WO2009131698A2

WO2009131698A2 - PHOSPHORYLATED RECOMBINANT N-ACETYL-alpha-D- GLUCOSAMINIDASE (NaGlu) AND USES THEREOF

Info

Publication number: WO2009131698A2
Application number: PCT/US2009/002529
Authority: WO
Inventors: Norman Matthew Ellinwood; Rafi Simon Awedikian
Original assignee: Iowa State University Research Foundation, Inc.
Priority date: 2008-04-23
Filing date: 2009-04-23
Publication date: 2009-10-29
Also published as: WO2009131698A3

Abstract

The present invention relates generally to phosphorylated recombinant NaGIu fusion proteins, the production of phosphorylated recombinant NaGIu fusion proteins and uses thereof.

Description

PHOSPHORYLATED RECOMBINANT N-ACETYL-alpha-D- GLUCOSAMINIDASE (NaGIu) AND USES THEREOF

Related Application

This application claims priority from U.S. Provisional Application Serial No. 61/047,362 filed April 23, 2008, which application is herein incorporated by reference. Background of the Invention

Lysosomal storage diseases form a group of more than 30 metabolic disorders in which the function of one or several lysosomal hydrolases is deficient. Although the prevalence of each disease is low, prevalence of lysosomal storage diseases as a whole may be equivalent to that of cystic fibrosis in the general population (1 :2500). With the exception of Gaucher type I disease, Pompe disease, Fabry disease, mild forms of MPS I, MPS II and MPS VI there is no etiological treatment available for lysosomal storage diseases so far. Bone marrow transplantation (BMT), which may be an option in some MPS I patients, is not believed to be effective in MPS III. Lysosomal enzyme deficiencies induce the accumulation of intermediate catabolites in lysosomes, which progressively alters cell function and survival. Although deficiencies affect every tissue, clinical expression varies depending on the missing enzyme. Neurological symptoms are often predominant. They include severe motor impairments and mental retardation. Histopathology reveals characteristic vacuolizations in both neurons, glia and perivascular cells, without known predominance in specific locations. Other frequent symptoms include hepatomegaly, skeletal abnormalities, corneal clouding and respiratory, cardiac or renal dysfunctions leading to premature death.

MPS HIB is an autosomal recessive lysosomal storage diseases classified among mucopolysaccharidosis. This disease is caused by a defect in the degradation pathway of glycosaminogl yeans (GAGs). In MPS IIIB, the degradation of heparan sulfates is interrupted by the deficiency of N-acetyl-α-D- glucosaminidase (NaGIu) (Figure 1). MPS IIIB is associated with mutations in the human gene located on Chromosome 17 (q21). The glycoprotein produced by the gene is 743 amino acids (~ 80 KDa) and is phosphorylated on the high mannose carbohydrate chains. Except for frequent hepatosplenomegaly, peripheral abnormalities are generally absent in MPS IIIB, but may include mild somatic manifestation such as minimal skeletal involvement and mild joint stiffness. Symptomatology appears in children between the age 2 and 6 as behavioral troubles (hyperactivity with aggressive behavior, sleep disorders, hearing loss, speech development delay), which progressively leads to a severe mental and motor degradation. Death usually occurs in the second or third decade of life.

Therapy for MPS IIIB will require achieving adequate levels of NaGIu in the central nervous system. To date, however, no effective treatment is available for MPS IIIB patients. Previous studies regarding the expression and purification of recombinant human NaGIu from secretions of cells, including CHO-Zecl, CHO-Kl, HeLa, 293, MPS IIIB skin fibroblasts or human embryonic kidney cells (Zhao and Neufeld. Protein Expr Purif. (2000) 19:202- 211; Weber et al. Protein Expr Purif. (2001) 21 :251-259; Yogalingam et al. Biochim. et Biophys. Acta. (2000); 415-425), yielded rNaGlu that lacked normal levels of mannose-6-phosphate moieties which was not taken up by cells via the mannose-6-phosphate receptor. Intravenous injection of rNaGlu into MPS IIIB mice (Yu et al. MoI Genet Metab. (2000) 71:573-580) yielded endocytosis by macrophage lineage cells via mannose receptors, but no enzyme was delivered to different tissues, including the central nervous system.

Summary of the Invention

One embodiment provides an isolated recombinant mannose-6- phosphorylated N-acetyl-α-D-glucosaminidase (NaGIu) fusion protein (wherein the isolated recombinant mannose-6-phosphorylated N-acetyl-α-D- glucosaminidase (NaGIu) fusion protein is phosphorylated to a greater degree than a recombinant NaGIu protein that is not a fusion protein or wherein the recombinant NaGIu fusion protein is taken up cells in manner similar to that of wild-type or endogenous NaGIu). Another embodiment provides an isolated recombinant mannose-6- phosphorylated N-acetyl-α-D-glucosaminidase (NaGIu) fusion protein, wherein the NaGIu protein comprises SEQ ID NO:1 or a protein sequence having at least 80% sequence identity with SEQ ID NO:1 (the DNA encoding a NaGIu protein is presented in SEQ ID NO: 2 (sequences 904-3135)). In one embodiment, the protein comprises a sequence that has at least about 50% or about 60% or about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, or about 79%, or at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, or at least about 90%, about 91%, about 92%, about 93%, or about 94%, or at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity, compared to SEQ ID NO:1 using one of alignment programs available in the art using standard parameters. In one embodiment, the differences in sequence are due to conservative amino acid changes, hi another embodiment, the protein sequence has at least 80% sequence identity with SEQ ID NO:1 and is bioactive (e.g., retains NaGIu activity).

Methods of alignment of sequences for comparison are available in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin, USA). Alignments using these programs can be performed using the default parameters.

In one embodiment, the NaGIu protein is expressed as a fusion protein comprising a NaGIu protein and at least one other peptide. In one embodiment the at least one other peptide comprises a peptide of about 6 to about 60 amino acids. In another embodiment, the at least one other peptide comprises 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 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200 or more amino acids. In another embodiment, the at least one other peptide comprises at least one acidic amino acid. In one embodiment, the at least one other peptide comprises an Apolipoprotein E (see, for example, SEQ ID NO:5 and SEQ ID NO:6 (3284-3316)) or Apolipoprotein B (see, for example, SEQ ID NO:3 and SEQ ID NO:4 (3257-3382)) ligand domain or a peptide that has at least about 50% or about 60% or about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, or about 79%, or at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, or at least about 90%, about 91%, about 92%, about 93%, or about 94%, or at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to ApoE or ApoB ligand domain.

In another embodiment, at least one other peptide is a signaling moiety. In one embodiment, the signaling moiety is EQKLISEEDL (SEQ ID NO:7; c- myc epitope tag; also presented in SEQ ID NO:3 (aa) and SEQ ID NO:4 (nt; 3218-3382)).

One embodiment provides a composition comprising an isolated recombinant mannose-6-phosphorylated N-acetyl-α-D-glucosaminidase (NaGIu) fusion protein and a pharmaceutically acceptable carrier and/or culture medium. One embodiment of the invention provides a method to produce a mannose-6-phosphorylated N-acetyl-α-D-glucosaminidase (NaGIu) fusion protein comprising operably linking a first DNA sequence coding for a NaGIu protein to a second sequence coding for a peptide and expressing the linked sequence in a cell so as to yield a recombinant mannose-6-phosphorylated NaGIu fusion protein.

In one embodiment, the peptide comprises 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 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200 or more amino acids. In one embodiment, the peptide comprises about 6 to about 60 amino acids, hi another embodiment, the peptide comprises at least one acidic amino acid, hi one embodiment, the second sequence (the peptide) codes for an Apolipoprotein E or Apolipoprotein B ligand domain or a peptide that has at least about 50% or about 60% or about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, or about 79%, or at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, or at least about 90%, about 91%, about 92%, about 93%, or about 94%, or at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to ApoE or ApoB ligand domain.

Another embodiment provides a method for increasing mannose-6- phosphorylation of a recombinant N-acetyl-α-D-glucosaminidase (NaGIu) protein comprising operably linking a first DNA sequence coding for a NaGIu protein to a second sequence coding for a peptide and expressing the linked sequence in a cell so as to yield a recombinant NaGIu fusion protein with increased phosphorylation as compared to a recombinant NaGIu protein (that is not linked to a another sequence coding for a peptide).

In one embodiment, the first sequence and said second sequence are linked at the 3' end of said first sequence, hi another embodiment, the first sequence and said second sequence are linked at the 5' end of said first sequence, hi another embodimet, the linked sequence further comprises a third sequence coding for a signaling (or trafficking) moiety. For example, in one embodiment, the signaling moiety is EQKLISEEDL (SEQ ID NO:7). One embodiment provides a method to treat mucopolysaccharidosis type

IIIB (MPS MB) comprising administering to a subject in need thereof an effective amount of a mannose-6-phosphorylated Naglu fusion protein produced by the methods disclosed herein, hi one embodiment, the treatment reduces lysosomal storge of glycosaminogl yeans (GAGs), including heparan sulfate, in a tissue, such as brain tissue, of said subject. The recombinant NaGIu fusion protein described herein is taken up by cells via the mannose-6-phosphate receptor.

Another embodiment provides a method to produce a mannose-6- phosphorylated N-acetyl-α-D-glucosaminidase (NaGIu) fusion protein comprising expressing a recombinant NaGIu fusion protein in cell culture, for example, in CHO-dhfr- cells, so as produce a mannose-6-phosphorylated N- acetyl-α-D-glucosaminidase. hi one embodiment, the cells are cultured in Ex- Cell® 325 PF CHO serum-free medium, hi another embodiment, the medium is supplemented by 4 mM L-glutamine, 10 mg/L each of guanosine, adenosine, uridine, thymidine, cytidine and hypoxanthine and 50 μg/mL gentamicin 50 ug/mL.

One embodiment provides an isolated recombinant mannose-6- phosphorylated N-acetyl-α-D-glucosaminidase (NaGIu) fusion protein according to claims 1-9 for use in medical therapy, including treating mucopolysaccharidosis type IIIB (MPS IIIB).

Another embodiment provide the use of a recombinant phosphorylated NaGIu protein to prepare a medicament for treating mucopolysaccharidosis type IIIB (MPS IIIB). In one embodiment, medicament includes a physiologically acceptable carrier.

Brief Description of the Figures

Figure 1 depicts the pathway leading to heparan sulfate accumulation in MPS IIIB disease. Figure 2 depicts several NaGIu fusion constructs and flow diagram of the in vitro characterization of the constructs.

Figures 3 A-B depict the phNaGlu vector and cDNA sequences (SEQ ID NOs: 1-2) used to generate recombinant human NaGIu.

Figures 4A-B depict the prhNaGlu-myc-ApoB vector and cDNA sequences used to generate recombinant fusion protein human NaGlu-myc- ApoB (SEQ ID NOs:3-4).

Figures 5A-B depict the prhNaGlu-myc-ApoE vector and cDNA sequences used to generate recombinant fusion protein human NaGlu-myc-ApoE (SEQ ID NOs:5-6). Figure 6 depicts the generation of CHO-NaGIu clones. CHO-dhfr(-) cells were stably transfected with various NaGIu fusion constructs. A. Western blot analysis with the cell culture media of the NaGIu CHO clones. B. NaGIu activity was measured using the 4-Methylumbelliferyl N-acetyl-glucosaminide (4MU-Naglu) substrate. NaGIu fusions secretion: NaGIu activity in the CHO clones cell culture media 6 days post-incubation.

Figure 7 depicts an uptake experiment using NaGIu containing media. MPS IIIB fibroblasts were incubated with recombinant NaGIu containing media (10 units). NaGIu fusions cellular uptake: NaGIu activity in the MPS IIIB cells incubated with media containing 10 units of NaGIu fusions. Cellular uptake with NaGIu- ApoE containing media was optimal and the NaGIu- ApoE was selected for purification and further characterization.

Figure 8 depicts the biochemical characterization of NaGIu- ApoE fusion. Recombinant NaGlu-ApoE purification from CHO-NaGIu- ApoE media and native NaGIu purified from normal human urine were compared. A. Western Blot of NaGlu-Urine and NaGIu- ApoE. B. Activity of NaGlu-Urine and NaGIu- ApoE. C. pH optimum of NaGlu-ApoE (pH 4.3) and NaGlu-Urine. D. Km and Vmax of NaGlu-urine and NaGIu- ApoE. Biochemical characterization determined that NaGIu- ApoE and the native NaGIu from urine have similar biochemical properties.

Figure 9 depicts the cellular uptake of NaGIu- ApoE. MPS HIB fibroblasts were incubated with 10 units of NaGIu- ApoE or native NaGIu for 5 hours. A. Depicts NaGIu activity in cells after uptake. B. Confocal microscopy of MPS IIIB fibroblasts incubated with NaGIu- ApoE or native NaGIu and stained with Anti-myc or Anti-Lamp 1 antibodies (third row is a merge of panels from columns one and two).

Figure 1OA provides a putative mechanism of NaGIu- ApoE internalization. It is believed that NaGIu- ApoE uses the same membrane receptor and endocytosis mechanism as the native NaGIu: clathrin mediated endocytosis mediated by the mannose-6-phosphate receptor. Figure 1OB provides a Table depicting results of the incubation of MPS IIIB fibroblasts with either the rhNaglu-ApoE or the native hNaglu in the presence of receptor or endocytosis inhibitors as described in Materials and Methods. Intracellular Naglu activity was measured and normalized to the activity obtained in rhNaglu-ApoE-treated cells in the absence of inhibitors (considered equal 100%).

Figure 11 depicts the phosphorylation of NaGlu-ApoE. Phosphoprotein SDS-PAGE staining demonstrates that NaGlu-ApoE is mannose-6- phosphorylated as is the case of native NaGIu from Urine.

Figure 12 depicts a graph and table showing the efficiency of cellular uptake of rhNaGlu-ApoE, rhNaGlu-ApoB and recombinant NaGIu using enzyme containing media and not purifed enzymes.

Figure 13 depicts the dose-dependent reduction of GAGs storage in MPS IIIB fibroblasts by rhNaglu-ApoE fusion. GAGs in human MPS IIIB fibroblasts were radiolabeled and incubated with 10, 50 or 160 U/ml of rhNaglu-ApoE for 72 h at 37⁰C. Radioactivity in cell extracts was then quantified and reported as cpm per mg of proteins.

Figure 14 depicts GAGs clearance after incubation with rhNaglu-ApoE. Detailed Description of the Invention The invention relates to the development of a treatment for mucopolysaccharidosis type MB (MPS IIIB) by developing an active form of the enzyme N-acetyl-α-D-glucosaminidase (NaGIu) with similar properties to the native NaGIu enzyme found in the human body. Herein is provided an engineered recombinant NaGIu enzyme that is mannose-6-phosphorylated and capable of uptake by somatic tissues and can be transported by active transcytosis from the circulation across the blood brain barrier (BBB) to the brain. This is accomplished by the production of a fusion protein, for example, a NaGIu fusion with the ligand domain of the low density lipoprotein receptor (LDLr), including Apolipoprotein E or B ligand domain (ApoE or B).

All previous work undertaken for the production of a recombinant N- acetyl-alpha-D-glucosaminidase (NaGIu) enzyme produced a protein with poor cellular uptake and failed to generate a mannose-6-phosphorylated protein (Zhao et al.; Weber et al.). This is a very important feature of the native NaGIu in that it allows for its correct functioning in the cells. The recombinant fusion protein described herein is the first mannose-6-phosphorylated NaGIu shown to have similar properties to the native enzyme. The discovery of the phosphorylated protein was unexpected, as well as the increased cellular uptake. Initially the enzyme was modified by fusion to a ligand domain that will target the enzyme to a type of receptor found on almost all cell types so as to produce a therapeutic enzyme that could be used to treat Sanfilippo Syndrome type B by enzyme replacement therapy. Surprisingly, it was found that recombinant fusion enzyme generated in this fashion is functioning as the native enzyme using similar mechanisms. Thus, the recombinant fusion enzyme is an agent for the treatment of Sanfilippo Syndrome type B by, for example, enzyme replacement therapy. All previous attempts by other investigators failed to generate a therapeutic enzyme for the treatment of this disease. Definitions As used herein, the terms below are defined by the following meanings:

A "subject" is a vertebrate, including a mammal, such as a human. Mammals include, but are not limited to humans, farm animals, sport animals and pets.

A "cell" is a prokaryotic or eukaryotic cell, such as a mammalian cell (e.g., a human cell).

The term "isolated" refers to a protein or proteins which are not associated with one or more proteins or one or more cellular components that are associated with the protein or proteins in vivo. As used herein, "treat," "treating" or "treatment" includes treating, preventing, ameliorating, or inhibiting a disease related condition and/or a symptom of a disease related condition.

An "effective amount" generally means an amount which provides the desired local or systemic effect, such as a decrease in GAG accumulation. For example, an effective dose is an amount sufficient to affect a beneficial or desired clinical result. Said dose could be administered in one or more administrations and could include any pre-selected amount of protein or DNA (e.g., vector). The precise determination of what would be considered an effective dose may be based on factors individual to each subject, including their size, age, disease being treated and amount of time since the disease began. One skilled in the art, specifically a physician, would be able to determine the amount of DNA and/or protein that would constitute an effective dose.

"Administration" can occur via any method available to an art worker, including enzyme replacement therapy by, for example, injection of purified NaGIu protein (e.g., a recombinant fusion protein) or gene therapy via, for example, injection of vectors, including viral vectors, carrying the NaGIu coding sequence. For example, NaGIu can be administered via direct injection into the central nervous system of either recombinant enzyme, gene therapy vectors, stem cells, or gene therapy treated stem cells. Another method is to genetically engineer the missing enzyme of a particular lysosomal storage disease, so that it is able to cross the blood brain barrier by an active process of uptake from the blood and deliver it across the blood brain barrier. This would allow for the treatment of the central nervous system either by intravenous delivery of the engineered recombinant enzyme, or alternatively, treatment with a liver directed gene therapy vector designed to deliver the gene for the engineered enzyme.

The skilled artisan can readily determine the amount of NaGIu protein/DNA and optional additives, vehicles, and/or carrier in compositions to be administered in methods of the invention. Typically, any additives (in addition to the active agent) are present in an amount of 0.001 to 50 wt % solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, including about 0.0001 to about 1 wt %, including about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, about 0.01 to about 10 wt %, or about 0.05 to about 5 wt %.

When administering a therapeutic composition of the present invention, it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion). The pharmaceutical formulations suitable for injection include sterile aqueous solutions and dispersions. The carrier can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.

Additionally, various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the agents utilized in practicing the present invention in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Recombinant NaGIu of the invention can be purified to a degree sufficient to produce a desired effect. Those skilled in the art can readily determine the percentage of recombinant NaGIu fusion protein in a sample using various methods available to an art worker. Ranges of purity in samples comprising recombinant NaGIu fusion protein are 50-55%, 55-60%, and 65-70%. Ranges of purity also include 70-75%, 75-80%, 80-85%; 85-90%, 90-95%, and 95-100%. However, samples with lower purity can also be useful, such as about <25%, 25-30%, 30-35%, 35-40%, 40-45% and 45-50%.

"Co-administer" can include simultaneous and/or sequential administration of two or more agents. The term "signal moiety" refers in a general sense to a detectable moiety, such as a radioactive isotope or group containing the same, and non-isotopic moieties, such as enzymes, biotin, avidin, streptavidin, digoxygenin, luminescent agents, dyes, haptens and the like. Luminescent agents, depending upon the source exciting the energy, can be classified as radioluminescent chemiluminescent, bioluminescent, or photoluminescent (fluorescent). Other signal moieties include those that are detectable via the use of an antibody (e.g., an anti-myc antibody). The signaling moiety aids in the tracking of the protein of interest, for example, as it is trafficking throughout the cell and during purification.

"Fusion protein," also known as a chimeric protein, is a protein created through the joining of two or more genes which originally coded separate proteins. Translation of this fusion gene results in a single polypeptide often with functional properties derived from each of the original proteins. "Recombinant" fusion proteins are created artificially by recombinant DNA technology created through genetic engineering of a fusion gene for use in biological research or therapeutics.

The phrase "operably linking" refers to linking at least two sequences present on the same nucleic acid molecule in a manner such that both sequences are expressed. For example, a polypeptide made from a recombinant gene that contains portions of two or more different genes (a product of recombinant DNA in which one gene product is juxtaposed ("fused") to either the carboxyl-terminal or amino-terminal portion of another polypeptide).

"Expression" of a sequence includes the production of mRNA and/or protein.

The terms "comprises", "comprising", and the like can have the meaning ascribed to them in U.S. Patent Law and can mean "includes," "including" and the like. As used herein, "including" or "includes" or the like means including, without limitation.

Phosphorylated Recombinant Fusions Proteins

Provided herein is a recombinant chimeric (fusion) form of NaGIu using C-terminal addition to NaGIu of the Apolipoprotein E (ApoE) or Apolipoprotein B (ApoB) ligand domains. In one embodiment, N-terminal addition of ApoE or ApoB is also provided. The resultant enzyme constructs were evaluated for their phosphorylation status, their ability to yield an active form of NaGIu, and their ability to be taken up by different types of receptors including the mannose 6- phosphate receptor and the LDL receptor. It was determined that addition of Apo E or Apo B ligand domains to NaGIu, in particular to the C-terminus of NaGIu, yielded a form of recombinant NaGIu which is phosphorylated, retains enzymatic activity, as well as gaining the ability to bind to the mannose 6- phosphate receptor. The invention is not limited to the presence of ApoE or ApoB fused to NaGIu.

Examples

The following examples are provided in order to demonstrate and further illustrate certain embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

Example 1

Material and Methods Cloning of the recombinant fusion construct

The phNaGlu-ApoE plasmid was prepared using two different plasmids: the phNaGlu plasmid (Zhao HG et al., 1996), a generous gift from Dr. Neufeld and the pcDNA-GFPmycApoEft plasmid (provided by the Dr. Verma laboratory). To generate the phNaGlu-ApoE recombinant fusion construct the pcDNA-GFPmycApoEft was digested with Nhel and EcoRI restriction enzymes to remove the green fluorescent protein (GFP) sequence and replace it with the human NaGIu (hNaGlu) cDNA obtained from the phNaGlu plasmid digested with EcoRI enzyme. Thus the hNaGlu cDNA in the phNaGlu- ApoE is in frame with the myc epitope and the ApoE ligand domain coding sequences, all under the control of the cytomegalovirus (CMV) promoter. Generation of a stable cell line expressing recombinant rhNaGlu-ApoE fusion

The phNaGlu- ApoE or phNaGlu plasmids were electroporated into Chinese hamster ovary cells deficient in dihydrofolate reductase (CHO-dhfr-) obtained from Dr. Patricia Dickson's lab. After transfection, cells were grown in Ham's F12 - DME medium (Irvine Scientific), 10% fetal bovine serum, 4 mM L-glutamine, nonessential amino acids, 1 mM Sodium pyruvate, penicillin/streptomycin and nucleosides (10 mg/L each of guanosine, adenosine, uridine, thymidine, cytidine and hypoxanthine) for 48 h, then 0.75 mg/ml G418 (Geniticin) was added to the media until resistant colonies emerged. Single cell clones were selected, grown and tested for expression and secretion of the rhNaGlu-ApoE enzyme by Western blot and enzymatic assay. NaGIu enzyme assay

The NaGIu activity was measured using the fluorogenic substrate 4- methylumbelliferyl-alpha-N-acetylglucosaminide (4-MU-Naglu, Calbiochem). 25 μl of the sample was mixed with 25 μl of 0.2 mM of the substrate in 0.1 M Na-acetate buffer pH 4.3, containing 0.5 mg/ml bovine serum albumin and incubated at 37°C for 1 h to measure the activity of the purified enzymes and for 4 h to test the activity in cell culture media and cell extracts. After incubation, the reaction was stopped with glycine-carbonate pH 10.5 and fluorescence was measured using a fluoremeter at an excitation wavelength of 360 run and an emission wavelength at 450 run. The NaGIu activity was normalized for protein concentration (nmoles/h/mg or unit/mg) as determined by the BCA protein assay (Pierce). A unit (U) of NaGIu is equal to one nmole of 4-methylumbelliferone released per hour. To determine the pH optimum of the purified hNaGlu enzymes the assay was performed in the same buffer with a pH ranging from 3.15 to 6. Western Blot

Proteins were separated on 10% SDS-polyacrylamide then transferred to nitrocellulose membranes according to the manufacturer's recommendations (Promega). The membranes were blocked for 1 h at room temperature in

PBS/0.1% Tween/5% dried milk, and then incubated for 2 h at room temperature (RT) with the appropriate antibody diluted in PBS/0.1% Tween/5% dried milk. For detection of hNaGlu, the rabbit anti-hNaGlu antiserum (Dr. Neufeld lab) and the mouse monoclonal anti-myc antibody (Invitrogen) were diluted 2000-fold into blocking buffer. Goat anti-mouse HRP-conjugated secondary antibody (Zymed Laboratories) were used at a 1 :2000 dilution in blocking buffer and incubated with the membrane for 1 h at RT. Blots were washed using PBS/0.1% Tween 20 and soaked in ECL reagent (enhanced chemiluminescence, Pierce) and exposed to a film. Purification of the recombinant rhNaGlu-ApoE fusion and urinary hNaGlu

Media from CHO clones expressing the rhNaGlu-ApoE or rhNaGlu were harvested, sterile filtered and proteins were precipitated with ammonium sulfate (70% saturation) for 1 to 2 h at 4°C in the presence of 0.1% Triton X-IOO. The pellet was collected by centrifugation at 12,000xg for 25 min, resuspended in Concanavalin A (ConA) binding buffer (20 mM sodium phosphate pH 6.8, 300 mM NaCl, 10 mM methyl-α-glucoside, ImM β-mercaptoethanol) and dialyzed against the same buffer at 4°C. A ConA column (15x500 mm) was packed and equilibrated with ConA binding buffer. Dialysate was sterile filtered and applied to column at lml/min at 4°C. Column was washed with 1 column volume of ConA binding buffer and several column volumes of ConA washing buffer (20 mM sodium phosphate pH 5.8, 300 mM NaCl, 10 mM methyl-α-glucoside, 10 mM methyl-mannoside, 1 mM /3-mercaptoethanol) at 20°C followed by loading of 1 bed volume of ConA elution buffer (20 mM sodium phosphate pH 5.8, 300 mM NaCl, 10 mM methyl-α-glucoside, 500 mM methyl-mannoside, 1 mM β- mercaptoethanol) with incubation for 6 to 12h at 20°C. Eluted fractions in which NaGIu activity was detected (active fractions) were pooled, dialyzed at 4°C against Q buffer (20 mM Tris-HCl, pH 7.5, 0.1% Triton X-100) and loaded on a 1 ml HiTrap Q Sepharose column. The column was then washed with 15 bed volumes of Q-buffer and enzymes were eluted by applying a NaCl step gradient (0.1 to 0.7M NaCl). Active fractions were pooled and loaded onto a size exclusion column (Sephacryl S-200, 26x600mm) followed by elution with S- buffer (20 mM sodium phosphate, pH 5.8, 300 mM NaCl, 10 mM methyl-α- glucoside, 1 mM jS-mercaptoethanol, 0.05% Triton X-100). Eluted active fractions were pooled and concentrated to ~lmg/ml by Amicon concentrator at 4°C. All operations if not specified were conducted at 20°C. Purified enzyme was verified by SDS-PAGE analysis and Western blot.

The purification of the urinary hNaGlu was done with minor modifications. Fresh human urine was used to precipitate proteins with 70% ammonium sulfate. The pellet was then collected by centrifugation at 8,000xg for 15min and resuspended in ConA binding buffer prior to dialysis against the same buffer. The urinary hNaGlu was then purified as previously described for the recombinant NaGIu proteins with the exception that buffers used did not contain Triton X-100. Immunofluorescence

MPS IIIB fibroblasts were grown on coverslips (22x22 mm) and incubated with 160 U of the purified rhNaGlu-ApoE in complete MEM for 72h at 37°C. Untreated cells were incubated with the same amount of enzyme storage buffer in MEM. After incubation cells were washed with PBS (3 x 5 min), fixed with PBS/4% paraformaldehyde then permeabilized with PBS/0.1% Triton X-IOO for 2 min. Cells were then blocked with PBS/10% normal goat serum (Southern Biotech) for 30 min followed by 1 h incubation with the mouse monoclonal anti-myc antibody (Santa Cruz Biotechnology) and the rabbit fluorescein-conjugated Lamp-1 (BDPharmingen) both diluted at 1:50 in

PBS/1%BSA. After washing three times with PBS, cells were incubated for Ih with the HRP-conjugated goat anti-mouse antibody (Molecular Probes) used at 1:100 dilution in PBS/1% blocking reagent from Tyramide Signal Amplification (TSA) kit (Molecular probes). After PBS washing (3 x 5 min) and PBS soaking (3h), the signal was amplified with TSA kit (Alexa Fluor 647 tyramide,

Molecular probes) and slides were mounted using anti-fading VECT ASHIELD hard-set mounting medium (VECTOR Laboratories). Images were collected on a Leica confocal microscope with an X 63/1.4 oil immersion lens. Cellular uptake and mechanism of internalization of the rhNaGlu-ApoE MPS IIEB fibroblasts were incubated with 1OU of the hNaGlu-containing media from CHO clones or the rhNaGlu-ApoE or hNaGlu purified enzymes at 37°C in a serum free medium. After 5 h cells were washed three times with PBS and harvested by trypsinization for 10 min. Cells were then resuspended in lysis solution (0.9% NaCl containing 0.2% Triton X-100) followed by six cycles of freeze-thawing. The cell lysates were clarified by centrifugation and stored frozen until use.

For studies of the inhibition uptake, 30 min prior to the incubation with the enzymes, MPS IIIB cells were incubated with 5 mM mannose-6-phosphate (M6P) or 5 mM mannose or 0.5 u/ml heparin or 50 mM ammonium chloride or 3 μg/ml filipin or 0.2 mM amiloride or 1 mg/ml low density lipoprotein (LDL). For the inhibition with sodium azide (5 mM) cells were incubated at 4°C. For the up-regulation of the LDL receptors, MPS IIIB fibroblasts were incubated with medium containing lipoprotein deficient serum (Kalen Biomedical, LLC) for 48h at 37⁰C prior to the incubation with the enzymes. Dephosphorylation of the hNaGlu enzymes

2 μg of rhNaGlu-ApoE or native hNaGlu from urine were digested with 1000 units of endoglycosidase H (Endo Hf, New England Biolabs) for 1 h at 37°C then analyzed on SDS-PAGE stained with Pro-Q Diamond phosphoprotein gel stain (Molecular Probes).

Quantification of GAGs Storage in MPS IIIB fibroblasts

Cultured MPS IIIB fibroblasts were labeled with H₂ ³⁵SO₄ as described by Zhao and Neufeld {Zhao et al., 2000} with the following modifications. Briefly, human MPS IIIB fibroblasts were grown to confluence in 6-well plates. Cells were labeled at 37°C in 2 ml sulfated deficient MEM containing 0.02% CaCl₂, pyruvate, nonessential amino acids, 8% dialyzed fetal bovine serum and 4 μCi/ml H₂ ³⁵SO₄. rh/Vαg/w-ApoE was added to the media as indicated in figure 7 and incubated for 72 hours. Cells were washed in three times with PBS then treated with trypsin and GAGs were extracted from cells by vortexing and boiling in 80% ethanol twice for 15 minutes. GAGs pellets were collected by centrifugation at 3000 g then resuspended in 10% sodium hydroxide and neutralized in 2 N acetic acid. Radioactivity was determined by scintillation counting and protein concentration measured according to the Bradford method using the Biorad protein assay kit.

Results/Discussion

In an attempt to target a wide range of cell types and tissues, especially the central nervous system, a rhNaGlu enzyme fused to the apolipoprotein E (rhNaGlu-ApoE) ligand domain that binds the low density lipoprotein receptor (LDL-R) family was designed. The LDL-R family members are expressed in all tissues and some are known to be involved in the transcytosis across the blood brain barrier (BBB). Biochemical characterization shows that the rhNaGlu- ApoE fusion has a Km toward the 4-methylumbelliferyl- a -N- acetylglucosaminide similar to the one of the native human NaGIu purified from urine (Km=O.6 mM). MPS IIIB fibroblasts were used to characterize the cellular uptake of the rhNaGlu- ApoE. Using different receptor competitors, the rhNaGlu-ApoE cellular internalization was surprisingly inhibited by mannose-6- phosphate (M6P) and not LDL. In addition, using endocytosis inhibitors, it is disclosed herein that rhNaGlu-ApoE enters the cells using the clathrin mediated endocytosis as is the case with native NaGIu. Moreover, using confocal microscopy, the rhNaGlu-ApoE was localized in the lysosomal compartment of the cells after uptake. The results show that the rhNaGlu-ApoE enters the cells using the same mechanism as the native NaGIu. Thus, recombinant NaGIu- ApoE fusion binds to the M6P receptor, enters cells via the clathrin mediated endocytosis and localizes in lysosomes. Therefore, use of the fusion can be the basis of recombinant protein based therapy, or a liver-targeted gene therapy.

Thus, an advantage of the recombinant enzyme disclosed herein is that it is mannose 6-phophorylated. This is an important property of the native enzyme that no one has previously been able to reproduce after overexpression of the enzyme. Furthermore, it was determined herein that the recombinant protein disclosed herein was able to enter the cells using the same receptor and mechanism as the native enzyme. Thus, we have provided herein a therapeutic cDNA/protein for the treatment of Sanfillipo Syndrome type B.

Example 2

The CHO cell line was CHO-dhfr-. The growth media was Ex-Cell 325 PF CHO serum-free, protein-free media and is manufactured by SAFC Biosciences (Sigma-Aldrich). It was supplemented by L-glutamine 4 mM, nucleosides (10 mg/L each of guanosine, adenosine, uridine, thymidine, cytidine and hypoxanthine) and gentamicin 50 ug/mL.

MPS IIIB fibroblasts were incubated in the presence or absence of M6P with: 10 units of rhNaGlu using the media of the CHO-NaGIu clone; 10 units of rhNaGlu-ApoE using the media of the CHO-NaGIu- ApoE clone; or 10 units of rhNaGlu- ApoB using the media of the CHO-NaGIu- ApoB clone. Cells were washed and collected after 5 or 24 hours and activity was measured in the cell extracts.

As depicted in Figure 12, the efficiency of the uptake of the recombinant hNaGlu-ApoE is higher than the recombinant NaGIu. Furthermore, the uptake of both the recombinant hNaGlu and hNaGlu-ApoE is inhibited by M6P suggesting mannose-6 phosphorylation of both enzymes. Furthermore, data (not shown) demonstrates that under these conditions, rhNaGlu-ApoE is phosphorylated. Example 3

The activity of NaGIu from the constructs and phorphorylated NaGIu disclosed in Examples 1 and 2 and throughout the specification can be further evaluated in vivo using the MPS IIIB mouse and liver directed gene therapy using an AAV8 vector (Wang Z, et al. Nature Biotechnology (2005) 23 :pp. 321 - 328). Expression of the NaGIu chimera in the liver of affected mice will lead to production of active enzyme capable of crossing the blood brain barrier, and will lead to clinical improvement and decreased pathological findings of MPS IIIB mice. The AAV8 vector with NaGIu can be further evaluated using the canine model of MPS IIIB. Short term evaluation for efficacy in MPS IIIB pups can be followed by evaluation of preclinical adult dogs to evaluate the chimeric enzymes ability to prevent the overt neurological phenotype seen in the canine form of MPS IIIB. The expression of the chimeric enzyme in the liver of affected MPS IIIB dogs will prevent neurological signs of disease from developing.

All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

Claims

WHAT IS CLAIMED IS:

1. A method to produce a recombinant mannose-6-phosphorylated N- acetyl-α-D-glucosaminidase (NaGIu) fusion protein comprising operably linking a first DNA sequence coding for a NaGIu protein to a second sequence coding for a peptide and expressing the linked sequence in a cell so as to yield a recombinant mannose-6-phosphorylated NaGIu fusion protein.

2. The method of claim 1, wherein the NaGIu protein comprises SEQ ID NO:1 or a protein sequence having at least 80% sequence identity with SEQ ID

NO:1.

3. The method of claim 1, wherein the linked sequences further comprise a third sequence coding for a signaling moiety.

4 The method of claims 1, wherein the second sequence comprises a peptide of about 5 to about 60 amino acids.

5. The method of claim 1, wherein the second sequence codes for an Apolipoprotein E or Apolipoprotein B ligand domain.

6. The method of claim 1, wherein the signaling moiety is EQKLISEEDL (SEQ ID NO:7).

7. A method to treat mucopolysaccharidosis type IIIB (MPS IIIB) comprising administering to a subject in need thereof an effective amount of a mannose-6-phosphorylated Naglu fusion protein produced by the method of claim 1.

8. The method of claim 7, wherein the treatment reduces lysosomal storage of heparan sulfate in a tissue of said subject.

9. The method of claim 8, wherein the tissue is brain tissue.

10. A method to produce a mannose-6-phosphorylated N-acetyl-α-D- glucosaminidase (NaGIu) fusion protein comprising expressing a recombinant NaGIu fusion protein in cell culture so as produce a mannose-6-phosphorylated N-acetyl-α-D-glucosaminidase fusion protein.

11. An isolated recombinant mannose-6-phosphorylated N-acetyl-α-D- glucosaminidase (NaGIu) fusion protein.

12. An isolated recombinant mannose-6-phosphorylated N-acetyl-α-D- glucosaminidase (NaGIu) fusion protein, wherein the NaGIu protein comprises SEQ ID NO: 1 or a protein sequence having at least 80% sequence identity with SEQ ID NO: 1.

13. The NaGIu fusion protein of claim 12, wherein the NaGIu protein is expressed as a fusion protein comprising a NaGIu protein and at least one other peptide.

14. The isolated recombinant phosphorylated NaGIu fusion protein of claim 13, wherein the at least one other peptide comprises a peptide of about 5 to about 60 amino acids.

15. The isolated recombinant phosphorylated NaGIu fusion protein of claim 13, wherein the at least one other peptide is an Apolipoprotein E or Apolipoprotein B ligand domain.

16. The isolated recombinant phosphorylated NaGIu fusion protein of claim 13, wherein the at least one other peptide is a signaling moiety.

17. The isolated recombinant phosphorylated NaGIu fusion protein of claim 14, further comprising a signaling moiety.

18. The isolated recombinant phosphorylated NaGIu fusion protein of claim 16, wherein the signaling moiety is EQKLISEEDL (SEQ ID NO: 7).

19. A composition comprising the isolated recombinant mannose-6- phosphorylated N-acetyl-α-D-glucosaminidase (NaGIu) fusion protein of claims 12 and a pharmaceutically acceptable carrier and/or culture medium.

20. An isolated recombinant mannose-6-phosphorylated N-acetyl-α-D- glucosaminidase (NaGIu) fusion protein according to claims 1 -9 for use in medical therapy.

21. The use of a recombinant phosphorylated NaGIu protein to prepare a medicament for treating mucopolysaccharidosis type MB (MPS MB).