Synthetic mammalian alpha-N-acetylglucosaminidase and genetic sequences encoding same

Abstract

An efficient architecture for an interpolator ( 100 ) disposed to process oversampled data is disclosed herein. The interpolator ( 100 ) includes an input divider circuit ( 104 ) configured to receive an input data word over an input data line. A register ( 108 ) is provided for latching the divided input data word from the divider ( 104 ). The divided input data word is added within a summer ( 112 ) to a latched divided data word from the register, thereby forming a summed data word. A multiplexer ( 116 ) produces an interpolated output by multiplexing the summed data word with an input data word. In a preferred implementation, the register ( 108 ) is latched at a first clock rate, and the multiplexer ( 116 ) is clocked at twice the first clock rate. The efficient filter architecture allows interpolation to be performed in the absence of multipliers, and in a manner using filter coefficients equivalent to powers of two. This enables the interpolator ( 100 ) to be realized inexpensively, and renders the filter particularly suitable for implementation within integrated circuits.

What is claimed is:  
     
         1 . An isolated nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes a mammalian α-N-acetylglucosaminidase or fragment or derivative thereof.  
     
     
         2 . The isolated nucleic acid molecule according to  claim 1  wherein the nucleotides are dcoxyribonucleoticles.  
     
     
         3 . The isolated nucleic acid molecule according to  claim 2  wherein said molecule is a cDNA.  
     
     
         4 . The isolated nucleic acid molecule according to  claim 2  wherein said molecule is a genomic DNA molecule.  
     
     
         5 . The isolated nucleic acid according to  claim 1  wherein the mammal is a human.  
     
     
         6 . The isolated nucleic acid according to  claim 5  wherein the α-N-acetylglucosaminidase is of liver, kidney or placenta origin.  
     
     
         7 . The isolated nucleic acid molecule according to  claim 1  having a nucleotide sequence substantially as set forth in SEQ ID NO: I or complementary thereto or having at least 40% similarity to all or part thereof.  
     
     
         8 . The isolated nucleic acid molecule according to  claim 1  having a nucleotide sequence substantially as set forth in SEQ ID NO:3 or complementary thereto or having at least 40% similarity to all or part thereof.  
     
     
         9 . The isolated nucleic acid molecule according to  claim 7  wherein the percentage similarity is at least 60%.  
     
     
         10 . The isolated nucleic acid molecule according to  claim 9  wherein the percentage homology is at least 80%.  
     
     
         11 . The isolated nucleic acid molecule according to  claim 1  wherein the α-N-acetylglucosaminidase or fragment or derivative thereof encoded by said molecule comprises an amino acid sequence substantially identical to SEQ ID NO:2 or is at least 0.40% similar to all or a part thereof.  
     
     
         12 . The isolated nucleic acid molecule according to  claim 11  wherein the percentage similarity to SEQ ID NO: 2 is at least 60%.  
     
     
         13 . The isolated nucleic acid molecule according to  claim 12  wherein the percentage similarity to SEQ ID NO:2 is at least 80%.  
     
     
         14 . The isolated nucleic acid molecule according to  claim 1  wherein said molecule is carried by a vector capable of replication in a eukaryotic cell and/or a prokaryotic cell.  
     
     
         15 . The isolated nucleic acid molecule according to  claim 14  wherein the vector is an expression vector.  
     
     
         16 . The isolated nucleic acid molecule according to  claim 15  wherein the expression vector is capable of being expressed in cells derived from a eukaryote.  
     
     
         17 . The isolated nucleic acid molecule according to  claim 16  wherein the expression vector is further capable of being expressed in cells derived from a mammal.  
     
     
         18 . The isolated nucleic acid molecule according to  claim 17  wherein the expression vector is further capable of being expressed in CHO cells.  
     
     
         19 . A recombinant mammalian α-N-acetylglucosaminidase or fragment or derivative thereof.  
     
     
         20 . The recombinant mammalian α-N-acetylglucosaminidase according to  claim 19  in substantially pure form.  
     
     
         21 . The recombinant mammalian α-N-acetylglucosaminidase according to  claim 19  when expressed in mammalian, yeast or insect cells.  
     
     
         22 . The recombinant mammalian α-N-acetylglucosaminidase according to  claim 21  when expressed in mammalian cells.  
     
     
         23 . The recombinant mammalian α-N-acetylglucosaminidase according to  claim 21 , wherein the cells are capable of glycosylating said recombinant mammalian α-N-acetylglucosaminidase.  
     
     
         24 . The recombinant mammalian α-N-acetylglucosaminidase according to  claim 23  wherein the cells are capable of N-glycosylating said recombinant mammalian α-N-acetylglucosaminidase.  
     
     
         25 . The recombinant mammalian α-N-acetylglucosaminidase according to  claim 24  wherein the cells are CHO cells.  
     
     
         26 . The recombinant mammalian α-N-acetylglucosaminidase according to  claim 19  wherein said recombinant α-N-acetylglucosaminidase is in a glycosylated form.  
     
     
         27 . The recombinant mammalian α-N-acetylglucosaminidase according to  claim 26  wherein the molecular weight of the glycosylated form as determined using SDS/PAGE is at least approximately 79 kDa.  
     
     
         28 . The recombinant α-N-acetylglucosaminidase according to  claim 26  wherein the molecular weight of the glycosylated form as determined using SDS/PAGE is at least approximately 79 kDa to 89 kDa.  
     
     
         29 . The recombinant mammalian α-N-acetylglucosaminidase according to  claim 19  comprising a sequence of amino acids substantially the same as a human α-N-acetylglucosaminidase.  
     
     
         30 . The recombinant mammalian α-N-acetylglucosaminidase according to  claim 19  when fused to another proteinaceous molecule.  
     
     
         31 . The recombinant mammalian α-N-acetylglucosaminidase according to  claim 30  wherein the other proteinaceous molecule is an enzyme, reporter molecule, purification site and/or a signal sequence.  
     
     
         32 . The recombinant mammalian α-N-acetylglucosaminidase according to  claim 19  comprising an amino acid sequence substantially as set forth in SEQ ID NO:2 or having at least 40% similarity to all or part thereof.  
     
     
         33 . The recombinant mammalian α-N-acetylglucosaminidase according to  claim 32  wherein the percentage similarity to SEQ ID NO:2 is at least 60%.  
     
     
         34 . The recombinant mammalian α-N-acetylglucosaminidase according to  claim 33  wherein the percentage similarity to SEQ ID NO:2 is at least 80%.  
     
     
         35 . A recombinant α-N-acetylglucosaminidase produced by expression of a nucleic acid molecule which encodes or is complementary to a sequence which encodes a mammalian α-N-acetylglucosaminidase or fragment thereof and wherein the molecule is carried by a vector capable of replication in a eukaryotic or prokaryotic cell.  
     
     
         36 . The recombinant α-N-acetylglucosaminidase according to  claim 35  when glycosylated.  
     
     
         37 . A method of diagnosing a mutation in a gene which encodes α-N-acetylglucosaminidase in a human patient said method comprising contacting genomic DNA or RNA derived from said patient with one or more isolated DNA molecules or oligonucleotides comprising at least 10 contiguous nucleotides derived from SEQ ID NO: 1 or SEQ ID NO:3 or a complementary strand thereof for a time and under conditions sufficient for hybridisation to occur and then detecting said hybridisation using a detection means.  
     
     
         38 . The method according to  claim 37  wherein the detection means is a reporter molecule covalently attached to the isolated DNA molecule or oligonucleotide.  
     
     
         39 . The method according to  claim 38  wherein the reporter molecule is  31 P,  35 S or biotin.  
     
     
         40 . The method according to  claim 37  wherein the detection means is a polyrnerase chain reaction format.  
     
     
         41 . The method according to  claim 40  wherein the polymerase chain reaction format is selected from the list comprising SSCP, AMD, AFLP, IRS-PCR, iPCR or RT-PCR, amongst others.  
     
     
         42 . The method according to  claim 41  wherein the polymerase chain reaction format is SSCP.  
     
     
         43 . The method according to  claim 42  wherein the isolated DNA molecule or oligonucleotide comprises at least 20 contiguous nucleotides derived from SEQ ID NO: 1 or SEQ ID NO:3 or a complementary strand thereof.  
     
     
         44 . The method according to  claim 43  wherein the isolated DNA molecule or oligonucleotide comprises at least 50 contiguous nucleotides derived from SEQ ID NO: 1 or SEQ ID NO: 3 or a complementary strand thereof.  
     
     
         45 . The method according to  claim 44  wherein the isolated DNA molecule or oligonucleotide comprises at least 7 contiguous nucleotides derived from SEQ ID NO: 1 or SEQ ID NO:3 or a complementary strand thereof.  
     
     
         46 . A method for treating a patient suffering from α-N-acetylglucosaminidase efficiency said method comprising administering to said patient an effective amount of recombinant mammalian α-N-acetylglucosaminidase or an active fragment or derivative thereof.  
     
     
         47 . The method according to  claim 46  wherein the mammalian α-N-acetylglucosaminidase comprises a sequence of amino acids substantially the same as the amino acid, sequence of human α-N-acetylglucosaminidase.  
     
     
         48 . The method according to  claim 47  wherein the patient is suffering from mucopolysaccharidosis type IIIB.  
     
     
         49 . The method according to  claim 46  wherein the recombinant α-N-acetylglucosaminidase is produced in mammalian cells.  
     
     
         50 . The method according to  claim 49  wherein the mammalian cells are capable of glycosylating the recombinant α-N-acetylglucosaminidase produced therein.  
     
     
         51 . The method according to  claim 50  wherein the recombinant α-N-acetylglucosaminidase is in a glycosylated form.  
     
     
         52 . The method according to  claim 51  wherein the glycosylated form of the recombinant α-N-acetylglucosaminidase has a molecular weight as determined using SDS/PAGE of at least approximately 79 kDa.  
     
     
         53 . The method according to  claim 52  wherein the glycosylated form of the recombinant α-N-acetylglucosaminidase has a molecular weight as determined using SDS/PAGE of at least approximately 79 kDa to 89 kDa.  
     
     
         54 . The method according to  claim 46  wherein the recombinant α-N-acetylglucosaminidase comprises a sequence of amino acids substantially as set forth in SEQ ID NO:2 or having at least 40% similarity to all or a part thereof.  
     
     
         55 . The method according to  claim 54  wherein the percentage similarity to SEQ ID NO:2 is at least 60%.  
     
     
         56 . The method according to  claim 55  wherein the percentage similarity to SEQ ID NO:2 is at least 80%.  
     
     
         57 . A method for treating a patient suffering from α-N-acetylglucosaminidase deficiency said method comprising administering to said patient an effective amount of recombinant mammalian α-N-acetylglucosaminidase or an active fragment or derivative thereof, wherein said recombinant mammalian α-N-acetylglucosaminidase is produced by expression of a nucleic acid molecule according to  claim 14 .  
     
     
         58 . The method according to  claim 46  wherein administration of the recombinant mammalian α-N-acetylglucosaminidase is by oral, intravenous, suppository, intraperitoneal intramuscular, intranasal, intradermal or subcutaneous administration by infusion or implantation or by enzyme replacement therapy or by gene therapy.  
     
     
         59 . The method according to  claim 58  wherein the method of administration is by enzyme replacement therapy.  
     
     
         60 . A pharmaceutical composition comprising a recombinant mammalian α-N-acetylglucosaminidase or an active fragment or derivative thereof and one or more pharmaceutically acceptable carriers and/or diluents.  
     
     
         61 . The pharmaceutical composition according to  claim 60  wherein the recombinant mammalian α-N-acetylglucosaminidase comprises a sequence of amino acids substantially the same as human α-N-acetylglucosaminidase.  
     
     
         62 . The pharmaceutical composition according to  claim 60  wherein the recombinant mammalian α-N-acetylglucosaminidase is produced in a mammalian cell.  
     
     
         63 . The pharmaceutical composition according to  claim 62  wherein the mammalian cell is a CHO cell line which is capable of glycosylating the recombinant mammalian α-N-acetylglucosaminidase.  
     
     
         64 . The pharmaceutical composition according to  claim 60  wherein the α-N-acetylglucosaminidase is glycosylated.  
     
     
         65 . The pharmaceutical composition according to  claim 64  wherein the recombinant α-N-acetylglucosaminidase has a molecular weight as determined using SDS/PAGE of at least approximately 79 kDa.  
     
     
         66 . The pharmaceutical composition according to  claim 65  wherein the recombinant α-N-acetylglucosaminidase has a molecular weight as determined using SDS/PAGE of approximately 79 kDa to 89 kDa.  
     
     
         67 . The pharmaceutical composition according to  claim 60  wherein the recombinant α-N-acetylglucosaminidase comprises a sequence of amino acids substantially as set forth in SEQ ID NO: 2 or having at least 40% similarity to all or part thereof.  
     
     
         68 . The pharmaceutical composition according to  claim 67  wherein the percentage similarity to SEQ ID NO:2 is at least 60%.  
     
     
         69 . The pharmaceutical composition according to  claim 68  wherein the percentage similarity to SEQ ID NO:2 is at least 80%.  
     
     
         70 . A pharmaceutical composition comprising recombinant mammalian α-N-acetylglucosaminidase or an active fragment or derivative thereof and one or more pharmaceutically acceptable carriers and/or diluents wherein said recombinant mammalian α-N-acetylglucosaminidase is produced by expression of a nucleic acid molecule according to  claim 35 .  
     
     
         71 . A pharmaceutical composition comprising recombinant mammalian α-N-acetylglucosaminidase or an active fragment or derivative thereof and one or more pharmaceutically acceptable carriers and/or diluents when used in the method for treating a patient suffering from α-N-acetylglucosaminidase.  
     
     
         72 . Use of recombinant mammalian α-N-acetylglucosaminidase or an active fragment or derivative thereof in the manufacture of a medicament for the treatment of α-N-acetylglucosaminidase deficiency in a patient.  
     
     
         73 . The use according to  claim 72  wherein the recombinant mammalian α-N-acetylglucosaminidase comprises a sequence of amino acids substantially the same as the amino acid sequence of human α-N-acetylglucosaminidase.  
     
     
         74 . The use according to  claim 72  wherein the patient is suffering from mucopolysaccharidosis type IIIB.  
     
     
         75 . The use according to  claim 74  wherein the recombinant is α-N-acetylglucosaminidase expressed in mammalian cells.  
     
     
         76 . The use according to  claim 75  wherein the cells are CHO cells.  
     
     
         77 . The use according to  claim 72  wherein the α-N-acetylglucosaminidase is glycosylated.  
     
     
         78 . The use according to  claim 77  wherein the recombinant α-N-acetylglucosaminidase has a molecular weight as determined using SDS/PAGE of at least approximately 79 kDa.  
     
     
         79 . The use according to  claim 78  wherein the recombinant α-N-acetylglucosaminidase has a molecular weight as determined using SDS/PAGE of approximately 79 kDa to 89 kDa.  
     
     
         80 . The use according to  claim 72  wherein the recombinant α-N-acetylglucosaminidase comprises a sequence of amino acids substantially as set forth in SEQ ID NO:2 or has at least 40% similarity to all or a part thereof.  
     
     
         81 . The use according to  claim 80  wherein the percentage similarity to SEQ ID NO:2 is at least 60%.  
     
     
         82 . The use according to  claim 80  wherein the percentage similarity to SEQ ID NO:2 is at least 80%.  
     
     
         83 . A nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide capable of hydrolyzing the terminal α-N-acetylglucosaminidase residues present at the non-reducing terminus of fragments of heparan sulphate and heparin and wherein said nucleotide sequence is capable of hybridising under at least low stringency conditions to the nucleotide sequence set forth in SEQ ID NO:1.  
     
     
         84 . A nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide capable of hydrolysing the terminal α-N-acetylglucosaminidase residues present at the non-reducing terminus of fragments of heparan sulphate and heparin and wherein said nucleotide sequence is capable of hybridising under at least low stringency conditions to the nucleotide sequence set forth in SEQ ID NO:3.  
     
     
         85 . A recombinant polypeptide comprising a sequence of amino acids corresponding to the amino sequence set forth in SEQ ID NO:2 or having at least 40% similarity thereto and encoded by a nucleic acid molecule which is capable of hybridising to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 under at least low stringency conditions.  
     
     
         86 . A genetic construct comprising the nucleic acid molecule according to  claim 1 , operably connected in the sense orientation to a promoter sequence such that said genetic construct is capable of being expressed in a eukaryotic or prokaryotic cell to produce a recombinant mammalian α-N-acetylglucosaminidase or a fragment or derivative thereof.  
     
     
         87 . The genetic construct according to  claim 86  wherein the promoter is capable of regulating expression of the recombinant α-N-acetylglucosaminidase in a mammalian cell.  
     
     
         88 . The genetic construct according to  claim 87  wherein the promoter is the CMV promoter sequence or a promoter derived therefrom.  
     
     
         89 . The genetic construct according to  claim 86  further comprising a transcription terminator sequence.  
     
     
         90 . The genetic construct according to  claim 86  when used to express or over-express α-N-acetylglucosaminidase in a eukaryotic or prokaryotic cell.  
     
     
         91 . An antibody to α-N-acetylglucosaminidase or a recombinant α-N-acetylglucosaminidase according to  claim 19  or an antigenic fragment thereof.  
     
     
         92 . The antibody according to  claim 91  further defined as a polyclonal antibody molecule.  
     
     
         93 . The antibody according to  claim 91  further defined as a monoclonal antibody molecule.  
     
     
         94 . The isolated nucleic acid molecule according to  claim 8  wherein the percentage similarity is at least 60%.  
     
     
         95 . The isolated nucleic acid molecule according to  claim 94  wherein the percentage homology is at least 80%.  
     
     
         96 . A recombinant mammalian α-N-acetylglucosaminidase or fragment thereof wherein the α-N-acetylglucosaminidase or fragment thereof is in glycosylated form and comprises a sequence of amino acids substantially the same as a human α-N-acetylglucosaminidase.  
     
     
         97 . The recombinant mammalian α-N-acetylglucosaminidase according to  claim 96  when fused to another proteinaceous molecule.  
     
     
         98 . The recombinant mammalian α-N-acetylglucosaminidase according to  claim 97  wherein the other proteinaceous molecule is an enzyme, reporter molecule, purification site and/or a signal sequence.  
     
     
         99 . The recombinant mammalian α-N-acetylglucosaminidase according to  claim 96  comprising an amino acid sequence substantially as set forth in SEQ ID NO:2 or having at least 40% similarity to all or part thereof.  
     
     
         100 . The method according to  claim 57  wherein administration of the recombinant mammalian α-N-acetylglucosaminidase is by oral, intravenous, suppository, intraperitoneal, intramuscular. intranasal. intradermal or subcutaneous administration by infusion or implantation or by enzyme replacement therapy or by gene therapy.  
     
     
         101 . A pharmaceutical composition comprising recombinant mammalian α-N-acetylglucosaminidase or an active fragment or derivative thereof and one or more pharmaceutically acceptable carriers and/or diluents when used in the method according to  claim 57 .  
     
     
         102 . A pharmaceutical composition comprising recombinant mammalian α-N-acetylglucosaminidase or an active fragment or derivative thereof and one ore more pharmaceutically acceptable carriers and/or diluents when used in the method according to  claim 58 .  
     
     
         103 . A genetic construct comprising the nucleic acid molecule according to  claim 83 . operably connected in the sense orientation to a promoter sequence such that said genetic construct is capable of being expressed in a eukaryotic or prokaryotic cell to produce a recombinant mammalian α-N-acetylglucosaminidase or a fragment or derivative thereof.  
     
     
         104 . A genetic construct comprising the nucleic acid molecule according to  claim 84 , operably connected in the sense orientation to a promoter sequence such that said genetic construct is capable of being expressed in a eukaryotic or prokaryotic cell to produce a recombinant mammalian α-N-acetylglucosaminidase or a fragment or derivative thereof.  
     
     
         105 . An antibody to α-N-acetylglucosaminidase or a recombinant α-N-acetylglucosaminidase according to  claim 28  or an antigenic fragment thereof.  
     
     
         106 . The antibody according to  claim 15  further defined as a polyclonal antibody molecule.  
     
     
         107 . The antibody according to  claim 15  further defined as a monoclonal antibody molecule.  
     
     
         108 . An antibody to α-N-acetylglucosaminidase or a recombinant α-N-acetylglucosaminidase according to  claim 35  or an antigenic fragment thereof.  
     
     
         109 . The antibody according to  claim 108  further defined as a polyclonal antibody molecule.  
     
     
         110 . The antibody according to  claim 108  further defined as a monoclonal antibody molecule.