NZ623690B2

NZ623690B2 - Recombinant human naglu protein and uses thereof

Info

Publication number: NZ623690B2
Application number: NZ623690A
Authority: NZ
Inventors: Markley C Leavitt; Anthony Quinn; Joseph Victor Rutkowski; Zhinan Xia
Original assignee: Synageva Biopharma Corp
Priority date: 2011-10-12
Filing date: 2012-10-11
Publication date: 2016-11-29

Abstract

The present disclosure provides compositions comprising an isolated mixture of recombinant human NaGlu proteins in which a substantial amount of the NaGlu proteins in the mixture has increased levels of phosphorylated mannose that confer the proteins to be efficiently internalized into human cells. The present disclosure also provides methods of producing such mixture of NaGlu proteins, vectors used in transgenesis and expression, host cells harboring such vectors, and methods of isolating and purifying the mixture of NaGlu proteins. The disclosure further provides methods of treating NaGlu associated diseases. The present disclosure also provides methods of producing such mixture of NaGlu proteins, vectors used in transgenesis and expression, host cells harboring such vectors, and methods of isolating and purifying the mixture of NaGlu proteins. The disclosure further provides methods of treating NaGlu associated diseases.

Description

RECOMBINANT HUMAN NAGLU PROTEIN AND USES THEREOF REFERENCE TO RELATED APPLICATIONS This application is related and claims priority to US. Provisional Application Serial No. 61/546,248, filed r 12, 2011, the entire contents of which are expressly incorporated herein by this reference.

BACKGROUND OF THE INVENTION Sanfilippo Syndrome B is an autosomal recessive lysosomal storage disease (LSD) caused by a deficiency in a lysosomal enzyme known as N-acetyl-alpha-D- glucosaminidase (NaGlu). NaGlu is required for the degradation of heparan sulfate as part of the se breakdown of glycosaminoglycans (GAG) in the lysosome. The deficiency or absence of NaGlu leads to accumulation and urinary excretion of heparan sulfate. With over 70 different mutations identified to date, Sanfilippo Syndrome B ts extensive molecular and genetic heterogeneity. imately 1 out of 200,000 births is ed by Sanfilippo Syndrome B and the deficiency mainly manifests in young children. After initial symptom-free interval, patients suffering from Sanfilippo Syndrome B usually present with a slowing of mental development and behavioral problems, followed by progressive intellectual e resulting in severe mental retardation, dementia and motor disease. Acquisition of speech is slow and incomplete. Profoundly affected patients may present d psychomotor and speech development as early as 2 years of age. The disease usually progresses to increasing behavioral disturbance and sleep disturbance. Although the clinical features are mainly neurological, ts often develop diarrhea, carious teeth, an enlarged liver and spleen, stiffjoints, hirsteness and/or coarse hair and may exhibit blood-clotting ms. In the final stage of the illness, ts become immobile and unresponsive and p swallowing difficulties and seizure. The life-span of an affected child typically does not extend beyond late teens to early twenties.

Different approaches have been attempted to e the missing enzyme in patients. To produce NaGlu for enzyme replacement therapy (ERT), human NaGlu has been expressed in various mammalian cell culture systems. However, in contrast to the naturally occurring NaGlu which trafficks to the lysosome intracellularly, inant NaGlu proteins produced and secreted from mammalian cells were found to contain no or only a trace amount of mannose 6—phosphate (M6P). The absence or scarcity of M6P moieties in the secreted NaGlu has been known to prevent its ent internalization into target cells (e. g., human skin lasts), which have M6P receptors on the surface on its plasma membrane (see, Zhao et al., Protein Expression and Purification, 19:202- 211 (2000); and Weber et al., Protein Expression and Purification, 21:251-259 (2001)).

The low degree of orylation was seen in secreted mouse NaGlu expressed in CHO cells, secreted human NaGlu expressed in HeLa cells, secreted human NaGlu expressed in human fibroblasts, and secreted human NaGlu expressed in human embryonic kidney (HEK) cell line 293 (see, Zhao et al., Protein Expression and Purification, 19:202-211 (2000); Yogalingam et al., Biochim Biophys. Acta 1502: 415- 425; and Weber et al., Protein Expression and Purification, 21:251-259 (2001)). No or weak phosphorylation of N-glycans in the NaGlu proteins secreted from the mammalian cells has posed a major obstacle for the development of a recombinant human NaGlu protein suitable for enzyme replacement therapy as all the aforementioned attempts has failed to produce an enzyme which is efficiently taken up by target cells as the tration of the internalized proteins, if able at all, was nearly a thousand times less than wild-type levels (see, Zhao et al., Protein Expression and Purification, 19:202-211 (2000)). To date, no approved product is available for the treatment of Sanfilippo Syndrome B.

Direct stration of mammalian cell—produced recombinant human NaGlu protein lu) having the native amino acid sequence into the central nervous system (CNS) (e. g., intrathecal administration into the cerebrospinal ﬂuid (CSF)) of NaGlu deficient mice has been attempted, but failed to trate successful biodistribution of the enzyme to the brain due to excessive accumulation of the protein on the ependymal ling of the ventricles as well as lack of requisite M6P residues for efficient ar uptake. Similarly, systemic administration (i.e., intravenous (IV) injection) of mammalian cell-produced rhNaGlu having the native amino acid sequence also failed to demonstrate sful localization of the protein to the brain. In addition to known risks associated with highly invasive intrathecal administration, these obstacles in targeting rhNaGlu to the brain have been too great a challenge to achieve effective therapy for the treatment of Sanfilippo Syndrome B.

Therefore, there is a need to provide a stable NaGlu protein which is enzymatically active and has physical properties that allow for the protein to cross the blood brain barrier (BBB) and for ive internalization of the protein into the lysosomes of target cells. There is also a need for a high expressing and robust protein production platform which can provide a recombinant human NaGlu that effectively crosses the blood brain barrier and is efficiently internalized into human target cells.

SUMMARY OF THE INVENTION The present invention is drawn to itions sing recombinant human NaGlu protein (rhNaGlu) useful for therapy, for example, in the treatment of Sanfilippo Syndrome B. The present invention is based on the surprising and unexpected ery that the rhNaGlu described herein has one or more glycosylation patterns that allow the rhNaGlu to efficiently cross the blood brain barrier (BBB), and be taken up into cells within the central s system (CNS) of animals deficient in the enzyme, resulting in a ic increase in (x-N-acetylglucosaminidase ty in the brain, as well as a reduction of substrate levels. Moreover, the rhNaGlu described herein is efficiently taken up into a mammalian cell (6g, human cell), resulting in an increased tic activity as compared to NaGlu proteins produced and secreted from unmodified mammalian cells that are not designed to produce specific glycosylation. The increased cellular uptake of the NaGlu protein also es benefits for the use in enzyme replacement therapy for a human patient ing from Sanfilippo Syndrome B by minimizing the need for an increased amount and frequency of dose, and thereby greatly reducing the potential risk of immunogenicity.

The rhNaGlu protein described herein contains sufficient amount of oligosaccharides (e. g., mannose and phosphorylated mannose (i. e., M6P)) to allow efficient cellular uptake via mannose and/or M6P receptor-mediated endocytosis and be correctly targeted into human cells. In one embodiment, the rhNaGlu contains at least one mole of n, for example, 1, 2, 3, 4, 5 or 6 moles of M6P per mole of protein. In one embodiment, rhNaGlu can be internalized into a NaGlu deficient human cell such that the internalized protein fully (100% or more) restores normal levels (i.e., wild-type levels) of NaGlu activity in the NaGlu deficient cell.

Also disclosed herein are methods for producing a transgenic avian that expresses rhNaGlu which benefits from orylation of mannose. In particular, a transgenic avian that expresses u protein in t cells, secretes into the lumen of the oviduct and ts the protein into egg white. Avian eggs that contain such rhNaGlu are also included in the present invention.

The present invention also contemplates s and host cells that contain a transgene ng u as well as ceutical compositions comprising rhNaGlu to be used in the application of such rhNaGlu for the treatment of Sanfilippo Syndrome B.

In one aspect, the invention es a composition comprising an isolated mixture of recombinant human N-acetyl-alpha—D-glucosaminidase (rhNaGlu) comprising the amino acid sequence 24-743 of SEQ ID NO:1, wherein at least 10 % of the rhNaGlu in the mixture comprises at least one glycan structure having mannose phosphate (M6P). In one embodiment, the rhNaGlu having M6P is capable of being taken up into a mammalian cell deficient in NaGlu such that internalized rhNaGlu restores at least 50%, 60%, 70%, 80%, 90% or 100% of normal NaGlu activity observed in a wild-type mammalian cell of the same type. In another embodiment, the glycan ure is an N-linked glycan.

In one embodiment, the rhNaGlu contains at least 1 mole of M6P per mole of protein. In another embodiment, the rhNaGlu contains between about 1 and about 6 moles of M6P per mole of protein. In another embodiment, the rhNaGlu contains about 2 moles of M6P per mole of protein. In yet another embodiment, the rhNaGlu contains about 3 moles of M6P per mole of n. In another embodiment, the rhNaGlu contains about 4 moles of M6P per mole of protein. In another embodiment, the u contains about 5 moles of M6P per mole of protein. In yet another embodiment, the rhNaGlu contains about 6 moles of M6P per mole of protein. ing to a first aspect of the present invention, there is provided a composition comprising an isolated mixture of recombinant human N-acetyl-alpha-D-glucosaminidase (rhNaGlu) whose amino acid sequence is set forth in 24-743 of SEQ ID NO:1, wherein said isolated mixture comprises a ient amount of rhNaGlu containing one or more glycan structures comprising mannosephosphate (M6P) such that said rhNaGlu containing M6P is internalized into a mammalian cell having NaGlu ency via M6P receptor-mediated endocytosis and restores at least 50% of NaGlu activity observed in a wild-type cell of the same type expressing nous NaGlu, wherein when the composition is administered intravenously to a subject having NaGlu deficiency, NaGlu activity is increased in the brain of the subject.

According to a second aspect of the present invention, there is provided the use of a composition in accordance with the first aspect of the present ion in the manufacture of a medicament for the treatment of NaGlu deficiency in a subject.

According to a third aspect of the present invention, there is provided a pharmaceutical formulation comprising a composition in accordance with the first aspect of the present invention in combination with a pharmaceutically acceptable carrier, t or excipient.

In the disclosure herein, the mammalian cell ent in NaGlu is a human cell. In r disclosure herein, the human cell deficient in NaGlu is a skin fibroblast, a hepatocyte or a macrophage. In one disclosure herein, the human cell deficient in NaGlu is a neuronal cell.

In one disclosure herein, the rhNaGlu is effectively delivered to the brain of a mammal having NaGlu deficiency when systemically administered. In one particular disclosure herein, the rhNaGlu is effectively delivered to the brain of a mammal having NaGlu deficiency when intravenously administered. In one disclosure herein, the u is ively delivered to the brain of a mammal having NaGlu deficiency when administered hecally.

In one disclosure herein, the rhNaGlu having M6P is alized by a NaGlu deficient cell and restores at least 100% of normal NaGlu activity in vivo. In one disclosure herein, the rhNaGlu having M6P contains at least 25 moles of mannose per mole of protein. 11307249:gcc In one disclosure herein, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the rhNaGlu in the mixture contains M6P. In another disclosure herein, at least 20% of the rhNaGlu in the mixture contains at least one M6P. In another disclosure herein, at least 30% of the rhNaGlu in the e contains at least one M6P. In another sure herein, at least 40% of the u in the mixture contains at least one M6P. In another sure herein, at least 50% of the rhNaGlu in the mixture contains at least one M6P. In another disclosure herein, at least 60% of the rhNaGlu in the mixture contains at least one M6P.

In another disclosure , the invention provides a composition comprising an isolated mixture of recombinant human N-acetyl-alpha-D-glucosaminidase (rhNaGlu) comprising the amino acid sequence 24-743 of SEQ ID NO: l, n the mixture comprises a sufficient amount of rhNaGlu containing one or more glycan structures comprising mannosephosphate (M6P) such that the rhNaGlu containing M6P is internalized into a mammalian cell having NaGlu deficiency via M6P receptor-mediated endocytosis and restores at least 50% of NaGlu activity observed in a ype cell of the same type sing endogenous NaGlu. In one disclosure herein, the rhNaGlu is N-linked glycosylated. In another disclosure herein, the rhNaGlu is O-linked glycosylated. 11307249:gcc In one embodiment, the rhNaGlu comprises at least 1 moles of M6P per mole of rhNaGlu. In another embodiment, the rhNaGlu comprises about 1, 2, 3, 4, 5 or 6 moles of M6P per mole of rhNaGlu. In another ment, the u ses about 3 moles of M6P per mole of rhNaGlu. In r embodiment, the rhNaGlu comprises about 4 moles of M6P per mole of rhNaGlu.

In one embodiment, the rhNaGlu comprises mannose. In another embodiment, the u ses N-acetylglucosamine (GlcNAc). In another embodiment, the rhNaGlu comprises galactose. In r embodiment, the rhNaGlu ses N- acetylgalactosamine (GalNAc). In another ment, the rhNaGlu contains no fucose. In another embodiment, the rhNaGlu contains no glucose. In one embodiment, the rhNaGlu restores at least 60, 70, 80, 90, 95 or 100% of normal NaGlu enzymatic activity.

In another embodiment, the rhNaGlu is effectively delivered to the brain of a mammal having NaGlu deficiency when administered systemically. In one embodiment, the rhNaGlu is effectively delivered to the brain of a mammal having NaGlu deficiency when administered intravenously. In another embodiment, the rhNaGlu is effectively red to the brain of a mammal having NaGlu deficiency when administered intrathecally.

In one embodiment, the mammalian cell deficient in NaGlu is a human cell. In another embodiment, the human cell is a skin fibroblast, a hepatocyte or a macrophage.

In one ment, the human cell deficient in NaGlu is a neuronal cell.

In one embodiment, the rhNaGlu is a fusion protein comprising a second moiety.

In one embodiment, the second moiety is a polypeptide. In another embodiment, the polypeptide is selected from the group consisting of transferrin receptor ligand (TfRL), insulin-like growth factor receptor (IGFZR) ligand, low density lipoprotein (LDL) receptor ligand and acidic amino acid (AAA) residues.

In one embodiment, the rhNaGlu is produced from a transgenic avian. In one embodiment, the transgenic avian is a chicken, a turkey, a duck or a quail. In one embodiment, the transgenic avian is a chicken. In one ment, the rhNaGlu is produced from an oviduct cell.

In another aspect, the ion provides a composition comprising an isolated recombinant human yl—alpha-D-glucosaminidase lu) comprising one or more glycan structures having sufficient amount of mannosephosphate (M6P) that allows for internalization of the rhNaGlu into a mammalian cell having NaGlu deficiency via M6P receptor—mediated endocytosis, such that when internalized in vivo, the rhNaGlu restores at least 50% of NaGlu activity observed in a wild—type cell of the same type expressing endogenous NaGlu.

In one embodiment, the rhNaGlu protein is N-linked glycosylated. In another embodiment, the rhNaGlu n is O-linked glycosylated. In one embodiment, the rhNaGlu ses about 2, 3, 4, 5 or 6 moles of M6P per mole of rhNaGlu.

In one embodiment, the rhNaGlu is ively delivered to the brain of a mammal having NaGlu deficiency when administered systemically. In another embodiment, the rhNaGlu is effectively delivered to the brain of a mammal having NaGlu deficiency when administered intravenously. In another embodiment, the rhNaGlu is effectively delivered to the brain of a mammal having NaGlu deficiency when administered hecally.

In another aspect, the invention provides a transgenic avian comprising a ene containing a promoter operably linked to a nucleic acid sequence ng a recombinant human NaGlu (rhNaGlu), wherein the transgene is contained in the genome of the transgenic avian and expressed in an oviduct cell such that the rhNaGlu is glycosylated in the oviduct cell of the transgenic avian, secreted into lumen of oviduct and deposited in egg white of an egg of the transgenic avian.

In one embodiment, the rhNaGlu comprises about 2, 3, 4 or 6 moles of M6P per mole of rhNaGlu. In another embodiment, the promoter component is an oviduct- specific promoter. In another ment, the t—specific er is an ovalbumin promoter. In yet another ment, the transgenic avian is selected from the group consisting of a chicken, a turkey, a duck and a quail.

In another aspect, the invention provides an egg produced by the transgenic avian of the invention.

In yet another aspect, the invention provides a method of producing a recombinant human NaGlu (rhNaGlu) comprising: a) producing a transgenic avian comprising a ene having a promoter component operably linked to a nucleic acid sequence encoding the rhNaGlu set forth in 24-?43 of SEQ ID NO:1, wherein the transgene is contained in the genome of the transgenic avian and expressed in an oviduct cell, such that the rhNaGlu is glycosylated in the oviduct cell of the transgenic avian, ed into lumen of oviduct and ted in egg white of an egg laid by the transgenic avian; and b) isolating the u from the egg white.

In one ment, the promoter component is an oviduct-specific promoter. In another embodiment, the t-specific promoter is an min er. In one embodiment, the avian is selected from the group consisting of a chicken, a turkey, a duck and a quail. In one embodiment, the avian is chicken.

In another aspect, the invention provides a vector comprising a nucleotide sequence encoding a human NaGlu operably linked to an ovalbumin promoter. In another aspect, the invention provides a host cell comprising the vector of the invention.

In another aspect, the invention provides an isolated nucleic acid comprising the c acid sequence of 5232-10248 of SEQ ID N024.

In one aspect, the invention provides a pharmaceutical formulation comprising a composition of the invention in combination with a pharmaceutically acceptable carrier, diluent or ent.

In another aspect, the invention provides a composition comprising recombinant human NaGlu protein that crosses the blood brain barrier of a mammal having NaGlu deficiency when administered intravenously.

In yet another aspect, the invention es a method of treating a subject suffering from NaGlu deficiency, the method comprising administering to the subject a therapeutically effective amount of the composition of the invention.

In yet another aspect, the invention provides a method of delivering recombinant human NaGlu protein to the brain of a subject suffering from NaGlu deficiency, the method comprising enously administering recombinant human NaGlu protein to the subject.

In another aspect, the invention provides a method of transporting a recombinant human NaGlu protein from the circulation across the blood brain barrier in a therapeutically effective amount, the method comprising intravenously administering a recombinant human NaGlu protein to a subject having NaGlu deficiency.

In one embodiment, the NaGlu deficiency is Sanfilippo Syndrome B. In another embodiment, the subject is a human.

In another embodiment, the recombinant human NaGlu protein is administered intravenously to the subject at a dosage of about 0.5 to about 50 mg/kg body weight. In another embodiment, the recombinant human NaGlu protein is administered enously to the subject at a dosage of about 1 to about 30 mg/kg body weight. In another embodiment, the recombinant human NaGlu protein is administered intravenously to the subject at a dosage of about 6 to about 27 mg/kg body .

In yet another ment, the recombinant human NaGlu protein is intrathecally administered to the t. In one embodiment, the recombinant human NaGlu protein is intrathecally administered at a dosage of at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 mg/kg body weight. In r embodiment, the recombinant human NaGlu protein is intrathecally administered at a dosage of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg body weight. In r embodiment, the recombinant human NaGlu protein is administered intrathecally at a dosage of about 10 to about 30 mg/kg body weight.

In another embodiment, the therapeutically effective amount is an amount effective to reduce heparan sulfate levels in the brain, the kidney, or the liver of the subject. In another ment, the therapeutically effective amount is an amount effective to increase NaGlu activity in the brain or the liver of the subject.

In r embodiment, the method further comprises administering a second therapeutic agent. In one embodiment, the second therapeutic is an immunosuppressant.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 depicts the amino acid ce of human recombinant NaGlu (amino acid residues 1-23, signal peptide).

Fig. 2 depicts the nucleic acid sequence (cDNA) of human recombinant NaGlu, including the nucleic acid sequence encoding the signal peptide.

Fig. 3 depicts the nucleic acid sequence of 1.1kb ovalbumin promoter.

Figs. 4A-D depict the nucleic acid sequence of V-l.1-I-rhNaGlu vector used in transgenesis of an avian.

Fig. 5 is a schematic representation of pSIN-OV-l.l-I-rhNaGlu .

Fig. 6 depicts Western analysis of rhNaGlu isolated and purified from egg white of a transgenic Gallus.

Fig. 7 depicts the average concentration of rhNaGlu deposited in egg white of transgenic Gallus.

Fig. 8 depicts an oligosaccharide profile of rhNaGlu produced from a transgenic Gallus using HPAEC-PAD.

Fig. 9 depicts uptake analysis of rhNaGlu by human skin fibroblasts (MPS IIIB, NaGlu deficient; Normal, wild—type human skin fibroblast; 1U of enzymatic activity = nmol of protein/hr).

Fig. 10 depicts uptake inhibition analysis of rhNaGlu s) using various concentrations of M6P monosaccharide (IU of enzymatic ty = lumol of n/min).

Fig. 11 depicts a schematic representation of pTT22 vector containing a recombinant human NaGlu fusion construct (AAA-NaGlu: acidic amino acid residues fused to N-terminus of the full length NaGlu).

Fig. 12 depicts a schematic representation of pTT22 vector containing a recombinant human NaGlu fusion construct (NaGlu-TfRL: transferrin receptor ligand fused to inus of the full length NaGlu).

Fig. 13 depicts enzymatic activity of AAA-NaGlu produced from HEK293 as compared to rhNaGlu produced from Callus.

Fig. 14 depicts enzymatic activity of TfRL produced from HEK293 as compared to AAA-NaGlu produced from HEK293.

Fig. 15 depicts uptake levels of rhNaGlu (Callus) into a macrophage cell line (NR8383) over time (48 hours). ar NaGlu activity was ed in units/mg of Fig. 16 depicts heparan sulfate substrate levels (ug/mg tissue) in the kidney of naglu (4') mice ing intravenous administration of vehicle (KO); rhNaGlu gallus at a dosage tration of 6.25 mg/kg; or rhNaGlu gallus at a dosage concentration of 27 mg/kg. Wild type (WT) mice were untreated.

Fig. 17 depicts heparan sulfate substrate levels (pg/mg tissue) in the brain of naglu (4') mice ing intravenous administration of vehicle (KO); rhNaGlu gallus at a dosage tration 6.25 mgikg; or rhNaGlu gallus at a dosage concentration of 27 mg/kg. Wild type (WT) mice were untreated.

Fig. 18 depicts heparan sulfate substrate levels (pg/mg tissue) in the liver of naglu (4‘) mice following intravenous administration of vehicle (KO); u gallus at a dosage concentration of 6.25 mg/kg; or rhNaGlu gallus at a dosage concentration of 27 mg/kg. Wild type (WT) mice were untreated.

Fig. 19 depicts n sulfate substrate levels (pg/mg tissue) in the brain of naglu (4') mice following intrathecal administration of vehicle (KO) or rhNaGlu gallus at a dosage concentration of 0.31 mg/kg. Wild type (WT) mice were untreated.

DETAILED DESCRIPTION OF THE INVENTION The present ion provides compositions sing recombinant human NaGlu protein lu) useful for therapy, for example, in the treatment of NaGlu associated es, e. g., Sanfilippo Syndrome B. The present invention is based on a discovery that the rhNaGlu protein described herein contains sufficient amount of oligosaccharides (e.g., mannose and phosphorylated mannose (i.e., M6P)) to allow efficient cellular uptake via mannose and/or M6P receptor-mediated endocytosis and be correctly targeted into human cells. Since the rhNaGlu of the invention is more efficiently taken up into a human cell, the rhNaGlu of the invention exhibits increased enzymatic activity as compared to NaGlu proteins produced and secreted from fied mammalian cells that are not designed to produce specific glycosylation.

Additionally, the rhNaGlu described herein has one or more glycosylation patterns that allow the rhNaGlu to efficiently cross the blood brain barrier (BBB) when administered intravenously. The increased cellular uptake of the rhNaGlu protein of the invention minimizes the need for large and frequent dosing, thereby greatly ng the potential risk of immunogenicity.

Some of the definitions and abbreviations used herein include the following: aa, amino acid(s); bp, base pair(s); CDS, coding sequence cDNA, DNA complementary to an RNA; GalNac, N-acetylgalactosamine; Gal, galactose; , N-acetylglucosamine; nt, nucleotide(s); kb, 1,000 base pairs; pg, microgram; mL, iter; ng, am; and nt, nucleotide Certain definitions are set forth herein to rate and define the meaning and scope of the various terms used to describe the invention herein.

The term “avian” as used herein refers to any species, subspecies or strain of organism of the taxonomic class ava, such as, but not limited to, chicken, turkey, duck, goose, quail, pheasants, parrots, finches, hawks, crows and ratites including h, emu and cassowary. The term includes the s known strains of Gallus gallus, or ns, (for example, White Leghorn, Brown Leghorn, Barred-Rock, Sussex, New Hampshire, Rhode Island, Ausstralorp, Minorca, Amrox, California Gray, Italian Partridge-colored), as well as strains of turkeys, pheasants, quails, duck, ostriches and other poultry commonly bred in commercial quantities.

The phrases “based on” and “derived from” typically mean obtained from, in whole or in part. For example, a retroviral vector being based on or derived from a particular irus or based on a nucleotide sequence of a particular retrovirus mean that the genome of the retroviral vector contains a substantial portion of the nucleotide sequence of the genome of the particular retrovirus. The substantial portion can be a particular gene or nucleotide sequence such as the nucleotide sequence encoding the gag, pol and/or env proteins or other structural or functional nucleotide sequence of the virus genome such as sequences encoding the long terminal s (LTRs) or can be substantially the complete retrovirus genome, for example, most (e.g., more than 60% or more than 70% or more than 80% or more than 90%) or all of the retrovirus genome, as will be apparent from the context in the specification as the dge of one skilled in the art. Examples of retroviral vectors that are based on or derived from a irus are the NL retroviral vectors (e. g., NLB) which are derived from the avian leukosis irus ) as disclosed in Cosset et (11., Journal of Virology (1991) vol. 65, p 3388-3394.

The term g sequence” and “coding ” as used herein refer to nucleotide sequences and nucleic acid sequences, including both RNA and DNA, that encode genetic information for the synthesis of an RNA, a protein, or any portion of an RNA or protein.

Nucleotide sequences that are not naturally part of a ular organism’s genome or are introduced at a non—native site in the organism’s genome are referred to as “foreign” nucleotide sequences, “heterologous” nucleotide sequences, “recombinant” nucleotide sequences or “exogenous” nucleotide sequences. In addition, a nucleotide sequence that has been isolated and then reintroduced into the same type (e.g., same species) of organism is not considered to be a naturally occurring part of a ular organism’s genome and is therefore considered exogenous or heterologous.

“Heterologous proteins” or “exogenous proteins” can be proteins encoded by n, heterologous or exogenous nucleotide sequences and ore are often not naturally expressed in a cell of the host organism.

As used herein, the terms “exogenous,” “heterologous” and “foreign” with reference to nucleic acids, such as DNA and RNA, are used interchangeably and refer to nucleic acid that does not occur naturally as part of a chromosome, a genome or cell in which it is present or which is found in a location(s) and/or in amounts that differ from the location(s) and/or amounts in which it occurs in nature. It can be nucleic acid that is not endogenous to the genome, chromosome or cell and has been exogenously introduced into the genome, chromosome or cell. Examples of heterologous DNA e, but are not limited to, DNA that s a gene product or gene product(s) of interest, for example, for production of an encoded protein. es of heterologous DNA include, but are not limited to, DNA that encodes traceable marker proteins, DNA that encodes therapeutic ns. The terms “heterologous” and “exogenous” can refer to a biomolecule such as a nucleic acid or a n which is not normally found in a n cell, tissue or substance produced by an sm or is not normally found in a certain cell, tissue or substance produced by an organism in an amount or location the same as that found to occur naturally. For example, a protein that is heterologous or exogenous to an egg is a protein that is not normally found in the egg.

The term “construct” as used herein refers to a linear or circular nucleotide sequence such as DNA that has been assembled from more than one segments of nucleotide ce which have been ed from a natural source or have been chemically synthesized, or combinations thereof.

The term “complementary” as used herein refers to two nucleic acid molecules that can form specific interactions with one another. In the specific interactions, an adenine base within one strand of a nucleic acid can form two hydrogen bonds with thymine within a second nucleic acid strand when the two nucleic acid strands are in opposing polarities. Also in the specific interactions, a guanine base within one strand of a nucleic acid can form three hydrogen bonds with cytosine within a second nucleic acid strand when the two nucleic acid strands are in opposing polarities.

Complementary nucleic acids as referred to , can further comprise modified bases wherein a modified adenine may form hydrogen bonds with a thymine or modified thymine, and a modified cytosine may form hydrogen bonds with a guanine or a ed e.

The term “expressed” or “expression” as used herein refers to the transcription of a coding ce to yield an RNA le at least complementary in part to a region of one of the two nucleic acid strands of the coding sequence. The term “expressed” or “expression” as used herein can also refer to the translation of an mRNA to produce a protein or peptide.

The term “expression vector” as used herein refers to a nucleic acid vector that comprises a gene expression controlling region, such as a promoter or promoter ent, operably linked to a nucleotide sequence ng at least one polypeptide.

The term “fragment” as used herein can refer to, for example, an at least about 10, 20, 50, 75, 100, 150, 200, 250, 300, 500, 1000, 2000, 5000, 6,000, 8,000, , ,000, 30,000, 40,000, 50,000 or 60,000 nucleotide long portion of a nucleic acid that has been ucted artificially (e.g., by chemical synthesis) or by cleaving a natural product into multiple pieces, using restriction endonucleases or mechanical shearing, or enzymatically, for example, by PCR or any other polymerizing que known in the art, or expressed in a host cell by recombinant nucleic acid technology known to one of skill in the art. The term “fragment” as used herein can also refer to, for example, an at least about 5, 10, 15, 20, 25, 30, 40, or 50 amino acid residues less than a full length amino acid sequence for NaGlu (526., amino acid sequence 24-743 of SEQ ID NO: 1), which portion is cleaved from a naturally occurring amino acid sequence by lytic cleavage by at least one protease, or is a portion of the lly occurring amino acid sequence synthesized by chemical methods or using recombinant DNA technology (e. g., expressed from a portion of the nucleotide sequence encoding the naturally occurring amino acid ce) known to one of skill in the art. ent” may also refer to a portion, for example, of about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 99% of a particular nucleotide sequence or amino acid sequence.

“Functional portion” and “functional fragment” can be used hangeably and as used herein mean a portion or fragment of a whole capable of performing, in whole or in part, a function of the whole. For example, a biologically functional portion of a molecule means a portion of the molecule that performs a biological function of the whole or intact molecule. Functional portions may be of any useful size. For example, a functional fragment may range in size from about 20 bases in length to a length equal to the entire length of the specified sequence minus one nucleotide. In another example, a functional fragment may range in size from about 50 bases in length to a length equal to the entire length of the specified sequence minus one nucleotide. In r example, a functional fragment may range in size from about 50 bases in length to about 20 kb in length. In another example, a functional fragment may range in size from about 500 bases in length to about 20 kb in length. In r example, a functional fragment may range in size from about 1 kb in length to about 20 kb in length. In r example, a functional fragment may range in size from about 0.1 kb in length to about 10 kb in length. In another example, a functional fragment may range in size from about 20 bases kb in length to about 10 kb in length.

The term “fully transgenic” or “germline transgenic” refers to an animal such as an avian that contains at least one copy of a ene in essentially all of its cells.

The term “gene expression lling region” as used herein refers to nucleotide sequences that are associated with a coding sequence and which regulate, in whole or in part, expression of the coding sequence, for example, regulate, in whole or in part, the transcription of the coding sequence. Gene sion controlling regions may be isolated from a naturally occurring source or may be chemically synthesized and can be incorporated into a nucleic acid vector to enable regulated transcription in appropriate cells. The “gene expression controlling regions” may precede, but is not limited to preceding, the region of a nucleic acid sequence that is in the region 5’ of the end of a coding sequence that may be transcribed into mRNA.

As used herein, “host cells” refers to cells that harbor vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene.

The term “isolated nucleic acid” as used herein , for example, (a) a DNA which has the sequence of part of a naturally ing genomic molecule but is not ﬂanked by at least one of the sequences that ﬂank that part of the molecule in the genome of the species in which it naturally occurs; (b) a nucleic acid which has been incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the ing vector or genomic DNA is not identical to naturally occurring DNA from which the c acid was obtained; (0) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), ligase chain reaction (LCR) or chemical synthesis, or a restriction fragment; (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene ng a fusion protein, and (e) a recombinant nucleotide sequence that is part of a hybrid sequence that is not naturally ing. Isolated nucleic acid molecules of the t invention can include, for example, natural allelic variants as well as c acid les modified by nucleotide deletions, insertions, ions, or substitutions.

The term “nucleic acid” as used herein refers to any linear or sequential array of nucleotides and nucleosides, for example cDNA, genomic DNA, mRNA, tRNA, oligonucleotides, oligonucleosides and derivatives thereof. For ease of discussion, non- naturally occurring nucleic acids may be referred to herein as constructs. Nucleic acids can include bacterial plasmid vectors including sion, cloning, cosmid and transformation vectors such as, animal viral vectors such as, but not d to, modified adenovirus, inﬂuenza virus, polio virus, pox Virus, retroviruses such as avian leukosis virus (ALV) retroviral vector, a murine leukemia virus (MLV) retroviral vector, and a lentivirus vector, and the like and fragments thereof. In addition, the nucleic acid can be an LTR of an avian leukosis virus (ALV) retroviral vector, a murine leukemia virus (MLV) retroviral vector, or a lentivirus vector and fragments thereof. Nucleic acids can also include NL vectors such as NLB, NLD and NLA and fragments thereof and synthetic oligonucleotides such as chemically synthesized DNA or RNA. Nucleic acids can include ed or derivatized nucleotides and nucleosides such as, but not limited to, halogenated tides such as, but not only, 5—bromouracil, and derivatized nucleotides such as biotin-labeled nucleotides.

As used herein, the terms “glycan,’9 66glycan structure, 99 CCglycan moiety,” “oligosaccharide,9’ 66oligosaccharide structure,99 66glycosylation pattern, 99 ‘6glycosylation e,” and “glycosylation structure” have essentially the same meaning and each refers to one or more structures which are formed from sugar residues and are attached to glycosylated n such as human NaGlu. For example, “N-glycan” or “N-linked glycan” refers to a glycan structure attached to a nitrogen of gine or arginine side- chain of the glycosylated n. “O—glycan” or “O—linked glycan” refers to a glycan structure attached to the yl oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side chain of the glycosylate n.

The term “vector” and “nucleic acid vector” as used herein refers to a natural or synthetic single or double stranded plasmid or viral nucleic acid molecule that can be transfected or transformed into cells and replicate independently of, or within, the host cell genome. A circular double stranded vector can be linearized by treatment with an appropriate restriction enzyme based on the nucleotide sequence of the vector. A c acid can be inserted into a vector by cutting the vector with restriction enzymes and ligating the desired pieces together, as is understood in the art. A typical vector can be comprised of the following elements operatively linked at appropriate distances for allowing functional gene expression: replication origin, promoter, enhancer, 5’ mRNA leader sequence, ribosomal binding site, nucleic acid cassette, ation and polyadenylation sites, and selectable marker sequences. One or more of these elements can be omitted in specific applications. The nucleic acid cassette can include a restriction site for insertion of the nucleic acid sequence to be expressed. In a onal vector the nucleic acid cassette ns the nucleic acid sequence to be sed including translation initiation and ation sites. An intron optionally can be included in the construct, for example, 5’ to the coding sequence. A vector is constructed so that the particular coding sequence is d in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control ces being such that the coding sequence is transcribed under the “control” of the l or tory ces. Modification of the ces encoding the particular protein of interest can be desirable to achieve this end.

For example, in some cases it can be necessary to modify the ce so that it can be attached to the control sequences with the appropriate orientation, or to maintain the reading frame. The control ces and other regulatory sequences can be ligated to the coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector which already ns the control sequences and an appropriate restriction site which is in reading frame with and under regulatory control of the control sequences.

The term “operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Gene expression controlling regions or promoter(s) (e. g., promoter components) operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The controlling sequence(s) or promoter need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, ening slated yet ribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.

“Overexpression”, as used herein, refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or ansformed organisms.

The term ct” or “oviduct tissue” refers to a tissue of an avian oviduct, such as the magnum, e. g., tubular gland cells, where proteins are produced with N-linked oligosaccharides that contain increased amounts of mannose and mammosephosphate (M6P) and substantially reduced amounts of galactose and/or sialic acid relative to that of proteins produced in other tissue of the avian such as liver or kidney tissue.

The term “oviduct-specific promoter” as used herein refers to promoters and promoter components which are functional, i.e., provide for transcription of a coding sequence, to a large extent, for example, primarily (i.e., more than 50% of the ription product produced in the animal by a particular promoter type being produced in t cells) or exclusively in oviduct cells of a bird. Examples of oviduct specific promoters include, but are not limited to, ovalbumin promoter, ovomucoid promoter, ovoinhibitor promoter, lysozyme promoter and ovotransferrin er and functional ns of these promoters, e.g., promoter components. By limiting the expression of NaGlu protein to the magnum using oviduct ic promoters, deleterious physiological effects to the bird as result of expression of these enzymes in other tissues of the bird can be minimized.

The terms “percent sequence identity,99 66percent identity,” “% identity,” nt 77 66 sequence homology, percent homology,” “% homology” and “percent sequence similarity” can each refer to the degree of sequence matching between two nucleic acid sequences or two amino acid sequences. Such sequence matching can be determined using the algorithm of Karlin & Altschul (1990) Proc. Natl. Acad. Sci. 87: 2264-2268, modified as in Karlin & ul (1993) Proc. Natl. Acad. Sci. 90: 5873-5877. Such an thm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) T. Mol. Biol. Q15: 403-410. BLAST nucleotide searches are performed with the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. BLAST n searches are performed with the XBLAST program, score = 50, ngth = 3, to obtain amino acid sequences homologous to a reference amino acid sequence. To obtain gapped alignments for ison purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucl. Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. Other algorithms, programs and default gs may also be suitable such as, but not only, the GCG—Sequence Analysis e of the UK. Human Genome Mapping Project Resource Centre that includes programs for nucleotide or amino acid sequence comparisons. A sequence may be at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to another sequence, e. g., the NaGlu protein sequence identified herein.

The term “avian derived” refers to a composition or substance produced by or obtained from a bird, poultry or avian. ” refers to birds that can be kept as livestock, including but not limited to, ns, duck, turkey, quail and ratites. For e, “avian d” can refer to chicken derived, turkey derived and/or quail derived.

The terms “polynucleotide,” 44oligonucleotide99 (Cnucleotide sequence” and “nucleic acid sequence” can be used interchangeably herein and include, but are not limited to, coding sequences, 226., polynucleotide(s) or nucleic acid sequence(s) which are transcribed and translated into polypeptide in vitro or in vivo when placed under the control of appropriate regulatory or control sequences; controlling sequences, e.g., translational start and stop codons, promoter sequences, ribosome binding sites, polyadenylation signals, transcription factor binding sites, transcription termination sequences, upstream and downstream regulatory domains, ers, silencers, DNA sequences to which a transcription factor(s) binds and alters the activity of a gene’s promoter either positively (induction) or vely (repression) and the like. No limitations as to length or to synthetic origin are suggested by the terms described herein.

As used herein the terms “polypeptide” and “protein” refer to a polymer of amino acids, for example, three or more amino acids, in a serial array, linked h peptide bonds. The term “polypeptide” includes proteins, protein fragments, protein analogues, oligopeptides and the like. The term “polypeptides” includes polypeptides as defined above that are encoded by nucleic acids, ed through recombinant technology (e.g., isolated from a enic bird), or synthesized. The term “polypeptides’ further contemplates polypeptides as defined above that e chemically modified amino acids or amino acids covalently or noncovalently linked to labeling ligands.

The term “promoter” as used herein refers to a DNA sequence useful to initiate transcription by an RNA polymerase in an avian cell. A ter component” is a DNA sequence that can, by itself or in ation with other DNA sequences, effect or facilitate transcription. Promoter ents can be functional fragments of promoters.

The terms binant nucleic acid” and “recombinant DNA” as used herein refer to ations of at least two nucleic acid ces that are not naturally found in a eukaryotic or prokaryotic cell. The nucleic acid sequences may include, but are not limited to, nucleic acid vectors, gene expression regulatory elements, origins of replication, suitable gene sequences that when expressed confer antibiotic resistance, n-encoding sequences and the like. The term binant polypeptide” is meant to include a polypeptide ed by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location, purity or structure.

Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature.

As used herein, the term atory” sequences or elements include promoters, enhancers, terminators, stop codons, and other elements that can control gene expression.

A “retrovirus77 66 CCtransducing particle,” or “transduction , retroviral particle,77 particle” refers to a replication-defective or replication-competent virus capable of transducing non-viral DNA or RNA into a cell.

A “SIN ” refers to a self-inactivating vector. In particular, a SIN vector is a retroviral vector having an altered genome such that upon integration into genomic DNA of the target cell (e.g., avian embryo cells), the 5’ LTR of the integrated retroviral vector will not on as a promoter. For example, a portion or all of the nucleotide sequence of the retroviral vector that results in the U3 region of the 5’ LTR of the retroviral vector once integrated can be deleted or altered in order to reduce or eliminate promoter activity of the 5’ LTR. In certain es, deletion of the CAAT box and/or the TAATA box from U3 of the 5’ LTR can result in a SIN vector, as is understood in the art.

The term “sense strand” as used herein refers to a single stranded DNA molecule from a genomic DNA that can be transcribed into RNA and translated into the l polypeptide product of the gene. The term “antisense strand” as used herein refers to the single strand DNA molecule of a genomic DNA that is complementary with the sense strand of the gene.

A “therapeutic n” or “pharmaceutical protein” is a substance that, in whole or in part, makes up a drug. In particular, “therapeutic ns” and “pharmaceutical proteins” include an amino acid sequence which in whole or in part makes up a drug.

The terms ter,3’ 44transcription regulatory sequence” and “promoter component” as used herein refer to nucleotide which regulates the transcriptional expression of a coding sequence. ary ription regulatory sequences include enhancer elements, hormone response elements, steroid se elements, negative regulatory elements, and the like. The “transcription tory sequence” can be isolated and incorporated into a vector to enable regulated transcription in appropriate cells of portions of the vector DNA. The cription regulatory sequence” can precede, but is not limited to, the region of a nucleic acid sequence that is in the region ’ of the end of a protein coding sequence that is transcribed into mRNA.

Transcriptional regulatory sequence can also be located within a protein coding region, for example, in regions of a gene that are identified as “intron” regions.

The terms “transformation” and “transfection” as used herein refer to the process of inserting a c acid into a host. Many techniques are well known to those skilled in the art to facilitate transformation or transfection of a nucleic acid into a prokaryotic or eukaryotic organism. These s involve a variety of techniques, such as treating the cells with certain concentrations of salt, for example, but without limitation, a calcium or magnesium salt, or ng the cells to an electric field, detergent, or liposome material, to render the host cell competent for the uptake of the c acid molecules.

As used herein, a “transgenic animal” is any non-human animal, such as an avian species, including the chicken, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic ques known in the art (see, for example, US. patent publication No. 2007/0243165, published October 18, 2007, the disclosure of which is incorporated in its entirety herein by reference) including those disclosed . The nucleic acid is introduced into an animal, directly or indirectly by introduction into a cell (e.g., egg or embryo cell) by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule can be integrated within a chromosome, or it may be hromosomally replicating DNA. In the typical transgenic animal, the transgene can cause cells to express a recombinant form of the target protein or polypeptide. The terms “chimeric ” or “mosaic animal” are used herein to refer to animals in which a transgene is found, or in which the recombinant nucleotide sequence is expressed, in some but not all cells of the animal. A germ-line chimeric animal contains a transgene in its germ cells and can give rise to an ing transgenic animal in which most or all cells of the offspring will contain the transgene.

As used herein, the term “transgene” means a nucleic acid sequence ing, for example, a human NaGlu protein) that is partly or entirely heterologous, i.e., n, to the animal or cell into which it is introduced, or, is partly or entirely homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be ed, or is inserted, into the animal or cell genome in such a way as to alter the genome of the organism into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).

As used herein, the term e replacement therapy (ERT)” refers to a eutic strategy for correcting an enzyme deficiency in a subject by administering the missing enzyme to a subject. For mal enzyme replacement therapy to be effective, the therapeutic enzyme must be delivered to lysosomes in the appropriate cells in tissues where the storage defect is manifested. In one embodiment, the enzyme may be administered to the subject intravenously, intrathecally, intracerebrally, intraventricularly, or intraparenchymaly. In one embodiment, the enzyme is able to cross the blood brain barrier (BBB). Without intending to be limited by mechanism, it is believed that as the blood es patient tissues, enzyme is taken up by cells and transported to the lysosome, where the enzyme acts to eliminate al that has accumulated in the lysosomes due to the enzyme deficiency. 1. Composition of NaGlu The present invention provides novel compositions of recombinant human NaGlu (rhNaGlu or NaGlu) (amino acid sequence 24-743 set forth in SEQ ID NO: 1) having patterns of glycosylation that confer an increased cellular uptake and an increased subcellular activity which are ularly useful for therapy, for example, in the treatment of Sanfilippo Syndrome B olysaccharidosis (MPS) IIIB).

In some aspects, the ition can be an isolated mixture of rhNaGlu comprising the amino acid sequence 24-743 of SEQ ID NO:1. In one embodiment, the mixture contains a sufficient amount of rhNaGlu having at least one glycan ure that contains phosphorylated mannose (e.g., M6P) or mannose such that the rhNaGlu containing M6P or mannose is internalized into a human cell deficient in NaGlu and restores at least 50 % of NaGlu activity observed in a wild-type human cell of the same type that actively expresses endogenous NaGlu. In one aspect, at least 10 %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 98% or 99% of rhNaGlu in the e ns at least one glycan structure having phosphorylated mannose and/or mannose. In one embodiment, at least 10 % of rhNaGlu in the mixture contains at least one glycan structure having phosphorylated mannose and/or mannose.

In one embodiment, at least 20 % of rhNaGlu in the mixture contains at least one glycan structure having phosphorylated mannose and/or mannose. In one embodiment, at least 30 % of rhNaGlu in the e ns at least one glycan structure having phosphorylated mannose and/or mannose. In one embodiment, at least 30 % of rhNaGlu in the mixture contains at least one glycan structure having phosphorylated mannose and/or e. In one embodiment, at least 40 % of rhNaGlu in the mixture contains at least one glycan structure having phosphorylated mannose and/or mannose. In one embodiment, at least 50 % of rhNaGlu in the mixture contains at least one glycan structure having phosphorylated mannose and/or mannose. In one embodiment, at least 60 % of rhNaGlu in the e contains at least one glycan structure having orylated mannose and/or mannose.

In some aspects, the NaGlu contains one or more N-linked glycan structure. The NaGlu contains at least one phosphorylated mannose (e.g., M6P or bis-M6P) which allows the protein to be recognized by the Mannose 6—phosphate receptor (M6P receptor), and subsequently taken up into a human cell, ing but not d to, a skin fibroblast, an endothelial, a neuronal cell, a hepatocyte, a macrophage or any cell that expresses M6P receptor on the cell surface via M6P receptor-mediated endocytosis.

In one embodiment, the NaGlu contains at least one mannose (Man). In another embodiment, the NaGlu contains at least one N—acetylglucosamine (GlcNAc).

In some s, the NaGlu contains a glycan structure comprising a phosphorylated mannose (M6P). As used herein, M6P can encompass any orylated mannose residue and includes mono- and bis-phosphorylated mannose.

In one ment, the M6P is present at a concentration that is about 1, about 2, about 3, about 4, about 5 or about 6 mole(s) per mole of protein. In one embodiment, the NaGlu contains M6P at a concentration that is about 2, about 3, about 4, or about 5 moles per mole of protein. In one embodiment, the NaGlu contains M6P at a concentration that is about 2 moles per mole of protein. In one embodiment, the NaGlu contains M6P at a tration that is about 3 moles per mole of protein. In one embodiment, the NaGlu contains M6P at a concentration that is about 4 moles per mole of protein. In one embodiment, the NaGlu contains M6P at a concentration that is about moles per mole of protein. In one embodiment, the NaGlu contains M6P at a concentration that is about 6 moles per mole of protein.

In some aspects, the rhNaGlu ns a sufficient amount of M6P for cellular uptake into a human cell having a M6P receptor on the cell e via M6P receptor- mediated endocytosis. In one ment, a sufficient amount of M6P for uptake into a human cell is about 1, 2, 3, 4, 5 or 6 moles per mole of protein. The rhNaGlu can be internalized into a human cell deficient in NaGlu such that the internalized protein fully (100% or more) restores a normal level of NaGlu activity in the human cell deficient in NaGlu. In one embodiment, the internalized u protein fully restores a normal level of NaGlu activity in the human cell at a concentration that is at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 ug/mL. In one embodiment, the internalized protein fully restores a normal level of NaGlu activity in the human cell deficient in NaGlu at a concentration that is at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 ug/mL. In one embodiment, the internalized protein fully restores a normal level of NaGlu activity in the human cell at a concentration that is at least 20, 30, 40, 50, 60, 70, 80, 90 or 100 ug/mL. As used herein, the normal level of NaGlu activity is a level of NaGlu ty measured in a wild-type human cell of the same type that actively expresses a normal NaGlu enzyme.

In some s, the rhNaGlu can be internalized into a human cell deficient in NaGlu such that the protein restores at least about 50%, about 60%, about 70%, about 80%, about 90% or about 95% of NaGlu activity of a normal human cell of the same type. In some embodiments, the u can be internalized into a human cell deficient in NaGlu such that the internalized rhNaGlu provides a higher enzymatic activity than that observed in a normal human cell of the same type. In one embodiment, the rhNaGlu is internalized into a human cell deficient in NaGlu such that the internalized rhNaGlu provides about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 and about d higher activity than that observed in a normal human cell of the same type. In one embodiment, the rhNaGlu is internalized into a human cell deficient in NaGlu such that the internalized rhNaGlu provides about 15, about 20, about 25, about , about 40, about 50, about 60, about 70, about 80, about 90 or about 100-fold higher activity than that observed in a normal human cell.

In one ment, the human cell deficient in NaGlu is any human cell deficient in NaGlu that expresses one or more M6P receptors on the cell surface. In one embodiment, the human cell deficient in NaGlu is a human mucopolysaccharidosis (MPS) IIIB fibroblast that accumulates heparan sulfate. In one embodiment, the human cell ent in NaGlu is a hepatocyte. In one embodiment, the human cell ent in NaGlu is a neuronal cell. In one embodiment, the human cell deficient in NaGlu is an endothelial cell. In one embodiment, the human cell deficient in NaGlu is a macrophage.

In some aspects, uptake of rhNaGlu into a human cell is inhibited by the presence of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10 mM of competing M6P ccharide. In some aspects, the cellular uptake of rhNaGlu is inhibited by the presence of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9 or about 1.0 mM of M6P monosaccharide. In one embodiment, the cellular uptake of rhNaGlu is inhibited by the presence of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, or about 0.09 mM of M6P monosaccharide.

In some aspects, the rhNaGlu contains mannose in its glycan structures at a concentration that is about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 moles per mole of protein. In one embodiment, the u has mannose at a concentration that is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 moles per mole of protein. The rhNaGlu contains mannose at a concentration that is about 22, 23, 24, 25, 26, 27 or 28 moles per mole of protein. The rhNaGlu contains mannose at a concentration that is about 24 moles per mole of protein. The u protein contains mannose at a concentration that is about 25 moles per mole of protein. The rhNaGlu contains mannose at a concentration that is about 26 moles per mole of n. The rhNaGlu contains mannose at a concentration that is about 27 moles per mole of protein.

In one embodiment, the rhNaGlu has mannose at a concentration that is between about and about 30 moles per mole of protein.

In some aspects, the rhNaGlu comprises N—acetylglucosamine (GlcNAc). In one embodiment, the rhNaGlu contains GlcNAc at a concentration that is between about 28 and about 42 moles per mole of protein. In one embodiment, the NaGlu protein has GlcNAc at a concentration that is between about 30 and about 40 moles per mole of protein. In one embodiment, the NaGlu protein comprises GlcNAc at a concentration that is between about 32 and about 38 moles per mole of protein. In one ment, the NaGlu protein comprises GlcNAc at a tration that is between about 34 and about 36 moles per mole of protein. In one ment, the NaGlu protein has GlcNAc at a concentration that is about 35 moles per mole of protein. In one embodiment, the rhNaGlu protein contains GlcNAc at a concentration that is about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 moles per mole of protein.

In some aspects, the rhNaGlu contains N-acetylgalactosamine (GalNAc) and/or ose (Gal). The ce of the GalNAc and Gal typically indicates that the NaGlu may contain one or more O—linked glycan structures which are added to the protein in the Golgi compartment. Accordingly, the present invention optionally includes a composition sing a recombinant human NaGlu that contains one or more 0- linked glycan structure.

In one embodiment, the rhNaGlu contains ose at a concentration that is about 1, 2, 3, 4, 5, 6 or 7 moles per mole of protein. In one ment, the rhNaGlu has galactose at a concentration that is about 2, 3, 4, 5 or 6 moles per mole of protein. In one embodiment, the rhNaGlu has galactose at a concentration that is about 3 moles per mole of protein. In one embodiment, the rhNaGlu has ose at a tration that is about 4 moles per mole of protein.

In one embodiment, the NaGlu comprises at least one GalNAc molecule per mole of protein. In one embodiment, the NaGlu comprises GalNAc at a concentration that is about 1 or 2 moles per mole of protein.

In one embodiment, the NaGlu contains no . In yet r embodiment, the NaGlu contains no glucose. In yet another embodiment, rhNaGlu contains neither fucose nor glucose.

The present invention also contemplates compositions of modified rhNaGlu proteins produced from modified nucleic sequences of rhNaGlu. The modified nucleic acid sequences include deletions, insertions, or substitutions of different nucleotides resulting in a polynucleotide that encodes a functionally equivalent polynucleotide or polypeptide. The encoded protein may also contain deletions, ions, or substitutions of amino acid residues that produce a silent change and result in a functionally lent protein or polypeptide. Deliberate amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the es as long as the ical activity of the NaGlu is retained. For example, negatively charged amino acids can include aspartic acid and glutamic acid; positively charged amino acids can include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values can include leucine, isoleucine, and valine; glycine and alanine; gine and glutamine; serine and threonine; phenylalanine and tyrosine.

In other aspects, the rhNaGlu can be modified such that it contains an additional moiety or second peptide. Although fied NaGlu protein may cross the blood brain r at a high serum concentration, modifications of the protein can be performed to increase the efficiency of central nervous system (CNS) ing. In one embodiment, errin receptor ligand (TfRL) can be attached to human NaGlu at N- or C-terminus of NaGlu protein. A non-limiting example of TrRL is THRPPMWSPVWP (SEQ ID N025). In one embodiment, the transferrin receptor ligand can be attached to human NaGlu C-terminus of the NaGlu protein. In another embodiment, human NaGlu is fused to insulin-like growth factor or (IGFZR) ligand at N- or C-terminus of the NaGlu protein. In yet another embodiment, the NaGlu protein is fused to low density lipoprotein (LDL) receptor ligand at N- or C-terminus of the NaGlu protein. In one ment, the NaGlu protein is fused to a stretch of five to ten consecutive acidic amino acid residues. The acidic amino acid residues can include aspartic acid (D) or glutamic acid (E).

In one embodiment, the u is produced in a transgenic avian that contains a transgene encoding the NaGlu protein. In one ment, the rhNaGlu is produced in an oviduct cell (e. g., a tubular gland cell) of a transgenic avian (e.g., n (Gallus)). In one embodiment, the rhNaGlu is glycosylated in the oviduct cell (e.g., tubular gland cell) of the transgenic avian. In one ment, the u has a glycosylation pattern resulting from the rhNaGlu being produced in an oviduct cell of a transgenic avian. In one embodiment, the rhNaGlu can be isolated and purified from the content of the hard shell eggs laid by the transgenic avian. In one embodiment, the rhNaGlu can be isolated and purified from egg white of the transgenic avian.

The present invention also includes itions of an isolated mixture of NaGlu proteins, such as a mixture of one or more fragments and full-length rhNaGlu (e.g., 24-743 set forth in SEQ ID NO: 1). In one embodiment, a substantial portion of the mixture contains orylated M6P. In one embodiment, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% 95%, 97%, 98% or 99% of the rhNaGlu in the mixture contains M6P. In yet r embodiment, at least 50% of the isolated rhNaGlu in the mixture contains M6P. In yet another embodiment, at least 60% of the isolated rhNaGlu in the mixture contains M6P. In yet another embodiment, at least 70% of the ed rhNaGlu in the e contains M6P. In yet another embodiment, at least 80% of the isolated rhNaGlu in the mixture contains M6P. In yet another embodiment, at least 90% of the isolated rhNaGlu in the mixture contains M6P. In yet another embodiment, at least 95% of the ed rhNaGlu in the mixture contains M6P. In yet another embodiment, at least 96% of the isolated rhNaGlu in the mixture contains M6P.

In yet another embodiment, at least 97% of the isolated rhNaGlu in the mixture contains M6P. In yet another embodiment, at least 98% of the ed rhNaGlu in the mixture ns M6P. In yet another embodiment, at least 99% of the isolated rhNaGlu in the mixture contains M6P.

Optionally, the rhNaGlu protein produced from an avian or mammalian expression system (e.g., CHO, HEK293, or human skin fibroblast cell-line) can be further modified to achieve a favorable glycosylation pattern (126., an increased amount of M6P) for cellular uptake while retaining the biological activity. Additional terminal M6P can be introduced to the rhNaGlu by the general methods applied to other hydrolases as described in US. Pat. No. 6,679,165, US. Pat. No. 7,138,262, or US.

Publication No. 2009/0022?02, the entire teachings of each of which are incorporated herein by reference. For example, a highly phosphorylated mannopyranosyl oligosaccharide compound can be derivatized with a al compound containing a carbonyl-reactive group, followed by ing the rhNaGlu n to te carbonyl (aldehyde) group on one glycan structure of the protein, and reacting the ed NaGlu n with the glycan with the derivatized highly phosphorylated mannopyranosyl oligosaccharide compound to form a new compound having hydrazine bond. 11. Vectors Methods which are well-known to those skilled in the art can be used to construct expression vectors containing sequences encoding NaGlu and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

Such techniques are described, for example, in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, iew, NY, and Ausubel, F. M. et al. (1989) Current Protocols in lar Biology, John Wiley & Sons, New York, N.Y., the entire teachings of which are incorporated herein by nce.

A variety of expression vector/host systems can be utilized to express c acid sequences encoding u. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant iophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e. g., baculovirus) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or ian cell culture systems (e. g., pTT22 vector). Non-limiting examples of the pTT22 vector containing human NaGlu cDNA fused to a c acid sequence encoding acidic amino acid residue and TfRL are shown in Figs. 11 and 12. cleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide d by a polynucleotide of the present invention. ing to the signal hypothesis, proteins secreted by vertebrate (e.g., avian or mammalian) cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing n chain across the rough endoplasmic reticulum (ER) has been ted. Those of ordinary skill in the art are aware that polypeptides produced in the ER by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or “full length” polypeptide to produce a secreted or “mature” form of the polypeptide. In certain embodiments, the native signal e, e.g., the MEAVAVAAAVGVLLLAGAGGAAG (1—23 of SEQ ID NO:1) signal peptide of human NaGlu is used, or a functional derivative of that ce that retains the ability to direct the secretion of the polypeptide that is operably associated with it.

Alternatively, a heterologous signal peptide (e.g., a heterologous mammalian or avian signal peptide), or a functional derivative thereof, may be used. For example, the wild- type leader sequence may be substituted with the leader sequence of, for example, human tissue plasminogen activator (tPA) or mouse B-glucuronidase.

The control elements or regulatory sequences can includes those non-translated regions of the vector-enhancers, promoters, 5’ and 3’ untranslated regions that interact with host cellular proteins to carry out transcription and translation. Such ts can vary in their strength and specificity. Depending on the vector system and host cell utilized, any number of le transcription and ation elements can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lac- Z promoter of the BluescriptTM phagemid (Stratagene, LaJolla, California) or pSportlTM plasmid (Gibco BRL) and the like can be used. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferred. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding NaGlu, vectors based on SV40 or EBV can be also used with an riate selectable marker such as puromycin and ampicillin (see, e.g., Figs. 11 and 12).

When the u is produced in a transgenic avian, the present invention contemplates that the rhNaGlu sequence be placed downstream of a promoter such that the sequence ng the rhNaGlu can be expressed in a tissue—specific manner in a transgenic avian. For example, the promoter can be an oviduct-specific promoter that is largely, but not entirely, specific to the , such as the oviduct-specific promoter, including but not limited to, ovalbumin, lysozyme, conalbumin, ovomucoid, ovomucoid, ovomucin and ovotransferrin promoters. In one embodiment, the promoter is an ovalbumin promoter, a lysozyme promoter, a conalbumin promoter, an ovomucoid promoter, an ovomucin promoter and/or an ovotransferrin promoter or any functional portion thereof.

Alternatively, a tutive promoter can be used to express the coding sequence of human NaGlu in an avian. In this case, expression is not limited to the magnum; expression also occurs in other tissues within the avian (e.g., blood). The use of such a ene, which includes a constitutive promoter and the coding sequence of NaGlu, is also suitable for effecting or driving the expression of a protein in the oviduct and the uent secretion of the protein into the egg. In one embodiment, the constitutive promoter can be, for example, a cytomegalovirus (CMV) promoter, a rous-sarcoma virus (RSV) er, a murine ia virus (MLV) er, and B-actin promoter. In one embodiment, the promoter is a CMV promoter, a MDOT promoter, a RSV promoter, a MLV promoter, or a mouse mammary tumor virus (MMTV) promoter of any functional n thereof.

The invention also plates any useful fragment or component of the promoters described herein. The promoter can be at least one segment, fragment or ent of a promoter region, such as a segment of the ovalbumin, lysozyme, conalbumin, ovomucoid, ovomucin, ovotransferrin, CMV, RSV or MLV promoter region. In a preferred embodiment, the promoter is a segment of the oviduct-specific promoter region which contains essential elements to direct expression of the coding sequence in the tubular gland cells. For example, included in the scope of the present invention is a segment, n or fragment of an oviduct-specific promoter and/or condensing the critical regulatory elements of the oviduct-specific er so that it retains sequences required for expression in the tubular gland cells of the magnum of the oviduct. In one embodiment, a segment of the ovalbumin promoter region is used. This segment comprises the 5 ing region of the ovalbumin gene.

A vector that contains a coding sequence for human NaGlu can be used for transfecting blastodermal cells of an avian or mammalian cell to generate stable integrations into the avian or mammalian genome and to create a germline transgenic avian or mammalian cell line. A non—limiting example of such vector is shown in Figs. 4A-D and 5. In the avian expression system, the human NaGlu coding sequence is operably linked to a promoter in a positional relationship to express the coding sequence in a transgenic avian, ularly in the tubular gland cell of the magnum of the avian oviduct, such that the recombinant human NaGlu n is expressed and deposited in egg white of a hard shell egg laid by the enic avian. onal suitable vectors and methods to making vectors for expressing rhNaGlu in an avian system are also disclosed in U.S. Pat. No. 6,730,822; U.S. Pat. No. 6,825,396; U.S. Pat. No. 6,875,588; U.S. Pat. No. 7,294,507; U.S. Pat. No. 7,521,591; U.S. Pat. No. 7,534,929; U.S. ation No. 2008/0064862A1; and U.S. Patent Publication No. 2006/0185024, the entire teachings of which are incorporated herein by reference. Non-limiting examples of other promoters which can be also useful in the present invention include Pol III promoters (for example, type 1, type 2 and type 3 Pol III promoters) such as H1 promoters, U6 promoters, tRNA promoters, RNase MPR promoters and functional portions of each of these promoters. Typically, functional terminator sequences are selected for use in the t invention in accordance with the er that is employed.

In one embodiment, the vector is a retroviral vector, in which the coding ce and the promoter are both positioned between the 5’ and 3’ LTRs of the retroviral vector. In one useful embodiment, the LTRs or retroviral vector is derived from an avian is virus (ALV), a murine leukemia virus (MLV) or a lentivirus.

One useful retrovirus for randomly introducing a transgene into the avian genome is the replication-deficient ALV, the replication-deficient MLV, or the replication-deficient irus.

The present invention also contemplates the use of self-inactivating (SIN) vectors. SIN vectors can be useful for increasing the quantity of human NaGlu produced in the oviduct of a transgenic avian. This effect can be further enhanced when the SIN vector does not contain any selectable marker cassette with a functional promoter (SIN/SC negative vector). In one ment, a SIN vector is a retroviral vector having altered genome so that the 5’ LTR of the integrated retroviral vector does not function as a promoter. In one particular embodiment, a portion or all of the nucleotide sequence of the retroviral vector that results in the U3 region of the 5’ LTR of the retroviral vector once integrated can be deleted or altered in order to reduce or eliminate promoter activity of the 5’ LTR. A non—limiting example of SIN vector which contains an ovalbumin er region fused to the coding sequence of human u is shown in Figs. 4A-D and 5. Functional components of the vector are also tabulated in Table 1.

Table 1. Functional components in pSIN-OV—1.1kb-I-rhNaGlu Functional components Nucleotide Sequence in SEQ ID NO:4 poly A site 634-639 Partial gag 692-945 LTR (RAV2) 1243-1588 l LTR (RAV2) 4691-4863 ALV CTE 4899-4986 1.1 kb Ovalbumin promoter 5232-6363 DHS II 5334-5714 DHS I 6064—6364 Exon L 6364—6410 Intron 1 6411—7999 NaGlu 8017— 10248 Any of the vectors described herein can include a sequence encoding a signal peptide that directs ion of the protein expressed by the ’s coding sequence from, for example, the tubular gland cells of the oviduct of an avian. Where a recombinant human NaGlu protein would not otherwise be secreted, the vector containing the coding sequence is ed to comprise a DNA ce comprising about 60 bp encoding a signal peptide from, for e, the lysozyme gene. The DNA sequence encoding the signal peptide is inserted in the vector such that it is located at the N-terminus of the rhNaGlu protein encoded by the DNA.

Further, the coding sequences of vectors used in any of the methods of the present invention can be ed with a 3’ untranslated region (3’ UTR) to confer stability to the RNA produced. When a 3’ UTR is added to a retroviral vector, the orientation of the promoter, the coding sequence and the 3’ UTR is preferably reversed with respect to the direction of the 3’ UTR, so that the addition of the 3’ UTR does not interfere with transcription of the full-length genomic RNA. In one embodiment, the 3’ UTR may be that of the ovalbumin gene, me gene or any 3’ UTR that is functional in a magnum cell, i.e., the SV40 late region. 111. Transgenic Avians enes described herein can be introduced into avian embryonic blastodermal cells to produce a transgenic chicken, transgenic turkey, transgenic quail and other avian s that carry the ene encoding recombinant human NaGlu in the genome of its germ-line tissue. In one aspect of the invention, a transgenic avian that produces rhNaGlu is created by transduction of embryonic blastodermal cells with replication-defective or replication—competent iral les carrying the transgene between the 5’ and 3’ LTRs of the retroviral vector. For instance, an avian leukosis virus (ALV) retroviral vector or a murine leukemia virus (MLV) retroviral vector can be used. An RNA copy of the modified retroviral vector packaged into viral particles can be used to infect embryonic blastoderms which develop into transgenic avians.

By the methods of the present invention, transgenes can be introduced into embryonic blastodermal cells of various avian species. For example, the methods can be applied to produce a transgenic chicken, transgenic turkey, transgenic quail, transgenic duct, and other avian species, that carry the transgene in the genome of its germ-line tissue in order to produce proteins of the invention. The dermal cells are typically stage VII-XII cells as defined by Eyal-Giladi and Kochav (1976), or the equivalent f. In a red embodiment, the blastoderm cells are at or near stage X.

In one method of transfecting blastodermal cells, a packaged iral-based vector can be used to deliver the vector into embryonic blastodermal cells so that the vector is ated into the avian genome. Such viral particles (i.e., transduction particles) are ed for the vector and d to determine the appropriate concentration that can be used to inject embryos. In one embodiment, avian eggs are windowed according to the procedure described in US. Pat. No. 5,897,998, the disclosure of which is incorporated herein by reference in its entirety, and the eggs are injected with transducing particles at or near stage X.

The transgenic avians of the invention which produce rhNaGlu are developed from the dermal cells into which the vector has been introduced. The resulting embryo is d to develop and the chick d to mature. At this stage, the transgenic avian produced from blastodermal cells is known as a founder and is chimeric with respect to the cells carrying the transgene and is referred to G0. G0 founder avians are typically chimeric for each inserted transgene. That is, only some of the cells of the G0 transgenic bird contain the transgene. Some founders carry the transgene in tubular gland cells in the magnum of their oviducts. These avians express the rhNaGlu protein encoded by the transgene in their oviducts. The NaGlu protein may also be expressed in other s (e.g., blood) in addition to the oviduct. Some founders are ine founders that carry the transgene in the genome of the germ-line tissues, and may also carry the transgene in oviduct magnum tubular gland cells that express the exogenous protein.

The transgenic avian can carry the transgene in its germ-line providing transmission of the exogenous transgene to the avian’s offspring stably in a Mendelian n. The G0 generation is typically hemizygous for the transgene encoding rhNaGlu. The G0 generation can be bred to non—transgenic animals to give rise to G1 enic offspring which are also hemizygous for the transgene and contain the transgene in ially all of the bird’s cells. The G1 hemizygous offspring can be bred to non-transgenic animals giving rise to G2 hemizygous offspring or may be bred together to give rise to G2 offspring homozygous for the transgene. Substantially all of the cells of avians which are positive for the transgene that are derived from G1 offspring contain the ene. In one embodiment, hemizygotic G2 offspring from the same line can be bred to produce G3 offspring homozygous for the transgene. In another embodiment, hemizygous G0 or G1 s, for example, are bred together to give rise to homozygous G1 offspring containing two copies of the transgene(s) in each cell of the animal. These are merely examples of certain useful breeding methods and the present invention contemplates the employment of any useful breeding method such as those known to duals of ordinary skill in the art.

IV. Production of rhNaGlu The rhNaGlu can be produced using a transgenic avian that contains in the genome a ene encoding rhNaGlu. In one embodiment, the transgenic avian is a germline transgenic chicken, quail, duck or . In one particularly useful embodiment, the invention is drawn to the production of NaGlu which can be produced in the oviduct of a chicken.

Production of rhNaGlu with or without modification in the avian system (e.g., in the avian oviduct) is within the scope of the invention. In one embodiment, the unmodified rhNaGlu comprises the wild-type amino acid sequence 3 of SEQ ID NO:1) with a glycosylation structure (i. e., M6P) that enables efficient uptake by human cells. In another ment, the modified protein can be an rhNaGlu fusion protein having a glycosylation pattern (i. e., M6P) that s efficient uptake by human cells.

A suitable avian vector that contains a nucleic acid sequence encoding a NaGlu protein, operably linked to a tissue—specific or constitutive promoter that drives expression of the ng sequence in the chicken oviduct are introduced into chicken embryonic cells at or near stage X as bed herein. The transformed embryonic cells are incubated under conditions conducive to hatching live chicks. Live chicks are nurtured into a mature chimeric chicken which are mated with a non-transgenic chicken naturally or via artificial nation. A transgenic chicken is identified by screening progeny for germline incorporation of the protein encoding sequence. The enic progeny can be mated with another transgenic or a non-transgenic n to produce a fully germline transgenic hen that lays eggs.

The rhNaGlu can be produced in a tissue-specific manner. For example, u can be expressed in the oviduct, blood andfor other cells or tissues of the enic avian. In one embodiment, the NaGlu is expressed in the tubular gland cells of the magnum of the oviduct of the transgenic avian, secreted into the lumen of the oviduct, and deposited into egg white. In one embodiment, egg white containing rhNaGlu is harvested and stored in bulk at a temperature ranging from 4°C to -20°C.

The NaGlu is then isolated and purified from the contents of the eggs using various s known in the art.

One aspect of the present invention relates to avian hard shell eggs (e.g., chicken hard shell eggs) which contain the rhNaGlu protein. The rhNaGlu produced and secreted by the transgenic avian is glycosylated in a manner favorable to cellular uptake by a human cell. The n may be present in any useful amount. In one embodiment, the protein is present in an amount in a range between about 0.01 pg per hard-shell egg and about 1 gram per hard-shell egg. In another embodiment, the protein is present in an amount in a range of n about 1 pg per hell egg and about 1 gram per hard- shell egg. For example, the protein may be present in an amount in a range of between about 10 pg per hard-shell egg and about 1 gram per hard-shell egg (e.g., a range of between about 10 ug per hard-shell egg and about 400 milligrams per hard-shell egg).

In one embodiment, the rhNaGlu is present in the egg white of the egg. In one embodiment, the u is present in an amount in a range of between about 1 ng per milliliter of egg white and about 0.2 gram per milliliter of egg white. For example, the rhNaGlu may be present in an amount in a range of between about 0.1 ug per milliliter of egg white and about 0.2 gram per milliliter of egg white (e.g., the rhNaGlu may be present in an amount in a range of between about 1 ug per milliliter of egg white and about 100 milligrams per milliliter of egg white. In one embodiment, the u is present in an amount in a range of between about 1 ug per milliliter of egg white and about 50 milligrams per iter of egg white. For example, the rhNaGlu may be present in an amount in a range of about 1 pg per milliliter of egg white and about 10 milligrams per iter of egg white (e.g., the rhNaGlu may be t in an amount in a range of between about 1 pg per milliliter of egg white and about 1 milligrams per milliliter of egg white). In one embodiment, the rhNaGlu is present in an amount of more than 0.1 ug per milliliter of egg white. In one embodiment, the rhNaGlu is present in an amount of more than 0.5 ug per milliliter of egg white. In one embodiment, the rhNaGlu is present in an amount of more than 1 pg per milliliter of egg white. In one embodiment, the protein is present in an amount of more than 1.5 pg per milliliter of egg white. In one embodiment, the rhNaGlu is present in an amount of more than 0.5 pg per milliliter of egg white. In one embodiment, the protein is present in an amount of more than 0.1 pg per milliliter of egg white.

In one embodiment, the rhNaGlu is present in an amount of 20 mg/L, 30mg/L, 40mg/L, 50 mg/L, 60 mg/L, 7‘0 mg/L, 80 mg/L, 90 mg/L, 100 mg/L, 120 mg/L, 130 mg/L, 140 mg/L, 150 mg/L, 160 mg/L, 170 mg/L, 200 mg/L, 300mg/L, 400 mg/L, 500 mg/L, 600 mg/L, 700 mg/L, 800 mg/L, 900 mg/L, or 1,000 mg/L egg white. In one embodiment, the rhNaGlu is present in an amount of about 100 mg/L of egg white. In one embodiment, the rhNaGlu is present in an amount of about 200 mg/L of egg white.

V. Host Cells The present invention also contemplates rhNaGlu produced in any useful protein expression system including, t limitation, cell culture (e.g., avian cells, CHO cells, HEK293 cells and COS cells), yeast, bacteria, and plants.

A host cell strain can be chosen for its ability to modulate the expression of the ed sequences or to process the expressed NaGlu in the desired fashion. Such modifications of the polypeptide of NaGlu include, without limitation, glycosylation, phosphorylation, or lipidation. Different host cells such as CHO, COS, HeLa, MDCK, HEK293 and W138, which have specific cellular ery and teristic isms for such post-translational activities, can be chosen to ensure the correct modification and processing of the fusion protein of the present ion. An avian tumor cell line is also contemplated as a host cell for expressing the polypeptide of the present invention. Examples of a useful avian cell line (e.g., an avian t tumor cell line) are described in US. Pat. Publication No. 2009/0253176, the entire teachings of which are incorporated herein by reference.

VI. Pharmaceutical Compositions The present invention also features pharmaceutical compositions comprising ed and substantially ed u or a pharmaceutically acceptable salt thereof.

The recombinant human NaGlu proteins may be administered using one or more carriers, e.g., as part of a pharmaceutical formulation, or without a carrier. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. itions comprising such carriers, including composite molecules, are ated by well—known conventional methods (see, for example, ton’s Pharmaceutical Sciences, 14th Ed., Mack Publishing Co., Easton, Pa.), the entire ngs of which are orated herein by reference. The carrier may comprise a diluent. In one embodiment, the ceutical carrier can be a liquid and the protein may be in the form of a solution. The pharmaceutical carrier can be wax, fat, or alcohol. In another embodiment, the pharmaceutically acceptable r may be a solid in the form of a powder, a lyophilized powder, or a tablet. In one embodiment, the carrier may comprise a liposome or a microcapsule.

In some embodiments, a pharmaceutical composition comprising recombinant human NaGlu protein r comprises a buffer. Exemplary buffers include acetate, ate, citrate and glutamate buffers. Exemplary buffers also include lithium citrate, sodium citrate, potassium citrate, calcium citrate, lithium lactate, sodium lactate, potassium lactate, calcium lactate, lithium phosphate, sodium phosphate, potassium phosphate, calcium phosphate, lithium maleate, sodium maleate, potassium maleate, calcium maleate, lithium tartarate, sodium tartarate, potassium tartarate, calcium tartarate, lithium succinate, sodium succinate, ium succinate, m ate, lithium acetate, sodium acetate, potassium acetate, calcium acetate, and mixtures thereof. In some embodiments, the buffer is trisodium citrate dihydrate. In some embodiments, the buffer is citric acid monohydrate. In some embodiments, a pharmaceutical composition ses trisodium citrate dehydrate and citric acid monohydrate.

In some embodiments, a pharmaceutical composition comprising recombinant human NaGlu protein further comprises a stabilizer. Exemplary stabilizers include albumin, ose, , amino acids, polyols, cyclodextrins, salts such as sodium chloride, magnesium de, and calcium chloride, lyoprotectants, and mixtures thereof. In some embodiments, a pharmaceutical composition comprises human serum In some embodiments, it is desirable to add a surfactant to the pharmaceutical ition. Exemplary surfactants include nonionic surfactants such as Polysorbates (e.g., Polysorbates 20 or 80); poloxamers (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; —, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl- dimethylamine; sodium methyl cocoy1—, or disodium methyl ofeyl-taurate; and the MONAQUATTM series (Mona Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., ics, PF68, etc). Typically, the amount of surfactant added is such that it reduces aggregation of the protein and minimizes the formation of particulates or effervescences. For example, a surfactant may be present in a formulation at a concentration from about 0.001 - 0.5% (e.g., about 0.005 - 0.05%, or 0.005 - . In particular, a surfactant may be present in a ation at a concentration of approximately 0.005%, 0.01%, 0.02%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%, etc. Ranges and values ediate to the above recited ranges and values are also plated to be part of the invention.

In some embodiments, suitable pharmaceutical compositions of the invention may further include one or more bulking agents, in particular, for lyophilized ations. A "bulking agent" is a nd which adds mass to the lyophilized mixture and butes to the physical structure of the lyophilized cake. For example, a bulking agent may improve the appearance of lyophilized cake (6. g., essentially uniform lized cake). Suitable bulking agents include, but are not limited to, sodium chloride, lactose, mannitol, glycine, sucrose, trehalose, hydroxyethyl starch. Exemplary trations of bulking agents are from about 1% to about 10% (e. g., 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, and 10.0%). Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention. The pharmaceutical compositions can be in the form of a sterile lyophilized powder for injection upon reconstitution with a diluent. The diluent can be water for injection, bacteriostatic water for ion, or sterile saline. The lyophilized powder may be produced by freeze drying a solution of the fusion protein to produce the protein in dry form. As is known in the art, the lyophilized protein generally has increased stability and a longer shelf-life than a liquid solution of the protein.

Pharmaceutical formulations include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), ] or parenteral administration. Preferably, the pharmaceutical formulations of the invention include those suitable for administration by injection including intrathecal, intraparenchymal, intracerebral, intraventricular, intramuscular, sub-cutaneous and intravenous administration. In one embodiment, the formulations of the invention are suitable for intravenous administration. In another embodiment, the formulations of the ion are suitable for intrathecal stration. The pharmaceutical formulations of the ion also e those suitable for administration by inhalation or insufﬂation. The formulations can, where appropriate, be conveniently presented in discrete dosage units and can be prepared by any of the methods well known in the art of pharmacy. The s of producing the pharmaceutical formulations typically include the step of bringing the therapeutic proteins into association with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

Recombinant human NaGlu proteins of the invention can also be formulated for parenteral administration (6. g., by injection, for example bolus injection or continuous infusion) and can be ted in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The eutic ns can be injected by, for example, subcutaneous injections, intramuscular ions, intrathecal injections, intracerebral injections, intraparenchymal injections, intraventricular injections, and intravenous (IV) infusions or injections.

In one embodiment, the recombinant human NaGlu n is stered intravenously by IV infusion by any useful method. In one e, the recombinant human NaGlu protein can be administered by intravenous infusion through a peripheral line. In another example, the recombinant human NaGlu protein can be administered by intravenous infusion through a peripherally inserted central catheter. In another example, the recombinant human NaGlu protein can be administered by intravenous infusion facilitated by an ambulatory infusion machine attached to a venous vascular access port. In one ment of intravenous infusion, the medication is stered over a period of 1 to 8 hours depending on the amount of medication to be d and the patient‘s previous infusion—related reaction history, as determined by a physician skilled in the art. In another embodiment, the recombinant human NaGlu protein is administered intravenously by IV injection. In another embodiment, the recombinant human NaGlu protein can be administered via intraperitoneal or intrathecal injection.

In some embodiments, the therapeutic proteins are administered by on, and the infusion can occur over an extended time period, for example, 30 minutes to 10 hours. Thus, the on can occur, for example, over a period of about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours. The infusion can also occur at various rates. Thus, for example, the infusion rate can be about 1 mL per hour to about 20 mL per hour. In some embodiments, the infusion rate is 5 mL to 10 mL per hour. In one embodiment, the infusion rate is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mL per hour. In one embodiment, the infusion rate is 0.1 to 5 hr. In one embodiment, the infusion rate is about 0.1, about 0.2, about 0.3, about 0.5, about 1.0, about 1.5, about 2.0, or about 3 mg/kg/hr. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

The therapeutic proteins can take such forms as suspensions, solutions, or emulsions in oily or aqueous es, and can contain formulatory agents such as suspending, stabilizing and/or sing agents. The recombinant human NaGlu proteins can be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from on, for constitution with a suitable vehicle, e. g., sterile, pyrogen-free water, before use.

Formulations in accordance with the present invention can be assessed based on product quality analysis, reconstitution time (if lyophilized), quality of reconstitution (if lyophilized), high lar weight, moisture, and glass transition temperature.

Typically, protein quality and product analysis include t degradation rate is using s including, but not limited to, size exclusion HPLC (SE-HPLC), cation exchange-HPLC (CEX-HPLC), X—ray diffraction (XRD), modulated differential scanning calorimetry (mDSC), reversed phase HPLC (RP-HPLC), multi-angle light scattering (MALS), ﬂuorescence, ultraviolet absorption, nephelometry, capillary electrophoresis (CE), SDS-PAGE, and ations thereof. In some embodiments, evaluation of product in accordance with the present invention may e a step of evaluating appearance (either liquid or cake appearance).

Generally, formulations (lyophilized or aqueous) can be stored for extended periods of time at room temperature. Storage temperature may typically range from 0°C to 45°C (e. g., 4°C, 20°C, 25°C or 45°C). Formulations may be stored for a period of months to a period of years. Storage time generally will be 24 months, 12 months, 6 , 4.5 months, 3 months, 2 months or 1 month. Formulations can be stored directly in the container used for administration, eliminating transfer steps. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

Formulations can be stored directly in the lyophilization container (if lyophilized), which may also function as the reconstitution vessel, eliminating transfer steps. Alternatively, lized product formulations may be ed into smaller increments for e. Storage should generally avoid circumstances that lead to ation of the proteins, including but not limited to exposure to sunlight, UV radiation, other forms of electromagnetic radiation, excessive heat or cold, rapid thermal shock, and mechanical shock. The ceutical compositions according to the invention can also contain other active ingredients such as immunosuppressive agents, antimicrobial agents, or preservatives, discussed in more detail below.

VII. Methods of Treatment The present invention also provides methods of treating associated diseases, e. g., Sanfilippo Syndrome B. Recombinant NaGlu employed in accordance with the invention includes recombinant NaGlu which can be produced in any useful protein expression system including, without tion, cell culture (e.g., CHO cells, COS cells), bacteria such as E. coii, transgenic animals such as mammals and avians (e. g., ns, duck, and turkey) and in plant systems (e.g., duck weed and tobacco plants). In one embodiment, the recombinant NaGlu is produced in a transgenic animal, such as an avian.

In one embodiment, the method comprises stering to the subject a recombinant human NaGlu protein (rhNaGlu), for ce a recombinant human NaGlu protein ning a sufficient amount of oligosaccharides (e. g., mannose and phosphorylated mannose (i.e., M6P)), in an amount sufficient to treat (e.g., , ameliorate) or prevent one or more symptoms of a NaGlu deficiency or NaGlu associated disease. The recombinant human NaGlu protein can be administered therapeutically or prophylactically, or both. The recombinant human NaGlu protein (rhNaGlu) can be administered to the subject, alone or in combination with other therapeutic modalities as described herein.

The terms “treat,’7 66treating,” and “treatment” refer to methods of alleviating, abating, or ameliorating a e or symptom, preventing an additional symptom, ameliorating or preventing an underlying cause of a symptom, inhibiting a disease or condition, arresting the development of a disease or ion, relieving a disease or condition, causing regression of a disease or condition, relieving a condition caused by the disease or condition, or stopping a m of the disease or condition either prophylactically and/or after the symptom has occurred.

“Therapeutically effective dose” as used herein refers to the dose (e.g., amount and/or interval) of drug required to e an intended therapeutic response (e.g., reduction of heparan e levels and/or increase in NaGlu activity in a target tissue).

A therapeutically effective dose refers to a dose that, as compared to a corresponding t who has not received such a dose, results in improved treatment, healing, prevention, or amelioration of a e, disorder, or side effect, or a decrease in the rate of the occurrence or advancement of a disease or disorder. The term also includes within its scope, doses effective to enhance physiological functions.

As used herein, the term “subject” or “patient” is intended to include human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, ns, amphibians, and es. Preferred subjects include human ts having a NaGlu deficiency or NaGlu associated disease.

As used herein a “NaGlu associated e” is a disease or condition which is mediated by NaGlu activity or is associated with aberrant NaGlu expression or activity.

An example of an NaGlu associated disease includes, but is not d to, NaGlu deficiency such as Sanﬂippo me B (also known as lysaccharidosis type IIIB).

The therapeutic methods of the present invention encompass any route of administration which facilitates the uptake or transport of the recombinant human NaGlu protein into the pertinent organs and tissues. In one embodiment, the methods of the invention e delivering the recombinant human NaGlu proteins of the invention to the CNS al nervous system), the kidney, or the liver of a subject for the treatment of a NaGlu associated disease (e. g., NaGlu deficiency). For example, the recombinant human NaGlu protein may be administered to the patient intravenously (e.g., via intravenous injection or intravenous infusion) and surprisingly crosses the blood brain barrier (BBB) of the subject having NaGlu ency. In another embodiment of the ion, the inant human NaGlu protein is administered to the t intrathecally.

A. Device for Intrathecal Delivery Various devices may be used for intrathecal delivery according to the present invention. In some embodiments, a device for intrathecal administration contains a ﬂuid access port (6. g., injectable port); a hollow body (e.g., catheter) having a first ﬂow orifice in ﬂuid communication with the ﬂuid access port and a second ﬂow orifice configured for insertion into spinal cord; and a securing mechanism for securing the insertion of the hollow body in the spinal cord. As a non—limiting example, a suitable securing mechanism contains one or more nobs mounted on the surface of the hollow body and a sutured ring able over the one or more nobs to prevent the hollow body (e.g., catheter) from slipping out of the spinal cord. In s embodiments, the ﬂuid access port comprises a reservoir. In some embodiments, the ﬂuid access port comprises a mechanical pump (e.g., an infusion pump). In some embodiments, an implanted catheter is connected to either a reservoir (e. g., for bolus delivery), or an infusion pump.

The ﬂuid access port may be implanted or external.

In some embodiments, intrathecal stration may be performed by either lumbar puncture (i.e., slow bolus) or via a port-catheter delivery system (i.e., infusion or bolus). In some embodiments, the catheter is inserted n the laminae of the lumbar vertebrae and the tip is threaded up the thecal space to the desired level ally L3-L4). ve to intravenous administration, a single dose volume suitable for intrathecal administration is typically small. Typically, intrathecal delivery according to the present invention maintains the balance of the composition of the CSF as well as the intracranial pressure of the subject. In some embodiments, intrathecal delivery is performed absent the corresponding removal of CSF from a subject. In some embodiments, a suitable single dose volume may be e.g., less than about 10 mL, 8 mL, 6 mL, 5 mL, 4 mL, 3 mL, 2 mL, 1.5 mL, 1 mL, or 0.5 mL. In some embodiments, a suitable single dose volume may be about 0.5—5 mL, 0.5-4 mL, 0.5-3 mL, 0.5-2 mL, 0.5- 1 mL, 1-3 mL, 1-5 mL, 1.5-3 mL, 1-4 mL, or 0.5—1.5 mL. In some embodiments, intrathecal delivery ing to the present ion involves a step of removing a desired amount of CSF first. In some embodiments, less than about 10 mL (e.g., less than about 9 mL, 8 mL, 7 mL, 6 mL, 5 mL, 4 mL, 3 mL, 2 mL, 1 mL) of CSF is first removed before intrathecal administration. In those cases, a suitable single dose volume may be e.g., more than about 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, or 20 mL. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

Various other devices may be used to effect intrathecal stration of a therapeutic composition. For example, formulations containing desired enzymes may be given using an Ommaya reservoir which is in common use for intrathecally administering drugs for meningeal carcinomatosis (Lancet 2: 983—84, 1963). More specifically, in this method, a ventricular tube is ed through a hole formed in the anterior horn and is connected to an Ommaya reservoir installed under the scalp, and the reservoir is subcutaneously punctured to intrathecally deliver the particular enzyme being ed, which is ed into the reservoir. Other devices for hecal administration of therapeutic compositions or formulations to an individual are described in U.S. Pat. No. 6,217,552, the entire contents of which, as they relate to these devices, are incorporated herein by nce. Alternatively, the drug may be intrathecally given, for example, by a single injection, or continuous infusion. It should be understood that the dosage treatment may be in the form of a single dose administration or multiple doses.

For injection, formulations of the invention can be formulated in liquid solutions.

In addition, the NaGlu enzyme may be formulated in solid form and re—dissolved or suspended ately prior to use. Lyophilized forms are also included. The injection can be, for example, in the form of a bolus injection or continuous infusion (e.g., using infusion pumps) of the NaGlu enzyme.

In one embodiment of the invention, the NaGlu enzyme is administered by lateral cerebro ventricular injection into the brain of a subject. The injection can be made, for example, through a burr hole made in the subj ect's skull. In another embodiment, the enzyme andfor other ceutical formulation is administered through a surgically ed shunt into the cerebral ventricle of a subject. For example, the injection can be made into the lateral ventricles, which are larger. In some ments, injection into the third and fourth smaller ventricles can also be made.

In yet another embodiment, the pharmaceutical compositions used in the present invention are administered by injection into the cistema magna, or lumbar area of a subject.

In another embodiment of the method of the invention, the pharmaceutically acceptable formulation es sustained delivery, e.g., "slow release" of the enzyme or other pharmaceutical composition used in the present invention, to a subject for at least one, two, three, four weeks or longer periods of time after the pharmaceutically acceptable formulation is stered to the t.

As used , the term “sustained delivery” refers to continual delivery of a pharmaceutical ation of the ion in viva over a period of time following administration, preferably at least several days, a week or l weeks. Sustained delivery of the composition can be demonstrated by, for example, the continued therapeutic effect of the enzyme over time (e.g., sustained delivery of the enzyme can be demonstrated by continued reduced amount of storage granules in the subject).

Alternatively, sustained delivery of the enzyme may be demonstrated by detecting the presence of the enzyme in viva over time.

B. Intravenous ry As discussed above, one of the surprising features of the present invention is that the recombinant human NaGlu ns of the invention are able to effectively and extensively diffuse across the blood brain barrier (BBB) and brain surface and penetrate various layers or regions of the brain, including deep brain regions, when administered intravenously. The methods of the present invention effectively deliver the rhNaGlu proteins to various tissues, neurons or cells of the central s system (CNS), which are hard to target by existing CNS delivery methods. Furthermore, the methods of the present invention r sufficient s of the recombinant human NaGlu proteins to the blood stream and various peripheral organs and tissues.

“Intravenous injection,” often medically referred to as IV push or bolus injection, refers to a route of administration in which a syringe is connected to the IV access device and the medication is injected directly, typically rapidly and occasionally up to a period of 15 minutes if it might cause tion of the vein or a too-rapid effect. Once a medicine has been injected into the ﬂuid stream of the IV tubing, there must be some means of ensuring that it gets from the tubing to the patient. Usually this is accomplished by allowing the ﬂuid stream to ﬂow normally and thereby carry the ne into the bloodstream. However, in some cases a second ﬂuid injection, sometimes called a ” is used following the first injection to facilitate the entering of the medicine into the bloodstream.

“Intravenous infusion” refers to a route of administration in which medication is red over an extended period of time. For example, the medication can be red to a patient over a period of time n 1 and 8 hours. The medication can also be delivered to a patient over a period of about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8 hours. To accomplish an intravenous infusion, an IV gravity drip or an IV pump can be used. IV infusion is typically used when a patient requires medications only at certain times and does not require additional intravenous ﬂuids (e. g., water solutions which can contain sodium, chloride, glucose, or any combination thereof) such as those that restore olytes, blood sugar, and water loss.

C. Target Tissues In some embodiments, the rhNaGlu of the invention is delivered to the central nervous system (CNS) of a subject. In some embodiments, the rhNaGlu of the invention is delivered to one or more of target s of brain, spinal cord, and/or peripheral organs. As used herein, the term t " refers to any tissue that is affected by the NaGlu associated disease to be treated or any tissue in which the deficient NaGlu is normally expressed. In some embodiments, target tissues include those tissues in which there is a detectable or abnormally high amount of enzyme substrate, for example stored in the cellular lysosomes of the tissue, in patients suffering from or susceptible to the NaGlu associated disease. In some embodiments, target tissues include those tissues that display a disease-associated pathology, symptom, or feature. In some embodiments, target tissues include those tissues in which the deficient NaGlu is normally expressed at an elevated level. As used herein, a target tissue may be a brain target tissue, a spinal cord target tissue and/or a peripheral target tissue. Exemplary target tissues are described in detail below.

D. Brain Target s In general, the brain can be d into different regions, layers and tissues. For example, meningeal tissue is a system of membranes which ps the central s system, including the brain. The meninges contain three layers, including dura matter, arachnoid matter, and pia matter. In general, the primary on of the meninges and of the cerebrospinal ﬂuid is to protect the central nervous system. In some embodiments, a therapeutic protein in accordance with the present invention is red to one or more layers of the meninges.

The brain has three primary subdivisions, including the cerebrum, cerebellum, and brain stem. The cerebral hemispheres, which are situated above most other brain structures and are covered with a cortical layer. Underneath the cerebrum lies the brainstem, which resembles a stalk on which the cerebrum is attached. At the rear of the brain, beneath the cerebrum and behind the tem, is the cerebellum.

The phalon, which is located near the midline of the brain and above the mesencephalon, contains the thalamus, metathalamus, hypothalamus, lamus, prethalamus, and pretectum. The mesencephalon, also called the midbrain, contains the tectum, tegumentum, ventricular mesocoelia, and cerebral peduncels, the red nucleus, and the cranial nerve III nucleus. The mesencephalon is associated with vision, g, motor control, sleep/wake, alertness, and ature regulation. s of tissues of the central s system, including the brain, can be characterized based on the depth of the tissues. For example, CNS (e.g., brain) tissues can be terized as surface or shallow tissues, mid—depth tissues, and/or deep tissues.

According to the present invention, the rhNaGlu of the invention may be delivered to any appropriate brain target tissue(s) associated with a particular disease to be treated in a subject. In some embodiments, the rhNaGlu of the invention is delivered to surface or shallow brain target tissue. In some embodiments, the rhNaGlu of the invention is delivered to mid-depth brain target . In some ments, the rhNaGlu of the invention is delivered to deep brain target tissue. In some embodiments, the rhNaGlu of the invention is delivered to a combination of surface or shallow brain target , mid-depth brain target tissue, and/or deep brain target tissue. In some embodiments, the rhNaGlu of the invention is delivered to a deep brain tissue at least 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm or more below (or internal to) the external surface of the brain. Ranges and values intermediate to the above recited ranges and values are also plated to be part of the invention.

In some embodiments, the rhNaGlu of the invention is delivered to one or more e or shallow tissues of cerebrum. In some embodiments, the targeted surface or shallow tissues of the cerebrum are located within 4 mm from the surface of the cerebrum. In some embodiments, the ed surface or shallow tissues of the cerebrum are selected from pia mater tissues, cerebral cortical ribbon tissues, hippocampus, Virchow Robin space, blood vessels within the VR space, the hippocampus, portions of the hypothalamus on the inferior surface of the brain, the optic nerves and tracts, the olfactory bulb and tions, and combinations thereof.

In some embodiments, the u of the ion is delivered to one or more deep tissues of the cerebrum. In some embodiments, the targeted surface or shallow tissues of the cerebrum are located at least 4 mm (e.g., 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm) below (or internal to) the surface of the cerebrum. In some embodiments, targeted deep tissues of the um include the cerebral cortical .

In some embodiments, targeted deep tissues of the cerebrum include one or more of the diencephalon (e. g., the hypothalamus, us, prethalamus or subthalamus), metencephalon, lentiform nuclei, the basal ganglia, caudate, putamen, amygdala, globus pallidus, and combinations thereof.

In some embodiments, the rhNaGlu of the invention is delivered to one or more tissues of the cerebellum. In certain embodiments, the targeted one or more tissues of the cerebellum are selected from the group ting of tissues of the molecular layer, tissues of the Purkinje cell layer, s of the Granular cell layer, cerebellar peduncles, and ation thereof. In some embodiments, therapeutic agents (e.g., enzymes) are delivered to one or more deep tissues of the cerebellum including, but not limited to, tissues of the Purkinje cell layer, tissues of the Granular cell layer, deep cerebellar white matter tissue (e. g., deep relative to the Granular cell layer), and deep cerebellar nuclei In some embodiments, the rhNaGlu of the invention is delivered to one or more tissues of the brainstem. In some embodiments, the targeted one or more s of the brainstem include brain stem white matter tissue and/or brain stem nuclei tissue.

In some embodiments, the rhNaGlu of the invention is delivered to various brain tissues including, but not limited to, gray matter, white matter, periventricular areas, pia- arachnoid, es, neocortex, cerebellum, deep tissues in cerebral cortex, molecular layer, caudate/putamen region, midbrain, deep regions of the pons or medulla, and combinations thereof.

In some embodiments, the rhNaGlu of the ion is delivered to various cells in the brain including, but not d to, neurons, glial cells, perivascular cells and/or meningeal cells. In some embodiments, a therapeutic protein is delivered to oligodendrocytes of deep white matter.

E. Spinal Cord Target Tissue In general, regions or tissues of the spinal cord can be characterized based on the depth of the tissues. For example, spinal cord s can be characterized as surface or w s, mid-depth tissues, and/or deep tissues.

In some embodiments, the rhNaGlu of the invention are delivered to one or more surface or shallow tissues of the spinal cord. In some embodiments, a targeted surface or shallow tissue of the spinal cord is located within 4 mm from the surface of the spinal cord. In some embodiments, a targeted surface or shallow tissue of the spinal cord contains pia matter and/or the tracts of white .

In some embodiments, the rhNaGlu of the invention are delivered to one or more deep tissues of the spinal cord. In some embodiments, a targeted deep tissue of the spinal cord is located internal to 4 mm from the e of the spinal cord. In some embodiments, a targeted deep tissue of the spinal cord contains spinal cord grey matter and/or ependymal cells.

In some embodiments, replacement enzymes (e.g., a NaGlu fusion protein) are delivered to neurons of the spinal cord.

F. eral Target s As used herein, peripheral organs or tissues refer to any organs or tissues that are not part of the central nervous system (CNS). Peripheral target tissues may include, but are not limited to, blood system, liver, , heart, endothelium, bone marrow and bone marrow derived cells, spleen, lung, lymph node, bone, cartilage, ovary and .

In some embodiments, the rhNaGlu of the invention is delivered to one or more of the peripheral target tissues.

G. Biodistribution and Bioavailability In various embodiments, once delivered to the target tissue, the rhNaGlu of the invention is localized intracellularly. For example, the rhNaGlu of the invention may be localized to exons, axons, lysosomes, mitochondria or vacuoles of a target cell (e.g., s such as Purkinje cells). For example, in some embodiments the rhNaGlu of the invention demonstrates translocation dynamics such that the rhNaGlu moves within the perivascular space (6. g., by pulsation—assisted convective mechanisms). In addition, active axonal transport mechanisms relating to the association of the administered protein or enzyme with neurofilaments may also contribute to or otherwise facilitate the distribution of the rhNaGlu proteins of the invention into the deeper tissues of the central nervous system.

In some ments, the rhNaGlu of the ion delivered according to the present invention may achieve therapeutically or clinically effective levels or activities in various targets tissues described herein. As used , a therapeutically or clinically effective level or activity is a level or activity sufficient to confer a therapeutic effect in a target tissue. The therapeutic effect may be objective (i. e., able by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). For example, a therapeutically or clinically effective level or activity may be an enzymatic level or activity that is sufficient to ameliorate symptoms associated with the disease in the target tissue (e. g., GAG storage).

In some embodiments, the rhNaGlu of the invention delivered ing to the present invention may achieve an enzymatic level or activity that is at least 5%, 10%, %, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the normal level or ty of the corresponding NaGlu enzyme in the target tissue. In some ments, the rhNaGlu of the invention delivered according to the t invention may achieve an tic level or activity that is increased by at least 1-fold, 2-fold, 3 -fold, 4-fold, 5—fold, 6-fold, , 8-fold, 9-fold or 10-fold as compared to a control (6. g., endogenous levels or activities without the treatment). In some embodiments, the rhNaGlu delivered according to the present invention may e an increased enzymatic level or activity at least approximately 10 nmol/hr/mg, 20 nmol/hr/mg, 40 nmol/hri'mg, 50 nmol/hr/mg, 60 r/mg, 70 nmol/hr/mg, 80 nmol/hr/mg, 90 nmol/hr/mg, 100 nmol/hr/mg, 150 nmol/hr/mg, 200 nmol/hr/mg, 250 nmol/hr/mg, 300 nmol/hr/mg, 350 nmol/hr/mg, 400 nmol/hr/mg, 450 nmol/hr/mg, 500 nmol/hr/mg, 550 nmol/hr/mg or 600 r/mg in a target tissue. Ranges and values intermediate to the above d ranges and values are also contemplated to be part of the invention.

In some ments, inventive methods according to the present invention are particularly useful for targeting the lumbar . In some embodiments, the rhNaGlu delivered according to the present invention may achieve an increased enzymatic level or activity in the lumbar region of at least approximately 500 nmol/hr/mg, 600 nmol/hr/mg, 700 nmol/hr/mg, 800 nmol/hr/mg, 900 nmol/hr/mg, 1000 r/mg, 1500 nmol/hr/mg, 2000 r/mg, 3000 nmol/hr/mg, 4000 nmol/hr/mg, 5000 nmol/hr/mg, 6000 nmol/hr/mg, 7000 nmol/hr/mg, 8000 r/mg, 9000 nmol/hr/mg, or 10,000 nmol/hr/mg. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

In general, therapeutic agents (e.g., the rhNaGlu) red according to the present invention have sufficiently long half time in CSF and target tissues of the brain, spinal cord, and peripheral organs. In some embodiments, the rhNaGlu delivered according to the present invention may have a half—life of at least imately 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 16 hours, 18 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, up to 3 days, up to 7 days, up to 14 days, up to 21 days or up to a month. In some embodiments, In some embodiments, the rhNaGlu red according to the present invention may retain detectable level or activity in CSF or bloodstream after 12 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours, 102 hours, or a week ing administration. Detectable level or activity may be determined using various methods known in the art. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

In certain embodiments, the rhNaGlu delivered according to the present invention achieves a concentration of at least L in the CNS tissues and cells of the subject following administration (e.g., one week, 3 days, 48 hours, 36 hours, 24 hours, 18 hours, 12 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 s, or less, following administration of the pharmaceutical composition to the subject). In certain embodiments, the rhNaGlu delivered according to the present invention achieves a concentration of at least 2 ugImL, at least 15 ugme, at leastl ug/mL, at least 7 ug/mL, at least 5 ug/mL, at least 2 ug/mL, at least 1 ugme or at least 0.5 ug/mL in the targeted tissues or cells of the subject(e.g., brain tissues or neurons) following administration to such subject (e.g., one week, 3 days, 48 hours, 36 hours, 24 hours, 18 hours, 12 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, or less following administration of such pharmaceutical compositions to the subject). Ranges and values intermediate to the above recited ranges and values are also plated to be part of the invention.

H. Treatment of Sanfilippo Syndrome Sanfilippo syndrome, or mucopolysaccharidosis III (MPS III), is a rare genetic disorder characterized by the ency of enzymes involved in the degradation of glycosaminoglycans (GAG). In the absence of enzyme, partially degraded GAG molecules cannot be cleared from the body and accumulate in lysosomes of various s, resulting in progressive widespread somatic dysfunction (Neufeld and Muenzer, 2001).

Four distinct forms of MPS III, designated MPS IIIA, B, C, and D, have been identified. Each represents a deficiency in one of four enzymes involved in the degradation of the GAG n sulfate. All forms e varying degrees of the same clinical symptoms, including coarse facial features, hepatosplenomegaly, corneal clouding and skeletal deformities. Most notably, however, is the severe and progressive loss of cognitive ability, which is tied not only to the accumulation of n sulfate in neurons, but also the subsequent elevation of the gangliosides GM2, GM3 and GD2 caused by primary GAG accumulation (Walkley 1998).

Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome B) is an autosomal recessive disorder that is characterized by a deficiency of the enzyme alpha- yl-glucosaminidase (NaGlu). In the e of this enzyme, GAG heparan sulfate accumulates in lysosomes of neurons and glial cells, with lesser accumulation e the brain.

A defining clinical feature of this disorder is central nervous system (CNS) degeneration, which s in loss of, or e to attain, major developmental milestones. The progressive ive decline culminates in dementia and premature mortality. The disease typically manifests itself in young children, and the lifespan of an affected individual generally does not extend beyond late teens to early es.

Compositions and methods of the present invention may be used to effectively treat individuals suffering from or susceptible to Sanfilippo syndrome B. The terms, “treat” or “treatment,” as used herein, refers to amelioration of one or more symptoms associated with the disease, prevention or delay of the onset of one or more symptoms of the disease, and/or lessening of the ty or frequency of one or more symptoms of the disease.

In some embodiments, treatment refers to partial or complete alleviation, amelioration, relief, inhibition, delaying onset, reducing severity and/or incidence of neurological impairment in a ippo syndrome B patient. As used herein, the term “neurological impairment” includes various symptoms associated with impairment of the central nervous system (e.g., the brain and spinal cord). ms of neurological impairment may include, for example, developmental delay, progressive cognitive impairment, hearing loss, impaired speech development, deficits in motor skills, hyperactivity, siveness and/or sleep disturbances, among others.

Thus, in some embodiments, treatment refers to decreased lysosomal storage (e.g., of GAG) in various tissues. In some embodiments, treatment refers to sed lysosomal storage in brain target tissues, spinal cord neurons, and/or peripheral target tissues. In certain embodiments, lysosomal storage is decreased by about 5%, 10%, 15%, %, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more as compared to a control. In some ments, lysosomal e is decreased by at least 1-fold, 2—fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold as compared to a control. In some embodiments, lysosomal e is determined by LAMP-1 staining. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

In some embodiments, treatment refers to d vacuolization in neurons (e. g., s containing Purkinje . In certain embodiments, ization in neurons is decreased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more as compared to a control. In some embodiments, vacuolization is decreased by at least l-fold, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold as compared to a l.

Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the ion.

In some embodiments, treatment refers to increased NaGlu enzyme activity in various tissues. In some ments, treatment refers to increased NaGlu enzyme activity in brain target tissues, spinal cord neurons and/or peripheral target tissues. In some embodiments, NaGlu enzyme activity is increased by about 5%, 10%, 15%, 20%, %, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% 1000% or more as compared to a control. In some embodiments, NaGlu enzyme activity is increased by at least 1-fold, 2-fold, 3 -fold, 4—fold, 5—fold, 6—fold, 7—fold, 8-fold, 9-fold or 10-fold as compared to a control. In some embodiments, increased NaGlu enzymatic activity is at least approximately 10 nmolx’hrx’mg, 20 nmol/hrfmg, 40 nmol/hr/mg, 50 nmol/hr/mg, 60 nmol/hr/mg, 70 nmol/hr/mg, 80 nmol/hr/mg, 90 nmol/hr/mg, 100 nmol/hr/mg, 150 nmol/hr/mg, 200 nmol/hr/mg, 250 nmol/hr/mg, 300 nmol/hr/mg, 350 nmol/hr/mg, 400 nmol/hr/mg, 450 nmol/hr/mg, 500 nmol/hr/mg, 550 nmol/hr/mg, 600 r/mg or more. In some embodiments, NaGlu tic activity is increased in the lumbar region. In some embodiments, increased NaGlu enzymatic activity in the lumbar region is at least approximately 2000 nmol/hr/mg, 3000 nmol/hr/mg, 4000 nmol/hr/mg, 5000 nmol/hr/mg, 6000 nmol/hr/mg, 7000 r/mg, 8000 nmol/hr/mg, 9000 nmol/hr/mg, ,000 nmol/hr/mg, or more. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

In certain embodiments, treatment according to the present invention results in a reduction (e.g., about a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 97.5%, 99% or more reduction) or a complete elimination of the presence, or alternatively the accumulation, of one or more pathological or biological markers which are associated with the NaGlu associated disease. Such ion or elimination may be particularly t in the cells and tissues of the CNS (e. g., neurons and oligodendrocytes). For example, in some embodiments, upon stration to a subject the pharmaceutical compositions of the t invention trate or achieve a reduction in the accumulation of the ker lysosomal associated membrane protein 1 (LAMPl) in the CNS cells and s of the subject (6. g., in the cerebral cortex, cerebellum, caudate nucleus and putamen, White matter and/or us). LAMPl is a glycoprotein highly expressed in lysosomal membranes and its ce is elevated many patients with a lysosomal storage disorder (Meikle et al., Clin. Chem. (1997) 43: 1325—1335). The presence or absence of LAMP 1 in patients (e.g., as determined by LAMP staining) with a lysosomal storage disease therefore may provide a useful indicator of lysosomal activity and a marker for both the diagnosis and monitoring of lysosomal storage diseases.

Accordingly, some ments of the present invention relate to s of reducing or otherwise eliminating the presence or accumulation of one or more pathological or ical markers associated with the NaGlu associated disease. rly, some embodiments of the invention relate to methods of increasing the degradation (or the rate of degradation) of one or more pathological or biological markers (e.g., LAMPl) associated with lysosomal storage diseases.

In some embodiments, treatment refers to decreased progression of loss of cognitive ability. In certain embodiments, progression of loss of cognitive ability is decreased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more as ed to a control. In some embodiments, treatment refers to sed developmental delay. In certain embodiments, developmental delay is decreased by about 5%, 10%, 15%, 20%, 25%, %, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more as compared to a control. Ranges and values ediate to the above d ranges and values are also contemplated to be part of the invention.

In some embodiments, ent refers to increased survival (e.g., survival time).

For example, treatment can result in an increased life expectancy of a patient. In some embodiments, treatment according to the present invention results in an increased life expectancy of a patient by more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65 %, about ?0%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 1 10%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, about 200% or more, as compared to the average life ancy of one or more control individuals with similar disease without treatment. In some embodiments, treatment according to the present ion results in an increased life expectancy of a patient by more than about 6 month, about 7 months, about 8 , about 9 months, about 10 months, about 11 months, about 12 , about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years or more, as compared to the average life expectancy of one or more control individuals with similar disease without treatment. In some ments, treatment according to the present invention results in long term survival of a patient. As used herein, the term "long term survival" refers to a survival time or life expectancy longer than about 40 years, 45 years, 50 years, 55 years, 60 years, or longer.

Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

The terms, “improve,” “increase” or “reduce,” as used herein, indicate values that are relative to a l. In some embodiments, a suitable control is a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein. A "control individual" is an individual ed with Sanfilippo syndrome B, who is about the same age and/or gender as the individual being treated (to ensure that the stages of the disease in the treated individual and the l individual(s) are comparable).

The individual (also referred to as “patient” or “subject”) being treated is an individual , infant, child, adolescent, or adult human) having Sanfilippo syndrome B or having the potential to develop Sanﬁlippo syndrome B. The individual can have residual nous NaGlu expression and/or activity, or no measurable activity. For example, the individual having Sanfilippo me B may have NaGlu expression levels that are less than about 30-50%, less than about 25-30%, less than about 20-25%, less than about 15-20%, less than about 10-15%, less than about 5-10%, less than about 0.1-5% of normal NaGlu expression levels. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

In some embodiments, the individual is an dual who has been recently sed with the disease. Typically, early ent ment commencing as soon as possible after diagnosis) is important to minimize the effects of the disease and to maximize the benefits of treatment.

I. Combination Therapies Recombinant human NaGlu proteins, for instance a recombinant human NaGlu protein ning a sufficient amount of oligosaccharides (e.g., mannose and phosphorylated mannose (i.e., M6P)), can be used alone or in combination to treat NaGlu associated diseases (e. g., Sanfilippo Syndrome B). It should be understood that the recombinant human NaGlu proteins of the invention can be used alone or in combination with an additional procedure, e.g., surgical procedure, or agent, e.g., therapeutic agent, the onal procedure or agent being selected by the skilled artisan for its intended purpose. For instance, the additional procedure or agent can be a therapeutic procedure or agent art-recognized as being useful to treat the disease or condition being d by the recombinant human NaGlu protein of the present invention. The additional ure or agent also can be an agent that imparts a beneficial attribute to the therapeutic composition, e. g., an agent which affects the viscosity of the composition.

It should also be understood that the combinations which are ed within this invention are those combinations useful for their intended purpose. The agents and procedures set forth below are for illustrative es and not intended to be limiting to the present invention. The combinations, which are part of this invention, can be the inant human NaGlu proteins of the present invention and at least one additional agent or procedure selected from the lists below. The combination can also include more than one additional agent or procedure, e.g., two or three additional agents if the combination is such that the formed composition can perform its intended function.

The combination therapy can include surgical procedures, gene therapy, or enzyme-replacement therapy. Additionally, the recombinant human NaGlu protein can be coformulated with one or more additional therapeutic agents, e.g., other recombinant proteins or antibodies or drugs capable of ting or reducing the accumulation of undegraded ates (e.g., ate reduction therapy).

In one or more embodiments, the combination therapy can include co- administration with immunosuppresants, as discussed in further detail below.

Immunosuppresants such as, but not limited to, antihistamines, corticosteroids, sirolimus, voclosporin, ciclosporin, methotrexate, IL—2 receptor directed antibodies, T- cell receptor directed dies, TNF-alpha directed antibodies or fusion proteins (e.g., inﬂiximab, etanercept, or adalimumab), CTLAIg (e.g., abatacept), anti-OX-40 antibodies can also be administered before, during, or after administration of a recombinant human protein, such as a recombinant human NaGlu protein, for example, if an anaphylactic reaction or adverse immune response is ed or experienced by a patient.

J. Immunogenicity The ceutical compositions of the present ion are characterized by their tolerability. As used herein, the terms “tolerable” and “tolerability” refer to the ability of the pharmaceutical compositions of the present invention to not elicit an adverse reaction in the subject to whom such composition is stered, or alternatively not to elicit a serious adverse reaction in the subject to whom such composition is administered. In some embodiments, the pharmaceutical compositions of the present invention are well tolerated by the t to whom such compositions is administered.

Generally, administration of a rhNaGlu protein according to the t invention does not result in severe adverse effects in the subject. As used herein, severe adverse effects induce, but are not limited to, substantial immune se, ty, or death. As used herein, the term antial immune se” refers to severe or serious immune responses, such as adaptive T-cell immune responses.

Thus, in many embodiments, inventive methods according to the present invention do not involve concurrent immunosuppressant therapy (i. 6., any immunosuppressant therapy used as pre-treatmentipre-conditioning or in parallel to the method). In some embodiments, inventive methods according to the present invention do not involve an immune tolerance induction in the subject being treated. In some embodiments, inventive methods according to the present invention do not involve a pre-treatment or ditioning of the subject using T-cell immunosuppressive agent.

However, in some embodiments, a subject mounts an immune response after being stered the rhNaGlu of the invention. Thus, in some embodiments, it may be useful to render the subject receiving the rhNaGlu of the invention tolerant to the enzyme replacement y. Immune tolerance may be induced using various methods known in the art. For example, an initial 30-60 day regimen of a T-cell immunosuppressive agent such as cyclosporin A (CsA) and an oliferative agent, such as, azathioprine (Aza), combined with weekly intrathecal ons of low doses of a desired replacement enzyme may be used.

Any immunosuppressant agent known to the skilled artisan may be employed together with a combination therapy of the invention. Such immunosuppressant agents include but are not limited to cyclosporine, FK506, rapamycin, CTLA4-Ig, and anti-TNF agents such as etanercept (see e. g., Moder, 2000, Ann. Allergy Asthma Immunol. 84, 280-284; , 2000, Curr. Opin. Pediatr. 12, 146-150; Kurlberg et al., 2000, Scand. J.

Immunol. 51, 224-230; Ideguchi et al., 2000, Neuroscience 95, 217-226; et al., 1999, Ann. NY. Acad. Sci. 875, 159-174; Slavik et al., 1999, l. Res. 19, 1-24; Gaziev et al., 1999, Bone Marrow Transplant. 25, 6; Henry, 1999, Clin.

Transplant. 13, 209-220; Gummert et al., 1999, J. Am. Soc. Nephrol. 10, 1366-1380; Qi et al., 2000, Transplantation 69, 1275—1283). The anti-1L2 receptor (or-subunit) antibody daclizumab (e. g., ZenapaxTM), which has been demonstrated effective in transplant patients, can also be used as an immunosuppressant agent (see e. g., Wiseman et al., 1999, Drugs 58, 1029-1042; Beniaminovitz et al., 2000, N. Engl J. Med. 342, 613-619; Ponticelli et al., 1999, Drugs R. D. 1, 55—60; Berard et al., 1999, Pharmacotherapy 19, 1 127-1 137; Eckhoff et al., 2000, Transplantation 69, 1867—1872; Ekberg et al., 2000, l. Int. 13, 15 1- 159). Additional immunosuppressant agents include but are not limited to anti-CD2 (Branco et al., 1999, lantation 68, 1588-1596; Przepiorka et al., 1998, Blood 92, 4066-40?1), anti-CD4 (Marinova-Mutafchieva et al., 2000, Arthritis Rheum. 43, 638-644; Fishwild et al., 1999, Clin. Immunol. 92, 138-152), and anti-CD40 ligand (Hong et al., 2000, Semin. Nephrol. 20, 108-125; Chirmule et al., 2000, J. Virol. 74, 3345-3352; Ito et al., 2000, J. Immunol. 164, 1230-1235).

In other embodiments, the invention es methods comprising co- administration of the NaGlu proteins of the present invention with agents which decrease or suppress an immune response to the NaGlu protein, e. g., immunosuppresants. Immunosuppresants such as, but not limited to, antihistamines, osteroids, sirolimus, voclosporin, ciclosporin, methotrexate, IL-2 receptor directed antibodies, T-cell receptor directed antibodies, TNF-alpha directed antibodies or fusion ns (e. g., inﬂiximab, cept, or umab), -Ig (e.g., ept), anti- OX-40 antibodies can also be administered before, , or after administration of a recombinant human protein, such as a recombinant human NaGlu protein, for example, if an anaphylactic reaction or adverse immune response is expected or experienced by a patient.

In one embodiment, the invention provides for a pretreatment procedure to minimize or prevent any potential anaphylactic reactions that can be incurred by administration of the recombinant protein in accordance with the invention. In one embodiment, to prevent a ial anaphylactic reaction, an H-1 receptor antagonist, also known as an antihistamine (e. g., diphenhydramine) is administered to the patient.

In one embodiment, the H-1 receptor antagonist is administered in a dose of about 1 mg to about 10 mg per kilogram of body weight. For example, an antihistamine can be administered in a dose of about 5 mg per kilogram. In one embodiment, the antihistamine is administered in a dose of between about 0.1 mg and about 10 mg per kilogram of body weight. In one ment, the antihistamine is administered in a dose between about 1 mg and about 5 mg per kilogram of body weight. For example the dose can be 1 mg, 2 mg, 3 mg, 4 mg, or 5 mg per kilogram of body weight. The antihistamine can be administered by any useful method. In one embodiment, the antihistamine is administered intravenously. In another embodiment, the antihistamine is stered in pharrnaceutically acceptable capsules.

Administration of the antihistamine can be prior to the stration of the recombinant NaGlu in accordance with the invention. In one embodiment, the H-1 receptor nist is administered about 10 to about 90 minutes, for example, about 30 to about 60 minutes prior to the stration of recombinant NaGlu. The H-l receptor antagonist can be administered using an ambulatory system connected to a ar access port. In one embodiment, the antihistamine is administered about 90 minutes prior to the administration of recombinant NaGlu. In one ment, the antihistamine is administered between about 10 and about 60 minutes prior to the administration of recombinant NaGlu. In another embodiment, the antihistamine is administered between about 20 and about 40 minutes prior to administering recombinant NaGlu. For example, the antihistamine can be administered 20, 25, 30, 35, or 40 minutes prior to the administration of recombinant NaGlu.

In one embodiment, the antihistamine administered is diphenhydramine. Any useful antihistamine can be used. Such antihistamines include, without limitation, clemastine, doxylamine, dine, atidine, fexofenadine, pheniramine, cetirizine, ebastine, promethazine, chlorpheniramine, levocetirizine, olopatadine, quetiapine, meclizine, dimenhydrinate, embramine, dimethidene, and dexchloropheniramine.

In another embodiment, with reference to intravenous infusion, the potential for anaphylactic reactions can be reduced by administering the infusions using a ramp-up protocol. In this context, a ramp-up protocol refers to slowly increasing the rate of the on over the course of the infusion in order to desensitize the patient to the infusion of the tion.

K. Administration The methods of the present invention contemplate single as well as multiple administrations of a therapeutically effective amount of the rhNaGlu of the invention described herein. The rhNaGlu of the invention can be administered at regular intervals, depending on the , severity and extent of the subject's condition. In some embodiments, a therapeutically ive amount of the rhNaGlu protein of the t invention may be administered intravenously or intrathecally periodically at regular intervals (e. g., once every year, once every six , once every five months, once every three months, bimonthly (once every two months), monthly (once every month), biweekly (once every two weeks) or weekly).

In some embodiments, intrathecal administration may be used in conjunction with other routes of administration (e.g., enous, subcutaneously, intramuscularly, parenterally, trans dermally, or transmucosally (e.g., orally or nasally)). In some embodiments, those other routes of administration (e.g., intravenous administration) may be performed no more frequent than biweekly, monthly, once every two months, once every three months, once every four months, once every five months, once every six months, annually stration.

As used herein, the term "therapeutically effective amount" is largely determined based on the total amount of the therapeutic agent contained in the pharmaceutical compositions of the present invention. lly, a therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject (e.g., treating, ting, , preventing and/or rating the underlying disease or condition). For example, a therapeutically effective amount may be an amount sufficient to achieve a desired therapeutic and/or prophylactic effect, such as an amount sufficient to modulate lysosomal enzyme receptors or their activity to thereby treat such lysosomal storage disease or the symptoms thereof (e.g., a reduction in or elimination of the ce or incidence of “zebra bodies” or cellular vacuolization following the administration of the compositions of the present invention to a subject). Generally, the amount of a therapeutic agent (e.g., the rhNaGlu of the invention) administered to a subject in need thereof will depend upon the characteristics of the subject. Such characteristics include the condition, disease severity, general health, age, sex and body weight of the subject.

One of ordinary skill in the art will be readily able to determine appropriate dosages depending on these and other d factors. In addition, both objective and subjective assays may optionally be employed to identify optimal dosage ranges.

A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular therapeutic n, a therapeutically ive amount (and/or an riate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, on combination with other pharmaceutical . Also, the specific eutically effective amount (and/or unit dose) for any particular patient may depend upon a y of factors including the disorder being treated and the severity of the disorder; the —67- activity of the specific pharmaceutical agent employed; the specific composition ed; the age, body weight, l health, sex and diet of the patient; the time of administration, route of administration, and/0r rate of excretion or metabolism of the specific fusion protein employed; the duration of the treatment; and like factors as is well known in the medical arts.

In some embodiments, the therapeutically effective dose ranges from about 0.005 mg/kg body weight to 500 mg/kg body weight, e.g., from about 0.005 mg/kg body weight to 400 mg/kg body weight, from about 0.005 mg/kg body weight to 300 mg/kg body , from about 0.005 mg/kg body weight to 200 mg/kg body weight, from about 0.005 mg/kg body weight to 100 mg/kg body weight, from about 0.005 mg/kg body weight to 90 mg/kg body weight, from about 0.005 mg/kg body weight to 80 mg/kg body weight, from about 0.005 mg/kg body weight to 70 mg/kg body weight, from about 0.005 mg/kg body weight to 60 mg/kg body weight, from about 0.005 mg/kg body weight to 50 mg/kg body weight, from about 0.005 mg/kg body weight to 40 mg/kg body weight, from about 0.005 mg/kg body weight to 30 mg/kg body weight, from about 0.005 mg/kg body weight to 25 mg/kg body weight, from about 0.005 mg/kg body weight to 20 mg/kg body weight, from about 0.005 mg/kg body weight to 15 mg/kg body weight, from about 0.005 mg/kg body weight to 10 mg/kg bra body in weight. Ranges and values intermediate to the above recited ranges and values (e.g., 10- 50 mg/kg, 1-5 mg/kg, 2-8 mg/kg, 5—10 mg/kg, 0.1-10 mg/kg, 0.3-30 mg/kg, 0.3-50 mg/kg, 0.5-10 mg/kg, 5-30 mgikg, or 6-27 mg/kg) are also contemplated to be part of the invention.

In some embodiments, the therapeutically effective dose is greater than or at least about 0.1 mg/kg body weight, greater than or at least about 0.2 mg/kg body , greater than or at least about 0.3 mg/kg body weight, greater than or at least about 0.4 mg/kg body weight, greater than or at least about 0.5 mg/kg body weight, greater than or at least about 1.0 mg/kg body , greater than or at least about 3 mg/kg body , greater than or at least about 5 mg/kg body weight, greater than or at least about 6 mg/kg body , greater than or at least about 7 mg/kg body weight greater than or at least about 10 mg/kg body weight, greater than or at least about 15 mg/kg body weight, greater than or at least about 20 mg/kg body weight, greater than or at least about 30 mg/kg body weight, greater than or at least about 40 mg/kg body weight, greater than or at least about 50 mg/kg body weight, greater than or at least about 60 mg/kg body weight, greater than or at least about 70 mg/kg body , greater than about or at least 80 mg/kg body weight, greater than or at least about 90 mg/kg body weight, greater than or at least about 100 mg/kg body weight. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

In some embodiments, the eutically ive dose may also be defined by mg/kg brain weight. As one skilled in the art would appreciate, the brain s and body s can be ated (see, e. g., Dekaban AS. "Changes in brain weights during the span of human life: relation of brain weights to body heights and body weights," Ann Neurol 1978; 42345—56).

In some embodiments, the therapeutically effective dose may also be defined by mg/15 cc of CSF. As one skilled in the art would appreciate, therapeutically effective doses based on brain weights and body weights can be converted to mg/15 cc of CSF.

For example, the volume of CSF in adult humans is approximately 150 mL (Johanson CE, et a1. "Multiplicity of cerebrospinal ﬂuid functions: New challenges in health and e," Cerebrospinal Fluid Res. 2008 May 14;5: 10). Therefore, single dose injections of 0.1 mg to 50 mg protein to adults would be approximately 0.01 mg/15 cc of CSF (0.1 mg) to 5.0 mg/15 cc of CSF (50 mg) doses in adults.

It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the enzyme replacement therapy and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed invention.

VIII. m The present invention further provides kits or other articles of manufacture which contain the recombinant human NaGlu of the t invention and provide instructions for its reconstitution (if lized) and/or use. Kits or other articles of manufacture may include a container, a catheter and any other articles, s or equipment useful in intrathecal administration and associated surgery. Suitable containers include, for example, bottles, vials, syringes (e.g., pre-filled syringes), s, cartridges, reservoirs, or lyo-j ects. The container may be formed from a variety of materials such as glass or c. In some embodiments, a container is a pre—filled syringe. Suitable pre- filled es include, but are not limited to, licate glass syringes with baked silicone coating, borosilicate glass syringes with sprayed ne, or plastic resin es without silicone.

Typically, a label on, or associated with, the container may te directions for use and/or reconstitution. For example, the label may indicate that the formulation is reconstituted to protein concentrations as described above. The label may further indicate that the formulation is useful or intended for, for example, intravenous or intrathecal administration. In some embodiments, a container may contain a single dose of a stable formulation containing a replacement enzyme (e.g., a recombinant NaGlu protein). In various embodiments, a single dose of the stable formulation is present in a volume of less than about 15 mL, 10 mL, 5.0 mL, 4.0 mL, 3.5 mL, 3.0 mL, 2.5 mL, 2.0 mL, 1.5 mL, 1.0 mL, or 0.5 mL. Alternatively, a container holding the ation may be a multi-use vial, which allows for repeat administrations (e.g., from 2-6 administrations) of the formulation. Kits or other articles of manufacture may further include a second container comprising a suitable diluent (e.g., BWPI, saline, buffered saline). Upon mixing of the diluent and the formulation, the final protein concentration in the reconstituted formulation will generally be at least 1 mg/mL (e. g., at least 5 mg/mL, at least 10 mg/mL, at least 25 mg/mL, at least 50 mg/ mL, at least 75 mg/mL, at least 100 mg/mL).

Kits or other articles of cture may further include other materials desirable from a commercial and user oint, including other buffers, diluents, filters, s, catheters, es, and package inserts with instructions for use.

EXAMPLES The following specific es are intended to illustrate the invention and should not be ued as limiting the scope of the claims. The contents of all figures and all references, patents and published patent applications cited throughout this application, as well as the Figures, are expressly incorporated herein by reference in their entirety.

Example 1 Purification of rhNaGlu u protein was purified by using methods known in the art. Egg white (EW) ning rhNaGlu was lized at pH 6 overnight and clarified through centrifugation and/or depth filtration. The EW was ed with l M NaOAc buffer (pH 4) to pH 6. For the depth tion process, T2600 filter (PallTM, 40 um) was used as a lSt filtration and then PDFl (PallTM, K200P,15 um + EKS, 0.22 um) as a 2m1 filtration step. The filters are single-use membrane with an optimized capacity 60 L EW/m2 for each filter. The hold volume of membrane is 2 L/m2 for T2600 and 4-5 L/m2 for PDFl. In the process, the hold volume was discarded before the filtered EW collected. The buffer (20 mM Phosphate/137 mM NaCl, pH 6) equivalent to the membrane hold volume was used to chase EW left on the filters.

A phenyl-HIC (hydrophobic interaction chromatography) column was applied as a e step. Since most of egg white proteins are hydrophilic, 99% of egg white proteins passed through the HIC column into flow through. rhNaGlu has a higher hydrophobicity binding to phenyl—HIC.

Egg white containing u was loaded onto the column with a ratio of 30:1.

After completion of loading, the column was washed with the equilibration buffer, 5 mM phosphate buffer, pH 6, and 5 mM Tris buffer, pH 7.2. rhNaGlu was eluted with % propylene glycol, pH 7.2. After the completion of loading, the column was washed with equilibration buffer and 5 mM phosphate buffer (pH 6). rhNaGlu was eluted with % propylene glycol with 5 mM Tris buffer (pH 7.2). The column binding capacity is approximately 4.5 mg/mL. The purity of rhNaGlu h the phenyl-HIC column can be reached to >95% (950 time increase). The recovery is approximately 80% with 30% of ene glycol elution.

The eluted u fraction was adjusted to pH 5 with l M acetic acid and then loaded onto a GigaCap S column (EW: column size=10:l). The column was equilibrated with 50 mM NaOAc buffer (pH 5). After completion of loading, the column was washed with the equilibration buffer. The rhNaGlu was eluted with 50 mM NaOAc/60 mM NaCl (pH 5).

The protein characterization was med using purified rhNaGlu. The molecular weight of rhNaGlu (~90 kDa) purified from egg white was analyzed on SDS- PAGE (Fig. 6). The average expression level of rhNaGlu in egg white is shown in Fig. 7. The characteristics of rhNaGlu produced from the transgenic avian are summarized in Table 2.

Table 2. —rhNaGlu (Galas) Apparent Molecular Weight ~90 kDa 6.1—6.9 l-H Stability pH 5-8 Stability in Egg White > 50 days Example 2 Stability ofrhNaGlu in egg white A single egg was cracked 7‘ days post-lay and analyzed for activity. Contents were divided in half and each half was subject to rd egg white clarification. Both untreated and clarified egg whites were ted and stored at 4°C and -20°C for enzyme activity stability. rhNaGlu in egg white showed stable enzyme activity at least up to 50 days.

Freeze/thaw cycle stability was assessed. The purified rhNaGlu was frozen in liquid nitrogen for 10 seconds and thawed at 37 0C for 2 min. The enzyme activity showed no change for 10 cycles.

The purified rhNaGlu was dialyzed into different pH buffers to measure the stability of pure enzyme. The results showed that pure rhNaGlu was stable between pH -8 for 12 days.

Example 3 Oligosaccharide Proﬁling ephosphate (M6P) is a terminal monosaccharide of N—linked oligosaccharides that is an important part of the tertiary structure of glycoprotein and, when incorporated in the glycoprotein’s final oligosaccharide, is recognized by and bound to the M6P receptors present on the cell surface, subsequently allowing internalization into the lysosomes. Thus, M6P is an ive epitope for the targeting of glycoproteins to the lysosomes.

Analysis of protein glycosylation is an important part of rotein characterization. Oligosaccharides can be linked to a protein through a serine or a threonine as O-lined glycans or through an asparagine as N-linked glycans.

To analyze the structure of oligosaccharides, various chromatographic and spectroscopic techniques were performed. erformance anion exchange chromatography with pulsed amperometric detection (HPAEC—PAD) was employed.

Using this technique, oligosaccharides were y separated into general groups based on charge (i.e., neutral, singly charged, or multiply charged) and their structures were determined by comparison to pure standards.

All methods were based on protocols bed by Hardy and nd (Hardy, M. R., and Townsend, R. R., “High—pH exchange chromatography of glycoprotein-derived carbohydrates”, 1994, Methods Enzymol. 230: 208-225). Purified samples of transgenic avian derived rhNaGlu were ed using a Tube-O-Dialyzer against nanopure water at 4°C for about 24 hours to remove salts and other contaminants. Nanopure water was replaced four times during the entire dialysis period.

After dialysis, each of the s was divided into three aliquots. The t intended for neutral and amino sugars analysis was hydrolyzed with 2 N triﬂuoroacetic acid (TFA) at 100°C for 4 hours and the aliquot for ephosphate analysis was yzed with 6.75 N TFA at 100°C for 1.5 hours. The hydrolysates were then dried under N2, re-dissolved with 50 uL H20, sonicated for 7 min in ice and transferred to an injection vial.

A mix of rds for neutral and amino sugars, and for mannose—6—phosphate with a known number of moles was hydrolyzed in the same manner and at the same time as the sample. Four ent concentrations of the neutral and amino sugar standard mix and ephosphate were prepared to establish a calibration equation. The number of moles of each sugar in the sample was quantified by linear interpolation from the calibration equation.

The oligossacharide profile and mannose—6—phosphate profile were analyzed separately by HPAEC-PAD. Instrument l and data acquisition were accomplished using Dionex chromeleon software. HPAEC—PAD analysis of hydrolyzed rhNaGlu ed M6P. The mean measured amount of M6P was 3.8 ug (CV 3.7%) per 210 ug of hydrolyzed protein. Converting to moles resulted in 13.4 nmol of M6P per 2.8 nmol of protein which was equivalent to a ratio of 3.2 moles of M6P per mole of protein.

The oligosaccharide profile was also obtained for rhNaGlu (Gallus) using HPAEC—PAD (see Fig. 8). The profiles demonstrated good repeatability of the PNGase F reaction on the single sample. Peak clusters were observed in s corresponding to neutral oligosaccharides (~10 min to ~20 min). A group of significantly smaller peaks eluting between ~25 and ~35 min were also observed, which were possibly attributed to singly charged species.

The monosaccharide composition analysis results ed from samples of rhNaGlu produced from a transgenic avian (Callus) are summarized in Table 3, which tabulates the average molar ratio of each monosaccharide analyzed for rhNaGlu.

Table 3. Monosaccharide Molar Ratios in rhNaGlu (Gallus) N-acetylgalactosamine (GalNAc) ‘ 1.1* N-acetylglucosamine (GlcNAc) 356* Galactose (Gal) 4* Mannose (Man) 255* ephosphate (M6P) 32* Fucose Not detected Glucose Not detected mo 6 O monosacc an C 1361‘ mo 6 0 protein Example 4 Cellular Uptake into Fibroblasts Wild-type human fibroblasts and mucopolysaccharidosis III B (NaGlu ent) human fibroblasts were placed in a 24—well plate (2.5 x 104 cells per well) and incubated for overnight at 37°C in 5% C02. Conditioned media containing fibroblast basal medium and fibroblast growth kit having low serum were used. Various amounts of rhNaGlu (30, 10, 3.0, 1.0, 0.3 and 0 pg/mL) were co-incubated for 24 hours at 370C with 5% C02 to determine levels of cellular uptake by the human lasts (see, Fig. 9). The wells were washed three times with PBS. 100pL1ysis buffer was added per well and the plate was incubated for 10 min at 37°C. Cell lysate was transferred into 1.5 mL centrifuge tube. One cycle of freezing and thawing was med. The cell lysate was centrifuged at 10,000 rpm for 10 min. 25pL of supernatants were used for the assay. The assay time was 2 hours. The enzyme activity was measured using the methods known in the art and according to the methods described in Marsh et al., Clinical Genetics (1985) 27: 258—262, Chow et al., Carbohydrate Research (1981) 96:87-93; Weber et al., Protein Expression and Purification, (2001)21:251-259).

As shown in Fig. 9, negative control (i.e., MPS IIIB) did not exhibit any NaGlu activity while positive l (i.e., wild—type human fibroblast) showed NaGlu activity.

MPS IIIB cells treated with 0.3 ug/mL of rhNaGlu exhibited approximately 50% of the normal ty level observed in wild-type last cells. MPS IIIB cells treated with 1 ug/mL of rhNaGlu demonstrated NaGlu ty that was approximately 4-fold higher than that ed in wild-type cells. Surprisingly, MPS IIIB cells treated with 30 pg/mL of rhNaGlu showed NaGlu activity that was at least 40-fold higher than that observed in wild-type cells. This result indicated that u produced from a enic avian (Gallus) was efficiently internalized into human fibroblasts at a high level.

To determine whether internalization of rhNaGlu is via M6P receptor mediate endocytosis, M6P inhibition assays were med. For the M6P inhibition assays, various concentrations of free M6P were added to human MPS IIIB fibroblasts treated with 30ug/mL of rhNaGlu and enzymatic activity was measured as described above As shown in Fig. 10, human MPS IIIB fibroblasts did not exhibit any NaGlu activity, suggesting effective inhibition of NaGlu uptake by free M6P. In contrast, MPSIII fibroblasts treated with 30 ugImL of rhNaGlu in the absence of free M6P exhibited a high level of tic ty, suggesting that the protein was efficiently internalized into the NaGlu deficient fibroblasts and retained activity. This tic activity was inhibited by the presence of M6P monosaccharide in the medium at the concentration 0.03 mM and higher. The presence of lmM of M6P monosaccharide in conditioned medium inhibited more than 90% of cellular uptake of the n.

These results indicated that the rhNaGlu produced from a transgenic avian was efficiently internalized into the MPS IIIB lasts via M6P receptor-mediated endocytosis and the rhNaGlu competed with M6P ccharides for the receptor recognition. The results were consistent with the glycan analysis that revealed the presence of the M6P structures on the rhNaGlu produced from the transgenic avian.

Example 5 Generation ofActive NaGlu Fusion Proteins Two different rhNaGlu fusion constructs were ed to validate the feasibility of expressing rhNaGlu fusion proteins in the avian expression .

In one construct, a c acid sequence encoding 8 consecutive aspartic acid residues (DDDDDDDD) was fused to the nucleic sequence encoding NaGlu protein at the 5’ end of the full-length NaGlu cDNA sequence (SEQ ID NO:2) using conventional PCR and DNA recombinant technology. In another construct, a nucleic acid ce encoding TfRL (i.e., THRPPMWSPVWP; SEQ ID NO:5) was fused to the nucleic sequence encoding NaGlu at the 3’ end of the full—length NaGlu cDNA sequence. The —76- each construct was inserted into the pTT22 expression vector using EcoRI and I restriction sites. The resulting vectors were each transfected into human embryonic kidney (HEK) 293 cells and stable clones expressing high levels of the fusion NaGlu proteins were obtained. An rhNaGlu protein fused to a stretch of 8 consecutive aspartic acid residues at N—terminus (AAA—NaGlu) and an rhNaGlu protein fused to transferrin receptor ligand (TfRL) at C—terminus (NaGlu—TfRL) were isolated from conditioned media.

The enzymatic activity of AAA-NaGlu and NaGlu-TfRL was measured using the methods known in the art (see, e.g., Marsh et al., Clinical Genetics (1985) 27:258-262; Chow et al., Carbohydrate Research, (1981) 96:87—93; Weber et al., Protein Expression and Puriﬁcation (2001) 21 1251—259; Neufeld et al., Protein Expression and cation (2000) 19:202-211; and Weber et al., Human Molecular Genetics (1996) 5:771-777.

As shown in Figs. 13 and 14, AAA—NaGlu and NaGlu-TfRL fusion proteins produced from HEK293 cells showed high levels of tic ty. These results med the possibility that these constructs can be used to produce NaGlu fusion proteins that have increased levels of phosphorylated mannose while retaining enzymatic activity from a transgenic avian expression system.

Example 6 Cellular Uptake into hages alization of u produced from Gallus into human hage cells was also measured. NR8383 macrophage cells were incubated with lOug/mL of rhNaGlu in F12 growth media for 0, 4, 8, 24, 32 and 48 hours at 37 0C with 5% C02.

Samples were red and washed with PBS prior to lysis. 2.5 x 105 cells were lysed in 1 mL of lysis buffer (10mM of Na Phosphate pH6.0, 0.05% NP40), and lysates transferred into 1.5 mL centrifuge tubes and centrifuged at 10,000 rpm for 10 min.

Protein concentration was determined by the Bradford assay and aliquots were frozen for NaGlu enzyme assays.

Enzyme activity was measured using standard methods. 25mM of substrate (4- methylumbelliferyl 2-Acetamido—2—deoxy-a-D-glucopyranoside) was diluted to 2mM in nanopure water to form a working substrate stock. ons of samples were prepared in assay buffer (1% bovine serum albumin). 25pL of 200mM sodium acetate was distributed to wells of a multi—well plate. 25pL of standard and 25uL of samples were added to designated wells. SOuL of the working substrate stock was added to each well and the plate was gently tapped to mix. The plate was sealed with ve film and incubated at 37°C for 30 minutes. The reaction was then terminated by addition of 50uL of stop solution (1M Glycine pH 12.5). The plate was placed on a microplate reader using a ﬂuorescence bottom and the intensity was measured at an excitation 360nm and an emission 460nm. The level of liberated 4-methylumbelliferone (4-MU) was measured by comparison with standards of 4-MU at 0.25mM, 0.125mM, 0.0625mM, 0.0312mM, 0.0156mM, and 0.0078mM.

As depicted in Fig. 15, levels of the NaGlu activity in macrophages incubated with 10ug/mL of rhNaGlu increased almost linearly over a 48 hour period: The u uptake by macrophages was rather slow, but steady throughout the entire time period measured. The relatively slow, extended uptake of NaGlu activity (as compared to other lysosomal enzymes containing M6P and/or mannose in their ylation structures) was unexpected and surprising. Equally surprising and unexpected was that a large amount of rhNaGlu proteins was taken up into the hages over the extended time period, resulting in intracellular tic ty levels at least 10, 50, 100, 200, 300, 500, or even 1,000—fold higher than the basal levels observed in wild-type macrophages not exposed to rhNaGlu. The results demonstrate that rhNaGlu is ely stable in extracellular as well as intracellular environments. Further, these results suggest that rhNaGlu may s physicochemical characteristics that allow for longer serum half- life (e.g., longer circulation) and high serum concentrations in vivo, properties which are ideal for ed uptake into the central nervous system (CNS). —78- Table 4. Summary of NaGlu Characteristics Avian (gallus) l produced human CHO produced rhNaGlu NaGlu human NaGlu Apparent lar Mass (kDa) ~85 — ~90 ~79 - ~89 Enzymatic Activity min/mg) >l,000 ~1,057 None or very Mannose3hos3hate High High Low Example 7 Administration of rhNaGlu into NaGlu deﬁcient mice Homozygous null mice were generated from breeding pairs of the strain B6. 129$6-NaGlutm1Efn/J. Control wild—type mice were generated in the same manner.

Genotyping was performed according to a standard PCR protocol. It is bed in the art that at birth, homozygous naglu(‘/') null mice are viable, normal in size, and do not display any gross physical or behavioral abnormalities, though they exhibited no NaGlu in all s (see, Li et al., (1999) Proc., Natl. Acad. Sci. USA 96:14505-14510). At one month of age, vacuolated macrophages are found in most tissues. Epithelial cells in kidney and neurons in some parts of the brain are also affected. The vacuolation becomes more prominent with age. At 4-5 months, the mice show al behavior in an open field test. Older animals may have urinary retention and difficulty walking.

Typical life span of the homozygous null nagla 4‘ mice is 8-12 months (see, Li et al., (1999) Proc., Natl. Acad. Sci. USA 96:14505— 14510).

Intravenous (IV) stration The intravenous administration of test article and vehicle by tail vein injection was accomplished as follows. Before injection vasodilation was achieved by gently warming the animal with an incandescent lamp or by soaking the tail in warm water, approximately 43°C. The animal was then placed in restraint device. The e of the tail was disinfected with 70% isopropanol prior to injection. The lateral veins of the tail are located just under the skin and are fied in the distal part of the tail with the application of tension. A 27G needle, bevel up, was inserted into the vein for 3 — 4 mm.

The test article or vehicle was then administered as a slow bolus injection over a period of ten seconds as evidenced by the ed clearing of the vein as the stered liquid momentarily occupies the vascular space. After removal of needle, gentle pressure was applied to the puncture site to provide hemostasis. The animal was monitored immediately ing procedure to assure normal activity.

Intrathecal (IT) administration The hecal administration of test e and vehicle by lumbar puncture injection was accomplished as follows. Before injection, animals were anesthetized using isoﬂurane that was maintained via nose cone throughout the procedure. The site of injection was prepared by shaving the fur, as necessary, prior to each injection. The animal was placed in a prone on on a rm, ensuring the hind limbs were straddling the platform forming a convex curve of the animals back. The surface of the back was swabbed with 70% isopropanol and allowed to dry prior to injection. Spinal column and hip bones were palpated to locate the L4-L5 or L5-L6 margin. A 30G needle, bevel facing cranially, was inserted into the intervertebral space. Placement was confirmed by the observation of a tail ﬂick. The test article or vehicle was then administered as bolus injection. The animal was d to recover from anesthesia and monitored ately following procedure to assure normal activity and use of limbs.

Results Twelve-week old naglu mice (B6.129S6-NaglumIEf”/J) were administered rhNaGlu (Gallus) at dose levels of 6.75 or 27 mg/kg via tail vein injection (IV administration), once every other day, for a total of 5 doses (at rhNaGlu concentrations of 1.125, or 4.5 mg/mL, respectively). Similarly, twelve—week old naglu mice were administered with rhNaGlu (Gaffus) at a dose level of 0.31 mg/kg via lumbar re injection (IT administration), once every other day, for a total of 5 doses at NaGlu concentrations of 1.54 mg/mL. Vehicle (10 mM phosphate buffer, 150 mM NaCl and 0.02% 0, pH 55-5.8) was administered to naglu '/' knock-out mice at the same dose concentration for 5 doses every other day. Untreated wild-type and naglu knock- out mice were also maintained for the duration of the study.

Animals were sacrificed 4 hours after the fifth and final injection. All animals were necropsied and the liver, brain, spleen, heart, lung and kidneys were excised. Each organ was divided sagittally, providing samples for both frozen (-80°C) and formalin- fixed storage.

Tissue samples were analyzed for: (1) heparan sulfate concentration using an analytical method based on SAX—HPLC analysis of heparan sulfate disaccharides; and (2) a-N—acetylglucosaminidase enzyme activity using a cell-based enzyme activity assay.

Histopathologic tion of brain, liver, kidney, spleen, heart and lung tissue was conducted using formalin—fixed tissue samples, embedded in paraffin, sectioned at 4 pm, mounted on glass slides and stained with xylin and eosin (H&E).

Following the repeated intravenous administration (5 doses over a 10 day period) of rhNaglu (Gallus) to naglu'l' mice at dose levels of 6.25 and 27 mg/kg body weight, there was an nt dose-dependent decrease in the concentration of Heparan Sulfate in the brain, liver and kidney of l' mice (Table 5; Figs. . The relative 0t-N- glucosaminidase activity was increased in the brain and liver following intravenous administration (Table 6). These results were unexpected and surprising because the NaGlu enzymatic activities and resulting substrate clearance were observed in the brain of the d naglu'/' mice with the IV administration, suggesting that rhNaGlu (Gallus) administered systemically was distributed to the brain of the naglu'/' mice and effective to elicit cy even in the present of the blood brain barrier (BBB).

Following the intrathecal administration (5 doses over a 10 day period) of rhNaGlu (Gallus) to naglu‘l‘ mice at a dose level of 0.31 mg/kg, there was a decrease in the concentration of Heparan Sulfate in the brain of naglu'/' mice (Table 5; Fig. 19), suggesting that rhNaGlu (Gallus) was targeted to the brain and effective in reducing the accumulated ate in the brain of naglu‘/‘ mice.

Table 5: Tissue Substrate Level (rhNaGlu Callus) Heparan Age at Animal sacrifice Tissue Number Genot ' ue (wks) ent - - KIDNEY 253 WT 4 na ‘ i 155 ‘ WT ‘ 12 na — i — i 0.045 ‘ 0.0725 ‘ 0.038891‘ ‘ ‘178‘K0 ‘ 12 na — ‘—‘1.882‘ ‘ ‘ 242 KO 4 na - — 1 .687 145 KO 13 na - — 1.904 474 KO 13 vehicle 0 IV 1.501 479 KO 13 vehicle 0 IV 1.983 484 K0 13 vehicle 0 IV 1.839 1.799333 0.175908 487 KO 13 625 IV 0.928 492 KO 13 625 IV 0737 0.8325 0135057 481 KO 13 27 IV 0.591 485 KO 1 3 27 IV 0.3 1 1 490 KO 13 27 IV 0585 0.495667 0.159954 86 KO 15 _ 0 IT 2105 91 KO 14 0 IT 1704 1.9045 0.28355 94 KO 14 0.31 IT 1324 101 KO 14 0.31 IT 2.233 1.7785 0.64276 LIVER 253 WT 4 n — — 0.045 155 WT 12 na — — 0.092 0.0685 0.033234 243 WT 4 na - - 0.045 178 KO 12 na — — 1 .8 5 242 KO 4 na — — 2.263 2.0565 0.292035 255 KO 4 na — — 1 .85 474 KO 13 0 IV 1.822 479 KO 13 0 IV 1.981 484 KO 13 0 IV 2.004 1.961667 0.165779 487 KO 13 6.25 IV 0.748 492 KO 13 6.25 IV 0.444 504 KO 13 6.25 IV 0.494 0.562 0.163009 481 KO 13 27 IV 0.491 485 KO 13 27 IV 0.172 0.3315 0.225567 BRAIN 253 WT 4 na - - 0.021 155 WT 12 — — 0.013 243 WT 4 — — 0.014308 WT 36 - - 0.012649 39 0003906 239 KO 4 n - - 0,095 178 KO 12 n - - 0.084 242 KO 4 n - - 0.099 255 KO 4 n - - 0.094538 165 KO 24 na — — 0084015 474 KO 13 e 0 IV 0085447 479 KO 13 vehicle 0 IV 0.072 484 K0 13 0 IV 0.073 0.085875 0009972 487 K0 13 6.25 IV 0.045 492 K0 13 6.25 IV 0044119 504 K0 13 6.25 IV 0.044 0.044373 0.000546 481 K0 13 rhNaGlu 27 IV 0.017796 485 KO 13 27 IV 0.016668 490 KO 13 rhNaGlu 27 IV 0.028 21 0.006242 86 KO 15 0 IT 0.094521 KO 14 0 IT 0.072623 0.083572 0.015484 94 KO 14 031 IT 0.038866 101 KO 14 031 IT 0.028229 0.033548 0.007521 na: Not applicable (mice were untreated).

Table 6: Tissue enzymatic activity (rhNaGlu Callus; U/ng protein) Enzymatic Age at ty Animal sacrifice (U/ug Tissue Number Genotype (wks) ent (mg/kg) protein) BRAIN 253 7.7 178 0 474 0 479 0 484 0.575 487 10.58 492 66667 504 4.033333333 481 87.91666667 485 90.15 490 17.35 LIVER 253 36.69 178 0 474 0 479 0 484 0 487_-512.92 492 K_—_—: 378.805 504 rhNaGlu 6.25 607.9225 481 KO 13 rhNaGlu 27 IV 25 485 KO 1 13 rhNaGlu 27 1 IV 654.2475 490 KO 1 13 rhNaGlu 27 1 IV 25 na: not applicable (mice were untreated).

Each example in the above specification is provided by way of explanation of the invention, not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications, combinations, additions, deletions and variations can be made in the t invention without departing from the scope or spirit of the invention. For instance, es illustrated or described as part of one embodiment can be used in another ment to yield a still further embodiment. It is intended that the present invention cover such modifications, combinations, additions, deletions, and variations.

All publications, patents, patent applications, intemet sites, and accession numbers/database sequences ding both cleotide and polypeptide sequences) cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, intemet site, or accession number/database sequence were specifically and individually indicated to be so incorporated by reference.

I/WE

Claims

CLAIM :

1. A composition comprising an isolated mixture of recombinant human yl-alpha-D- glucosaminidase (rhNaGlu) whose amino acid sequence is set forth in 24-743 of SEQ ID NO:1, wherein said ed mixture comprises a sufficient amount of rhNaGlu containing one or more 5 glycan structures comprising mannosephosphate (M6P) such that said u containing M6P is internalized into a mammalian cell having NaGlu deficiency via M6P receptor-mediated endocytosis and restores at least 50% of NaGlu activity observed in a wild-type cell of the same type sing endogenous NaGlu, wherein when the composition is administered intravenously to a subject having NaGlu deficiency, NaGlu activity is increased in the brain of 10 the subject.

2. The ition of claim 1, wherein said rhNaGlu comprises at least 1 moles of M6P per mole of rhNaGlu.

3. The composition of claim 1, wherein said rhNaGlu comprises mannose.

4. The composition of claim 1, wherein said rhNaGlu comprises N-acetylglucosamine 15 (GlcNAc).

5. The composition of claim 1, wherein said rhNaGlu comprises galactose.

6. The composition of claim 1, wherein said rhNaGlu comprises N-acetylgalactosamine (GalNAc).

7. The composition of claim 1, wherein said mammalian cell ent in NaGlu is a human 20 cell.

8. The composition of claims 7, wherein said human cell is a skin fibroblast, a neuronal cell, a hepatocyte or a macrophage.

9. The ition of claim 1, wherein said rhNaGlu is ed from an oviduct cell of a transgenic avian. 11307249:gcc

10. A composition of Claim 1, wherein, when internalized in vivo, said u restores at least 50% of NaGlu activity observed in a wild-type cell of the same type expressing endogenous NaGlu.

11. The composition according to claim 1 or 10, n said rhNaGlu protein is N-linked 5 glycosylated.

12. The composition according to claim 1 or 10, wherein said rhNaGlu protein is O-linked glycosylated.

13. The composition according to claim 1 or 10, wherein said u comprises about 1, 2, 3, 4, 5 or 6 moles of M6P per mole of u. 10

14. Use of a composition according to any one of claims 1 to 13 in the manufacture of a medicament for the treatment of NaGlu deficiency in a t.

15. The use according to claim 14 wherein said medicament is suitable for stration systemically such that said rhNaGlu is effectively delivered to the brain of a mammal having NaGlu deficiency. 15

16. The use according to claim 14 wherein said medicament is suitable for administration intravenously such that said rhNaGlu is effectively delivered to the brain of a mammal having NaGlu deficiency.

17. A pharmaceutical formulation comprising a composition according to claim 1 or 10 in combination with a pharmaceutically acceptable carrier, diluent or ent. 20

18. The use ing to claim 14, wherein said NaGlu deficiency is Sanfilippo B.

19. The use according to claim 14, wherein the subject is a human subject.

20. The use according to claim 14, wherein said medicament is suitable for administration intravenously to a human subject at a dosage of about 5 to about 30 mg/kg body weight. 11307249:gcc

21. The use according to claim 14, wherein said medicament is suitable for administration intrathecally to a human subject.

22. The use according to claim 14, n, in said treatment, said medicament is for stration in a therapeutically effective amount to reduce heparan sulfate levels in the brain, the kidney, or the liver of the subject.

23. The use according to claim 14, wherein, in said treatment, said medicament is for administration in a eutically effective amount to increase NaGlu activity in the brain or liver of the subject.

24. The use according to claim 14, wherein said treatment r encompasses use of a second therapeutic agent.

25. The use according to claim 24, wherein the second therapeutic is an immunosuppressant. Synageva BioPharma Corp. By the Attorneys for the Applicant SPRUSON & FERGUSON Per: 11642805