ceramidase

Fabry a-galactosidase

Gaucher's (types i, 2, and 3) β-glucoceramidase Krabbe β-galactoceramidase

Metachromatic leukodystrophy arylsulfatase A (cerebroside sulfatase)

Saposin deficiency saposin precursor

Table 4: Examples of other types LSDs

10046] The lysosomal protein may have 1 , 2, 3, 4, 5 or more consensus sites for - lmked or O-liriked glycosylation, each of which may or may not be glycosylated, For example, human glucocerebrosidase has five N-glycosylation amino acid consensus sequences (Asn-X- Ser/Thr), four of which are normally glycosylated (Asn 19, Asn59, Asnl46 and Asn270).

[0047] The lysosomal protein may be an enzyme or a transporter protein that has optimal activity, as measured by an activity assay, at a pH ranging from 1-7, such as, a pH ranging from 1-3, 2-5, 3-6, 4-5, 5-6, or 4-6. Preferably, the lysosomal protein has optimal activity at a pH ranging between 3 to 5,

(1) glucocerebrosidase

[0048] In certain embodiments, the lysosomal protein is glucocerebrosidase. In certain embodiments, the glucocerebrosidase is a human glucocerebrosidase.

[0049] The invention provides a composition comprising a glucocerebrosidase wherein (a) at least about 60% of the glucocerebrosidase molecules are glycosylated; (b) the glycosylated molecules comprise at least two glycoforms; (c) each of the glycoforms comprises a glycan that comprises: (i) at least one terminal mannose, (ii) no more than 5 mannose residues total; and (iii) does not comprise a xylose or an a(l ,3)-fucose. [0050] In certain embodiments, of the two or more glycoforms, at least one glycoform comprises a giycan that comprises: (i) a terminal mannose, and (ii) 2, 4, or 5 mannoses total, For example, the composition may comprise a glucocerebrosidase that comprises a N-glycan that is Man₃GlcNAc₂, and a glucocerebrosidase that comprises a N-glycan that is Mar GlcNAc?. In certain embodiments, of the two or more glycoforms, at least one glycoform does not comprise a giycan that comprises: a terminal mannose and 3 mannoses total. For example, the composition may comprise a glucocerebrosidase that comprises a N-glycan that is Man₄GlcNAc₂, and a glucocerebrosidase that comprises a N-glycan that is Man₅GlcNAc₂.

[0051 ] Preferably, the giycan is an N-glycan. Preferably, the giycans are selected from any 2 (or more) of the following: Man₂GlcNAc₂, Man₂Glc Ac₂Fuc, Man₃GlcNAc₂, an₃GlcNAc₂Fuc, an₄GleNAc₂, Man₄GlcNAc₂Puc, Man₅GlcNAc₂, or MarisGlcNAc^Fue,

[0052] In certain embodiments, at least about 60%, at least about 65%, at least about 70%), at least about 75%, at least about 80%), at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the glucocerebrosidase molecules are glycosylated.

[0053] In certain embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%>, at least about 90%, at least about 95%, about 96%>, at least about 97%, at least about 98%), or at least about 99% of the glycosylated glucocerebrosidase molecules comprise a giycan that comprises (i) at least one terminal mannose, (ii) no more than 5 mannose residues total; and (iii) does not comprise a xylose or an a(l,3)-fucose.

[0054] In certain embodiments, from about 5% to about 95% of the glycosylated glucocerebrosidase molecules comprise a giycan that comprises a terminal mannose and 2 mannoses total; e.g., from about 5% to about 95%, from about 10% to about 95%, from about 15% to about 95%, from about 20%) to about 95%), from about 25% to about 95%, from about 30% to about 95%, from about 35% to about 95%), from about 40% to about 95%, from about 45°/» to about 95%, from about 50% to about 95%>, from about 55% to about 95°/», from about 60%) to about 95%), from about 65% to about 95%, from about 70% to about 95%), from about 75% to about 95%, from about 80% to about 95°/», from about 85% to about 95%>, or from about 90% to about 95% of the glycosylated glucoeerebrosidase molecules comprise a glycan that comprises a terminal mannose and 2 mannoses total.

[0055] Alternatively or in addition, from about 5% to about 95% of the glycosylated glucoeerebrosidase molecules comprise a glycan tha comprises a terminal mannose and 3 mannoses total; e.g., from about 5% to about 95%, from about 10% to about 95%, from about 15% to about 95%, from about 20% to about 95%, from about 25% to about 95%, from about 30°/» to about 95%, from about 35% to about 95%, from about 40% to about 95°/», from about 45% to about 95%, from about 50% to about 95%, from about 55% to about 95%, from about 60% to about 95%, from about 65% to about 95°/», from about 70% to about 95%, from about 75% to about 95%, from about 80% to about 95%, from about 85% to about 95%, or from about 90% to about 95% of the glycosylated glucoeerebrosidase molecules comprise a glycan that comprises a terminal mannose and 3 mannoses total.

[0056] Alternatively or in addition, from about 5% to about 95% of the glycosylated glucoeerebrosidase molecules comprise a glycan that comprises a terminal mannose and 4 mannoses total; e.g., from about 5% to about 95%, from about 10% to about 95%, from about 15% to about 95%, from about 20% to about 95%, from about 25% to about 95%, from about 30°/» to about 95%, from about 35% to about 95%, from about 40% to about 95°/», from about 45% to about 95%, from about 50% to about 95%, from about 55% to about 95%, from about 60% to about 95%, from about 65% to about 95%, from about 70% to about 95%, from about 75% to about 95%, from about 80% to about 95%, from about 85% to about 95%, or from about 90% to about 95% of the glycosylated glucoeerebrosidase molecules comprise a glycan that comprises a terminal mannose and 4 mannoses total.

[0057] Alternatively or in addition, from about 5% to about 95% of the glycosylated glucoeerebrosidase molecules comprise a glycan that comprises a terminal mannose and 5 mannoses total; e.g., from about 5% to about 95%, from about 10% to about 95%, from about 15% to about 95%, from about 20% to about 95%, from about 25% to about 95%, from about 30°/» to about 95%, from about 35% to about 95%, from about 40% to about 95°/», from about 45% to about 95%, from about 50% to about 95%, from about 55% to about 95%, from about 60% to about 95%, from about 65% to about 95%, from about 70% to about 95%, from about 75% to about 95%, from about 80% to about 95%, from about 85% to about 95%, or from about 90% to about 95% of the glycosylated glucoeerebrosidase molecules comprise a glycan that comprises a terminal mannose and 2 mannoses total.

[0058] Alternatively or in addition, no more than about 50%) of the glycosylated glucoeerebrosidase molecules comprise a glycan that comprises 6 mannoses or more; e.g., no more than about 50%, no more than about 45%), no more than about 40%», 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%, no more than about 10%, or no more than about 5%> of the gly cosylated

glucoeerebrosidase molecules comprise a glycan that comprises 6 mannoses or more.

[0059] Glucoeerebrosidase (also called acid β-glucosidase, D-glucosyi-N- acylsphingosme glucohydrolase, or GCase) is an enzyme with glucosylceramidase activity (EC 3.2.1.45) that is needed to cleave the β -glucosidic linkage of the chemical glucocerebroside. Mature human glucoeerebrosidase is a 497 amino acid membrane-associated monomelic glycoprotein of about 67 kDa and has a pH optimum for enzymatic activity of about 5.5.

Glucoeerebrosidase has five N-glycosylation amino acid consensus sequences (Asn-X-Ser/Thr), four of which are normally glycosylated (Asnl9, Asn59, Asnl46 and Asn270). Recombinant- produced glucoeerebrosidase (Cerezyme®; SEQ ID NO:2) differs from placenta

glucoeerebrosidase (Ceredase®; SEQ ID NO:3) in position 495, in which an argi ine is substituted with a histidine.

[0060] In certain embodiments, the lysosomal protein comprises an amino acid 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 Imiglucerase (Cerezyme®; SEQ ID NO: 2). In certain embodiments, the lysosomal protein comprises an amino acid sequence that is at least 75%», at least 8G%>, 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 Alglucerase (Ceredase©; SEQ ID NO: 3).

[0061] The glycan-modified glucoeerebrosidase can be used to treat Gaucher s (types 1 , 2, and 3).

(2) Acid a-glucosid se [0062] In certain embodiments, the lysosomal protein is an acid a-glucosidase (E.C. 3,2. 1.20). In certain embodiments, the acid a-glucosidase is a human acid a-ghicosidase.

[0063] The invention provides a composition comprising an acid a-glucosidase, wherein the composition is characterized by: (a) at least about 60% of the acid a-glucosidase molecules are glycosylated; and (b) at least about 50% of the glycosylated acid a-giucosidase molecules comprise a glycan that comprises: (i) at least one terminal mannose, and (ii) no more than 5 mannose residues total. For example, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the acid α-giucosidase molecules are glycosylated; and at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%», about 96%», at least about 97%, at l east about 98%, or at least about 99% of the glycosylated acid α-glucosidase molecules comprise a glycan that comprises (i) at least one terminal mannose, (ii) no more than 5 mannose residues total.

[0064] Preferably, at least about 50% (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%>, about 96%>, at least about 97%. at least about 98%, or at least about 99%) of the glycosylated acid a-glucosidase molecules comprise a glycan that comprises (i) at least one terminal mannose, (ii) no more than 5 mannose residues total; and (iii) does not comprise a xylose or an a(l ,3)-fucose.

[0065] Acid a-glucosidase is a lysosomal enzyme essential for the degradation of glycogen to glucose in lysosomes, and catalyzes the hydrolysis of a- 1,4- and a- 1,6- glycosidic linkages of glycogen. Human acid α-glucosidase is encoded by the GAA gene,

[0066] In certain embodiments, the lysosomal protein comprises an amino acid 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 Alglucosidase alfa (Lumizyme® or Myozyme©; SEQ ID NO: I). Commercially available Alglucosidase alfa is produced by recombinant DNA technology in a Chinese hamster ovary cell line. [0067] The glycan-modified acid a-gtucosidase can be used to treat Pompe disease, (3) Protease Inhibitors

[0068] in certain embodiments, the lysosomal protein is protease inhibitor. In certain embodiments, the protease inhibitor is a human protease inhibitor.

[0069] The invention provides a composition comprising a protease inhibitor, wherein the composition is characterized by: (a) at least about 60% of the protease inhibitor molecules are glycosylated; and (b) at least about 50% of the glycosylated protease inhibitor molecules comprise a glycan that comprises: (i) at least one terminal mannose, and (ii) no more than 5 mannose residues total. For example, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%», at least about 90%», at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the protease inhibitor molecules are glycosylated; and at least about 50%), at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%», about 96%», at least about 97%), at least about 98%, or at least about 99%) of the glycosylated protease inhibitor molecules comprise a glycan that comprises (i) at least one terminal mannose, (ii) no more than 5 mannose residues total.

[0070] Preferably, at least about 50%) (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%», about 96%», at least about 97%, at least about 98%, or at least about 99%) of the glycosylated protease inhibitor molecules comprise a glycan that comprises (i) at least one terminal mannose, (ii) no more than 5 mannose residues total; and (iii) does not comprise a xylose or an a(l,3)-fucose.

[0071] In certain embodiments, the lysosomal protein is a trypsin inhibitor, such as alphaj -antitrypsin (also known as the serum trypsin inhibitor), ovomucoid, basic pancreatic trypsin inhibitor (e.g., aprotinin), a soybean trypsin inhibitor (SBTI, such as a Kunitz trypsin inhibitor, or a Bowman-Birk trypsin and chymotrypsin inhibitor). [0072] Alpha 1 -Antitrypsin (A1AT, or a 1 -antitrypsin) is a protease inhibitor belonging to the serpin superfamily. It protects tissues from enzymes of inflammatory cells, especially neutrophil elastase. In its absence, neutrophil elaslase is free to break down eiastin, which contributes to the el asticity of the lungs, resulting in respiratory complications such as emphysema, or COPD (chronic obstructive pulmonary disease) in adults and cirrhosis in adults or children. Mature al -antitrypsin is a single-chain glycoprotein of 394 amino acids, and has a number of glycoforms. The three N-linked glycosvlation sites are mainly biantennary (complex) N-glycans. However, one particular site, Asparagine 107 (ExPASy amino acid nomenclature), shows a considerable heterogeneity, since tri- and even tetra-antennary N-glycans can be attached to Asnl07. These glycans also cany different amounts of sialic acids. In addition, ai,3 inked fucosylated triantennary N-glycans, which forms part of the "Sialyl Lewis x epitope," also exist.

[0073] Three al -antitrypsin products derived from human plasma have been approved by the FDA, Prolastin® (Talecris Biotherapeutics), Zemaira® (Aventis Behring LLC), and Aralast® (Baxter International). All three products showed minor differences compared to the normal human plasma A 1 AT, and are introduced during the specific purifications procedures. See, Kolarich D. et al., Transfusion 46 (11): 1959-77 (2006). Recombinant alpha 1 -antitrypsin is not yet commercially available.

[0074] In certain embodiments, the lysosomal protein comprises an amino acid 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 al -antitrypsin (SEQ ID NO: 4).

[0075] The gly can-modified alpha 1 -antitrypsin can be used to treat alpha 1 ~ antitrypsin deficiency.

[0076] In certain embodiments, the lysosomal protein is an esterase inhibitor, such as a CI Esterase Inhibitor, or a Ubiquitin C-Terminal Hydrolase Esterase (UCH) inhibitor (e.g., UCH-L1 Inhibitor, UCH-L2 Inhibitor, or UCH-L3 Inhibitor).

[0077] CI -inhibitor (also called CI esterase inhibitor) is a protease inhibitor belonging to the serpin superfamily. Its main function is to inhibit the complement system to prevent spontaneous activation. CI -inhibitor has a 2-domain structure. The C-terminal serpin domain is similar to other serpins, and is responsible for its mhibitory activity. The N-terminal domain is not essential for its inhibitor}' activity. CI -inhibitor is highly glycosylated, bearing both N- and O-glycans. The N-terminal domain is especially heavily glycosylated. Human CI - inhibitor is encoded by the SERPING1 gene.

[0078] Deficiency of CI -inhibitor is associated with hereditary angioedema

(hereditary angioneurotic edema, or HAE), or swelling due to leakage of fluid from blood vessels into connective tissue.

10079] In certain embodiments, the lysosomal protein comprises an amino acid 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 human CI -inhibitor (Cinryze®, SEQ ID NO: 5). Cinryze© is a pharmaceutical-grade CI -inhibitor approved for the use of HAE.

[0080] The glycan-modified CI -inhibitor can be used for treating CI -inhibitor deficiency (Types I, II, III), as well as sepsis, vascular leak syndrome, acute myocardial infarction, pancreatitis, thermal injury, and for the management of xenotransplantation. See, Caliezi C, et al, Pharmacol. Rev. 52 (1): 91-112 (2008).

(4) a-galactosidase A

[0081] In certain embodiments, the lysosomal protein is an a-galactosidase A (E.G. 3.2.1.22). In certain embodiments, the a-galactosidase A is a human a-galactosidase A.

[0082] The invention provides a composition comprising an α-galactosidase A, wherein the composition is characterized by: (a) at least about 60%) of the a-galactosidase molecules are glycosylated; and (b) at least about 50% of the glycosylated a-galactosidase molecules comprise a glycan that comprises: (i) at least one terminal mannose, and (ii) no more than 5 mannose residues total. For example, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the α-galactosidase molecules are glycosylated; and at least about 50%, at least about 55%), at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85°/», at least about 90%, at least about 95%, about 96%, at least about 97%, at least about 98%, or at least about 99% of the glycosylated a-galaetosidase molecules comprise a giycan that comprises (i) at least one terminal mannose, (ii) no more than 5 mannose residues total,

[0083] Preferably, at least about 50% (e,g,, at least about 50%, at least about 55%, at least about 60%), at least about 65%), at least about 70%), at least about 75%, at least about 80%, at least about 85%), at least about 90%, at least about 95%, about 96%, at least about 97%, at least about 98%>, or at least about 99%) of the glycosylated acid a-glucosidase molecules comprise a glycan that comprises (i) at least one terminal mannose, (ii) no more than 5 mannose residues total; and (iii) does not comprise a xylose or an cx(l ,3)-fucose.

[0084] a-galactosidase A (a-D-galactoside galactohydrolase, E.C. 3.2, 1.22) is a lysosomal glycoprotein of about 101 kDa and has a homodimeric structure, It contains a 5-15% N-linked complex and high-mannose oligosaccharide chains. It hydrolyses the terminal a- galactosyl moieties from glycolipids and glycoproteins, It predominantly hydrolyzes ceramide trihexoside, and it can catalyze the hydrolysis of melibiose into galactose and glucose, a- galactosidase A is encoded by the GLA gene.

[0085] In certain embodiments, the lysosomal protein comprises an amino acid 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 Agalsidase (Replagal® or

Fabrazyme®; SEQ ID NO: 10).

[0086] Replagal© ( Agalsidase alpha) is produced by human fibroblasts that have been modified by gene activation; Fabrazyme® (Agalsidase beta) is produced by CHO cells that are transduced with human a-galactosidase A cDNA, Agalsidase alpha and beta both have the same amino acid sequence as the native enzyme,

[0087] The glycan-modified α-galactosidase A can be used to treat Fabry's disease.

4. METHODS FOR MODIFYING PROTEIN GLYCOSYLATION

[0088] The invention also provides methods for producing glycan-modified lysosomal proteins, 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 lysosomal protein.

[0089] In one aspect, the invention provides a method for producing a composition comprising a lysosomal protem, the method comprises: culturing a large scale host ceil culture in a medium that comprises an effective amount of an RNA effector molecule; fa) wherein the host ceil (i) expresses the lysosomal protein, and (ii) comprises a target gene that encodes Mannose-P-dolichol utilization defect 1 protein (MPDUl); (b) wherein the RNA effector is substantially complementary to the target gene that encodes MPDU 1 , and reduces or prevents the expression of the target gene; and (c) wherein the host ceil is cultured for a period of time sufficient for the production of the iyososomal protein.

[0090] MPDUl (also known as Lec35) is an endoplasmic reticulum membrane protein that is required for the utilization of the mannose donor mannose-P-dolichol in the synthesis of lipid-linked oligosaccharides and giycosylphosphatidyiinositols {see, e.g., Wopereis et ai,, Clinical Chemistry 52: 574-600, 2006). Mutations in MPDUl result in congenital disorder of glycosylation type I F. The sequence of hamster gene that encodes MPDUl is provided in Appendix II.

[0091] If desired, two or more genes may be targeted. For example, the host cell may further comprises a second target gene that encodes a mannosyl (a- 1 ,3-)-glycoprotein beta- 1,2-N-acetylglucosaminyltransferase (MGAT); and the method may further comprise: culturing the large scale host ceil culture in a medium that comprises an effective amount of a second RNA effector molecule, wherein the second RNA effector is substantially complementary to the second target gene that encodes MGAT, and wherein the RNA effector reduces or prevents the expression of the second target gene that encodes M GAT.

[0092] MGATs are a family of enzymes that are responsible for the biosynthesis of hybrid-type and complex-type N-glycans (Figure 1). Known members of MGATs include, e.g., MGAT1 (EC 2.4.1.101), MGAT2 (EC 2.4.1.143), MGAT3 (EC 2.4.1.144), GAT4A and MGAT4B (EC 2.4.1.144), MGATS A and MGAT5B (EC 2.4.1.155). The sequences of hamster gene that encodes MGATl , MGAT2, and MGAT4B are provided in Appendix II, In certain embodiments, MGATl , MGAT2, or MGAT4B, is targeted.

[0093 j In certain embodiments, MGAT 1 , MGAT2, or MGAT4B, is targeted.

[0094] In one aspect, the invention provides a method for producing a composition comprising a lysosomal protein, the method comprises: culturing a large scale host cell culture in a medium that comprises an effecti ve amount of an RNA effector molecule; (a) wherein the host cell (i) expresses the lysosomal protein, and (ii) comprises a target gene that encodes a mannosyl (a-1 ,3-)-giycoprotein beta-1 ,2-N-acetylglucosaminyltransferase (MGAT); (h) wherein the RNA effector is substantially complementary to the target gene that encodes MGAT, and reduces or prevents the expression of the target gene; and (c) wherein the host cell is cultured for a period of time sufficient for the production of the lyososomal protein. In certain embodiments, MGATl , MGAT2, or MGAT4B, is targeted, In one embodiment, MGAT2 is targeted.

[0095] In certain embodiment, the lysosomal protein is a human lysosomal protein. When the lysosomal protein is a human lysosomal protein, and the host cell is not a human host ceil, it may be desirable to also reduce or prevent the expression of the corresponding host lysosomal protein. For example, if CHO cells (or CHO-derived ceils) are used to express human glucocerebrosida se, it may be desirable to also reduce or prevent the expression of the hamster glucocerebrosidase. Otherwise, the endogenous hamster glucocerebrosidase expressed by CHO cells could co-purify with exogenous human glucocerebrosidase.

[0096] Therefore, in one aspect, the invention provides a method for producing a composition comprising an exogenous lysosomal protein, the method comprises: culturing a large scale host cell culture in a medium that comprises an effective amount of an RNA effector molecule; (a) wherein the host cell comprises (i) an exogenous nucleic acid that expresses the exogenous lysosomal protein, and (ii) an endogenous target gene that encodes an ortholog of the lysosomal protein; (b) wherein the RNA effector is substantially complementary to the endogenous target gene that encodes the ortholog, and reduces or prevents the expression of the endogenous target gene; and (c) wherein the host cell is cultured for a period of time sufficient for the production of the lysosomal protein. [0097] In certain embodiments, the host cell is a CHO cell, or CHO-derived cell. In certain embodiments, the exogenous lysosomal protein is a human lysosomal protein, and the endogenous gene encodes the hamster ortholog of the human lysosomal protein,

[0098] In certain embodiments, the exogenous lysosomal protein is huma

glucocerebrosidase, and the endogenous target gene encodes hamster glucocerebrosidase. In certain embodiments, the exogenous lysosomal protein is human acid a-glucosidase. and the endogenous target gene encodes hamster acid a-glucosidase. in certain embodiments, the exogenous lysosomal protein is a human trypsin inhibitor, and the endogenous target gene encodes the corresponding hamster trypsin inhibitor, in certain embodiments, the exogenous lysosomal protein is a human esterase inhibitor, and the endogenous target gene encodes the corresponding hamster esterase inhibitor. In certain embodiments, the exogenous lysosomal protein is human a-galactosidase A, and the endogenous target gene encodes hamster ct- galactosidase A.

[0099] The R A effector can be an siRNA, shRNA, or antisense RNA.

[00100] Additional genes that encode a protein that is involved in lysosome targeting may also be targeted, such as, e.g., a protein that is involved in the synthesis of mantiose-6- phosphate.

[00101] In certain embodiments, the RNA effector transiently reduces the expression of its target gene.

[00102] In certain embodiments, the method further comprising harvesting said glycoprotein from said large scale culture.

B. RNA effector molecules

[00103] 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.

[00104] RNA effector molecules are ribonucleotide agents that are capable of reducing or preventing the expression of a target gene within a host cel l, 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 18, 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 degradation or otherwise prevent their translation.

[00105] 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 RN A

(dsRNA), microRNA (miRNA), antisense RNA, promoter-directed RNA (pdRNA), Piwi- interacting RNA (piRNA), expressed interfering RNA (eiRNA), short hairpin RNA (shRNA), antagomirs, decoy RNA, DNA, piasmids and aptamers. The RNA effector molecule can be single-stranded or double-stranded. A single-stranded RNA 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.

[00106] 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.

[00107] In some embodiments, the RNA effector molecule comprises a single- stranded oligonucleotide that interacts with and directs the cleavage of RN A 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, a antisense nucleic acid can form hydrogen bonds with a sense nucleic acid target.

[00108] 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 mles 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 deoxyribonucleotides and ribonucleotides. For example, an antisense agent consisting only of ribonucleotides can hybridize to a complementary RNA and prevent access of the translation machinery to the target RNA transcript, thereby preventing protein synthesis. An antisense molecule including only deoxyribonucleotides, or deoxyribonucleotides and ribonucleotides, can hybridize to a complementar RNA and the RN A 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 RNAseH activity is desired.

[00109] 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 substantial ly complementary to one of the target genes, and wherein said region of complementarity is from 10 to 30 nucleotides in length.

[00110] 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.

[00111] 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, 11, 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.

[00112] 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."

[00113] 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). [00114] 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 overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucieotide(s) of an overhang can be present on the 5' end, 3' end, or both ends of either an antisense or sense strand of a dsRNA.

[00115] In one embodiment, at least one end of a dsR A has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides.

[00116] The overhang can comprise a deoxyribonucleoside or a nucleoside analog. Further, one or more of the mternucloside l inkages 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 dT, 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 intemucleoside linkage.

[00117] 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 RN A effector molecule contains one or more mismatches to a target sequence, it is preferable that the rnismatch(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 RN A effector molecule agent RNA, the antisense strand generally does not contain any mismatch within the central 13 nucleotides.

[00118] The dsRNA can be synthesized by standard methods known in the art as further discussed belo w, e.g., by use of an automated D A synthesizer, such as are

commercially available from, for example, Biosearch Technologies (Novato, CA).

[00119] In some embodiments, the RNA effector molecule is a promoter-directed

RNA (pdRNA) which is substantially complementary to a noncoding region of an mRNA 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 than 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^* MOE, 2'OMe, or Locked Nucleic Acid bases (LNA), which protect the gapmer from cellular nucleases.

[00120] 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 RNA transcripts which overlap with noncoding regions of a target gene mRNA transcript, and recruiting Argonaute proteins (in the case of dsRNA) or host cell nucleases (e.g., RNase H) (in the case of gapmers) to selectively degrade the endogenous antisense RNAs. In some embodiments, the endogenous antisense RNA negatively regulates expression of the target gene and the 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 RNA. Methods for identifying antisense transcripts encoded by promoter sequences of target genes and for making and using promoter- directed RNAs are known, see, e.g., WO 2009/046397.

[00121] 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 structure that directs the recognition of a targeted binding site on the non-nucleic acid ligand. Aptamers can contain any of the modifications described herein. [00122] In some embodiments, the RNA effector molecule comprises an antagomir. Antagomirs are single stranded, double stranded, partially double stranded or hairpin structures that target a micro R A. 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 mi RNA. or pre-miRNA nucleotide sequence. Antagomirs preferably have a nucleotide sequence sufficiently complementary to a miRNA target sequence of abou t 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.

[00123] In some embodiments, antagomirs are stabilized against nucleo lytic 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 firs t, second, and/or third internucleotide linkages at the 5' or 3' end of the nucleotide sequence. In further embodiments, antagomirs include a 2 '-modified nucleotide, e.g., a 2'-deoxy, T- deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2 ' -O-dimethy laminoethy 1 (2 ' -O-DMAOE), 2 ' -O-dimethylaminopropyl f 2 '-0-DM AP ), 2 ' -O- dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0-N-methylacetamido (2'-0-NMA). In some embodiments, antagomirs include at least one 2'-0-metbyl-modified nucleotide.

[00124] In some embodiments, the RNA 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 complementar to the 3'-UTR of a target gene mR 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 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 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.

[00125] Expressed interfering RNA (eiRNA) can be used to selectively increase, decrease, or otherwise modulate expression of a target gene. Typically, for eiRNA, the dsRNA is expressed in the first transfected cell from an expression vector. In such a vector, the sense strand and the antisense strand of the dsRNA can be transcribed from the same nucleic acid sequence using e.g., two convergent promoters at either end of the nucleic acid sequence or separate promoters transcribing either a sense or antisense sequence. Alternatively, two plasmids can be cotransfected, with one of the plasmids designed to transcribe one strand of the dsRN A 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.

[00126] In some embodiments, the RNA effector moiecule comprises a small single- stranded Piwi-interacting RNA (piRNA effector molecule) which is substantially

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

Aubergine subclasses of Argonaute proteins. A piRNA effector molecule can be about 10 to 50 nucleotides in length, about 25 to 39 nucleotides in length, or about 26 to 31 nucleotides in length. See, e.g., U.S. Patent Application Pub. No. 2009/0062228.

[00127] MicroRNAs are a highly conserved class of small RNA. molecules that are transcribed from DN A in the genomes of plants and animals, but are not translated into protein. Pre-microR As 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 mRNAs, MicroRNAs cause post- transcriptional silencing of specific target genes, e.g., by inhibiting translation or initiating degradation of the targeted mRNA. in some embodiments, the miRNA is completely complementary with the 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 bul ge) is flanked by regions of sufficient complementarity, e.g., complete complementarity, to allow duplex formation. For example, the regions of complementarity are at least 8 to 10 nucleotides long (e.g., 8, 9, or 10 nucleotides long).

|0012S] miRNA can inhibit gene expression by, e.g., repressing translation, such as when the miRNA is not completely complementary to the target nucleic acid, or by causing target RNA degradation, when the miRNA binds its target with perfect or a high degree of complementarity .In further embodiments, the RNA effector molecule can include an

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

[00129] The miRN A can comprise naturally occurring nucleobases, sugars, and covalent internucleotide (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 miRN A target, and/or increased stability in the presence of nucleases. In some embodiments, an miRNA designed to bind to a specific endogenous miRNA has substantial complementarity, e.g., at least 70%, 80%, 90%, or 100% complementary, with at least 10, 20, or 25 or more bases of the target miRNA. Exemplary 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.

100130] A miRNA or pre-miRNA can be 10 to 200 nucleotides in length, for example from 16 to 80 nucleotides in length. Mature miRNAs 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 have 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 Drosha. miRNAs or pre-miRNAs can be synthesized in vivo by a cell-based system or can be chemically synthesized. mi RNAs 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.

[00131] Upon contact with a ceil 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 immunoblot.

[00132] Optionally, an RNA effector may bechemically modified to enhance stability or other beneficial characteristics.

[00133] 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, nc., NY). Modifications include, for example, (a) end modifications, e.g., 5 ' end modifications (phosphorylation, conjugation, inverted linkages, etc.), or 3 ' end modifications (conjugation, DN A 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) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. Specific examples of

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

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

100134] Modified intemucieoside linkages include (e.g., RNA backbones) include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, ammoaikyiphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphmates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,

thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 '-5' linkages, 2'-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3 '-5' to 5 '-3' or 2'-5' to 5 '-2'. Various salts, mixed salts and free acid forms are also included.

[00135] 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. RE39464.

[00136] Additionally, both the sugar and the intemucieoside 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 he found, for example, in Nielsen et al., 254 Science 1497-1500 (1991).

[00137] 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- alkynyi; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and aikynyl can be substituted or unsubstituted Q to Cio alkyl or C₂ to Cio alkenyl and aikynyl. Exemplary suitable modifications include 0[(CH₂)_nO]_raCH , 0(CH₂)_nOCH₃, G(CH₂)_nNH₂, 0(CH₂)_:1CH₃, 0(CH₂)_nONH₂, and 0(CH₂)_nON[(CH₂)_nCH₃)]₂, where n and m are from 1 to 10, inclusive. In some embodiments, oligonucleotides include one of the following at the 2' position: Ci to Cio lower alkyl, substituted lower alky], alkaryl, aralkyl, Q~alkaryl or O-aralkyl, SH, 8CH₃, OCN, CI, Br, CN, CF₃, OCF3. SOCH₃, S0₂CH₃, ON0 , N0₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalky] amino, substituted silyl, a RNA cleaving group, a reporter group, an intercalator, 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- CH₂CH₂OCH₃, 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(CH₂)₂ON(CH₃)₂ group, also known as 2 -D AOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethylaminoethoxyethyl or 2 -DMAEOE), i.e., 2'-0-CH₂-0-CH₂-N(CH₂)₂.

[00138] Other modifications include 2'-methoxy (2'-OCH ), 2'-aminopropoxy (2'-OCH₂CH₂CH₂NH₂) and 2'-fiuoro (2'-F). Similar modifications can also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar 011 the 3' terminal nucleotide or in 2'-5' linked oligonucleotide and the 5' position of 5' terminal nucleotide.

Oligonucleotides can also ha ve sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative patents that teach the preparation of such modified sugar stmctures include, but are not limited to, U.S. Patents No. 4,981,957; No. 5,1 18,800;

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, 5,567,811; 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.

[00139] 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 (Li). Modified nucleobases include other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubuiarine, isoguanisine, tubercidine, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine₅ 2

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

(isopentenyl)adenine, 6 (alkyl)ademne, 6 (methyl)adenine, 7 (deazajadenine, 8 (alkerryl)adenine, 8-(alkyl)adenme, 8 (alkynyl)adenine, 8 (amino )adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8 (thioalkyl)adenine, 8-(thiol)adenine, N6-(isopentyl)adenine, N6 (methyl)adenine, N6, N6 (dimethyl)adenme, 2-(alkyl)guanine,2 (propyl)guanine, 6-(alkyl)guanine, 6 (methyl)guanine,

7 (alkyl)guaiiine, 7 (methyl)guanine, 7 (deaza)guanine, 8 (alkyl)guanine, 8-(alkenyl)guanine,

8 (alkynyl)guanine, 8-(amino)guanine, 8 (halo)guanine, 8-(hydroxyl)guanine,

8 (thioalkyl)guaniiie, 8-(thiol)guanine, N (methyi)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 (niethyl)cytosine, 5 (propynyi)cytosine, 5 (propynyi)cytosine,

5 (triiluoroniethy])cytosine, 6-(azo)cytosine, N4 (aceiyl)cytosine, 3 (3 amino-3

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

(thio)uracii, 4-(thio)uracil, 5 (methyl) 4 (thio)uracil, 5 (methylammomethyl)-4 (thio)uracil, 5 (methyl) 2,4 (dithio)uracil, 5 (methylaminomethyl)-2,4 (dithio)uracil, 5 (2-aminopropyl)uracil, 5-(aikyi)uracil, 5-(alkynyl)uracil, 5-(allyla.miiio)uracil, 5 (aminoaliyl)uracil,

5 (aminoalkyl)uracii, 5 (guanidiniumaikyi)uracil, 5 (l ,3-diazole-] -alkyl)uracil,

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

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

(triiluoromeihyi)uracii, 6 (azo)uracil, dihydrouracil, N3 (methyl)uracil, 5 -uracil (i.e.,

pseudouracil), 2 (thio)pseudouracil,4 (thio)pseudouracil,2,4-(dithio)psuedouracil,5- (alkyl)pseudouracil, 5~(methy])pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2- (thio)pseudouracil, 5-(alkyl)-4 (thio)pseudouracil, 5-(methyl)-4 (thio)pseudouracil, 5-(alkyi)~2,4 (dithio)pseudouracii, 5-(methyl)-2,4 (dithio)pseudouracii, 1 substituted pseudouracil,

1 substituted 2(thio)-pseudouracil, 1 substituted 4 (thio)pseudouracil, I substituted 2,4- (dithio)pseudouracil, 1 (aminocarbonylethylenyl)-pseudouracil, 1 (aminocarbonylethylenyl)- 2(thio)-ps udouracil, 1 (aminocarbonylethylenyl)-4 (thio)pseudouracil,

1 (aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1 (aminoalkylaminocarbony!ethylenyi)- pseudouracil, 1 (aniinoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil,

1 (aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil,

1 (amiiioalkylamiiiocarbonylethyienyl)-2,4-(dithio)pse 1 ,3-(diaza)-2-(oxo)- phenoxazin- 1 -yl, 1 -(aza)-2-(thio)-3-(aza)-phenoxazin- 1 -yl, 1 ,3-(diaza)-2-(oxo)-phenthiazin- 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)-phenoxazin- 1 -yl, 7-substituted 1 ,3~(diaza)-2-(oxo)- phenthiazin- 1 -yl, 7-substituted 1 -(aza)-2-(thio)-3 -(aza)-phenthiazin- 1 -y 1,

7-(aminoalkylhydroxy)- 1 ,3-(diaza)-2-(oxo)-phenoxazin- 1 -yl, 7-(aminoalkylhydroxy)- 1 -(aza)-2- (thio)-3-(aza)-phenoxazin- 1 -y ί , 7-(aminoalkylhydroxy)- 1 ,3-(diaza)-2-(oxo)-phenthiazin- 1-yl, 7-(aminoalk lhydroxy)- 1 -(aza)-2-(thio)-3 -(aza)-phenthiazin- 1 -yl, 7-(guanidiniumalkylliydroxy)- l,3-(diaza)-2-(oxo)-phenoxazm-l-yi, 7-(guamdiniumalkylhydroxy)-l -(aza)-2-(thio)-3-(aza)- phenoxazin- 1 -yl, 7-(gimiiidimumalkyl-hydroxy)- 1 , 3-(diaza)-2-(oxo)-phenthiazin- 1-yl,

7-(guanidiniumalkylhydroxy)- 1 -(aza)-2-(thio)-3-(aza)-phen.thiaziii- ] -yl, l,3,5-(triaza)-2,6- (dioxa)-naphthaiene, inosine, xanthine, hypoxanthme, nubuiarine, tubercidme, isoguanisine, inosinyi, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazoly], nitroindazolyi, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrily[,

5- (methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7~(aza)indolyl, 6-(methy{)- 7-(aza)indolyl, imidizopyridinyl, 9-(niethyl)-imidizopyridinyl, pyrrolopyrizvnyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl, 2,4,5-(trinietiiyl)plienyl,

4-(methyl)indolyl, 4,6-(dimethyi)indoiyl, phenyl, napthalenyl, anthracenyl, phenanthracenyi, pyrenyl, stilbenyi, tetracenyl, pentacenyi, difluorotoiyl, 4-(fluoro)-6-(niethyl)benzimidazole, 4-(methyi)berizimida.zoie, 6~(azo)thymine, 2-pyridinone, 5 nitroindoie, 3 nitropyrrole,

6- (aza)pyriniidine, 2 (animo)purine, 2,6-(diamino)purine, 5 substituted pyriniidines,

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-pyrrolo-pyrimidin-2-on-3-yl, ortho-substituted-6-pheny3-pyrrolo-pyrimidm-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- 3, 2-oxo-pyridopyrimidme-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.

[00140] 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 Mol. Cancer Ther. 833-43 (2007); Grunweller 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.

100141] In certain instances, the oligonucleotides of a RNA effector molecule can be modified by a non-ligand group. A number of non-iigaiid 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 al, 86 PNAS 6553 (1989)); choiic acid (Manoharan et al., 1994); a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., 1992; Manoharan et al, 1993); a thiocholesteroi (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); Svinarchuk et al., 75 Biochimie 75 (1993)); a phospholipid, e.g., di-hexadecyi- rac-glyceroi 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 adaniantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995); a palmityl moiety (Mishra et al,, 1995); or an octadecylamine or hexyiamino-carbonyl-oxycholesierol moiety (Crooke et al., 1996). Representative United States patents that teach the preparation of such RNA conjugates have 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 wit 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

[00142] 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 2011/005786, which discloses exemplary delivery methods that can be used in this invention at pages 187-219, the teachings of which are incorporated herein by reference, For example, deliver}' 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.

100143] 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 lipophillic group, etc.

[00144] For example, RNA effector molecules can be delivered using a drug deliver}' system such as a nanoparticie, a dendrimer, a polymer, a liposome, or a cationic delivery system. Positively charged cationic deliver}' 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 caiionic-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); Verrna et al., 9 Clin. Cancer Res. 1291-1300 (2003); Arnold et al, 25 J. Hypertens. 197-205 (2007).

[00145] In one embodiment, the reagent that facilitates RNA effector molecule uptake used herein comprises a charged lipid as described in international Application Ser.

No, PCT/US 10/59206, filed 7 December 2010.

[00146] 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 arachidomc acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, iinoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1 -monocaprate,

1 -dodecylazacycloheptan-2-one, an acylcarnitine, an acyicholine, or a C I -20 alkyl ester (e.g., isopropylmyri state 1PM), monoglyceride, diglyceride, or acceptable salts thereof.

[00147] 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. 2009/0291 131; U.S. Patent Applications Ser. No, 12/343,342; No.12/424,367. The term "SPLP" refers to a nucleic acid- lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate), 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 degradati on 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.

[00148] 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.

[00149] A cationic lipid of the formulation can comprise at least one protonatable group having a pKa of from 4 to 15. The cationic lipid can be, for example, N,N-dioleyl-N, - dimethyiammonium chloride (DODAC), N.N-distearyl-N,N-dimethylammomum bromide (DDAB), N-(I-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(i- (2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3- dioleyioxy)propylamine (DODMA), 1 ,2-DiLinoleyloxy-N,N-dimethylaminopropane

(DLinDMA), 1 ,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLeiiDMA), 1 ,2- Dilinoiey]carbamoyloxy~3-dimethylaminopropane (DLin-C-DAP), l,2~Dilinoleyoxy~3~ (dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-Dilinoleyoxy-3-morpholinopropane (DLin- MA), l,2-Dilinoleoyi-3-dimediylaminopropane (DLinDAP), l,2-Dilinoleyithio-3- dimethyiaminopropane (DLin-S-DMA), l -Linoleoyl-2-linoleyioxy-3-diniethylaniinopropane (DLin-2-DMAP), l,2-Dilinoleyloxy-3-trimediylaminopropane chloride salt (DLin-TMA.Ci), 1 ,2-Dilinoieoy3-3-trimethylammopropane chloride salt (DLin-TAP.Q), 1 ,2-Dilinoleyioxy-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-Diiinoieyloxo~3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]- dioxolane (DLin- -DMA), 2,2-Dilinoleyl-4-dimethylaminoethyl-[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. f 00150] A non-cationic lipid can be an anionic lipid or a neutral lipid, such as distearoyl-phosphatidylcholine (DSPC), dioieoylphosphatidyicholine (DOPC), dipalmitoyl- phosphatidylcholine (DPPC), dioieoylphosphatidylglycerol (DOPG), dipalmitoyl- phosphatidylglycerol (DPPG), dioleoyi-phosphatidylethanolamine (DOPE), palmitoyioleoyl- phosphatidylcholine (POPC), palmitoyloleoyl- phosphatidylethanolamme (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-I- carboxylate (DOPE-mal), dipalrnitoyi phosphatidyl ethanolarnine (DPPE), dimyristoylphosphoethanola ine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-0-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 moi% to about 90 mol%, inclusive, of about 10 mol%, to about 58 mol%, inclusive, if cholesterol is included, of the total lipid present in the particle.

[00151 ] 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-phospho!ipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA can be, for example, a PEG-dilauryloxypropyl (CI 2), a PEG- dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (CI 6), or a PEG- distearyloxypropyl (CI 8), The lipid that prevents aggregation of particles can be from 0 mo I % to about 20 mol % or about 2 moi % of the total lipid present in the particle. In one embodiment, PEG lipid can be further conjugated to a ligand.

100152] In some embodiments, the nucleic acid-iipid particle further includes a steroid such as, cholesterol at, e.g., about 10 mol% to about 60 moi%, inclusive, or about 48 mol% of the total lipid present in the particle.

[00153] 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 CI -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 , or S; R¹ is alkyl alkenyl or alkynyl; each of which is optionally substituted with one or more substituents; and R² is H, alkyl alkenyl or alkynyl; each of which is optionally substituted each of which is optionally substituted with one or more substituents,

[00154] In one example, the lipidoid ND98-4HC1 (MW 1487) (Formula 2),

Cholesterol (Sigma- Aldrich), and PEG-Ceramide CI 6 (Avanti Polar Lipids) ca be used to prepare lipid RNA effector molecule nanoparticies (e.g., LNPOl particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg mL; Cholesterol, 25 mg mL, PEG- Ceramide C16, 100 mg/mL. The ND98, Cholesterol, and PEG-Ceramide CI 6 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 mM to 300 mM, inclusive. Lipid RNA effector molecule nanoparticies 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 imi 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.

D98 isomer

Formula 2 [00155] In some embodiments, the nucleic acid-Iipid 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.

[00156] LNPOl formulations are described elsewhere, e.g., WO 2008/042973.

[00157] In one embodiment, the reagent that facilitates RNA effector molecule uptake used herein comprises a cationic lipid as described in e.g., in International Application Ser. No. PCT/US 10/59206, filed 7 December 2010.

[00158] In various embodiments, the RNA effector molecule composition described herein comprises a cationic lipid selected from the group consisting of: "Lipid H", "Lipid "; "Lipid L", "Lipid M"; "Lipid P"; or "Lipid R", whose formulas are indicated as follows:

100159] Also contemplated herein are various formulations of the lipids described above, such as, e.g., K8, P8 and L8 which refer to formulations comprising Lipid K, P, and L, respectively. Some exemplary lipid formulations for use with the methods and compositions described herein are found in e.g., Table 5:

[00160] 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 approximately 25 mol % - 100 mol% of the lipid. In another embodiment, the lipid formulation comprises between 0 mol% - 50 mol% cholesterol, In still another embodiment, the lipid formulation comprises between 30 mol% - 65 mol% of a neutral lipid. In particular

embodiments, the lipid formulation comprises the relative mol% of the components as listed in Table 6 as follows:

5 52.73 37.27 10

6 52.92 42.08 5

53.01 44.49 2.5

8 47.94 47.06 5

[00161] Additional exemplary lipid-siRNA. formulations are as shown below in Table

TABLE 7: lipid-siRNA formulations

[00162] LNP09 formulations and XTC comprising formulations are described, e.g., in PCX Publication No. WO 2010/088537, which is hereby incorporated by reference,

[00163] LNP11 formulations and MC3 comprising formulations are described, e.g., in PCX Publication No. WO 2010/144740, which is hereby incorporated by reference.

[00164] L.NP12 formulations and XechGl comprising formulations are described, e.g., in International Application Ser. No. PCT/USIO/ 33777, filed May 5, 2010, which is hereby incorporated by reference.

[00165] Formulations prepared by either the standard or extrusion- free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, PA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. 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 RNA effector molecule content. Percent 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 110 nm, or at least 120 nm. The suitable range is typically about at least 50 nm to about at least 1 10 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm, inclusive.

[00166] 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 tha 50 ran, 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.

[00167] 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 drags; and liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation. See, e.g., Wang et ai., DRUG DELIV.

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

[00168] Liposomes are useful for the transfer and delivery of active ingredients to the site of action , Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act. Liposomal formulations have been the focus of extensive investigation as the mode of deliver}' for many drugs. There is growing evidence tha t 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.

[00169] 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 cell surface and is internalized in an endosome, Due to the acidic pH within the endosome, the liposomes are aiptured, releasing their contents into the cell cytoplasm. Wang et ai., 147 Biochem. Biophys. Res, Commun., 980-85 (1987), [00170] Liposomes which are pH-sensitive or negatively-charged, entrap polynucleotide rather than complex with it. Because both the polynucleotide and the lipid are similarly charged, repulsion rather than comple formation occurs. Nevertheless, some polynucleotide is entrapped within the aqueous interior of these l iposomes. 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).

[00171] 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

phosphatidvlglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolarmne (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.

[00172] 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 GM1 , or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES). Alien et al, 223 FEBS Lett. 42 (1987); Wu et al., 53 Cancer Res. 3765 (1993).

[00173] Various liposomes comprising one or more glycolipids are known in the art.

Papahadjopoulos et al, (507 Arm. NY Acad. Sci. 64 (1987)), reported the ability of

monosialoganglioside GM1 , galactocerebroside sulfate and phosphatidyl inositol 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 a!.).

100174] 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., PCX 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

distearoylphosphatidylethanolamine (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,

[00175] 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-modifted 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.

[00176] 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 can 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.

[00177] 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 covaiently 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),

[00178] 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. [00179] Transfersomes are yet another type of liposomes, and are highly deforrnable lipid aggregates which are attractive candidates for drag 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.

[00180] Encapsulated nanoparticles can also be used for delivery ofRNA effector molecules. Examples of such encapsulated nanoparticles include those created using yeast cell wall particles (YCWP). For example, glucan-encapsulated siRNA particles (GeRPs) are payload 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

[00181] Methods described herein use host cells to produce glycosylated lysosomal proteins having modified glycans. A host cell can be derived from a yeast, insect, amphibian, fish, reptile, bird, mammal or human, or can be a hybrid cell, such as 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 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.

[00182] A mammalian host cell can be advantageous where the glycoprotein is a mammalian glycoprotein, particularly if the glycoprotein is a biotherapeutic agent or is otherwise intended for administration to or consumption by humans. In some embodiments, the host cell is a CHO cell, which is a ceil line used for the expression of many recombinant proteins. Additional mammalian cel l lines used commonly for the expression of recombinant proteins include 293 HEK cells, HeLa cells, COS cells, NIH/3T3 cells, Jurkat Cells, NSQ cells, and HUVEC cells,

[00183] 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 selec tion of cells exhibiting high level expression of the recombinant protein. Examples of CHO cell derivatives useful in methods provided herein include, but are not limited to, CHO-K1 ceils, CHO-DUKX, CHO-DUKX Bl , CHO-DG44 ceils, CHO-ICAM-1 cells, and CHO-hlFNy ceils. 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,

[00184] 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-1 (pancreatic), CCRF-CEM (leukemia), COLO 205 (colon), DLD-1 (colon), DMS 114 (small cell lung), DU145 (prostate), EKVX (non-small cell lung), HCC-2998 (colon), HCT-15 (colon), HCT-116 (colon), HT29 (colon), HT-1080 (fibrosarcoma), HEK 293 (embryonic kidney), HeLa (cervical carcinoma), HepG2 (hepatocellular carcinoma), HL-60(TB) (leukemia), HOP-62 (non- small cell lung), HQP-92 (non-small cell lung), HS 578T (breast), HT-29 (colon

adenocarcinoma), IGR-OVl (ovarian), IMR32 (neuroblastoma), Jurkat (T lymphocyte), K-562 (leukemia), KM 12 (colon), KM20L2 (colon), LANS (neuroblastoma), LNCap.FGC (Caucasian prostate adenocarcinoma), LOX IMVI (melanoma), i .XI i 529 (non-small cell lung), Ml 4 (melanoma), M19-MEL (melanoma), MALME-3M (melanoma), MCFIOA (mammary epithelial), MCF7 (mammary), MDA-MB-453 (mammary epithelial), MDA-MB-468 (breast), MDA-MB-231 (breast), MDA-N (breast), MOLT-4 (leukemia), NCI/ADR-RES (ovarian), NCI- H226 (non-small cell lung), NC1-H23 (non-small cell lung), NCI-H322M (non-small cell lung ), NCI-H460 (non-small cell lung), NCI-H522 (non-small cell lung), OVCAR-3 (ovarian), OVCAR-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 (CNS), 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).

100185] 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).

[00186] Examples of rodent cell lines useful in methods provided herein include the cell lines baby hamster kidney (BHK) (e.g., BHK21 , BHK 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).

100187] 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-Il) cells, human

mesenchymal stem (hMSC) cells, and induced pluripotent stem (iPS) cells.

[00188] 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 Trichoplusla ni egg ceil homogeiiates); or S2 ceils (from Drosophila melanogaster).

[00189] 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 ceils include ceils 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 cells (e.g., epithelial cells, fibroblasts),

[00190] In some embodiments, the host ceil is an attachment dependent ceil which is grown and maintained in adherent culture, Exampl es of human adherent cell 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 (2931^'), and human breast carcinoma (S -B 3).

100191] 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 huma Nanialwa Burkitt lymphoma cell (BLcl-kar-Namaiwa), baby hamster kidney fibroblast (BH ), CHO cell, Murine myeloma ceil (NS0, SP2/0), hybridoma cell, human embryonic kidney cell (293 HE ), human retina-derived cell (PER.C6© cells, U.S. Patent No. 7,550,284), insect cell line (Sf9, derived from pupal ovarian tissue of Spodopiera 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 (MDCK), 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 cell (WI-38, MRC-5), African green monkey kidney epithelial cell (Vero, CV-1), Rhesus monkey kidney cell (LLC-MK2), or yeast cell. Additional mammalian cell 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) cells.

[00192] 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, [00193] The host cells may express the glycoprotein of interest endogenous!}', or alternatively, the host cell 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, one or more recombinant expression vectors encoding a lysosomal protein may be transfected, such that the lysosomal protein is expressed in the host ceil. Alternatively, the host ceil can be engineered such that that the gene encoding the lysosomal protein is activated by an exogenous promoter. This way, a host cell that does not normally express the lysosomal protein (or expresses the lysosomal protein at low level) can be modified to promote the production the lysosomal protein.

[00194] 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.

[00195] 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 Laborator 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.

[00196] For example, to express a lysosomal protein, nucleic acids encoding the lysosomal protein may be first obtained. These nucleic acids can be obtained by amplification and modification of a gene that encodes the lysosomal protein, using e.g., PGR.

[00197] 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 Enzymology 185, Academic Press (1990)). it will he 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 level of expression of protein desired, etc. Exemplary regulatory sequences for mammalian host cell expression include viral elements that direct high levels 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.

[00198] In addition to sequences encoding the glycoprotein and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e. g. , origins of replication) and selectable marker genes,

[00199] 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 transfection. 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).

[00200] The methods described hrerein 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 L to 20,000 L, from 10 L to 50,000 L, from 40 L 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 1 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. [00201] 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), HYCLONE 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,

[00202] 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.

[00203] 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 amount of an RNA effector is added to the cell culture such that the expression level of its target gene is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25°/», at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%.

[00204] 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 than about 50nM. [00205] The composition can be added to the ceil 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 RNA effector molecule over a several-day-period.

[00206] 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.

[00207] 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 biological 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.

[00208] 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 RN A effector molecule as such can ensure that modul ation of target gene expression is maintained even at high cell densities.

[00209] 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 fmol/10⁶ cells, at least 0.1 fmol 10⁶ cells, at least 0.5 fmol/10⁶ cells, at least 0.75 fmol/10⁶ cells, at least 1 fmol/10⁶ ceils, at least 2 fmol/10⁶ cells, at least 5 fmol/10⁶ ceils, at least 10 fmol/10⁶ ceils, at least 20 fmol/10⁶ cells, at least 30 fmol/10⁶ cells, at least 40 fmol/10⁶ cells, at least 50 fmol/10⁶ cells, at least 60 fmol/10⁶ cells, at least 100 fmol/10⁶ cells, at least 200 fmol/10⁶ cells, at least 300 fmol/10⁶ cells, at least 400 fmol/10⁶ cells, at least 500 fmol/10⁶ ceils, at least 700 fmol/10⁶ ceils, at least 800 fmol/10⁶ cells, at least 900 fmol/10⁶ cells, or at least 1 pmol/10⁶ cells, or more.

[00210] 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,

[00211] 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/cell, 10-50 molecules/cell, 10-40 molecules/cell, 10-30 molecules/cell, 10-20 molecules/ceil, 90-100 molecules/cell, 80-100 molecules/ceil, 70-100 molecules/cell, 60-100 moiecuies/celi, 50-100 molecules/cell, 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/cell, or any range there between.

100212] 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 Lysosomal Glycoproteins

[00213] The lysosomal glycoproteins produced in accordance with the methods described herein can be harvested from host cells, and purified using any suitable methods. For example, methods for purifying polypeptides by immune-affinity chromatography are known in the art. Ruiz-Argue Ho et al, J, Gen. Virol, 55:3677-3687 (2004). Suitable methods for purifying desired lysosomal 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 he 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.

[00214] For example, normal phase liquid chromatography can be used to separate glycans and/or glycoproteins based on polarity. Reverse-phase chromatography can be used, e.g., with derivatized sugars. Amon-exchange columns can be used to purify sialylated, phosphoryiated, and sulfated sugars. Other methods include high pH anion exchange chromatography and size exclusion chromatography can be used and is based on size separation.

[00215] Affinity based methods can be selected that preferentially bind certain chemical units and glycan structures. Matrices such as m-ammophenylboronic acid,

immobilized lectins and antibodies can bind particular glycan structures. M- aminophenylboromc acid matrices can form a temporary covalent bond with any molecule (such as a carbohydrate) that contains a 1,2-cis-dioi group. The covalent bond can be subsequently disrupted to eiute 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/giucose, galactoses-acetylgalactosamine, N-acetylglucosamine, fucose, and sialic acid (QProteome Glycoarray Handbook, Qiagen, September 2005, available at:

http://wolfson.huji.ac.iI/urification/PDF/Lectins/Q] GlycoArrayHa-ndbook.pdf) 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 concavalin 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.

[00216] 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 http://wv^.ncbi,nlm.mh.gov^ 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 Lysosomal glycoproteins

1. Analysis of the stroetisre and composition of glycans

100217] The gly can structure of the lysosomal glycoproteins described herein can be determined using art-known methods for analyzing glycan staictures 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).

|0021S] 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., sialidase, galactosidase, hexosaminidase, fucosidase, mannosidase etc.) can be used cleave terminal glycosidic bonds from the non-reducing end of glycans.

Alternatively, acid hydrolysis (e.g., trifiuoroacetic acid) can be used to release neutral saccharides (e.g., galactose, mannose, fucose) or amino saccharides (e.g., N-acetylglucosamine) from a glycan. The cleaved or hydrolyzed saccharides can be analyzed using chromatography spectrometry, or electrophoresis methods described above.

[0021 ] 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.

[00220] 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 absorption ionisation mass spectrometry (MALDI- S), Fourier transform mass spectrometry (FTMS), ion mobility separation with mass spectrometry (IMS-MS), electron transfer dissociation (ETD-MS), or combinations thereof.

[00221] Another method to analyze giycan structure and composition is

electrophoresis, including, e.g., capillary electrophoresis (CE), CE-MS, gel electrophoresis, agarose gel electrophoresis, aciyiamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting using antibodies that recognize specific giycan 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. Biochern., 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.

[00222] Another method to analyze giycan 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- NMR), total correlated spectroscopy NMR (TOCSY-NMR), heteronuclear single-quantum coherence NMR (HSQC-NMR}, heteronuclear multiple quantum coherence (HMQC-NMR), rotational nuclear overhauser effect spectroscopy NMR (RQESY -NMR), nuclear overhauser effect spectroscopy (NOESY-NMR), or combinations thereof.

[00223] Saccharide composition of a giycan can also be analyzed by fluorescence labeling. For example, acid-hydrolyzed glycans can be labeled with 2-aminopyridine and then analyzed by HPLC.

[00224] Immunological methods (e.g., antibody staining, lectin staining) may also be used to determine the structures of N-glycan. For example, lectin molecul es 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 glycoforms 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), Con A

(coeanavalin 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 aurantia lectin), ACL (Amaranthus caudatus lectin), BPL (Bauhinia purpurea lectin), DSL (Datura stramonium lectin), DBA

(Dolichos bin or as agglutinin), EBL (elderberry balk lectin), ECL (Erythrina cristagaili lectin), EEL (Euonymus eoropaeus lecin), GNL (Galanthus nivalis lectin), GSL (Griffonia siniplicifolia lectin), HPA (Helix pomatia agglutinin), HHL (Hippeastrum hybrid lectin), Jacalin, LTL (Lotus tetragonolobus lectin), LEL (Lycopersicon esculentum lectin), MAL (Maackia amurensis lectin), PL (Madura pomifera lectin), NPL (Narcissus pseudonarcissus lectin), PNA (peanut agglutinin), E-PHA (Phaseolus vulgaris erythroagglutmin), PTL (Psophocarpus tetragonolobus lectin), RCA (Ricinus communis agglutinin), STL (Solanum tuberosum lectin), SJA (Sophora japonica agglutinin), SBA (soybean agglutinin), UEA (Ulex europaeus agglutinin), VVL (Vicia viilosa lectin) and WFA (Wisteria fioribunda agglutinin).

[00225] For example, a lectin that specifically recognizes a complex N-glycan in which a fucose residue is linked to the N-acetylglucosamine 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 bean lectin VFA (agglutinin derived from Vicia faba) and Aleuria aurantia lectin AAL (lectin derived from Aleuria aurantia).

[00226] Another method to analyze glycan structure and composition is by capillary electrophoresis (CE), which is described e.g., in Szabo et a!., Electrophoresis, 2010 April; 31(8): 1389-1395. doi: 10.1002/elps.201000037.

[00227] Techniques described herein may be combined with one or more other technologies for the detection, analysis, and or isolation of glycans or glycoproteins. For example, any combination of NMR, mass spectrometry, liquid chromatography, 2-dimensional chromatography, SDS-PAGE, antibody staining, lectin staining, monosaccharide quantitation, capillary electrophoresis, fluorophore-assisted carbohydrate electrophoresis (FACE), micellar elecirokinetic chromatography (ME C), 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-quadrupole 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 anihranilic acid, and fractionated by norma] 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 MALDI TOF S analysis of the resulting products yields information about structural features of the giycan.

[00228] 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 lysosomal glycoproteins

[00229] The biological activity of the glycoprotein compositions described herein may be assessed using any art known method. Such biological activities include, e.g., bioavailability, pharmacokinetics, pharmacodynamics, enzymatic activity etc. Additionally, therapeutic activity of a glycoprotein may be assessed (e.g., efficacy of a lysosomal protein in decreasing severity or symptom of a disease or condition, or in delaying appearance of a symptom of a disease or condition).

[00230] Methods of analyzing bioa vailability, 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(l):l-4, 1996; and Srinivas et al., Pharm. Res. 14(7):91 1-6, 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, 151 1 (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.

100231] The potential adverse activity or toxicity (e.g., propensity to cause

hypertension, immimogenicity/allergic reactions, thrombotic events, seizures, or other adverse events) of glycoprotein preparations can be analyzed by any available method. For example, immunogenicity 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), or by in vivo administration to determine whether the composition elicits an antibody response in a subject.

5. PHARMACEUTICAL COMPOSITIONS, METHODS OF ADMINISTRATION, A D KITS

A. Pharmaceutical Compositions

[00232] In one aspect, the invention relates to pharmaceutical compositions comprising the lysosomal proteins described herein.

[00233] 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 will vary according to the route of administration selected. [00234] Optionally, the glycoprotein can be lyophilized for storage and reconstituted in a suitable carrier prior to use. Any suitable lyophilization 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.

[00235] 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 histidine buffer, or a citrate buffer. Butter salts will typically be included in the 5-20mM range.

[00236] The pharmaceutical compositions are preferably sterile, and may be sterilized by conventional sterilization techniques.

[00237] 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.

[00238] Pharmaceutical compositions of the invention may include sodium salts (e.g. sodium chloride) to give tonicity. A concentration of 10+2 mg/ml NaCl is typical e.g. about 9 mg/ml.

[00239] 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

[00240] In another aspect, the invention provide a method for treating Gaucher' s disease (types 1, 2, or 3), comprising administering to a subject in need thereof a therapeutically effectively amount of a glucocerebrosidase as described herein. The subject is preferably human.

[00241] Subjects in need of treatment of Gaucher's disease include those that demonstra te pre-symptomatic phases of the disease, as well as those that demonstrate various symptoms of Gaucher's disease. Typically, the pre-symptomatic patient can be diagnosed with Gaucher's disease by genetic analysis known to the skilled artisan.

[00242] Gaucher's disease is a heterogeneous disease, and has been subdivided into three different types on the basis of age of onset, clinical signs and involvement of neurologica! symptoms. Type 1 (adult type, chronic, non-neuronopathic; MIM# 230800) is the most common form and is characterized by hematological abnormalities with hypersplenism, bone lesions, skin pigmentation, Pingueculae (brown spots of Gaucher cells at corneoscleral limbus) and the lack of central nervous system involvement. It is heterogeneous in its clinical features (Beutler and Grabowski, 1995, Gaucher Disease, in Scriver et aL (eds.), The Metabolic and Molecular Bases of Inherited Diseases. Seven ed. McGraw Hill, Vol. II, pp. 2641-2663). Type 2 (infantile, acute neuronopathic; M 1 M# 230900) is a rare and lethal form of the disease. It is characterized by early appearance of visceral signs, enlargement of the abdomen from hepatosplenomegaly and central nervous system involvement such as retroflexion of the head, strabismus, dysphagia, choking spells, and hypertonicity. Type 3 juvenile, subacute

neuronopathic; MIM #321000) is characterized by early onset of visceral impairment (e.g., hepatosplenomegaly) and a later appearance of central nervous system symptoms.

[00243] A therapeutically effectively amount of a glucocerebrosidase is administered that such that symptoms of Gaucher's disease are ameliorated, or the onset of symptoms is delayed. A therapeutically effective amount will, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms associated with gl cocerebrosidase deficiency.

[00244] In another aspect, the invention provide a method for treating Pompe disease (also known as acid a-glucosidase deficiency, acid maltase deficiency, glycogen storage disease type II, glycogenosis I I, and lysosomal -g!ucosidase deficiency), comprising administering to a subject in need thereof a therapeutically effectively amount of an acid a-glucosidase as described herein. The subject is preferably human,

[00245] A therapeutically effectively amount of an acid α-glucosidase is administered that such that symptoms of Pompe disease are ameliorated, or the onset of symptoms is delayed, A therapeutically effective amount will, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms associated with acid a-glucosidase deficiency.

100246] The therapeutic efficacy of the administered acid α-glucosidase may be determined by biochemical (see, e.g., Zhu et al,, J, Biol, Cheni, 279: 50336-50341 (2004)) or histological observation of reduced lysosomal glycogen accumulation in, e.g., cardiac myocytes, skeletal myocytes, or skin fibroblasts. Acid α-glucosidase activity may also be assayed in, e.g., a muscle biopsy sample, in cultured skin fibroblasts, in lymphocytes, and in dried blood spots. Dried blood spot assays are described in e.g., Umpathysivam et al., Clin, Chem, 47: 1378-1383 (2001) and Li et al, Clin. Chem, 50: 1785-1796 (2004). The therapeutic efficacy of the administered acid α-glucosidase may also be assessed by, e.g., serum levels of creatinine kinase, gains in motor function (e.g., as assessed by the Alberta Infant Motor Scale), changes in left ventricular mass index as measured by echocardiogram, and cardiac electrical activity, as measured by electrocardiogram. Administration of acid a-glucosidase as described herein may result in a reduction in one or more symptoms of Pompe disease such as cardiomegaly, cardiomyopathy, daytime somnolescence, exertional dyspnea, failure to thrive, feeding difficulties, floppiness, gait abnormalities, headaches, hypotonia, organomegaly (e.g., enlargement of heart, tongue, liver), lordosis, loss of balance, lower back pain, morning headaches, muscle weakness, respiratory insufficiency, scapular winging, scoliosis, reduced deep tendon reflexes, sleep apnea, susceptibility to respiratory infections, and vomiting.

[00247] In another aspect, the invention provide a method for treating alpha 1 - antitrypsin deficiency, comprising administering to a subject in need thereof a therapeutically effectively amount of an alpha 1 -antitrypsin as described herein. The subject is preferably human. [00248] A therapeutically effectively amount of an alpha 1 -antitrypsin is administered that such that symptoms of alpha 1 -antitrypsin deficiency are ameliorated, or the onset of symptoms is delayed. A therapeutically effective amount will, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms associated with alpha 1 -antitrypsin deficiency.

[00249] In another aspect, the invention provide a method for treating CI -inhibitor deficiency (Types I, II, or II I ), comprising administering to a subject in need thereof a therapeutically effectively amount of a CI -inhibitor as described herein. The subject is preferably human.

[00250] A. therapeutically effectively amount of a C I -inhibitor is administered that such that symptoms of CI -inhibitor deficiency are ameliorated, or the onset of symptoms is delayed. A therapeutically effective amount will, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms associated with CI -inhibitor deficiency.

[00251] In another aspect, the invention provide a method for treating sepsis, vascular leak syndrome, acute myocardial infarction, pancreatitis, or thermal injury, comprising administering to a subject in need thereof a therapeutically effectively amount of a CI -inhibitor as described herein. The subject is preferably human.

[00252] In another aspect, the invention provide a method for reducing or preventing the adverse effect of xenotransplantation, comprising administering to a subject in need thereof a therapeutically effectively amount of a C I -inhibitor as described herein. The subject is preferably human.

[00253] The CI -inhibitors described herein can also be used to treat other diseases in which classical pathway complement activity (activated CI component) and/or contact system

(factor I la, kallikrein, factor XIa) activity contributes to undesired immune or inflammatory responses. Such diseases include myocardial infarction (WO 95/06479); acquired systemic inflammatory responses among which severe sepsis, septic shock, ARDS (Adult Respiratory

Distress Syndrome), multiple organ failure and preeclampsia (WO 92/22320); capillary leakage syndrome and circulatory failure in cases of severe bums, polytraumata, operations with extracorporeal circulation (EP 0586909), therapeutic cytokine (e.g. IL2) infusion, acute graft versus host disease after allogeneic (or autologic) bone marrow transplantation. Other indications may be disorders in which excess classical route complement and/or contact activation, and/or CI mhibitor consumption or (relative) functional CI -inhibitor deficiency has been implicated in the pathophysiology, such as meningitis, rheumatoid arthritis, hyper acute graft rejection after alio- and xenotransplantation and pancreatitis.

100254] In another aspect, the invention provide a method for treating Fabr disease comprising administering to a subject in need thereof a therapeutically effectively amount of an a-galactosidase A as described herein. The subject is preferably human.

[00255] A. therapeutically effectively amount of an a-galactosidase A is administered that such that symptoms of Fabry disease are ameliorated, or the onset of symptoms is delayed. A therapeutically effective amount will, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms associated with α-galactosidase A deficiency,

[00256] Subjects in need of treatment of various lysosomal storage diseases described herein include those that demonstrate pre-symptomatic phases of the disease, as well as those that demonstrate various symptoms of LSD (which can be diagnosed e.g., by genetic analysis known to the skilled artisan).

[00257] 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.

[00258] The amounts of a glycoprotein in a given dosage will vary according to the size of the indi vidual 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 mg/day, about 50 mg/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.

100259] 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.).

[00260] Standard dose-response studies, first in animal models and then in clinical testing, can reveal optima] dosages for particular diseases and patient populations.

[00261] 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 (NSAlDs), 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.

3. Kits

[00262] In another aspect, the invention provides kits that comprise the lysosomal glycoprotein compositions described herein packaged in a manner that facilitates their use for therapy. For example, such a kit includes a lysosomal glycoprotein as described herein, packaged in a container such as a sealed bottle or vessel, with a label affixed to the contamer 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. [00263] 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 lysosomal glycoprotein by the host cell, where the kits comprise a substrate having one or more assay surfaces suitable for culturing cel ls 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.

[00264] In some embodiments, the assay surfaces on the substrate are sterile and are suitable for culturing host cel ls 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 cul ture conditions to be established prior to full-scale production of the glycoprotein.

[00265] 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.

[00266] In some embodiments, kits provided herein further comprise cell culture media suitable for culturing 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 reconstit table form.

[00267] In further embodiments, kits provided herein further comprise one or more reagents suitable for detecting production of the lysosomal 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.

[00268] 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 RNA effector molecules are known in the art and are described herein. For example, in some embodiments, the earlier is a lipid formulation such as Lipofectamine™ transfection reagent (Invitrogen; Carlsbad, CA) or a related formulation. Examples of such carrier formulations are described herein. In some embodiments, the reagent that facilitates RNA effector molecule uptake comprises a charged lipid, an emulsion, a liposome, a cationic or non-cationic lipid, an anionic lipid, a transfection reagent or a penetration enhancer as described throughout the application herein. In particular embodiments, the reagent that facilitates RNA effector molecule uptake comprises a charged lipid as described in U.S. Application Ser.

No. 61/267, 19, filed on December 7, 2009.

[00269] 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 molecule(s) and the carrier effective for facilitating uptake of the RNA effector molecule(s) by the cells and modulation of the expression of one or more genes targeted by the RNA effector molecules.

[00270] 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, methylcelrulose. 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 vitrigel, PHEMA (poly(2-hydroxylniethacrylate)) hydrogels, PVP/PEQ hydrogels, BD PuraMatrix™ hydrogels, and copolymers of 2-methacryloyloxyethyl phophorylcholine (MPC). [00271] In some embodiments, the substrate comprises a microarray plate, a bioehip, 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 bioehip having m columns and n rows of assay surfaces (e.g., residing within wells) which allow for the testing of ra 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.

[00272] 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 RNA effector molecules, or for the selection of a concentration or concentration range from a wider range of concentrations of a given RN A effector molecule, In some embodiments, the kits allow for selection of one or more RNA effector molecules from a series of candidate RNA effector molecules directed against a common target gene. In further embodiments, the kits allo w 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 cel l glycosylation pathway.

[00273] 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.

[00274] 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 material 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. [00275] 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, Suc equivalents are intended to be encompassed by the following embodiments.

Appendix I: Sequences of Exemplary Lysosomal Proteins

Alglucosidase alia (SEQ ID NO:

1 eee visins Igqlyfehlq ilhkqraake neeeasvdts qenqedlglw eekfgkfvdi 61 kangpssigl dfslhgfehl ygipqhaesh qlkntgdgda yrlynlavyg yqiydkmgiy 121 gsvpyllahk: Igrt ig i fwl nasetl ein tepaveyt 1 t qmgpva .kqk rsrthvhwra 181 sesgiidvf 1 ltgptpsdvf kqyshltgtq arapplfslgy hqcrwnyede qdvkavdagf 241 dehdipydam wldiehtegk ryftwdknrf pnpkrmqell rskkrkiwi sdphikidpd 301 ys y kakdq gffvknqege dfegvcwpgl ssyldftnpk vrewysslfa fpvyqgstdi 361 Iflwndmnep svfrgpeqtm qknaihhgnw ehrelhniyg fyhqmataeg likrskgker 421 pf 1 Crsffa gsqkygavwt gdntaevjstil kisiprrilltl sitgisfega digg igripe 481 tel Ivrwyqa gayqpffrgh atmntk ep wl fgeehtrl ireaireryg llpywysl fy 541 hahvasqpvm rpl vefpde lktfdmedey mlgsallvhp vtepkattvd vfipgsnevw 601 ydyktfahwe ggctvkipva ldtipvfqrg gsvipikttv gkstgwmtes syglrvalst 661 kgssvgelyl ddghsfqylh qkqfIhrkfs fcssvlinsf adqrghypsk cvvekil lg 721 frkepssv c t hssdg k.dqpv af ycaktsi isl.eklsl.ni atdwevri i

Smiglucerase (SEQ ID NO: 2) Arg495His Ghicocerebrosidase

ARPCIPKSFGY3SVVCVCNATYCDSFDPPTFPALGTFSRYESTR3GRRMELSMGPIQANH

GTGLLLTLQPSQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNI IR VPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWT SPTWLKTNGAVNGKG3LKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEP3AGL

LSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPH AKVVLTDPE AAKYVHGIAVHWYLDFLAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRG QYSHSI I NLLYH G TDW LALNPEGGPNWVRNFVDSPI IV ITKDTFYKQPMFYHL GHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFL ETISPGYSIHTYL HRQ

Alglucerase (SEQ ID NO: 3)

ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANH TGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIR VP ASCDFSTRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLA3PWT

SPTWLKTNGAVNGKG3LKGQPGDIYHQT ARYFVKFLDAYAEHKLQFWAVTAENEP3AGL

LSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPE AAKYVHGIAVHWYLDFLAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRG MQYSHSI ITNLLYHVVG TDWNLALNPEGGPNWVRNFVDSPI IVDITKDTFYKQPMFYHL GHF3KFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRS3KDVPLTIKDPAVGFL

ETI3PGYSIHTYL RRQ a 1 -antitrypsin (SEQ ID NO: 4)

EDPQGDAAQKTDTSKHDQDHPTFNKITPNLAEFAFSLYRQLAHQ3NSTNIFF3PVSIATA

FAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTL QPDSQLQLTTGNGLFL SEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDT VFALVNYIFFKGK ERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVL LMKYLGNATAIFFLPDEGKLQHLENELTHDI ITKFLE EDRRSASLHLPKLSITGTYDLK SVLGQLGI KVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIP SI

PPEVKFNKPFVFLMIEQN KSPLFMGKVVNPTQK

Human CI -inhibitor (SEQ ID NO: 5)

1 masrltllcl II.1.1 lagdra ssnpn.atsss sqdpesIqdr gegkva tt i s krol f epi 1

61 evsslpttns ttnsatkita nttdepttqp fctepttqpfci qptqp tqlp tdsptqpttg

121 sfcpgpvtlc sdleshstea vlgdalvdfs Iklyhafsam kkvetnmafs pfsiaslltq

181 vllgagentk tnlesilsyp kdftcvnqal kgft kgvts vsqifhspdl airdtfvnas

241 rtlysssprv Isnnsdanle lintwvaknt nnkisrllds lpsdtrlvll naiylsakwk

301 tt dpkktrm epfhfknsvi kvpmmns kky pvahfidqt1 ka kvgqlq Is hnlsIvi 1 ρ

361 qn1 khr1edm eqalsps f k. aimeklernsk fqptlltlpr ikvttsqdral simekieffd

421 fsydlnlcgl tedpdlqvsa mqhqtvlelt etgveaaaas aisvartllv fevqqpfIfv

481 Iwdqqhkfpv fmgrvydpra

Agalsidase (SEQ ID NO: 510)

LD GLAR PTMGWLH EP.FMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCT

DDC MAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGY YDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSI YSCEWPLYMWPF QKPNYTEIRQYCNHWRNFADIDDSWKSIKSILD TSFNQERIVDVAGPGGWNDPDMLVIG NFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQL RQGDNFEV ERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFI QLLPVK RKLGFYEWTSRLRSHI PTGTVLLQLE TMQM3LKDLL

Appendix O: nucleotide sequences of exemplary target genes from Chinese Hamster

SEQ ID NO: 6: mannose-P-dolichol utilization defect 1 (Mpdul)

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNN N G ACG G G CCGTTCAAAG G G CTG CTG GTG CCAATTC

TTTTACCTG AG AAATG TTACG ACCAG CTCTTCGTG CACTG G G ACTTCCTTCATGTTCCCT

G CCTCAAG ATTCTCCTCAG CAAAG G CCTTG G ACTG G G CATCGTG G CTG G GTCACTTCTCG

TCAAGCTGCCCCAGGTATTTAAACTCTTGGGCGCCAAGAGTGCCGAAGGACTGAGTCTCC

AGTCAGTAATG CTG G AG CTG GTAG CATTG ACAG G AACTGTG ATCTACAG CATCACCA ACA

ACTTCCCCTTCAG CTCTTG G G GTG AAG CG CTGTTCCTG AC ACTACAG ACAATTACC ATCT

GCTTCCTGGTCCTGCACTACAGAGGAGACACTGTGAAAGGAGTTGCTTTACTTGCCTGCT

ATG CG ACACTCCTG CTG G CTCTG CTTTCTCCACTCACG CCTCTG G CTGTAGTCACCATG C

TCCAGGCCTCCAATGTACCTGCTGTGGTGGTGGGCAAGTTGCTCCAGGCAGCCACCAACT

ACCACAACG G G CACAC AG G CC AG CTTTCAG CT ATCACAGTGTTTATG CTGTTTG GGGG CT

CCTTGGCCAGAATCTTCACTTCTGTTCAGGAAACTGGAGACCCCCTCATGGCTGGAGTCT

TTGTGGTCTCTTCCCTCTGCAATGGCCTCATTGCCGCCCAGGTCCTCTTCTACTGGAATG

CAAAG CCTCCCCACAAACATAAAAAG GAG CAATAGTG CTG AG CTAG CTTCTAG AATCATT

CCATTCCACTCATCCN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

SEQ ID NO: 7: mannosyi (a- 1 ,3~)-glycoprotein beta- 1 ,2-N-acet lgiucosaminyltransferase (MGAT2)

NNNNNNNNNNNGCCGGAGGGCCCGGGCGCTCGCAGGCCAGTCGCGGCTTGTCAGNNNNNTCGCCGCCGGAGCGGGGCG AGGCTGCGGCGCTCGGTGTCGCCG NNNiNiNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN N NGGGTGCTGCATGG A

GAGGACCGAGGCGACGCTGAGCCGCGGCTCGTAGCGGCGGGGCGGCTGCTGGAGCTGCAACTGCCAGAGAGGATGCGCGG

AGCCCGGGCGGCGCGAGGCCGTTGAGAGCCTTCGGGCCCCAGGACGCCGGGGCCCGGGATGAGTTAGCGAGGGCCGCCGC

GGGGGCCAGTTCCTAGGGCTACAGGCCAAGGCGACGGCGCCGCCCGCCCGCCCCTTCCGTGCAGAGGCCGCCAGCTGCTTTC

CGCGCCCGCGCTCCCGGCCCCGGAGACCATGAGGTTCCGCATCTACAAACGGAAGGTGCTGATCCTGACGCTCGTGGTGGCC

GCCTGCGGCTTCGTCCTCTGGAGCAGCAATGGGCGACAAAGGAAGAACGACGCCCTTGGCCCGCCGCTGCTGGACGCGGAG

CCCGTACGGGGTGCGGGCCACCTTGCTGTGTCCGTAGGCATCCGCAGGGTCTCAAACGAATCGGCCGCTCCTCTGGTTCCCGC

GGTCCCGCGGCCCGAGGTGGACAACCTAACGCTGCGGTACCGGTCGCTGGTGTACCAGTTGAACTTCGATCAGATGCTGAGG

AACGTCGGTGATGACGGCACCTGGCGCCCCGGGGAGCTGGTGCTGGTGGTCCAGGTGCATAACCGGCCGGAATACCTCAGG

CTGCTGCTAGACTCGCTTCGCAAAGCCCAGGGTATTCAGGAAGTCCTAGTCATCTTCAGCCATGACTTCTGGTCCGCAGAGAT

CAACAGTCTGATCTCCAGGGTGAACTTCTGCCCGGTTCTGCAAGTGTTCTTTCCAmAGCATTCAGCTGTACCCGAGTGAGTT

TCCGGGTAGTGACCCCAGAGACTGCCCCAGAGACCTAAAGAAGAATGCAGCTCTGAAGTTGGGGTGCATCAATGCCGAATAC CCTGACTCCTTTGGCCATTACAGAGAGGCCAAATTCTCGCAAACCAAACATCATTGGTGGTGGAAGCTGCACTTTGTGTGGGA

GAGAGTCAGAGTTCTTCAGGATTACACTGGCC rATACTTTTC GGAAGAGGACCACTACTTAGCCCCAGACTTTTACCATGT

CTTCAAAAAGATGTG G AAGTTG AAG CAGCAG G AGTGTCCTGG GTGTG ATGTCCTCTCCCTAG GG ACCTACACTGCCAGTCG G

AGTTTCTATGGCATTGCTGACAAGGTAGATGTGAAAACTTGGAAATCCACAGAGCACAATATGGGGTTAGCCTTGACCCGAG

ATGCC ATCAGAAGTTGATCGAGTGCACAGACACTTTCTGTACTTATGATGA1TATAACTGGGACTGGACTGTCAATATTTGA

CTGTATCTTGTCTTCCTAAATTCTGGAAAGTCTTAGTTCCTCAAGCTCCGAGGATTTTTCATGCTGGAGACTGTGGTATGCATCA

CAAGAAAACATGTAGGCCATCCACCCAGAGTGCCCAGATCGAGTCATTCTTAAATAATAACCAACAGTACATGTTTCCAGAAA

CTCTAGTTATCAGTGAGAAATTTTCTATGGCAGCCATTTCCCCACCTAGGAAAAATGGTGGGTGGGGAGATATTAGGGACCAT

GAACTCTGTAAAAGTTATAGAAGACTGCAGTGAAAACAAAAATCACAATTGCAAAAGAAAGATGACAGTCTTCTGTTTTTTAT

ATTTCTTTCAATGGGATATACAAITGAATAAAGGTATAGGAACTGGTTTCTGCTTTAATACACAGATTTGTGTAACAGGTGTC

CAAATATATAGTAATCCTATTCAGTTAGTCTGATTAAAATTTCAAACTGAAATTTTCATTTTGGGTCTTGGGTTTTAAAATTCAG

TCTATCTGCTTAAGGGTAATGATTTGATAATTATTGATTAGAAAAGGAAATTTTATTTAAATCGCATCTGTTAATCTTTCTATCT

AAAACTTTGTATAGTTTCCAClTTCAGAAGTATTTTAAGTACAGCAAGAGTATTrAAAACTGTCACAACAGTAAAAAGTATT

ACAAGACTATTAGAGTATTGATGGGACAAACCCAGTGTCACTATTGACTTTTATTTGGAGGAATTGTCTGACCGGTTTAATCAC

TCAGAAGTTCCGTTTGTTAAACGCCCTGTCAGAAGAGTCATTTCAGTATTGCTGATTCCTGTGCTATTGTGTTAAGATTTGCCTG

TGCTTTCAGCCTTCATACTACATGATTTATGTTGGAATGTAITTGGTTAATAAGAAAGTTTAAACACTGTTTTCACCTCAATGTA

GAAATACAGTGGT! m i l l ! ! ! ! ! ! ! ! ! ITAGTGCTGCCAAAATAAAATACTCATTTTTTCATAAAATTCCCTAATCAT7TTGCAG

AATCTTCTATTTGTATCAATAAAGGCATTCTAGGAGTTTTGAGAATGAAAAA

SEQ ID NO: 8: mannoside acetylglucosaminyltransferase 1 (MGAT1 )

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN N N CG G G CCG CAG G G G GTCG AG G G G GTG G G G CCTG CG G G N HNNH NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNN N N AAGCCAGCTGGATGAAGGGATGTAATAGGCGCCTGGG AGGG ACACTGAAGTCAGG AGTG

GGGGGGGAGGGGCCCGGACACACCCGTCCCCCCCAGACTCCTCCCNNNNNNNNNNCACGTGGCCATGGACTTTGGACCTG

G ATAAGTG G AG AG AAG CTGTG CTTTG G G G G ATCT N N NNhiNNN^NNNNNNhiNNN^NNNNNNhiNNhiNNN^NNNhiNNiNiN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNTGGGGAAGTGAGCCAGGCAAGCCAAAGGCAGCCTTGAGCCCTCCCNNNCCTGCCCTCCCCTGTGGGGGCCAGGATGC

TGAAGAAGCAGTC GCAGGGCTTGTGCTTTGGGGTGCTATCCTCTTTGTGGGCTGGAATGCCCTGCTGC CCTCTTCTTCTGGA

CACGCCCAGCCCCTGGCAGGCN NNNNNNNNNNNNNNNNNNNN NTGATGACCCTGCCAGCCTCACCCGTG AGGTGTTCCN

CCTGGCTGAGGACGCTGAGGTGGAGTTGGAGCGGCAGCGGGGGCTGTTGCAGCAAATCAGGGAGCATCATGCTTTGTGGAG

Nhi Nhi N ^ NAGTTCCCACCGTGGCCCCTCCAGCCTGGCCCCGTGTGCCTGCGACCCCCTCACCAGNN N NGATCCCCAT

CCTGGTCATTGCCTGTGACCGCAGCACTGTCCGGCGCTGCTTGGATAAGTTGTTGCACTATCGGCCCTCAGCTGAGCATTTCCC

CATCATTGTCAG NNNNNNNN NNGGGCACGAGGAGACAGCACAGGTCATTGCTTCCTATGGCAGTGCAGTCACACACATCCG

GCAGCCAGACCTGAGTAACATCGCCGTGCAGCCAGACCACCGCAAGTTCCAGGGTTACTACAAGATCGCCAGGCACTACCGC

TGGGCACTGGGCCAGATCTTCAACAAGTTCAAGTTCCCAGCAGCTGTGGTAGTGGAGGACGATCTGGAGGTGGCACCAGACT

TCTTTGAGTACTTCCAGGCCACCTACCCACTGCTGAGAACAGACCCCTCCCTTTGGTGTGTGTCTGCTTGGAATGACAATGGCA

AGGAGCAGATGGTAGACTCAAGCAAACCTGAGCTGCTCTATCGAACAGACTTTTTTCCTGGCCTTGGCTGGCTGCTGATGGCT

GAGCTGTGGACAGAGCTGGAGCCCAAGTGGCCCAAGGCCTTCTGGGATGACTGGATGCGCAGACCTGAGCAGCGGAAGGG

GCGGGCCTGTATTCGTCCAGAAATTTCAAGAACGATGACCTTTGGCCGTAAGGGTGTGAGCCATGGGCAGTTCTTTGATCAGC

ATCTTAAGTTCATCAAGCTGAACCAGCAGTTCGTGTCTTTCACCCAGTTGGATTTGTCATACTTGCAGCGGGAGGCTTATGACC

GGGATTrCCTTGCCCGTGTCTATAGTGCCCCCCTGCTACAGGTGGAGAAAGTGAGGACCAATGATCAGAAGGAGCTGGGGGA GGTGCGGGTACAGTACACTAGCAGAGACAGCTTCAAGGCCTTTGCTAAGGCCCTGGGTGTCATGGATGACCTCAAGTCTGGT GTCCCCAGAGCTGGCTACCGGGGCGTTGTCACTTTCCAGTTCAGGGGTCGACGTGTCCAC GGCACCCCCACAAACCTGGGA AGGCTATGATCCTAGCTGGAATTAGCAGCACCTGCCTTTCCCTGCTGGATCTGCTTGTCATATCATGAGCTGAGACAGGCCTGC AGTCCCTG AGCTGTACCATCCTGTCCCTGTTTCCCTCTN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNGGGCATATTGAACAAGAAACCACTGTGTGGTATGGGGAGGCTTGGGCTTGTTGGGGCCN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN^

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

ACCAGGACATCTCTGGCCCCAAAGCAGCTCCTGCCCTGCCTTCCTCACCCTTCCCCCACCCCCACCAAGATGCAGGTAGCTGGT TGGAGAGAAATCTGTGTGTTTTTGTTTGTTGCCTGACCTTAGTTTCATGGAAGAAAATGGAATCTACAGAATTATTTTCAAAAA TAAAGTAAAGGCTGAATTGTCTGAAAAAAAAAAAAAA

SEQ ID NO: 9: mannoside acetylglucosaminyltransferase 4, isoenzyme B (MGAT4B)

NNNNNNNNNNNNNNNNNNNNNNGGATCCAGCCCGGCCGCGGCTCTTGCCGCCTTGCCNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNGAGGCCGCCAGCACCGCGCACCGCGGAGGAGCAGCNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCCGCAGCTCCTCCGCGCTCCGGGGCCGGGGCCCCNNNNNN

NNNNNGGCCCCCGCGCCCCGGCCCCGGTCCCCCGGGCCATGCGGCCTCGGCCCCGCCGGCGCCCGCCGCGCACCGGAGGAG

ATGAGGCTCCGCAATGGCACCTTCCTGACGCTGCTGC CTTCTGCTTGTGCGC iTCCTCTCGCTCTCCTGGTACGCGGCGCTC

AGCGGCCAGAAAGGTGACGTGGTGGACATTTACCAGAGGGAGTTCCTGGCTCTGCGAGACCGTTTGCACGCTGCTGAGCAG

GAGAGCCTGAAGCGCTCTAAGGAGCTAAACCTGGTGCTGGACGAAATCAAGAGGGCAGTGTCCGAGAGGCAGGCGCTGCG

GGACGGAGAGGGCAATCGCACTTGGGGCCGCCTTACTGAGGATCCGCGACTGAAGCCGTGGAACGTCTCGCACAGGCACGT

GCTTCATCTGCCCACCGTCTTCCACCATCTGCCGCACCTGCTGGCCAAGGAGAGCAGTCTGCAACCCGCAGTGCGGGTGGGCC

AGGGCCGCACCGGAGTGTCCGTGGTGATGGGCATTCCAAGCGTGAGGCGCGAGGTGCACTCGTACCTGACTGACACATTGC

ACTCGCTCATCTCGGAGCTGAGCCCGCAGGAGAAGGAAGACTCAGTCATCGTGGTGCTGATCGCCGAGACGGACCCACAGTA

CACCTCGGCAGTGACAGAAAACATCAAGGCC TGTTCCCCACAGAGATCCATTCTGGGCTCCTGGAAGTCATCTCCCCTTCCCC

TCACTTCTACCCTGACTTCTCCCGCCTTCGAGAGTCCTTCGGGGACCCCAAGGAGAGAGTCAGGTGGAGGACCAAACAGAA.ee

TGGATTACTGCTTCCTCATGATGTATGCACAGTCCAAAGGCATCTACTATGTGCAGCTGGAGGATGACATTGTAGCCAAGCCC

AACTACTTGAGCACTATGAAGAACTTTGCCCTCCAGCAGCCCTCTGAGGACTGGATGATCCTGGAGTTCTCCCAGCTGGGCTT

CATCGGGAAGATGTTCAAGTCATTGGACCTGAGCCTGATTGTGGAATTCATCCTTATGTTCTACCGAGACAAGCCTATTGACTG

GCTCCTGGACCACATCCTGTGGGTGAAAGTCTGCAACCCTGAGAAAGATGCGAAACATTGTGACCGGCAGAAGGCCAACCTT

CGGATCCGCTTCAAGCCGTCCCTCTTCCAGCATGTGGGCACTCACTCCTCACTGGCGGGCAAGATCCAGAAACTGAAGGATAA

GGATTTTGGAAAGCATGCTCTCCGGAAGGAGCATGTGAACCCCCCAGCGGAGGTGAGCACAAGCCTCAAGACGTACCAGCAT

TTCACCCTGGAGAAGGCCTACTTGCGAGAGGATTTCTTCTGGGCCTTTACACCTGCAGCAGGAGACTTCATCCGCTTCCGCTTC

mCAGCCACTGCGCCTTGAGCGGTTCTTCTTCCGCAGTGGGAACATTGAGCACCCAGAAGATAAACTCTTCAACACTTCrGTG

GAGGTGTTGCCTTTTGACAATCCCCAGTCAGAGAAGGAGGCCCTTCAAGAGGGCCGCTCAGCCACTCTACGGTACCCTAGGA

GCCCTGATGGTTACCTCCAGATCGGCTCCTTCTACAAGGGTGTAGCCGAAGGGGAAGTGGATCCTGCCTTTGGGCCCCTGGA

AGCACTGCGCCTCTCCATCCAGACGGACTCCCCAGTGTGGGTCATCTTGAGTGAGATCTTTCTGAAAAAGGCTGACTAAATGG

AAGGCTTCCAAGGGTGCTCCGTGGCCGGCTCTGGAGTCCACGGTTCCAAGGGTGTTGCTGCTGCTGCCGCCGATCCAAGAGG

ACCAGGCATATCCACCCCACCTGAAGGGTTCTGCCTGGCAGGCGGCTCGGGCTGGCCTGGGGTCCACCGCTGGCCCGGAGGC

CCCAGAAGCTGGTGCTGCGCCGGGCCGCGGGAGGAGGCAGGCGGCCCCCACTCTGTGCCTGAGGGCCGGCCTGCTGCCGCC

CAAAAAGAACCGAACCGAACCGmGCCCCTGGCCGGTCGGCTTGAGCCAGGCCATmAGAAGAGCTTTTTCTTGGGCGCCC

GCTGTGTGCGGCGCGAACACTGGAATGCATACACTACTTTATGTGCTGTGTT^

AAGTTCGCATATACTTCTATAAGAGCGTGACTTGTAATAAAGGGATAATGAAG Table 8

CAAGCUG C C C C AGGUAUUU 82 AAAUACCUGGGGCAGCUUG 83

CUGUGAAAGGAGUUGCUUU 84 AAAGCAACUCCUUQCACAG 85

ACACUACAGACAAUUACCA 86 UGGUAAUUGUCUGUAGUGU 87

GUUCCUGACAGUACAGACA 88 UGUCUGUAGUGUCAGGAAC 8 9

CUGAGAAAUGUUACGACCA 90 UGGUCGUA C UUUCUCAG 91

CAGAAUCUUCACUUCUGUU 92 A C GAAGUG AAGAUUCUG 93

CCAAUUCUUUUACCUGAGA 94 UCUC AGGUA AAGAAUUG G 95

C AAUUCUUUUAC CUGAGAA 96 UUCUCAGGUAAAAGAAUUG 97

C G C C AAG AGUG C C G AAGG A 98 UCCUUCGGCACUCUUGGCG 99

CUACCACAACGGGCACACA 100 UGUGUGCCCGUUGUGGUAG 101

GGGUGAAGCGCUGUUCCUG 1 02 CAGGAACAGCGCUUCACCC 103

GAGCUAGCUUCUAGAAUCA 1 04 UGAUUCUAGAAGGUAGCUC 105

GGCUGGAGUCUUUGUGGUC 1 06 GAG C CAAAGACUC C GC C 107

AAUG C AA GC CUC C C C C A 1 08 UGUGGGGAGGCUUUGCAUU 109

GAAUCUUC ACUUCUG UUC A 110 UGAACAGAAGUGAAGAUUC 111

UC AC AGUG UUU AUGC UGUU 112 AACAGCAUAAACACUGUGA 113

CUAGAAUCAUUCCAUUCCA 114 UGGAAUGGAAUGAUUCUAG 115

GC CUC C C CACAAACAQAAA 116 UUUAUGUUUGUGGGGAGGC 117

CAGCCACCAACUACCACAA 118 UUGUGGUAGmJGGUGGGUG 119

GGGGUGAAGCGCUGUUCCU 12 0 AGGAAC GCG CUUC CCCC 121

GCUGGAGUCUUUGUGGUCU 122 AGACCACAAAGACUCCAGC 123

GGCUGCUGGUGCCAAUUCU 124 AG AAUUGGC AC C AGC AG C C 125

GCUGGUGCCAAUUCUUUUA 126 UAAAAGAAUUGGCACCAGC 127

GC AGC C AC CAAC UAC C AC A 128 UG UGGUAGU UGGU GG CUG C 12 9

CAAUGUACCUGCUGUGGUG 13 0 CACCACAGCAGGUACAUUG 131

ACUGGGA CUUC C UUC AUGU 132 ACAUGAAGGAAGUCCCAGU 133

C CAACU C C C ACGG GC A 1 34 UGCCCGUUGUGGUAGUUGG 135

AGCUAGCUUCUAGAAUCAU 1 36 AUGAUUCUAGAAGCUAGCU 137

GUUACGACCAGCUCUUCGU 138 ACGAAGAGCUGGUCGUAAC 13 9

GGCUCUGCUUUCUCCACUC 14 0 G AGUG G AGAAAG C AG AG C C 141

GAGUCUC C AGU CAG^'UAAUG 142 C AUUAC UGACUGGAGACU C 143

GACAGGAACUGUGAUCUAC 144 GUAGAUCACAGUUCCUGUC 145

AAUC UUC C UUC C ACUCA 146 UGAGUGGAAUGGAAUGAUU 147

GC G C C GGUAUUUAAACUC 148 GAGUUUAAAUACCUGGGGC 14 9

ACAGGAACUGUGAUCUACA 150 UGUAGAUCACAGUUCCUGU 151

GUAG C AUUGAC AGGAACUG 152 C AGUU C CUG UC AAU GCUAC 153

AGGCCAGCUUUCAGCUAUC 154 G AUAG CUGAAAG CUGG C C U 155

GAi^CUGUGAUCUACAGCAU 156 AUGCUGUAGAUCACAGUUC 157

CCUGACACUACAGACAAUU 158 AAUUGUCUG[JAGUGUCAGG 159

UACCTJGAGAAAUGUUACGA 1 6 0 UCGUAACAUUUCUCAGGUA 1 1

G 'CU'GGUA G C AUt JG A G AGG A 1 62 UCCUGUCAAUGCUACCAGC 163

AGC CUC C C C C A CAUAA 1 64 UUAUGUUUGUGGGGAGGCU 165 CAAGAGUGCCGAAGGACUG 166 CAGUCCUUCGGCACUCUUG 167

GGCUGGGQCACDUCUCG^'uC 168 GACGAGAAGUGACCCAGCC 16 9

C CU CUGC AAUGGC CUC AUU 170 AAUGAGGCCAUUGCAGAGG 171

GUGCUGAGCUAGCUUCUAG 172 GUAGAAGCUAGCUCAGGAC 173

GGAGCAAUAGUGCUGAGCU 174 AGCUCAGCACUAUUGCUCC 175

CUG CUAUGCGA C ACUC CUG 176 CAGGAGUGUCGCAUAGCAG 177

GCUUUACUUGCCUGCUAUG 178 CAUAGCAGGCAAGUAAAGC 179

AG C ΑΑϋ AGOG C UG AG CUAG 18 0 CUAGCUCAGCACUAUUGCU 181

AGCUGCCCCAGGUAUUUAA 182 UUAAAUAC C rjGGGGC AG CU 183

UGGCUGGGUCACUUCUCGU 184 ACGAGAAGUGACCCAGCCA 185

AGACACUGUGAAAGGAGUU 1 86 AACUGCtnJUCACAGUGUCU 187

GCUCTJUGGGGUGAAGCGCU 1 88 AGCGCUUCACCCCAAGAGC 18 9

GC C AG AAUCUUC ACUUCUG 1 90 CAGAAGUGAAGAUUCUGGC 191

GC C AAUUCUUUU C CUGAG 1 92 CUCAGGUAAAAGAAUUGGC 193

GUGC C AAUUCUUUUAC CUG 194 CAGGUAAAAGAAUUGGCAC 195

GGAACUG UGAU CUAC AGC A 196 UGCUGUAGAUCACAGUUCC 197

GGAGUCUUUGUGGUC UCUU 198 AAGAGAC C AC AAA GACU C C 199

GUGAAAGGAGUUGCUUUAC 2 00 GUAAAGCAAOJCCUUUCAC 2 01

DGGCCAGAADCUDCACUDC 2 02 GAAGUGAAGAUiJCUGGGCA 2 03

GGCUGUAGUCACCAUGCUC 2 04 GAGCAUGGUGACUACAGCC 2 05

CCAAGAGUGCCGAAGGACU 2 06 AGUCCUUCGGCACUCUUGG 2 07

CCUUCAGCUCUUGGGGUGA 2 08 UC AC C C C AAG AGCUGAAG G 2 09

Table 9

AAACUGUC AC AAC AG UAAA 238 UUUACUGUUGUGACAGUUU 23 9

CUGUGCUAUUGUGUUAAGA 24 0 UCUUAACACAAUAGCACAG 241

UUUUC AUAAAAUUC C C UAA 242 UUAGGGAAUDUUAQGAAAA 243

AGUUGAACTJUCGAUCAGAU 244 AUCUGAUCGAAGUUCAACU 245

GACUAUUAGAGUAUUGAUG 246 CAUCAAUACUCUAAUAGUC 247

CCAGAAACUCUAGUUAUCA 248 UGAUAACUAGAGUUUCUGG 24 9

CUUGUAAC AGGUGUC C AA 250 UUUGGACACCUGUUACAAG 251

UUGAUAAUUAUUGAUUAGA 252 UCUAAUCAAUAAUUAUCAA 253

CCCGGUUCUGCAAGUGUUC 254 G AAC ACUUG C AGAAC CGG G 255

UGUAUUUGGUUAAUAAGAA 256 UUCUUAUUAACCAAAUACA 257

GCCCGGUUCUGCAAGUGUU 258 AACACUDGCAGAACCGGGC 259

AGACUAUUAGAGUAUUGAU 26 0 AUCAAUACUCUAAUAGUCU 261

CAAUUGAAUAAAGGUA.UAG 262 CUAU C CUUU UUC AAUUG 263

GUUGGAAUGUAUUUGGUUA 264 UAAC C AAUAC UUC C AAC 265

C C AC CUUG CUGUGUC CGUA 266 UACGGACACAGCAAGGUGG 267

UGAUAAUUAUUGAUUAGAA 268 UUCUAAUC AAUAAUUAU C A 26 9

ACUUCUGGUCCGCAGAGAU 270 AUCUCUGCGGACCAGi¾AGU 271

GGAGAUAUUAGGGACCAUG 272 C AUGG UC C CU AAUAUCU C C 273

CCAGUGUCACUAUUGACUU 274 AAGUCAAUAGUGACACUGG 275

CAAGAAAACAUGUAGGCCA 276 UG GC CU C UGUUUUCUUG 277

AAA CUGUC CAACAGUAA 278 UUACUGUUGUGACAGUUUU 279

CCUAGUCAUCUUCAGCCAU 28 0 AUGGCUGAAGAUGACUAGG 281

UCCCUAAUCAUUUUGCAGA 282 UCUGCAAAAUGAUUAGGGA 283

GCUGAUUCCUGUGCUAUUG 284 C/iAUAGCAC/iGGAAUCAGC 285

C AG UUGAACUUCGAU C AGA 286 UCUGAUCGAAG^'uUCAACQG 287

GUGGGUGGGGAGAUAUUAG 288 CUAAUAUCUC C C GAG C C AC 28 9

GGGAG AUAUUAGGG C C AU 2 90 AUGGUC C CUAAUAUCUC C C 2 91

UAUUCAGUUAGUCUGAUUA 2 92 UAAUCAGACUAACUGAAUA 2 93

ACACUUUCUGUACUUAUGA 2 94 UCAUAAGUACAGAAAGUGU 2 95

AGGCCAAAUUCUCGCAAAC 2 96 GUUUGCGAGAAUUUGGCCU 2 97

GCGACAAAGGAAGAACGAC 2 98 GUCGUUCUUCCUUUGUCGC 2 99

CGCAAACCAAACAUCAUUG 3 00 CAAUGAUGUUUGGUUUGCG 3 01

AAUUCAGUCUAUCUGCTJUA 3 02 UAAGCAGAUAGACUGAAUU 3 03

G AA GUUG AUC G AGUG C AC A 3 04 UGUGCACUGGAUCAACUUC 3 05

GGAUUACACUGGCCUUAUA 3 06 UAUAAGGC CA GUGU AUC C 3 07

GAAUAAAGGUAUAGGAACU 3 08 AG UUC CUAUAC CUUUAUUC 3 09

Table 10

Mga l CAGGGAGCAUCAUGCUUUG 310 CAAAGCAUGAUGCUCCCUG 311

AGAAAAUGGAAUCUACAGA 312 UCUG [JAGAUUC C AUDUDCU 313

GGAGGUGCGGGUACAGUAC 314 GUAC DGDAC C CGC AC CDC C 315

AGGCUGAAUUGUCUGAAAA 316 DDUUCAGACAAUUCAGCCU 317

GGCGCUGCUUGGAUAAGUU 318 AACUUAUCCAAGCAGCGCC 319

GGGUGUC AUGGA.UGAC CDC 32 0 GAGGUCAUC C AUG C C C C 32

GCACCAGACUUCUUUGAGU 322 CUCAAAGAAGUCUGGUGC 323

C CG C AGUUC CAGGGUUAC 324 G UAAC C CUG GAACUUG CGG 32 5

GAAAUUUCAAGAACGAUGA 326 UCAUCGUUCUDGAAAUUUC 327

CCAAGAUGCAGGUAGCUGG 328 CCAGCUACCUGCAUCUUGG 32 9

CCAGAAAUUUCAAGAACGA 33 0 UCGUUCUU'GAAAUUUCUGG 331

GCUGCUUGGAUAAGUUGUU 332 AACAACUUAUCGAAGCAGC 333

GGGGCGGGCCUGUAUUCGU 334 ACGAAUACAGGCCCGCCCC 335

GUGUGUGUCUGCUUGGAAU 336 AUUC CAAGC G C AC AC C 337

GCUCUAUCGAACAGACUUD 338 AAAGUCDGDUCGAUAGAGC 33 9

GGADCUGCUUGDCAUAUCA 34 0 DGAUAUGAC AAG CAGAUC C 341

AGUAAAGGCDGAAUUGUCU 342 AGAC AADU C AGC C UDD ACU 343

C AC C C AG UUGGAUUUGUC A 344 UGACAAAU C CAAC UGGGUG 345

UCUGCUUGUCAUAUCAUGA 346 DCAUGAUADGACAAGGAGA 347

UCUA CAGAAUUAUUUUC A 348 DUGAAAAUA AUUCUGU GA 34 9

GGAUAAGUUGUUGCACUAU 350 AUAGUGCAACAACUUAUCC 3 51

GGUACAGUACACUAGCAGA 352 UCDGCUAG UGDACUGUAC C 353

AAUG G AAUCUAC AGAAUUA 354 UAAUUCUG UAGAUUC CAUD 355

CCAGCAGUUCGUCUCUUUC 356 GAAAGACACGAACUGCQGG 357

GCCUGDAUUCGUCCAGAAA 358 UUDCUGGACGAAUACAGGC 359

GGAGCAGAUGGUAGACUCA 36 0 UGAGUCUACCAUCUGCUCC 361

GGAUGAAGGGAUGUAAUAG 362 CUAUUAC AUG C CUUCAUC C 363

GGAAGGGGCGGGCCUGUAU 364 AUACAGGCCCGCCCCUUCC 365

CUADGAUCCUAGCUGGAAU 366 AUUCCAGCUAGGAUCAUAG 367

GGGCGGGCCUGDAUUCGUC 368 GACGAADACAGGCCCGCCC 36 9

UDGD C AUAUC AD GAGC UGA 370 DCAGCUCADGAUAUGACAA 371

GAD C AGC ADCDD AAG DC A 372 DGAACUDAAGAUGCDGAUC 373

GCAGUUCUUUGAUCAGCAU 374 AUGCUGAUCAAAGAAGDGC 375

GGGCCDCAGCDGAGCATJUD 376 AAAUGCUCAGCUGAGGGCC 377

GUCAUAUCAUGAGCUGAGA 378 UCUCAGCUCAUGAUAUGAC 379

UGGAUGAAGGGAUGUAAUA 38 0 UADDACAUC C CDDCAUC C A 381

CUUUC AC C C AG UUGGAUUU 382 AAAD C C AACUGGGUGAAAG 383

GGACCAADGADCAGAAGGA 384 UC CUD CUGAUC ADUGG C C 385

AGCAGADGGUAGACDCAAG 386 CUDGAGUCtJACCAUCUGCD 387

UCAGGGAGCAUCAUGCUUU 388 AAAGCAUGAUGGUCCCUGA 38 9

UGGAUAAGUUGUUGCACUA 3 90 UAGUGCAACAACmJAUCCA 3 91

CCDCUUUGUGGGCUGGAAU 3 92 AUUC C AG C C C C A AAG AGG 3 93 CUUGGAAUG ACAAUGG CAA 3 94 UUGCCAUUGUCAUUCCAAG 3 95

GGACUUUGGAC CUGGAUAA 3 96 UUAUC C AGGUC C AAAGUC C 3 97

GGCUUAUGACCGGGAUUUC 3 98 GAAAQCCCGGUCAUAAGCC 3 99

C CUGUAUUCGUC C AGAAAU 4 00 AUUUCUGGACGAAUACAGG 4 01

C CUUAGUUU CAUGGAAGAA 4 02 UUCUUCCAUGAAACUAAGG 4 03

UGGAAUCUACAGAAUUAUU 4 04 AAUAAUUCUGUAGAUUCCA 4 05

GCUUGUCAUAUCAUGAGCU 4 06 AGCUCAUGAUAUGACAAGC 4 07

GGGAAGGCUAUGAUCCUAG 4 08 CUAGGAUCAUAGCCUUCCC 4 09

Table 1 1

GAAUGCAUACACUACUUUA 466 OAAAG UAGUGUAUGC AUUC 467

GCCCCUGGCCGGUCGGCUU 468 AAGCCGACCGGCCAGGGGC 46 9

GCGACUGAAGCCGUGGAAC 470 GUUCCACGGCUUCAGUCGC 471

GUUCGCAUAUACUUCUAUA 472 UAUAGAAGUAUAUGCGAAC 473

GAAGGAAGACUCAGUCAUC 474 GAUGACUGAGUCUUCCmJC 475

CCGAGACAAGCCUAUUGAC 476 GUCAAUAGG CUUGUCUCGG 477

ACGUAAGUUCGCAUAUACU 478 AGUAUAUGCGAACUUACGU 479

ACCCUGAGAAAGAUGCGAA 48 0 UUCGCAUCUUUCUCAGGGU 481

UGGAAUU C AUG CUUAUGUU 482 AACAOAAGGAUGAAUUCCA 483

GAGACAAGCCUAtfUGACUG 484 CAGU C AAUAGGCUUGUC UC 485

GGAUCCGCUUCAAGCCGUC 4 86 GACGGCiJCJGAAGCGGAUCC 4 87

GGCUCUGGAGUCCACGGUU 4 88 AACCGUGGACUCCAGAGCC 4 8 9

GAAAGAUG CGAAACAUUGU 4 90 ACAAUGUUUCGCAUCUUUC 4 91

AGCACAAGCCUCAAGACGU 4 92 ACGUCUUGAGGCUUGUGCU 4 93

CACGUAAGUUCGCAUAUAC 4 94 GUAUAUG C G AAC UUAC GUG 4 95

AG C G C U C U AAG G AG C U AAA 4 96 UUUAGCUCCUUAGAGCGCU 4 97

CUCAAGACGUACCAGCAUU 4 98 AAUGCUGGUACGUCUUGAG 4 99

CACUCGUACCUGACUGACA 500 UGUCAGUCAGGUACGAGUG 501

GGGUGUAGCCGAAGGGGAA 502 UUC C C CUUCGGCUAC C C C 503

C CUUCGGAUC CG CUUC AG 504 CUUGAAGCGGAUCCGAAGG 505

ACUUGCGAGAGGAUUUCUU 506 AAGAAAUCCUCUCGCAAGU 507

GACACAUUGCACUCGCUCA 508 UG AG C GAG UG C AAUGUG UC 509

Claims

What is claimed is:

A method for producing a composition comprising a lysosomal protein, comprising: culturing a large scale host cell culture in a medium that comprises an effective amount of an RN A effector molecule,

(a) wherein said host cell: i) expresses the lysosomal protein; and ii) comprises a target gene that encodes Mannose-P-dolichoi utilization defect 1 protein (MPDU 1);

(b) wherein said RNA effector is substantially complementar}_' to said target gene that encodes MPDU1 , and reduces or prevents the expression of said target gene; and

(c) wherein said host cell is cultured for a period of time sufficient for the

production of said lyososomal protein.

The method of claim 1, wherein said RNA effector transiently reduces the expression of said target gene that encodes MPDU1.

The method of claim 1 or 2, wherem said host cell further comprises a second target gene that encodes a mannosyl (a-1 ,3-)-glycoprotein beta-l,2-N- acetylglucosaminyltransferase (MGAT), and wherein the method further comprises: culturing said large scale host cell culture in a medium that comprises an effective amount of a second RNA effector molecule, wherein said second RNA effector is substantially complementary to said second target gene that encodes MGAT, and wherein said RNA effector reduces or prevents the expression of said target gene that encodes MGAT,

4. The method of claim 3, wherein said target gene that encodes MGATl, MGAT2, or MGAT4B.

5. The method of claim 3 or 4, wherein said R A effector transiently reduces the

expression of said target gene that encodes MGAT,

6. A method for producing a composition comprising a lysosomal protein, comprising: culturing a large scale host cell culture in a medium that comprises an effective amount of an RNA effector molecule,

(a) wherein said host cell: i) expresses the lysosomal protein: and ii) comprises a target gene that encodes alpha- 1 ,6-mamiosyl-glycoprotein 2- beta-N-acetyiglucosaminyltransferase (MG AT2);

(b) wherein said RN A effector is substantially complementary to said target gene that encodes MGAT2, and reduces or prevents the expression of said target gene; and

(c) wherein said hos t cell is cultured for a period of time sufficient for the

production of said iyososomal protein.

7. The method of claim 6, wherein said RNA effector transiently reduces the expression of said target gene that encodes MGAT2,

8. The method of any one of claims 1-7, further comprising harvesting said lysosomal protein from said large scale culture.

9. The method of any one of claims 1 -8, further comprising isolating said lysosomal protein.

10. The method of any one of claims 1-9, wherein said lysosomal protein is secreted into the medium.

11. The method of any one of claims 1-10, wherein said RNA effector molecul e is an siRNA.

12. The method of any one of claims 1-1 1, wherein said RNA effector molecule is a shRNA.

13. The method of any one of claims 1-11 , wherein said RNA effector molecule is an antisense molecule.

14. The method of any one of claims 1-13, wherein said RNA effector molecule that is substantially complementary to a target gene that encodes MPDU1 comprises a sequence selected from the group consisting of SEQ ID NOs: 10-209.

15. The method of any one of claims 3-13, wherein said RN A effector molecule that is substantially complementary to a target gene that encodes MGAT comprises a sequence selected from the group consisting of SEQ ID NOs: 210-509.

16. The method of any one of claims 6-13, wherein said RNA effector molecule that is substantially complementary to a target gene that encodes MGAT2 comprises a sequence selected from the group consisting of SEQ ID NOs: 210-309:

17. A composition comprising a lysosomal protein produced according to the method of any one of claims 1-16.

18. The composition of claim 17, wherein said lysosomal protein is selected from the group consisting of glucosidase, glucocerebrosidase, galactocerebrosidase, galactosidase, iduronidase, hexosaminidase, marmosidase, fucosidase, aryisulfatase, V-acetylgalactosamine-6-sulfate sulfatase, acteylgalactosaminidase,

aspartyigiucosaminidase, iduronate-2-sulfatase, a-glucosaminide-N-acetyltransferase acetyl-CoA: a-glucosaminide N-acetyltransferase, β-D-glucoronidase, hyaluronidase mannosidase, neuraminidase, phosphotransferase, acid lipase, acid ceramidase, sphinogmyelinase, thioesterase, cathepsin , sialidase and lipoprotein lipase.

19. The composition of claim 17, wherein said lysosomal protein is selected from the group consisting of glucocerebrosidase, idursulfase, alglucosidase alia, galsulfase, agalsidase beta, and laronidase.

20. The composition of claim 17, wherein said lysosomal protein is an acid a- glucosidase.

21. The composition of claim 20, wherem said lysosomal protein comprising a sequence that is at least 90% identical to Alglucosidase alia.

22. The composition of claim 21, wherein said lysosomal protein comprises SEQ ID NO:

1 .

23. The composition of claim 17, wherein said lysosomal protein is a glucocerebrosidase,

24. The composition of claim 23, wherein said lysosomal protein comprising a sequence that is at least 90% identical to imiglucerase.

25. The composition of claim 24, wherein said lysosomal protein comprises SEQ ID NO:

2 or SEQ ID NO: 3.

26. The composition of claim 17, wherein said lysosomal protein is a protease inhibitor.

27. The composition of claim 26, wherein said lysosomal protein is a trypsin inhibitor.

28. The composition of claim 27, wherein said lysosomal protein comprising a sequence that is at least 90% identical to alphai -antitrypsin.

29. The composition of claim 28, wherem said lysosomal protein comprises SEQ ID NO:

4.

30. The composition of claim 26, wherein said lysosomal protein is an esterase inhibitor.

31. The composition of claim 30, wherein said lysosomal protein comprising a sequence that is at least 90% identical to CI Esterase Inhibitor,

32. The composition of claim 31 , wherein said lysosomal protein comprises SEQ ID NO: 5.

33. The composition of claim 17, wherem said lysosomal protein is an a-galactosidase A.

34. The composition of claim 33, wherein said lysosomal protein comprising a sequence that is at least 90% identical to Agalsidase beta.

35. The composition of claim 34, wherein said lysosomal protein comprises SEQ ID NO:

510.

36. A eel! comprising an RNA effector molecule substantially complementary to a target gene encoding MPDU1.

37. An RNA effector molecule substantially complementary to a target gene encoding MPDU! .

38. The RN A effector agent of claim 37, wherein the RNA effector agent comprises a sequence selected from the group consisting of: SEQ ID NO: 10-209.

39. A cell comprising an RNA effector molecule substantially complementary to a target gene encoding MGAT2.

40. An RNA effector molecule substantially complementary to a target gene encoding MGAT2.

41. The RNA effector agent of claim 40, wherein the RNA effector agent comprises a sequence selected from the group consisting of: SEQ ID NO: 210-309.

42. A composition comprising a lysosomal protein,

(a) wherem at least about 80% of the lysosomal protein molecules are

glycosylated, and (b) wherein at least about 70% of the glycosylated lysosomal protein molecules comprise a glycan that comprises at least one terminal mamiose, and no more than 5 marmose residues total, with the proviso that said lysosomal protein is not a glucocerebrosidase,

43. The composition of claim 42, wherein said lysosomal protein is selected from the group consisting of glucosidase, galactocerebrosidase, galactosidase, iduronidase, hexosaminidase, mannosidase,, fucosidase, arylsulfatase, N-acetylgalactosamiiie-6- sulfate sulfatase, acteylga!actosaminidase, aspartylg!ucosaminidase, iduronate-2- sulfatase, cx-glucosaminide-N-acetyltransferase, acetyl-CoA:a-glucosaminide N- acetyltransferase, β-D-giucoronidase, hyaiuronidase, mannosidase, neurominidase, phosphotransferase, acid lipase, acid ceramidase, sphinogmyelinase, thioesterase, cathepsin K, sialidase and lipoprotein lipase.

44. The composition of claim 42, wherem said ly sosomal protein is selected from the group consisting of: idursulfase, alglucosidase alfa, galsulfase, agalsidase β, laronidase, acid a-glucosidase, and a protease inhibitor.

45. The composition of claim 42, wherem said lysosomal protein is selected from the group consisting of idursulfase, alglucosidase alfa, galsulfase, agalsidase β, and laronidase,

46. The composition of claim 42, wherein said lysosomal protein is an acid a- glucosidase.

47. The composition of claim 46, wherein said lysosomal protein comprising a sequence that is at least 90% identical to Alglucosidase alfa.

48. The composition of claim 47, wherein said lysosomal protein comprises SEQ ID NO:

1.

49. The composition of claim 42, wherein said ly sosomal protein is a protease inhibitor.

50. The composition of claim 49, wherein said ly sosomal protein is a trypsin inhibitor.

51. The composition of claim 50, wherein said lysosomal protein comprising a sequence that is at least 90% identical to alphai-antitrypsin.

52. The composition of claim 51 , wherem said lysosomal protein comprises SEQ ID NO:

4,

53. The composition of claim 49, wherein said lysosomal protein is an esterase inhibitor,

54. The composition of claim 53, wherein said lysosomal protein comprising a sequence that is at least 90% identical to CI Esterase inhibitor.

55. The composition of claim 54, wherem said lysosomal protein comprises SEQ ID NO:

5.

56. The composition of claim 49, wherein said lysosomal protein is an a-galactosidase A,

57. The composition of claim 56, wherein said lysosomal protein comprising a sequence that is at least 90% identical to Agalsidase beta,

58. The composition of claim 57, wherein said lysosomal protein comprises SEQ ID NO:

510.

59. The composition of any one of claims 42-58, wherein at least about 80% of the

lysosomal protein molecules are N-glycosylated.

60. The composition of any one of claims 42-58, wherein at least about 70% of the

glycosylated lysosomal protein molecules comprise a glycan that is: Man₂GlcNAc₂, Man₂GlcNAc₂Fuc, Man₃GlcNAc₂, MaihGic.VNoi c. Man₄GlcNAc₂,

Man₄GlcNAc₂F c, an₅GlcNAc₂, or Man₅GlcNAc₂Fuc.

61. A composition comprising a glucocerebrosidase

(a) wherein at least about 80% of the glucocerebrosidase molecules are

glysocylated; (b) wherein the glycosylated glucocerebrosidase molecules comprise at least two glycoforms, each of the glycoforms comprises a glycan that comprises: (i) at least one terminal ma mose, (ii) no more than 5 mannose residues total; and (iii) does not comprise a xylose or an a(l,3)-fucose; and

(c) wherein at least one of the glycoform comprises a glycan that comprises: (i) a terminal mannose, and (ii) 2, 4, or 5 mannoses total.

62. The composition of claim 61 , wherein said glycan is an -glycan.

63. The composition of claim 61 or 62, wherein said ly sosomal protein comprising a sequence tha is at least 90% identical to imiglucerase or Aiglucerase.

64. The composition of claim 63, wherein said lysosomal protein comprises SEQ ID NO:

2 or SEQ ID NO: 3.

65. The composition of any one of claims 61-64, wherein from about 5% to about 95% of the glycosylated glucocerebrosidase molecules comprise a glycan that comprises a terminal mannose and 2 mannoses total.

66. The composition of any one of claims 61-65, wherein from about 5% to about 95% of the gly cosylated glucocerebrosidase molecul es comprise a glycan that comprises a terminal mannose and 3 mannoses total.

67. The composition of any one of claims 61-66, wherein from about 5% to about 95% of the glycosylated glucocerebrosidase molecules comprise a glycan that comprises a terminal mannose and 4 mannoses total.

68. The composition of any one of claims 61-67, wherein from about 5% to about 95% of the glycosylated glucocerebrosidase molecules comprise a glycan that comprises a terminal mannose and 5 mannoses total.

69. The composition of any one of claims 61-68, wherein no more than about 50%» of the glycosylated glucocerebrosidase molecules comprise a glycan that comprises 6 mannoses or more. A method for producing a composition comprising a human lysosomal protein, comprising: culturing a large scale host cell culture in a medium that comprises an effective amount of an RNA effector molecule,

(a) wherein said host cell is a non-human ceil, and wherein said host ceil: i) comprises an exogenous nucleic acid that expresses said human lysosomal protein; and ii) comprises an endogenous target gene that encodes an ortholog of said human lysosomal protein;

(b) wherein said RNA effector is substantially complementary to said

endogenous target gene that encodes said ortholog, and reduces or prevents the expression of said endogenous target gene; and

(c) wherein said host cell is cultured for a period of time sufficient for the

production of said human lyososomal protein,

The method of claim 70, wherein said host cell is a CHO cell.

The method of claim 70 or 71, wherein said human lysosomal protein is selected from the group consisting of: glucocerebrosidase, glueosidase, galactocerebrosidase, gaiactosidase, iduronidase. hexosaminidase, mannosidase, fucosidase, arylsuifatase, -acetylgalactosamine-6-sulfate suifatase, acteylgalactosaminidase,

aspartylglucosammidase, iduronate-2-sulfatase, a-glucosamiiiide-N-acetyltraiisferase, acetyl-CoA:a-glucosaminide N-acetyitransferase, β-D-glucoromdase, hyaluronidase, mannosidase, neuraminidase, phosphotransferase, acid lipase, acid ceramidase, sphinogmyelinase, thioesterase, cathepsin K, sialydase and lipoprotein lipase.

The method of claim 70 or 71 , wherein said lysosomal protein is selected from the group consisting of: glucocerebrosidase, idursulfase, alglucosidase alfa, galsulfase, agalsidase β, laronidase, acid a-glucosidase, and a protease inhibitor. The method of claim 70 or 71, wherein said lysosomal protein is selected from the group consisting of: glucocerebrosidase, idursulfase, alglucosidase alfa, galsulfase, agalsidase β, and laronidase.

The method of claim 70 or 71 , wherein said lysosomal protein is Imiglucerase, Aigiucerase, Alglucosidase alfa, Agalsidase, a phai-antitrypsin, or CI Esterase Inhibitor.

The method of any one of claims 70-75, wherem said RNA effector molecule is an siRNA.

The method of any one of claims 70-75, wherein said RNA effector molecule is a shRNA.

The method of any one of claims 70-75, wherein said RNA effector molecule is an antisense molecule.