CN101636200A - Methods for treating pompe disease - Google Patents

Abstract

The present invention provides methods for treating Pompe disease in a subject by administering to the subject a therapeutically effective amount of a fusion protein which includes human acid alpha-glucosidase (GAA), or a fragment thereof, and a lysosomal targeting domain. The lysosomal targeting domain binds the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner.

Description

The method that is used for the treatment of Pompe

Quoting of related application

The application requires in the U.S. Provisional Patent Application No.60/900 of submission on February 7th, 2007,187, the U.S. Provisional Patent Application No.60/879 that submits on January 5th, 2007,255, the U.S. Provisional Patent Application No.60/858 that submits on November 13rd, 2006,514 priority is incorporated into this with the content of its each application in full with it.The application also is involved in the U.S. Patent application No.11/057 that submitted on February 10th, 2005, and 058, its full content is hereby expressly incorporated by reference.

Technical field

The present invention relates to be used for the treatment of the method and composition of Pompe (huge skin disease, Pompe disease).More specifically, the present invention relates to be used for by acid alpha-Glucosidase targeting lysosome being treated the Therapeutic Method of Pompe in non-Man-6-P dependent form (mannose-6-phosphate-independent) mode.

Background technology

Pompe is the autosomal recessive hereditary diseases (genetic disorder, genetic disorder) that is caused by the acid alpha-Glucosidase (GAA) of lysosomal hydrolase, glycogen degraded lysosomal enzyme shortage or dysfunction.The shortage of GAA causes the accumulation of lysosome glycogen takes place in the many tissues of Pompe patient, follows cardiac muscle and skeletal muscle tissue to suffer to have a strong impact on most.The total incidence rate of the Pompe of form of ownership is estimated as 1: 40, and 000, and this disease influences all colonies and does not have race tendency simultaneously.According to estimates, nearly 1/3rd crowds that suffer from Pompe have the mortality child who develops rapidly the form of falling ill, and Most patients shows as the few phase of green grass or young crops or the more late mode of onset of slower development.

Drug therapy strategy, diet control and bone marrow transplantation are used the means as the treatment Pompe, but all not have significantly successful.In recent years, algucerase (ERT) provides the new hope of treatment Pompe.For example, A kind of reorganization GAA protein drug was used to suffer from the patient of Pompe in 2006 in the US and European official approval.

Depend on the Man-6-P (M6P) that is used on the GAA protein surface that lysosome is sent.

Summary of the invention

The invention provides the novel and improved method of treatment Pompe.Particularly, the invention provides and be used in non-Man-6-P dependency mode the lysosomal method and composition of acid alpha-Glucosidase (GAA) targeting.Therefore, method of the present invention simpler, more effective, have more potentiality and have more cost effectiveness.Thereby the present invention has advanced the progress of the algucerase that is used for Pompe significantly.

In one aspect, the invention provides a kind of method for the treatment of the Pompe of main body by the fusion rotein for the treatment of effective dose to main body.This fusion rotein comprises acid alpha-Glucosidase (GAA) (the perhaps fragment of people GAA) of people and lysosome targeting domain.This lysosome targeting domain connects people's non-cationic dependent form Man-6-P receptor in non-Man-6-P dependent form mode.

In one embodiment, lysosome targeting domain comprises ripe human insulin-like growth factor II (IGF-II), or becomes fragment or the sequence variants (sequencevariant) of acquaintance IGF-II.In one embodiment, lysosome targeting domain comprises into the aminoacid 8-67 of acquaintance IGF-II.Preferably, lysosome targeting domain comprises into the amino acid/11 of acquaintance IGF-II and 8-67 (acquaintance GAA Δ 2-7).In another embodiment, fusion rotein comprises the aminoacid 70-952 of people GAA.

In one embodiment, than wild type people GAA, be applicable to that fusion rotein of the present invention has Man-6-P (M6P) level of reduction on this protein surface.In another embodiment, be applicable to that fusion rotein of the present invention does not have functional M6P level on this protein surface.

In another embodiment, the treatment effective dose is in the scope of every kilogram of main body body weight about 2.5～20 milligrams (mg/kg).

In one embodiment, fusion rotein is via intravenous administration.In other embodiments, fusion rotein with per the bimester, every month, per three weeks, per two weeks, weekly, administration every day or interval administration to change.As used in this article, term " per bimester administration " is meant per two months and is administered once (be per two months once); Term " administration in every month " is meant that each month is administered once; Term " per three all administrations " is meant that per three week is administered once; Term " per two all administrations " is meant that per fortnight is administered once; Term " administration weekly " is meant that each week is administered once; And term " administration every day " is meant that be administered once every day.

In further embodiment, fusion rotein combined immunization inhibitor carries out administration.Immunosuppressant can be carried out administration before any administration of fusion rotein.In some embodiments, the method that is used for the treatment of Pompe further comprises the additional step that makes main body immunologic tolerance (tolerizing).

Another aspect of the present invention provides a kind of method for the treatment of the Pompe of main body by the fusion rotein for the treatment of effective dose to main body.This fusion rotein comprises the aminoacid 70-952 of the amino acid/11 of ripe human insulin-like growth factor II (IGF-II) and 8-67 (acquaintance GAA Δ 2-7) and the acid α glucosidase of people (GAA).One preferred embodiment in, fusion rotein is included in the aminoacid of people GAA and becomes intervening sequence Gly-Ala-Pro between the aminoacid of acquaintance IGF-II.

In one embodiment, than wild type people GAA, be applicable to that fusion rotein of the present invention has Man-6-P (M6P) level of reduction on this protein surface.In another embodiment, the fusion rotein that is applicable to this aspect of the present invention does not have functional M6P level on this protein surface.

Another aspect of the present invention provides a kind of method that reduces glycogen levels in the body by the fusion rotein for the treatment of effective dose to the main body of suffering from Pompe.This fusion rotein comprises acid alpha-Glucosidase (GAA) (the perhaps fragment of people GAA) of people and lysosome targeting domain.This lysosome targeting domain in non-Man-6-P dependent form mode in conjunction with people's non-cationic dependent form Man-6-P receptor.

In one embodiment, lysosome targeting domain comprises ripe human insulin-like growth factor II (IGF-II), or becomes acquaintance's IGF-II fragment or sequence variants.In one embodiment, lysosome targeting domain comprises into the aminoacid 8-67 of acquaintance IGF-II.Preferably, lysosome targeting domain comprises into the amino acid/11 of acquaintance IGF-II and 8-67 (acquaintance GAA Δ 2-7).In another preferred embodiment, fusion rotein comprises the aminoacid 70-952 of people GAA.

In one embodiment, than wild type people GAA, be applicable to that the fusion rotein of this respect of the present invention has Man-6-P (M6P) level of reduction on this protein surface.In another embodiment, be applicable to that fusion rotein of the present invention does not have functional M6P level on this protein surface.

In another embodiment, effective dose is in the scope of every kilogram of main body body weight about 2.5～20 milligrams (mg/kg).

In some embodiments, fusion rotein is via intravenous administration.In other embodiments, fusion rotein administration with per bimester the, administration in every month, per three all administrations, per two all administrations, administration weekly, administration every day or interval administration to change.

In yet another aspect, the invention provides a kind of method that reduces glycogen levels in the mammal lysosome by fusion rotein to lysosome targeted therapy effective dose.This fusion rotein comprises acid alpha-Glucosidase (GAA) (the perhaps fragment of people GAA) of people and lysosome targeting domain.This lysosome targeting domain in non-Man-6-P dependent form mode in conjunction with people's non-cationic dependent form Man-6-P receptor.

In one embodiment, lysosome targeting domain comprises ripe human insulin-like growth factor II (IGF-II), or becomes acquaintance's IGF-II fragment or sequence variants.In one embodiment, lysosome targeting domain comprises into the aminoacid 8-67 of acquaintance IGF-II.Preferably, lysosome targeting domain comprises into the amino acid/11 of acquaintance IGF-II and 8-67 (acquaintance GAA Δ 2-7).In another preferred embodiment, fusion rotein comprises the aminoacid 70-952 of people GAA.

In yet another aspect, the invention provides the method for glycogen levels in a kind of muscular tissue that reduces the main body of suffering from Pompe by fusion rotein to muscular tissue delivery treatments effective dose.This fusion rotein comprises acid alpha-Glucosidase (GAA) (the perhaps fragment of people GAA) of people and lysosome targeting domain.This lysosome targeting domain in non-Man-6-P dependent form mode in conjunction with people's non-cationic dependent form Man-6-P receptor.In one embodiment, muscular tissue is skeletal muscle.

Another aspect of the present invention provides a kind of method for the treatment of cardiomyopathy relevant with Pompe in this main body (cardiomyopathy) by the fusion rotein for the treatment of effective dose to main body.This fusion rotein comprises acid alpha-Glucosidase (GAA) (the perhaps fragment of people GAA) of people and lysosome targeting domain.This lysosome targeting domain in non-Man-6-P dependent form mode in conjunction with people's non-cationic dependent form Man-6-P receptor.

In yet another aspect, the invention provides a kind of method for the treatment of myopathy relevant in this main body (myopathy) with Pompe by the fusion rotein for the treatment of effective dose to main body.This fusion rotein comprises acid alpha-Glucosidase (GAA) (the perhaps fragment of people GAA) of people and lysosome targeting domain.This lysosome targeting domain in non-Man-6-P dependent form mode in conjunction with people's non-cationic dependent form Man-6-P receptor.

It is a kind of by give the method that fusion rotein increases this main body inner acidic alpha-glucosidase activity of suffering from Pompe to main body that another aspect of the present invention provides, and this fusion rotein comprises acid alpha-Glucosidase (GAA) (the perhaps fragment of people GAA) of people and lysosome targeting domain.This lysosome targeting domain in non-Man-6-P dependent form mode in conjunction with people's non-cationic dependent form Man-6-P receptor.

Another aspect of the present invention provides a kind of pharmaceutical composition that is applicable to the treatment Pompe.This pharmaceutical composition comprises the fusion rotein for the treatment of effective dose, and this fusion rotein comprises acid alpha-Glucosidase (GAA) (the perhaps fragment of people GAA) of people and lysosome targeting domain.This lysosome targeting domain in non-Man-6-P dependent form mode in conjunction with people's non-cationic dependent form Man-6-P receptor.

In one embodiment, lysosome targeting domain comprises ripe human insulin-like growth factor II (IGF-II), or becomes acquaintance's IGF-II fragment or sequence variants.In one embodiment, lysosome targeting domain comprises into the aminoacid 8-67 of acquaintance IGF-II.Preferably, lysosome targeting domain comprises into the amino acid/11 of acquaintance IGF-II and 8-67 (acquaintance GAA Δ 2-7).In another preferred embodiment, fusion rotein comprises the aminoacid 70-952 of people GAA.

In another embodiment, fusion rotein comprises the aminoacid 70-952 and amino acid/11 that becomes acquaintance IGF-II and 8-67 (that is, becoming the Δ 2-7 of acquaintance GAA) of people GAA.In another embodiment, fusion rotein further is included in into the fragment (amino acid/11 and 8-67) of acquaintance IGF-II and the intervening sequence Gly-Ala-Pro between the people GAA fragment (aminoacid 70-952).

In one embodiment, than wild type people GAA, be applicable to that the fusion rotein of this aspect of the present invention has Man-6-P (M6P) level of reduction on this protein surface.In another embodiment, the fusion rotein that is applicable to this aspect of the present invention does not have functional M6P level on this protein surface.

In another embodiment, this pharmaceutical composition comprises pharmaceutical carrier.

As used in this application, term " people acid alpha-Glucosidase (GAA) " is meant the precursor wild type form of people GAA or can reduces the functional variant that one or more Pompe symptoms maybe can be saved or alleviate to glycogen levels in the mammal lysosome.

As used in this application, term " about " and " approximately " are equal to the replacement use.In this application be with or without approximately or about situation under any numeral of using all mean and contain any normal fluctuation range that various equivalent modifications is understood.

Will become clearly in other features of the present invention, purpose and the advantage detailed description hereinafter.Yet, should be appreciated that although pointed out the specific embodiment of the present invention, these detailed descriptions only are that the mode that illustrates by way of example provides, but not limit.According to following detailed description, to those skilled in the art, the variations and modifications in the scope of the invention will become apparent.

Description of drawings

Accompanying drawing only is for illustrational purpose, and nonrestrictive.

Fig. 1 shows the sketch map of the GAA ZC-701 of GILT-labelling.

Fig. 2 A-C shows SDS-PAGE and the Western blotting of the GAAZC-701 of unlabelled GAA of wild type and GILT-labelling.Fig. 2 A shows the SDS-PAGE before the silver dyeing.Fig. 2 B shows the Western blotting that uses anti--GAA antibody.Fig. 2 C shows the Western blotting that uses anti--IGF-II antibody.

Fig. 3 A shows the sketch map of p1288 and p1355, and p1288 and p1355 are two kinds and contain the biotinylation of wild type CI-MPR domain 10-13 and point mutation variant and the recombiant protein of His-labelling respectively.

Fig. 3 B has described the expression by silver painted 1288 and 1355.

It is interactional that Fig. 4 A-B has described the GAA ZC-701 and the CI-MPR of GILT-labelling

The example results of analyzing.Fig. 4 A has described the exemplary combination curve of IGF-II.Fig. 4 B has described the exemplary combination curve of the GAA ZC-701 of GILT-labelling.

Fig. 5 has described the labelling of the GAA ZC-701 of GILT-labelling-dependent form picked-up has been entered the myoblastic example results of rat L6.

GAA ZC-701 and the unmarked GAA picked-up of wild type that Fig. 6 has described purification GILT-labelling enter the myoblastic exemplary saturation curve of rat L6.

Fig. 7 has described the GAA ZC-701 and the example results of the half-life of the unmarked GAA of wild type (ZC-635) in rat L6 sarcoplast of reflection GILT-labelling.

Fig. 8 A-B is presented at picked-up to enter the rat L6 sarcoplast exemplary Western blotting of the Proteolytic enzyme processing of the GAA ZC-701 of GILT-labelling afterwards.Fig. 8 A is the exemplary Western blotting that is presented at the GILT label forfeiture of picked-up back.Fig. 8 B is that the GAA that is presented at picked-up back wild type and GILT-labelling enters the exemplary Western blotting of the processing in the various peptide species.

Fig. 9 has described the example results of the serum half-life in wild type 129 mices of the GAA ZC-701 that is reflected in the GILT-labelling that produces in three different tissues culture medium.Red line is corresponding to PF-CHO culture medium, t1/2=43min; The orange line is corresponding to CDM4 culture medium, t1/2=38min; And green line is corresponding to the CD17 culture medium, t1/2=52min.

Figure 10 A-D has described the exemplary decay curve in the various tissues of Pompe mice of GAA ZC-1026 of the GAA ZC-701 of the unlabelled GAA of wild type (ZC-635), GILT-labelling and GILT-labelling.Figure 10 A has described the exemplary decay curve in the musculus quadriceps tissue.Figure 10 B has described the exemplary decay curve in heart tissue.Figure 10 C has described the exemplary decay curve in diaphragm tissue.Figure 10 D has described the exemplary decay curve in liver organization.

Figure 11 has described the colocated (co-localization) of the GAA and the lysosome label LAMP1 of GILT-labelling.

Figure 12 has described the example results that confirms to remove glycogen in the heart tissue sample of taking from the Pompe mice of the GAA albumen ZC-701 of single injection GILT-labelling or unlabelled GAA treatment.

Figure 13 A-H is presented at after the GAA ZC-701 of injection unmarked GAA of wild type or GILT-labelling, removes the exemplary graph of glycogen in the various muscular tissues of Pompe mice.

Figure 14 shows the detail flowchart of clinical research program.

The specific embodiment

The invention provides the method and composition that is used for the treatment of Pompe based on non-glycosylated dependent form lysosome targeting technology (GILT).Particularly, the invention provides by acid alpha-Glucosidase targeting lysosome being used for the treatment of the method and composition of Pompe in non-Man-6-P-dependent form mode.

Various aspects of the present invention will at length be described with the lower part.Use these parts and do not mean that restriction the present invention.Each part can be applicable to any aspect of the present invention.In this application, unless otherwise noted, the meaning of " or " all be meant " and/or " of use.

Pompe

Pompe is a kind of rare hereditary, is because to lack the acid alpha-Glucosidase (GAA) need be used to destroy glycogen (a kind of storage form that is used for the sugar of energy) in the enzyme caused.Pompe is also referred to as glycogen storage disease II type (GSD II), II type glycogen storage disease, glycogenosis II type, acid maltase deficiency, α-1,4-glucosidase deficiency disease, the heart disease form of diffusivity glycogen megalocardia (cardiomegalia glycogenic diffusa) and generalized glycogenosis.The gathering the gradual myasthenia (myopathy) that causes whole health and influence each soma, particularly heart, skeletal muscle, liver, breathing and nervous system of glycogen.

The clinical manifestation that Pompe presents is active and extensively change according to age of onset and residual GAA.Residual GAA is active relevant with the severity of the cumulative quantity of glycogen and tissue distribution and this disease.It is the most serious form that the children sends out a property Pompe (being lower than normal GAA active 1%), and it is characterized in that tension force is low excessively, general myasthenia and hypertrophic cardiomyopathy, and a large amount of glycogens accumulations that in heart and other muscular tissue, occur.Because the heart respiratory disorder, so can just take place in back 1 year dead in birth usually.Hirschhorn?et?al.(2001)″Glycogen?Storage?Disease?Type?II：Acid?Alpha-glucosidase(Acid?Maltase)Deficiency，″in?Scriver?et?al.，eds.， The?Metabolic?and Molecular?Basis?of?Inherited?Disease.8th?Ed.，New?York：McGraw-Hill，3389-3420。Blue or green few phase-ictal (normal GAA active 1%～10%) and the manhood-ictal (normally GAA active 10%～40%) Pompe is clinical more multiple class, has bigger variation aspect age of onset, clinical manifestation and the progression of disease.Ictal and ictal Pompe general characteristic of manhood of blue or green few phase be lack that serious heart is got involved, more late age of onset and slower progression of disease,, but finally breathe or limb flesh is got involved and caused significant M ﹠ M.Although life expectancy can change, dead generally meeting is owing to respiratory failure takes place.Hirschhorn?et?al.(2001)“Glycogen?StorageDisease?Type?II：Acid?Alpha-glucosidase(Acid?Maltase)Deficiency，”inScriver?et?al.，eds.， The?Metabolic?and?Molecular?Basis?of?Inherited Disease.8th?Ed.，New?York：McGraw-Hill，3389-3420。

Algucerase

Algucerase (ERT) is that a kind of enzyme that lacks by perfusion in blood flow is corrected enzymoprivic therapeutic strategy.As the patient tissue of hemoperfusion, enzyme is by cellular uptake and be transported to lysosome, wherein play a role and remove since azymia and in lysosome cumulative material.In order to make the lysosomal enzyme alternative medicine effective, treatment must be delivered to the lysosome in the suitable cell that shows the tissue that stores defective therein with enzyme.Traditional lysosomal enzyme alternative medicine adopts and naturally is connected to that proteic carbohydrate sends to cooperate the lip-deep special receptor of target cell.A kind of receptor, non-cationic dependent form M6P receptor (CI-MPR) is specially adapted to targeting and substitutes lysosomal enzyme, because CI-MPR is present on the surface of most cells type.

Term " non-cationic dependent form Man-6-P receptor (CI-MPR) ", " M6P/IGF-II receptor " and " CI-MPR/IGF-II receptor " can exchange use in this article, are meant the two the cell receptor in conjunction with M6P and IGF-II.

Non-glycosylated dependent form lysosome targeting

The present invention has developed a kind of non-glycosylated effect dependent form lysosome targeting (GILT) technology, is used for using the enzyme targeting in lysosome treatment.Particularly, the present invention has adopted peptide tag to replace M6P to be used for the CI-MPR of lysosome targeting with cooperation.Typically, the GILT label is a kind of with albumen, peptide or other part of non-Man-6-P dependent form mode in conjunction with CI-MPR.Advantageously, this technical modelling the standard biological of picked-up lysosomal enzyme learn mechanism, also carry out in non-Man-6-P dependent form mode.

Preferred GILT label derives from human insulin-like growth factor II (IGF-II).People IGF-II is the high-affinity part of CI-MPR, and it is also referred to as the IGF-II receptor.The treatment of GILT-labelling makes this albumen via endocytosis approach targeting lysosome with enzyme and combining of M6P/IGF-II receptor.This method has lot of advantages with respect to relating to glycosylated method, comprises simple and cost effectiveness, in case because albumen is separated, just no longer need to do further modification.

The detailed description of GILT technology and GILT label can find in the open No.20030082176,20040006008,20040005309 and 20050281805 of the U.S., and its full content is hereby expressly incorporated by reference.

The GAA of GILT-labelling

A box by the appropriate GILT label of will encoding is fused to the GAA-coded sequence, the invention provides a kind of GAA of GILT-labelling, and it can be with high-affinity in conjunction with CI-MPR, and does not rely on the M6P content on this albumen.In addition, the invention provides a kind of GAA preparation, wherein each enzyme molecule has the high-affinity part that is used for CI-MPR.As described in the embodiment part, by

Analyze, the GAA of GILT-labelling has high-affinity to CI-MPR, and more effective than traditional lysosomal enzyme alternative medicine from treatment in vivo.

The excellent usefulness of the GAA of GILT-labelling provides many clinical benefits.The usefulness that increases will cause simply identical or more favourablely under the low dosage clinically prejudge.The GAA of GILT-labelling can more effectively be delivered to a plurality of tissues that are subjected to sickness influence.For example, the GAA of GILT-labelling can have to sending that skeletal muscle increases, especially under lower dosage.Thereby the usefulness that increases also can be permitted harmful situation that the enough low patient of minimizing of dosage often suffers, and reduces the production of antibodies at patient's drug disposition.The usefulness that increases also can permit utilizing the therapeutic scheme of the interval that increases between inculcating.

One preferred embodiment in, the GAA of GILT-labelling comprises people GAA or its fragment or the sequence variants of the ability that keeps the α 1-4 keyed jointing in the linear oligosaccharide of cutting, and with the lysosome targeting domain of non-Man-6-P dependent form mode in conjunction with people CI-MPR.Suitable lysosome targeting domain comprises sophisticated people IGF-II or its fragment or sequence variants.

IGF-II is targeting CI-MPR specifically preferably.Useful especially is sudden change in the IGF-II polypeptide, and it causes producing with the albumen of high-affinity in conjunction with CI-MPR, simultaneously no longer with obvious affinity in conjunction with other IGF-II receptor.IGF-II also can modify to minimize with serum I GF-is protein-bonded and combine (Baxter (2000) Am.L PhysiolEndocrinol Metab.278 (6): 967-76), thereby avoid the chelating of IGF-II/GILT construct.Many researchs all be positioned at for the essential IGF-II of igf binding protein in residue.The construct that has sudden change at these residue places can screen maintenance to the high-affinity of M6P/IGF-II receptor in conjunction with and reduce the protein-bonded affinity of IGF-.For example, it is reported that the Phe 26 that substitutes IGF-II with Ser can reduce the affinity of IGF-II to IGFBP-1 and IGFBP-6, and can not influence combination (Bach etal. (1993) J.Biol.Chem.268 (13): 9246-54) the M6P/IGF-II receptor.Other substitutes, and for example Lys substitutes Glu 9, also can be favourable.Utilize the similar sudden change in the zone of IGF-I of IGF-II high conservative, independent or combination, cause bonded (Magee et al. (1999) Biochemistry 38 (48): 15863-70) that reduces greatly of IGF-BP.

A kind of method of replacing is to identify the Minimum Area that can be attached to the IGF-II of M6P/IGF-II receptor with high-affinity.In the IGF-II that is attached to the M6P/IGF-II receptor implicit residue great majority all on the face of IGF-II cluster (Terasawa et al. (1994) EMBO is (23) J.13: 5590-7).Although the IGF-II tertiary structure is generally kept by three intramolecular disulfide bonds, the peptide that is incorporated into the lip-deep aminoacid sequence of M6P/IGF-II receptors bind of IGF-II can be designed to correctly fold and have in conjunction with active.This minimum binding peptide is highly preferred lysosome targeting domain.For example, preferred lysosome targeting domain is exactly the aminoacid 8-67 of people IGF-II.Based on the zone around the aminoacid 48-55, the designed peptide in conjunction with the M6P/IGF-II receptor also is desired lysosome targeting domain.Alternately, can the random library of peptide be screened, with screening via twice hybridization assays of yeast or the ability measured via the phage display type in conjunction with the M6P/IGF-II receptor.

The GILT label can be fused to the N-end or the C-end of GAA polypeptide.The GILT label can be fused to directly that the GAA polypeptide maybe can pass through bonding pad (linker) or spacer (spacer) separates with the GAA polypeptide.The aminoacid bonding pad is incorporated into and is different from the aminoacid sequence that occurs in this position of native protein, and is usually designed to and is flexible or inserts structure, for example a α-Luo Xuanjiegou between two protein parts.The bonding pad can be relatively short, and for example sequence Gly-Ala-Pro or Gly-Gly-Gly-Gly-Gly-Pro perhaps can be long, for example 10～25 amino acid whose length.The position of merging joint should conscientiously be selected promoting the correct folding and active of fusion partner, and prevents peptide tag and GAA polypeptide premature disengagement.In a preferred implementation, the bonding pad sequence is Gly-Ala-Pro.

Proteic other construct of GAA that can be used in the GILT-labelling in the inventive method and the compositions is described in detail in the open No.20050244400 of the U.S., and its full content is hereby expressly incorporated by reference.

The GAA of GILT-labelling can express in various mammal cell lines, includes but not limited to human embryo kidney (HEK) (HEK) 293, Chinese hamster ovary (CHO), monkey kidney (COS), HT1080, C10, HeLa (draw in the sea), hamster baby kidney (BHK), 3T3, C127, CV-1, HaK, NS/O and L-929 cell.The GAA of GILT-labelling also can express in various nonmammalian host cells, for example insecticide (for example Sf-9, Sf-21, Hi5), plant are (for example, leguminous plant (Leguminosa), cereal or tobacco plant), yeast (for example, saccharomyces cerevisiae (S.cerivisae), methanol type yeast (P.pastoris)), prokaryote (for example, escherichia coli (E.coli), bacillus subtilis (B.subtilis) and other Bacillus, Rhodopseudomonas, streptomyces) or fungus.

In some embodiments, the GAA of GILT-labelling can utilize short secreting signal peptide to produce, to help the secretion of fusion rotein.For example, the GAA of GILT-labelling can utilize the IGF-II signal peptide to produce.Generally speaking, compare, utilize the GAA of the GILT-labelling of IGF-II signal peptide production on this protein surface, to have mannose-6 phosphoric acid (M6P) level of reduction with wild type people GAA.As indicated in the embodiment part, oligosaccharide analysis that connects by N-and functional picked-up measured and confirmed, do not have detectable M6P in exemplary treatment of the present invention on fusion rotein.

The enzyme-specific activity that GILT-GAA of the present invention has usually is about 150,000～600, in the proteic scope of 000nmol/h/mg, preferably about 250,000～500, in the proteic scope of 000nmol/h/mg.In one embodiment, it is about 150 that the enzyme-specific activity that GAA has is at least, 000nmol/h/mg albumen; Preferably, it is about 300 that the enzyme-specific activity is at least, 000nmol/h/mg albumen; More preferably, it is about 400 that the enzyme-specific activity is at least, 000nmol/h/mg albumen; And even preferably, it is about 600 that the enzyme-specific activity is at least, 000nmol/h/mg albumen.GAA is active in the GAA4MU unit definition.

The treatment of Pompe

Method of the present invention treatment by children's property sent out, the blue or green few phase is ictal or ictal Pompe invasion and attack of manhood individual aspect be equivalent.Usually, herein the Treatment and composition for of Miao Shuing treatment suffer from the blue or green few phase ictal-or ictal Pompe of manhood individual aspect may be more effective, because these individualities have higher remaining GAA activity level (being respectively 1%～10% and 10%～40%), therefore, may have more immunologic tolerance to the GAA of giving GILT-labelling.Do not expect to be bound by theory, these patients generally are intersection reactive immune material (CRIM)-positive for endogenous GAA.Therefore, their immune system may not can partly be identified as " external " albumen with the GAA of the GAA of GILT-labelling, and can not form at the antibody to the GAA part of the GAA of GILT-labelling.

As used in this article, term " treatment " or " processing " are meant one or more symptoms of improving with this disease association, prevent or postpone the outbreak of these one or more symptoms of disease, and/or reduce the severity or the frequency of these one or more symptoms of disease.For example, the improvement that treatment can be meant heart state (for example, the increase of end-diastolic dimension and/or end-systolic volume, the perhaps myocardiac reduction of progressivity, improvement or the prevention of usually in Pompe, finding) or the improvement of pulmonary function (for example, crying vital capacity increases than baseline vital capacity, and/or oxygen desaturation standardization during crying); Improvement aspect neurodevelopment and/or acrobatics and tumblings (for example, aspect the AIMS scoring, increasing); Reduced by the glycogen levels in the patient tissue of this disease infringement; Perhaps any combination of these effects.One preferred embodiment in, treatment comprises the improvement that glycogen is removed, and especially reduces or the relevant cardiomyopathy of prevention Pompe.As used in this article, term " improvement ", " increase " or " reduction " are meant the value with respect to baseline measures, example is treated the measured value in the beginning same individual before as described in this article, perhaps the measured value in the contrast individuality under not having treatment described herein (perhaps a plurality of contrasts are individual)." contrast individual " is and the individuality of the individual Pompe of suffering from same form (children sends out few phase property, blue or green paroxysmal or the manhood is ictal) treat by institute, its with the institute Individual Age for the treatment of roughly the same (with guarantee to be treated individuality be suitable with the disease stage that contrasts in the individuality).

The individuality of being treated (being also referred to as " patient " or " main body ") is to suffer from Pompe (promptly children's property sent out, blue or green few paroxysmal or ictal Pompe of manhood of phase) or have to develop into the possible individuality of Pompe (fetus, baby, child, teenager or adult).Individual can have remaining endogenous GA A activity, perhaps do not have measurable activity.For example, suffers from the GAA activity that the individuality of Pompe can have, for being lower than about 1% normal GAA activity (promptly usually with children's relevant GAA activity of property Pompe), being active about 1%～10% (the promptly common and blue or green few relevant GAA activity of ictal Pompe of phase) of normal GAA, perhaps is active about 10%～40% (the promptly common GAA activity relevant with ictal Pompe of manhood) of normal GAA.Individuality can be that endogenous GAA is the CRIM-positive or CRIM-feminine gender.In one embodiment, individuality is the CRIM-positive to endogenous GAA.In another embodiment, individuality is the individuality that diagnosis recently suffers from this disease.Early treatment's (after diagnosis just immediately begin treatment) as much as possible is very important for influence that minimizes disease and maximization treatment benefit.

The administration of the GAA of GILT-labelling

In the method for the invention, the GAA of GILT-labelling normally gives individuality separately, perhaps with the compositions of the GAA that comprises the GILT-labelling or medicament administration (for example in the medicament of this disease treatment is produced), as described in this article.Compositions enough physiology's acceptable carriers of energy or excipient are prepared to make pharmaceutical composition.Carrier and compositions can be aseptic.Preparation should be suitable for administering mode.

Suitable pharmaceutical carrier includes but not limited to water, saline solution (for example NaCl), saline, buffer saline, alcohol, glycerol, ethanol, Radix Acaciae senegalis, vegetable oil, benzylalcohol, Polyethylene Glycol, gelatin, carbohydrate such as lactose, amylose or amylopectin, sugar is as mannose, sucrose or other sugar, glucose, magnesium stearate, Talcum, silicic acid, viscous paraffin, aromatic oil, fatty acid ester, hydroxy methocel, polyvinylpyrrolidone etc., and their combination.If desired, pharmaceutical preparation can mix (for example lubricant, antiseptic, stabilizing agent, wetting agent, emulsifying agent, the salt that influences osmotic pressure, buffer agent, coloring agent, fragrance and/or fragrance matter etc.) with auxiliary agent, and auxiliary agent can not react or disturb its activity nocuously with reactive compound.One preferred embodiment in, adopted the water-solubility carrier that is applicable to intravenous administration.

If desired, compositions or medicament also can contain a spot of wetting agent or emulsifying agent, perhaps the pH buffer agent.Compositions can be liquid solution, suspension, emulsion, tablet, pill, capsule, slow releasing preparation or powder.Compositions also can be mixed with suppository with traditional binding agent and carrier such as triglyceride.Oral formulations can comprise standard vector such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharinate, cellulose, magnesium carbonate etc.

Compositions or medicament can be mixed with the pharmaceutical composition that is applicable to human administration according to conventional program.For example, a preferred implementation, the compositions that is used for intravenous administration is normally at the solution of sterile isotonic water-containing buffering liquid.If desired, compositions also can comprise solubilizing agent and local anesthetic, to alleviate the pain of injection site.Generally speaking, each component can provide separately or mix with unit dosage forms, for example, as the airtight sealing container as the indication activating agent amount ampoule bottle or the dry freeze-dried powder among the sachette or do not have aqueous concentrate.By the perfusion administration composition, it can be disperseed with the perfusion bottle that contains aseptic pharmaceutical grade water, saline or glucose/water if desired.By the drug administration by injection compositions, sterilized water that can be provided for injecting or brinish ampoule bottle are so that each component can be mixed before administration if desired.

The GAA of GILT-labelling can be mixed with neutrality or salt form.Pharmaceutical salts comprises the salt that those and free amine group form, for example be derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid etc., the salt that forms with those and free carboxy, for example be derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, hydrated ferric oxide., 2-aminopropane., triethylamine, 2-ethyl amido alcohol, histidine, procaine etc.

The GAA of GILT-labelling (compositions or the medicament that perhaps contain the GAA of GILT-labelling) is by any suitable path administration.One preferred embodiment in, the GAA of GILT-labelling is via intravenous administration.In other embodiments, the GAA of GILT-labelling is administered directly to target tissue, and for example heart or muscle (for example intramuscular) or nervous system (for example directly inject brain; In the ventricle; In the sheath).Alternately, the GAA of GILT-labelling (or contain the GAA of GILT-labelling compositions or medicament) can or stride mucosa delivery (for example, oral or nasal meatus) via parenteral administration, percutaneous dosing.If desired, can adopt multiple path simultaneously.

The GAA of GILT-labelling (or contain the GAA of GILT-labelling compositions or medicament) can be individually dosed, perhaps unite other reagent, as hydryllin (for example diphenhydramine (diphenhydramine)) or immunosuppressant or other antagonism anti--immunotherapeutic agent of the GAA antibody of GILT-labelling.Term " associating " be meant reagent before the GAA of GILT-labelling (or contain the GAA of GILT-labelling compositions or medicament) administration, carry out administration in the administration or after the administration.For example, reagent may be mixed in in the compositions of the GAA that contains the GILT-labelling, thus with GILT-labelling GAA administration simultaneously; Alternately, reagent can not mix and administration simultaneously (for example, send by " piggybacking " of the reagent on the intravenous route, the also GAA of administration GILT-labelling in this way, perhaps vice versa).In another embodiment, reagent can separate administration (for example, without blending), but is in the short duration of the GAA of GILT-labelling administration (for example in the 24h).One preferred embodiment in, if individual endogenous GAA is the CRIM-feminine gender, then the GAA of GILT-labelling (or contain the GAA of GILT-labelling compositions or medicament) be designed for the GAA antibody quantity that reduces anti--GILT-labelling or stop the immunosuppressant scheme of its generation or immunization therapy scheme associating and carry out administration.For example, be similar to scheme (the Nilsson et al (1988) that is used for hemophiliac N.Engl.J.Med., 318:947-50) can be used to reduce the GAA antibody of anti--GILT-labelling.But this scheme also can be used for the be positive individuality of the GAA antibody danger that GAA antibody with anti--GILT-labelling or existence have anti--GILT-labelling to endogenous GAA.In a particularly preferred embodiment, immunosuppressant scheme or immunization therapy scheme began before the GAA of GILT-labelling first administration, so that minimize the probability of the GAA antibody that produces anti--GILT-labelling.

The GAA of GILT-labelling (or contain the GAA of GILT-labelling compositions or medicament) with the treatment effective dose (promptly when the time with the administration of rule interval, be enough to treat the dosage of this disease, for example by the symptom of improvement with this disease association, prevent or postpone the outbreak of this disease, and/or also reduce the severity or the frequency of this disease, as mentioned above) carry out administration.Be used for the treatment of the nature and extent that the effective dosage of this treatment of diseases will depend on this sickness influence, and can determine by standard clinical techniques.In addition, body is interior or external test can adopt alternatively to help to determine best dosage range, for example following the examples given dosage.The exact dose that adopts also depends on the administration path, and severity of disease, and should judge with each patient's the patient's condition according to the doctor and determine.Effective dose can be inferred by external or the deutero-dose response curve of animal model test system.The treatment effective dose can for example be about 0.1～1mg/kg, about 1～5mg/kg, about 5～20mg/kg, about 20-50mg/kg or 20～100mg/kg.Effective dose for particular individual can change (for example, increase or reduce) in time, and these needs according to individuality are decided.For example, body illness or stress time in, if the GAA antibody of perhaps anti--GILT-labelling begins to occur or increase, if perhaps disease symptoms worsens, then dosage can increase.

The treatment effective dose of the GAA of GILT-labelling (or contain the GAA of GILT-labelling compositions or medicament) is with the administration of rule interval, and this depends on the nature and extent of sickness influence, and depends on occurent basis.As used in this article, be meant with certain " interval " administration and periodically treat effective dose (this is different from disposable dosed administration).Interval can be determined by standard clinical techniques.In preferred embodiment, the administration bimester that the GAA of GILT-labelling can be per, administration in every month, twice administration in every month, per three all administrations, per two all administrations, administration weekly, twice administration weekly, time administration or administration every day on every Wendesdays.Administration time for single individuality needs not to be regular time at interval at interval, but can change in time, and this depends on individual required.For example, body illness or stress time in, if the GAA antibody of anti--GILT-labelling begins to produce or increase, if perhaps disease symptoms worsens, then the interval between the dosage just may reduce.

As used in this article, term " per bimester administration " is meant per two months and is administered once (bimester of being per 1 time); Term " administration in every month " is meant every month and is administered once; Term " per three all administrations " is meant and is administered once in per three week (be per three week 1 time); Term " per two all administrations " is meant and is administered once per fortnight (be per two week 1 time); Term " administration weekly " is meant that each week is administered once; And term " administration every day " is meant that be administered once every day.

The present invention relates in addition and is a kind ofly having the container of label (liquid medicine bottle for example, vial, the bag that is used for intravenous administration, syringe etc.) pharmaceutical composition as described herein in, container are equipped with the administration (for example by method as herein described) that the is used for said composition description with the treatment Pompe.

The present invention will further and more specifically be described by following examples.Yet embodiment is just for illustrational purpose, but not limitation of the present invention.

Embodiment

Embodiment 1: the production of the GAA of reorganization wild type GAA and GILT-labelling

Plasmid

The DNA of the wild type people GAA of separation coding total length also is inserted into the expression vector that is used for production recombined human GAA.The DNA box (hereinafter referred to as " box 635 ") of complete people GAA amino acid/11-952 of encoding derives from IMAGE clone body 4374238 (OpenBiosystems), wherein utilizes following PCR primer to derive:

GAA13:5 '-GGAATTCCAACCATGGGAGTGAGGCACCCGCCC (SEQ ID NO:1) and

GAA27：5’-GCTCTAGACTAACACCAGCTGACGAGAAACTGC(SEQ?ID?NO：2)。

Box 635 utilizes EcoRI and XbaI digestion, by handling and passivation with Ke Lunnuo (Klenow) archaeal dna polymerase, and the HindIII site after colligation is handled to the Ke Lunnuo that expresses vehicle pCEP4 (Invitrogen) then, thus produce plasmid p635.Hereinafter, ZC-635 is meant the unlabelled GAA albumen of wild type.

The DNA box (hereinafter referred to as " box 701 ") that is used to produce the GAA ZC-701 of reorganization GILT-labelling is similar to box 635 and is prepared, and following N-terminal sequence just, its joint are corresponding to the upstream of the GAA sequence of aminoacid A70:

GAATTCACACCAATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCCGTCGCAGCCGTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCG(SEQ?ID?NO：3)。

Box 701 utilizes EcoRI and XbaI digestion, by handling and passivation with the Ke Lunnuo archaeal dna polymerase, and the HindIII site after colligation is handled to the Ke Lunnuo of expression vector pCEP4 then, thus produce plasmid p701.Hereinafter, ZC-701 is meant the GAA albumen by the plasmid-encoded GILT labelling of p701.Fig. 1 shows the diagram of the GAA ZC-701 of GILT-labelling, the IGF-II signal peptide of forfeiture when being included in secretion.Therefore, with excretory form (that is, because its will be administered into main body), ZC-701 comprises the aminoacid 70-952 of the amino acid/11 of people IGF-II and 8-67 (acquaintance IGF-II Δ 2-7), intervening sequence Gly-Ala-Pro and people GAA.Below show the aminoacid sequence of total length.Intervening sequence is added with underscore.The amino acid/11 of the terminal reflection of the sequence N-of intervening sequence people IGF-II and 8-67 (arrow points amino acid/11) and sequence C-end of intervening sequence reflects the aminoacid 70-952 of people GAA.

↓

MGIPMGKSMLVLLTFLAFASCCIAALCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSE GAPAHPGRPRAVPTQCDVPPNSRFDCAPDKAITQEQCEARGCCYIPAKQGLQGAQMGQPWCFFPPSYPSYKLENLSSSEMGYTATLTRTTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRRYEVPLETPRVHSRAPSPLYSVEFSEEPFGVIVHRQLDGRVLLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPIEALGSLPPPPAAPREPAIHSEGQWVTLPAPLDTINVHLRAGYIIPLQGPGLTTTESRQQPMALAVALTKGGEARGELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRVTSEGAGLQLQKVTVLGVATAPQQVLSNGVPVSNFTYSPDTKVLDICVSLLMGEQFLVSWC(SEQ?ID?NO：4)

The GAA box (ZC-1026) of the 2nd GILT-labelling makes up similarly.ZC-1026 comprises the amino acid/11 of people IGF-II and the aminoacid 70-952 of 8-67, intervening sequence Thr-Gly and people GAA.

These plasmids are used for the suspension HEK293 cell that transient transfection is used to produce recombiant protein.Description according to manufacturer (Invitrogen) is arrived suspension FreeStyle with plasmid transfection ^TMIn the 293-F cell.In brief, the Opti-of cell in Merlon vibration bottle

Be positioned over orbital shaker in the I medium (Invitrogen) in 37 ℃ and 8% CO ₂Under grow.Cell concentration is adjusted to 1 * 10 ⁶Individual cell/mL is 1: 1: 1 ml cell according to the description ratio of manufacturer (Invitrogen): μ g DNA: μ l 293fectin then ^TMCarry out transfection.Culture is gathered in the crops after 5～10 days in transfection, and removes cell by centrifugal and 0.2 μ m bottle top filter filtration.Supernatant stores down in-80 ℃.

Alternately, box 701 introducings (integration) are arrived

Retroviral vector (retrovector) expression system (Cardinal Health).This method is described in U.S. Patent No. 6,852, and in 510, its disclosure is hereby expressly incorporated by reference.Contain box 701

The retroviral vector expression system is used for being formed for producing the stable Chinese hamster ovary celI system of the GAA of reorganization GILT-labelling.Box 701 also can be incorporated into

Retroviral vector expression system and being used for is formed for producing the stable HEK293 cell line of the GAA of reorganization GILT-labelling.

The purification of the GAA of GILT-labelling

Initiation material is the mammalian cell cultures supernatant, as described above, thaws under-80 ℃ storage.Add sodium acetate (pH 4.6) so that ultimate density reaches 100mM, and add ammonium sulfate so that ultimate density reaches 0.75M.Material is centrifugal to remove precipitation and with 0.8/0.2 μ m AcroPak ^TM500 pod membranes (Pall, catalog number (Cat.No.) #12991) filter.

Material after the filtration is loaded into HIC and loads buffer (50mM NaAc pH 4.6,0.75M AmSO ₄) preparation Phenyl-SepharoseTM 6 Low-Sub Fast-Flow (GEHealthcare) posts.HIC dcq buffer liquid (50mM NaAc pH5.3,0.75M AmSO with 10 column volumes ₄) the flushing pillar, and with HIC elution buffer (50mM NaAc pH 5.3, the 20mM AmSO of 5 column volumes ₄) eluting.

The fraction that merges extensively carries out dialysis QXL and loads in the buffer (20mM histidine pH 6.5,50mM NaCl), is loaded into then on the Q SepharoseTM XL post (GE Healthcare).With the flushing of the QXL level pad of 10 column volumes, and with QXL elution buffer (20mM histidine pH 6.5, the 150mM NaCl) eluting of 10 column volumes.In some cases, being concentrated into protein concentration from the merging fraction of QXL post is 30～40mg/mL, is loaded into then in PBS pH 6.2 on equilibrated 2.6 * 90cm Ultrogel AcA, 44 posts.Loaded volume is 5～7.5mL (column volume 1%～1.5%).Speed with 0.4mL/min in PBS pH 6.2 is walked post, and collects the 4mL fraction.

The unmarked GAA of purification and the GAA of GILT-labelling illustrate at Fig. 2 A～C.Fig. 2 A shows the SDS-PAGE before the silver dyeing.Fig. 2 B shows the Western blotting that utilizes anti--GAA antibody.Fig. 2 C shows the Western blotting that utilizes anti--IGF-II antibody.

The GAA of embodiment 2:GILT-labelling is to the affinity of CI-MPR

The GAA ZC-701 of GILT-labelling is to the binding affinity utilization of CI-MPR

Surface plasma resonance is measured (surface plasmon resonance assay) and is determined.According to the standard molecule technology, form the recombiant protein (containing wild type CI-MPR domain 10-13 and a point mutation variant) of two biotinylations and His-labelling respectively.The sketch map of two recombiant proteins as shown in Figure 3A.And then the IGF-II signal peptide that plasmid p1288 contains has: poly His (poly-His) label; Biotin AS domain; And the sequence of encoding wild type CI-MPR domain 10-13.Plasmid p1355 contains the IGF-II signal peptide, and then has: poly His (poly-His) label; Biotin AS domain; And coding has the sequence of effective reduction receptor to the CI-MPR domain 10-13 of the point mutation I1572T of the affinity of IGF-II.Below show specific DNA and the aminoacid sequence relevant with these two recombiant proteins.

HIS-biotin-CI-MPR domain 10-13

ATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTGGCGCGCCGACCGGTCACCATCACCATCACCACGCGCCGGGCCTGAACGACATCTTCGAGGCCCAGAAGATCGAGTGGCACGAACCTTTCGATCTGACTGAATGTTCATTCAAAGATGGGGCTGGCAACTCCTTCGACCTCTCGTCCCTGTCAAGGTACAGTGACAACTGGGAAGCCATCACTGGGACGGGGGACCCGGAGCACTACCTCATCAATGTCTGCAAGTCTCTGGCCCCGCAGGCTGGCACTGAGCCGTGCCCTCCAGAAGCAGCCGCGTGTCTGCTGGGTGGCTCCAAGCCCGTGAACCTCGGCAGGGTAAGGGACGGACCTCAGTGGAGAGATGGCATAATTGTCCTGAAATACGTTGATGGCGACTTATGTCCAGATGGGATTCGGAAAAAGTCAACCACCATCCGATTCACCTGCAGCGAGAGCCAAGTGAACTCCAGGCCCATGTTCATCAGCGCCGTGGAGGACTGTGAGTACACCTTTGCCTGGCCCACAGCCACAGCCTGTCCCATGAAGAGCAACGAGCATGATGACTGCCAGGTCACCAACCCAAGCACAGGACACCTGTTTGATCTGAGCTCCTTAAGTGGCAGGGCGGGATTCACAGCTGCTTACAGCGAGAAGGGGTTGGTTTACATGAGCATCTGTGGGGAGAATGAAAACTGCCCTCCTGGCGTGGGGGCCTGCTTTGGACAGACCAGGATTAGCGTGGGCAAGGCCAACAAGAGGCTGAGATACGTGGACCAGGTCCTGCAGCTGGTGTACAAGGATGGGTCCCCTTGTCCCTCCAAATCCGGCCTGAGCTATAAGAGTGTGATCAGTTTCGTGTGCAGGCCTGAGGCCGGGCCAACCAATAGGCCCATGCTCATCTCCCTGGACAAGCAGACATGCACTCTCTTCTTCTCCTGGCACACGCCGCTGGCCTGCGAGCAAGCGACCGAATGTTCCGTGAGGAATGGAAGCTCTATTGTTGACTTGTCTCCCCTTATTCATCGCACTGGTGGTTATGAGGCTTATGATGAGAGTGAGGATGATGCCTCCGATACCAACCCTGATTTCTACATCAATATTTGTCAGCCACTAAATCCCATGCACGGAGTGCCCTGTCCTGCCGGAGCCGCTGTGTGCAAAGTTCCTATTGATGGTCCCCCCATAGATATCGGCCGGGTAGCAGGACCACCAATACTCAATCCAATAGCAAATGAGATTTACTTGAATTTTGAAAGCAGTACTCCTTGCTTAGCGGACAAGCATTTCAACTACACCTCGCTCATCGCGTTTCACTGTAAGAGAGGTGTGAGCATGGGAACGCCTAAGCTGTTAAGGACCAGCGAGTGCGACTTTGTGTTCGAATGGGAGACTCCTGTCGTCTGTCCTGATGAAGTGAGGATGGATGGCTGTACCCTGACAGATGAGCAGCTCCTCTACAGCTTCAACTTGTCCAGCCTTTCCACGAGCACCTTTAAGGTGACTCGCGACTCGCGCACCTACAGCGTTGGGGTGTGCACCTTTGCAGTCGGGCCAGAACAAGGAGGCTGTAAGGACGGAGGAGTCTGTCTGCTCTCAGGCACCAAGGGGGCATCCTTTGGACGGCTGCAATCAATGAAACTGGATTACAGGCACCAGGATGAAGCGGTCGTTTTAAGTTACGTGAATGGTGATCGTTGCCCTCCAGAAACCGATGACGGCGTCCCCTGTGTCTTCCCCTTCATATTCAATGGGAAGAGCTACGAGGAGTGCATCATAGAGAGCAGGGCGAAGCTGTGGTGTAGCACAACTGCGGACTACGACAGAGACCACGAGTGGGGCTTCTGCAGACACTCAAACAGCTACCGGACATCCAGCATCATATTTAAGTGTGATGAAGATGAGGACATTGGGAGGCCACAAGTCTTCAGTGAAGTGCGTGGGTGTGATGTGACATTTGAGTGGAAAACAAAAGTTGTCTGCCCTTGA(SEQ?ID?NO：5)

HIS-biotin-CI-MPR domain 10-13 protein sequence

MGIPMGKSMLVLLTFLAFASCCIAAGAPTGHHHHHHAPGLNDIFEAQKIEWHEPFDLTECSFKDGAGNSFDLSSLSRYSDNWEAITGTGDPEHYLINVCKSLAPQAGTEPCPPEAAACLLGGSKPVNLGRVRDGPQWRDGIIVLKYVDGDLCPDGIRKKSTTIRFTCSESQVNSRPMFISAVEDCEYTFAWPTATACPMKSNEHDDCQVTNPSTGHLFDLSSLSGRAGFTAAYSEKGLVYMSICGENENCPPGVGACFGQTRISVGKANKRLRYVDQVLQLVYKDGSPCPSKSGLSYKSVISFVCRPEAGPTNRPMLISLDKQTCTLFFSWHTPLACEQATECSVRNGSSIVDLSPLIHRTGGYEAYDESEDDASDTNPDFYINICQPLNPMHGVPCPAGAAVCKVPIDGPPIDIGRVAGPPILNPIANEIYLNFESSTPCLADKHFNYTSLIAFHCKRGVSMGTPKLLRTSECDFVFEWETPVVCPDEVRMDGCTLTDEQLLYSFNLSSLSTSTFKVTRDSRTYSVGVCTFAVGPEQGGCKDGGVCLLSGTKGASFGRLQSMKLDYRHQDEAVVLSYVNGDRCPPETDDGVPCVFPFIFNGKSYEECIIESRAKLWCSTTADYDRDHEWGFCRHSNSYRTSSIIEKCDEDEDIGRPQVFSEVRGCDVTFEWKTKVVCP(SEQ?ID?NO：6)。

HIS-biotin-CI-MPR domain 10-13 I1572T (sequence variation of underscore causes point mutation I1572T and forms the silent mutation in diagnosis SpeI site)

ATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTGGCGCGCCGACCGGTCACCATCACCATCACCACGCGCCGGGCCTGAACGACATCTTCGAGGCCCAGAAGATCGAGTGGCACGAACCTTTCGATCTGACTGAATGTTCATTCAAAGATGGGGCTGGCAACTCCTTCGACCTCTCGTCCCTGTCAAGGTACAGTGACAACTGGGAAGCCATCACTGGGACGGGGGACCCGGAGCACTACCTCATCAATGTCTGCAAGTCTCTGGCCCCGCAGGCTGGCACTGAGCCGTGCCCTCCAGAAGCAGCCGCGTGTCTGCTGGGTGGCTCCAAGCCCGTGAACCTCGGCAGGGTAAGGGACGGACCTCAGTGGAGAGATGGCATAATTGTCCTGAAATACGTTGATGGCGACTTATGTCCAGATGGGATTCGGAAAAAGTCAACCACCATCCGATTCACCTGCAGCGAGAGCCAAGTGAACTCCAGGCCCATGTTCATCAGCGCCGTGGAGGACTGTGAGTACACCTTTGCCTGGCCCACAGCCACAGCCTGTCCCATGAAGAGCAACGAGCATGATGACTGCCAGGTCACCAACCCAAGCACAGGACACCTGTTTGATCTGAGCTCCTTAAGTGGCAGGGCGGGATTCACAGCTGCTTACAGCGAGAAGGGGTTGGTTTACATGAGCATCTGTGGGGAGAATGAAAACTGCCCTCCTGGCGTGGGGGCCTGCTTTGGACAGACCAGGACTAGTGTGGGCAAGGCCAACAAGAGGCTGAGATACGTGGACCAGGTCCTGCAGCTGGTGTACAAGGATGGGTCCCCTTGTCCCTCCAAATCCGGCCTGAGCTATAAGAGTGTGATCAGTTTCGTGTGCAGGCCTGAGGCCGGGCCAACCAATAGGCCCATGCTCATCTCCCTGGACAAGCAGACATGCACTCTCTTCTTCTCCTGGCACACGCCGCTGGCCTGCGAGCAAGCGACCGAATGTTCCGTGAGGAATGGAAGCTCTATTGTTGACTTGTCTCCCCTTATTCATCGCACTGGTGGTTATGAGGCTTATGATGAGAGTGAGGATGATGCCTCCGATACCAACCCTGATTTCTACATCAATATTTGTCAGCCACTAAATCCCATGCACGGAGTGCCCTGTCCTGCCGGAGCCGCTGTGTGCAAAGTTCCTATTGATGGTCCCCCCATAGATATCGGCCGGGTAGCAGGACCACCAATACTCAATCCAATAGCAAATGAGATTTACTTGAATTTTGAAAGCAGTACTCCTTGCTTAGCGGACAAGCATTTCAACTACACCTCGCTCATCGCGTTTCACTGTAAGAGAGGTGTGAGCATGGGAACGCCTAAGCTGTTAAGGACCAGCGAGTGCGACTTTGTGTTCGAATGGGAGACTCCTGTCGTCTGTCCTGATGAAGTGAGGATGGATGGCTGTACCCTGACAGATGAGCAGCTCCTCTACAGCTTCAACTTGTCCAGCCTTTCCACGAGCACCTTTAAGGTGACTCGCGACTCGCGCACCTACAGCGTTGGGGTGTGCACCTTTGCAGTCGGGCCAGAACAAGGAGGCTGTAAGGACGGAGGAGTCTGTCTGCTCTCAGGCACCAAGGGGGCATCCTTTGGACGGCTGCAATCAATGAAACTGGATTACAGGCACCAGGATGAAGCGGTCGTTTTAAGTTACGTGAATGGTGATCGTTGCCCTCCAGAAACCGATGACGGCGTCCCCTGTGTCTTCCCCTTCATATTCAATGGGAAGAGCTACGAGGAGTGCATCATAGAGAGCAGGGCGAAGCTGTGGTGTAGCACAACTGCGGACTACGACAGAGACCACGAGTGGGGCTTCTGCAGACACTCAAACAGCTACCGGACATCCAGCATCATATTTAAGTGTGATGAAGATGAGGACATTGGGAGGCCACAAGTCTTCAGTGAAGTGCGTGGGTGTGATGTGACATTTGAGTGGAAAACAAAAGTTGTCTGCCCTTGA(SEQ?ID?NO：7)

HIS-biotin-CI-MPR domain 10-13 I1572T protein sequence (the I1572T sudden change has added underscore)

MGIPMGKSMLVLLTFLAFASCCIAAGAPTGHHHHHHAPGLNDIFEAQKIEWHEPFDLTECSFKDGAGNSFDLSSLSRYSDNWEAITGTGDPEHYLINVCKSLAPQAGTEPCPPEAAACLLGGSKPVNLGRVRDGPQWRDGIIVLKYVDGDLCPDGIRKKSTTIRFTCSESQVNSRPMFISAVEDCEYTFAWPTATACPMKSNEHDDCQVTNPSTGHLFDLSSLSGRAGFTAAYSEKGLVYMSICGENENCPPGVGACFGQTR TSVGKANKRLRYVDQVLQLVYKDGSPCPSKSGLSYKSVISFVCRPEAGPTNRPMLISLDKQTCTLFFSWHTPLACEQATECSVRNGSSIVDLSPLIHRTGGYEAYDESEDDASDTNPDFYINICQPLNPMHGVPCPAGAAVCKVPIDGPPIDIGRVAGPPILNPIANEIYLNFESSTPCLADKHFNYTSLIAFHCKRGVSMGTPKLLRTSECDFVFEWETPVVCPDEVRMDGCTLTDEQLLYSFNLSSLSTSTFKVTRDSRTYSVGVCTFAVGPEQGGCKDGGVCLLSGTKGASFGRLQSMKLDYRHQDEAVVLSYVNGDRCPPETDDGVPCVFPFIFNGKSYEECIIESRAKLWCSTTADYDRDHEWGFCRHSNSYRTSSIIFKCDEDEDIGRPQVFSEVRGCDVTFEWKTKVVCP(SEQ?ID?NO：8)

Recombiant protein is transient expression (referring to Fig. 3 B) in suspension HEK293 cell.Protein is collected from culture supernatants, and by nickel agarose purification, carries out biotinylation then.Particularly, the use by oneself supernatant of plasmid p1288 and p1355 cells transfected instructs according to manufacturer and is applied to 1mL His Gravitrap ^TMIn the post (GE Healthcare), be used for purification His ₆The receptor domain albumen of-labelling.Concentrate eluant and exchange to 10mMTris pH8 and 25mM NaCl buffer in, then according to the description of manufacturer (Avidity), in the reaction system of the receptor in containing the solution that cumulative volume that 70 μ g have 25 μ L BiomixA, 25 μ L BiomixB and 4 μ LBirA enzymes is 205 μ L, with this albumen of BirA enzyme biotinylation.The BirA enzyme is handled and carry out 1.5h under 30 ℃.The reaction system dilution is arrived His Gravitrap for 20 times then ^TMBinding buffer liquid (GE Healthcare), and be in application to His Gravitrap ^TMPost is to remove BirA enzyme and free biotin.Eluent stores down at 4 ℃.

Surface plasma resonance is analyzed

The all surface plasma resonance detects all and adopts down at 25 ℃

3000 instruments carry out.(Piscataway NJ) obtains from Biacore for SA sensor chip and surfactant P20.All buffer agent utilizations

Filter element (0.2 μ m) filters, and balance is to room temperature, and the degassing immediately before using.Protein sample at centrifugal 15min under 13,000 * g to remove any particle that may in sample, exist.

Biotinylated wild type (1288FS) behind the purification or saltant (1355FS) recombinant C I-MPR domain 10-13 (Dom 10-13) proteopexy are arrived

On streptavidin (SA) chip, it contains the covalently bound glucosan substrate to it of streptavidin.After the SA sensor chip docks, the SA chip is washed twice with deionized water.Link coupled flow cell is treated in the buffer adjusting that contains 50mM NaOH/1M NaCl by the flow velocity injection 60mL with 20mL/min.Water washes chip as mentioned above.Chip is used coupling buffer (10mM HEPES pH 7.4/100mM NaCl) flushing as mentioned above then.Biotinylated Dom 10-13 albumen 1355FS and 1288FS are diluted to 20ng/mL and 4ng/mL in coupling buffer.Flow cell (FC) 1 and 2 is to be higher than the density coupling of FC 3 and 4.FC 1 and FC 3 usefulness sudden change Dom 10-13 construct are fixed, and as (that is, deducting the response that obtains by FC 1 from FC 2 with reference to the surface; Deduct the response that obtains by FC 3 from FC 4).FC 2 and FC 4 usefulness wild type Dom 10-13 constructs are fixed.FC 1 carries out coupling by the 1355FS (20ng/ml) that the flow velocity with 10mL/min injects 50mL, and FC 2 carries out coupling by the 1288FS (20ng/ml) that the flow velocity with 10mL/min injects 50mL, to reach the coupling level of final about 5,000 resonance units (RU).FC 3 carries out coupling by the 1355FS (4ng/ml) that injects 50mL with the flow velocity of 10mL/min, and FC 2 carries out coupling by the 1288FS (4ng/ml) that the flow velocity with 10mL/min injects 50mL, to reach finally about 1, the coupling level of 000RU.These coupling levels have provided 800RU (FC2-1) for IGF-II or the theoretical R of 160RU (FC4-3) _MaxWith for 10 of GAA-GILT, 000RU (FC2-1) or 2, the theoretical R of 000RU (FC4-3) _MaxAfter injection 50ml contained the coupling buffer of Dom 10-13 construct, chip was separately with coupling buffer flushing (flow velocity with 10mL/min injects 10mL).Remaining unconjugated streptavidin binding site is undertaken saturated with biotin by continuous two the 10mL biotin of injection of flow velocity (10mM) with 10 μ L/min.

After the coupling, flow cell is balance in electrophoretic buffer (10mM HEPES pH 7.0,150mM NaCl and 0.005% (v/v) surfactant P20).The activity of fixed Dom10-13 construct by detect separately receptor to the affinity-plus of IGF-II to determine.IGF-II (134mM) at first is diluted to 1,5,10,25,50,75,100,250 and the ultimate density of 500nM in electrophoretic buffer.The IGF-II of each concentration is injected into (that is, the association phase) on the chip with the flow velocity of 40mL/min in 2min, then carry out separately 2min electrophoretic buffer injection (flow velocity=40mL/min) (that is, dissociate phase).Regenerate with the flow velocity injection 10mL of 10mL/min with 10mM HCl in the surface.After the regeneration, flow velocity is increased to 40mL/min, and next allows balance 1min before injecting and chip is in beginning.

Similarly, the GAA construct 701 of GILT-labelling is analyzed the binding affinity of itself and Dom 10-13 recombinant receptor.This structure is diluted to 1,5,10,25,50,75,100,250 and the ultimate density of 500nM in electrophoretic buffer, and injects according to above description to IGF-II.After the GAA of GILT-labelling construct, move the IGF-II concentration curve to test the integrity on fixed Dom 10-13 surface.

For each analyte concentration, determine to be in equilibrated response meansigma methods, the balance resonant element of gained is mapped to the concentration described point.Utilize BIAevaluation ^TMSoftware (edition 4 .1) fits data to stable state affinity model.Dissociation constant also utilizes 1: 1 temperature models such as combination to determine.All response datas are according to Myszka (2000) Methods Enzymo.323:325-3401 dual benchmark (double-referenced) is carried out in the description in, and the immobilized flow cell of Dom 10-13 is parallel to carry out and compose (sensorgram) from all in conjunction with sensing deducting wherein to be used for contributing the contrast (promptly injecting electrophoretic buffer separately) of variation to suddenly change with utilization to the body refractive index.Fig. 4 A-B is the GAA ZC-701 that shows the IGF-II be attached to CI-MPR and GILT-labelling

The exemplary concentration curve of analyzing.Fig. 4 A shows the binding curve of IGF-II.Fig. 4 B shows the binding curve of the GAAZC-701 of GILT-labelling.Two kinds of flow cells compare (Fig. 4 B) to the result of (that is, FC2-1 and FC4-3).

These experimental results show that the GAA ZC-701 of GILT-labelling has affinity for CI-MPR, are about 0.8 (tables 1) of IGF-II.These data show, with IGF-II the affinity of CI-MPR are compared, and the GAA of GILT-labelling has high-affinity for CI-MPR.Although measured IGF-II is 27nM to the absolute value of CI-MPR affinity, in previous document, the affinity of domain 10-13 that IGF-II is attached to receptor is lower about 10 times than the affinity that is attached to natural receptor.Linnell?et?al.(2001) J Biol?Chem.Jun?29；276(26)：23986-91。Therefore, the GAA of GILT-labelling also expects high 10 times to the binding affinity of natural receptor.

Table 1

Protein	??K d	Relative affinity
			??IGF-II	??27nM	??1
??ZC-701	??33nM	??0.8
			??ZC-1026	??43nM	??0.6

The oligosaccharide that embodiment 3:N-connects the analysis showed that ZC-701 lacks M6P

Utilize PNG enzyme de-glycosylation, the then combination of analyzing with HPLC (Blue Stream Laboratories) with fluoroscopic examination, the oligosaccharide analysis of implementing the N-connection is to determine the oligosaccharide distribution profile of ZC-701.

Utilize about 100 μ g albumen of each sample, implement the sugar (carbohydrate) that connects from glycoprotein sample cutting N-by the N-dextranase with the ratio of 1: 100 (enzyme is to substrate).In case discharge, just utilize cold ethanol extraction polysaccharide and dry by centrifugal realization.The oligosaccharide that reclaims is used 2-aminobenzamide (2-AB) labelling in the presence of sodium cyanoborohydride under acid condition.After the derivatization step, by

S sample filtering conduit (Prozyme) is removed remaining excess dye and other reaction reagents in the sample.

Use following condition by the N-connection oligosaccharide analysis that HPLC-FLD carries out: mobile phase A: the Mobile phase B of 65% acetonitrile/35%; Mobile phase B: 250mM ammonium formate, pH4.4; Detect: fluorescence (Ex:330nm, Em:420nm); With the HPLC gradient.

Chromatographic peak carries out integration, and compares based on the peak retention time.The result is reported as the area % of each sugared type of each total peak area.Analyze according to these, determined that the main oligosaccharide structure that exists is high mannose structures and some complicated feeler structures on ZC-701.Yet, do not detect the Man-6-P structure.

Embodiment 4: picked-up is measured and confirmed to lack functional M6P on the ZC-701 surface

It is by with the interaction of CI-MPR mediation that reorganization GAA absorbs in the mammalian cell, and CI-MPR is present on the surface of most of mammalian cell types.Depend on the oligosaccharide of protein surface and have M6P.

On the contrary, owing to hold the label that has the IGF-II source at proteic N-, so ZC-701 has high-affinity to CI-MPR.Verified in many experiments, ZC-701 shows does not have tangible M6P-dependent form to absorb in the mammalian cell, and this has confirmed the functional shortage of M6P on the ZC-701 surface.

Implement the cell based picked-up and measure, with confirm the GILT-labelling or unlabelled GAA enter the ability of target cell.Rat L6 sarcoplast the picked-up before 24h at the 24-orifice plate with every hole 1 * 10 ⁵The density lay of individual cell.When the experiment beginning, to remove medium from cell, and replace with 0.5mL picked-up medium, this picked-up medium comprises labelling or the unlabelled GAA that concentration range is 2～500nM.For the specificity that confirms to absorb, some holes are contained competitor M6P (5mM ultimate density) and/or IGF-II (ultimate densities of 18 μ g/mL) in addition.After the 18h, medium is from the cell sucking-off, and cell washes 4 times with PBS.Then, cell is with 200 μ L CelLytic M ^TMThe dissolving of cytolysis buffer, the cytolysis thing utilizes the 4MU substrate according to analysis GAA activity described below.Albumen adopts PierceBCA ^TMProtein determination kit is determined.

The ZC-701 typical case picked-up experimental result that produces in Chinese hamster ovary celI as shown in Figure 5.As viewed, ZC-701 absorbs the influence of the M6P that in fact is not subjected to adding big molar excess in the rat L6 sarcoplast, and picked-up is eliminated by excessive IGF-II fully.On the contrary, the picked-up of wtGAA (ZC-635) is added into excessive M6P fully and eliminates, but in fact is not subjected to the influence of IGF-II competition.The ZC-701 that the CHO-cell produces absorbs the insensitivity of mammalian cell to the inhibition of excessive M6P, shows the functional shortage of M6P on the ZC-701 surface that produces in the Chinese hamster ovary celI.

The GAA of embodiment 5:GILT-labelling shows the more effective picked-up than unlabelled GAA

Fig. 6 shows the GAA (ZC-701) of GILT-labelling of purification and unmarked GAA (ZC-635) picked-up of wild type enters the myoblastic saturation curve of L6.In the experiment of example, the K that the GAA of GILT-labelling has _Picked-up=7nM, and the K that wt GAA has _Picked-up=354nM.This explanation, the GAA of GILT-labelling demonstrates the more effective picked-up than unlabelled GAA, because compare with unlabelled GAA, the GAA that needs remarkable lower level GILT-labelling is to realize entering sarcoplast via the maximum ingestion of CI-MPR.

This proves that also the GILT label can not disturb the enzymatic activity of GAA.

GAA PNP measures

The GAA enzyme is hatched in 50 μ L contain the reactant mixture of 100mM sodium acetate pH 4.2 and 10mM paranitrophenol (PNP) α-glucoside substrate (Sigma N1377).Be reflected at 37 ℃ and hatch 20min, and stop with 300 μ L 100mM sodium carbonate.On the microtitre orifice plate of 96-hole, under 405nm, measure absorbance, and contrast with the standard curve of setting up by paranitrophenol (Sigma N7660).1GAA PNP unit definition is the 1nmolPNP/h of hydrolysis.

GAA 4MU measures

The GAA enzyme contains at 20 μ L in the reactant mixture of 123mM sodium acetate pH 4.0 and 10mM 4-methyl umbelliferone (4-methylumbelliferyl) α-D-glucosidase substrate (Sigma, catalog number (Cat.No.) #M-9766) hatches.Be reflected at 37 ℃ and hatch 1h, and stop with the pH of buffer 10.7 that 200 μ L contain 267mM sodium carbonate, 427mM glycine.On the microtitre orifice plate of 96-hole, excite and 460nm light filter mensuration fluorescence, and contrast with the standard curve of setting up by 4-methyl umbelliferone (Sigma, catalogue #M1381) with 355nm.The 1GAA4MU unit definition is the 1nmol 4-methyl umbelliferone/h of hydrolysis.

The given activity of the GAA of exemplary GILT-labelling and the unmarked GAA of wild type is as shown in table 2.The enzymatic activity of the GAA of GILT-labelling may be suitable with unlabelled GAA.

The GAA ZC-701 of table 2:GILT-labelling and given activity and the K of the unlabelled GAA of wild type _m

	??ZC-701	??WtGAA
			Activity specific *??4MU(nmol/h/mg)	??315,000	??346,000
??K m4MU(mM) **	??1.47	??1.41
			??K m?PNP(mM) **	??3.29	??9.21

^*For the definite meansigma methods of 3 preparations

^*For the definite meansigma methods of two preparations

The half-life of embodiment 6:GAA in rat L6 sarcoplast

According to above description (referring to embodiment 4), utilize GILT-labelling GAA and unlabelled GAA in rat L6 sarcoplast to absorb experiment.After the 18h, the medium that contains enzyme is from the cell sucking-off, and with PBS flushing cell 4 times.At this moment, double-hole (duplicate well) carries out cytolysis (time 0), and the cytolysis thing is freezing down at-80 ℃.After this every day, carry out the double-hole cytolysis, and storage is used for analyzing.After 14 days, all cytolysis things carry out the GAA activity analysis.Data based 1 grade of decay equation: ln C _t=-kt+ln C _oMap, wherein C is a compound concentration, t be in hour time, and k is 1 stage speed constant.Fig. 7 just shows the GAA and the graphical representation of exemplary of the half-life of unlabelled GAA (ZC-635) in rat L6 sarcoplast of GILT-labelling.Result shown in Fig. 7 shows that labelling and unlabelled albumen have the very similarly half-life, are respectively 6.5 and 6.7 days.This shows that in case in cell, the enzyme of GILT-labelling continues to keep having similar kinetics with unlabelled GAA.

Embodiment 7: the GAA after the picked-up handles or processing

According to document Moreland et al. (2005) J .Biol.Chem..280:6780-6791 and the description of the list of references that is comprised, the mammal GAA successive proteolytic treatment of experience in lysosome usually.Albumen after the processing produces 70kDa, 20kDa, the peptide of 10kDa and the spectrogram of the peptide that some are littler.Whether similar processed for the GAA that determines the GILT-labelling with unlabelled GAA, analyze by Western blotting from the aliquot of the cytolysis thing of above-mentioned picked-up experiment.Fig. 8 A～B shows that the GAA of GILT-labelling absorbs the Western blotting of rat L6 sarcoplast proteolytic treatment afterwards.Fig. 8 A is the Western blotting that shows the peptide spectrogram of identifying by monoclonal antibody (identification has the 70kDa IGF-II peptide and the bigger intermediate of IGF-II label).Show that in the result shown in Fig. 8 A the picked-up back just loses the GILT label immediately.Fig. 8 B shows the Western blotting of handling by monoclonal antibody (identification 70kDa peptide and the bigger intermediate) wild type of identifying that enters 76kDa and 70kDa species after picked-up and the GAA of GILT-labelling.In fact the polypeptide spectrogram of identifying in this experiment is identical for labelling with unlabelled enzyme.Enter cell in case this shows, the GILT label just loses, and that the GAA of GILT-labelling is similar to unlabelled GAA is processed.Therefore, possible is that in case enter in the cell, the GILT label has seldom or not influence the behavior of GAA.

Embodiment 8: pharmacokinetics

Pharmacokinetics at the GAA of the GILT-labelling of producing under the different condition of culture detects in wild type 129 mices.The GAA ZC-701 of GILT-labelling produces under three kinds of different condition of culture.Three groups of three 129 mices use the 10mg/kg ZC-701 of single dose to inject jugular vein, and this ZC-701 obtains from the culture supernatants purification of replacing the cell of growing the media at 3 kinds.Carry out the serum sampling respectively before injection and when injection back 15min, 30min, 45min, 60min, 90min, 120min, 4h and 8h.Kill this animal then.Blood serum sample is analyzed by the quantitative protein trace.Data based 1 grade of decay equation: ln C _t=-kt+ln C _oMap, wherein C is a compound concentration, t be in hour time, and k is 1 stage speed constant.The linear segment of half-life from loaarithmic curve shown in Figure 9 (log figure) obtains.The proteic half-life of the GAA of GILT-labelling is: red line, PF-CHO, t1/2=43min; The orange line, CDM4, t1/2=38min; Green line, CD17, t1/2=52min.Based on these results, the albumen that produces in CDl 7 media has the most favourable half-life.These results show that the GAA of GILT-labelling does not promptly excessively remove from blood circulation.

Embodiment 9:GAA organizes the half-life

In case the purpose of this experiment is the speed of the GAA loss of activity of GILT-labelling when determining that enzyme arrives its target tissue.In the Pompe mouse model,

Seem in various muscular tissues, to have about 6～7 days organizing the half-life (application number 125141/0, belong to drug evaluation and research center and biological assessment center, materia medica summary (the Center forDrug Evaluation and Research and Center for Biologies Evaluation andResearch, Pharmacology Reviews)).

The Pompe mice is (according to the Pompe mouse model 6 of Raben (1998) JBC.273:19086-19092 description ^Neo/ 6 ^Neo, its disclosure is hereby expressly incorporated by reference), with unmarked GAA (ZC-635) or the GAA ZC-701 of GILT-labelling or the GAA ZC-1026 injection jugular vein of GILT-labelling of 10mg/kg.After injection, killed mice then in 1,5,10 and 15 day.Tissue sample homogenizes and detects the GAA activity according to standardization program.The half-life of organizing of the GAA ZC-701 of GILT-labelling and ZC-1026 and unlabelled GAA ZC-635 is calculated (Figure 10 A, musculus quadriceps tissue according to the decay curve in different tissues; Figure 10 B, heart tissue; Figure 10 C, diaphragm tissue; And Figure 10 D, liver organization).The elimination half life values of calculating is summarised in the table 3.

The half-life of organizing of unlabelled protein (ZC-635) is 9.1～3.9 days in different tissues, and the half-life of the GAA ZC-701 of GILT-labelling is 8.5～7.4 days (table 3) in different tissues.These scopes possibilities are owing to relatively little sample size (3 animal/points) reflection is statistic bias, rather than significant difference.

For relatively, be respectively 6.5 days and 6.7 days according to the ZC-701 in rat L6 sarcoplast of the calculating of the decay curve shown in Fig. 9 and the half-life of unlabelled wild type GAA (ZC-635).

Table 3 the organizing the half-life of labelling and unlabelled GAA in various tissues

Tissue	ZC-701 T1/2, day	ZC-635 T1/2, day
			Musculus quadriceps	??8.5	??9.1
Heart	??7.4	??3.9
			Diaphragm	??7.5	??4.6
Liver	??8.2	??7.9
			Rat L6 sarcoplast	??6.5	??6.5

These data show that in case enter in the cell of Pompe mice, the GAA of GILT-labelling seems to keep and the similar kinetics of unlabelled GAA.And dynamic (dynamical) cognition that decays can help to design suitable administration time at interval to the GAA of GILT-labelling.

The GAA of embodiment 10:GILT-labelling absorbs the lyase of C2C12 mouse muscle-forming cell In the body

The C2C12 mouse muscle-forming cell is gone up growth and is had (panel A) or do not exist under the GAA of (panel B) 100nMGILT-labelling in 37 ℃ and 5%CO at the microscope slide (BDBiosciences) of polylysine coating ₂Under hatch 18h.Then cell is hatched 1h in somatomedin, then with after the D-PBS flushing 4 times, under room temperature with the fixing 15min of methanol.Ensuing hatching all at room temperature carried out, and hatches each time by three times to separate with the D-BPS flushing.Unless spell out, hatch and carry out 1h.Microscope slide is with 0.1%triton X-100 infiltration 15min, then with sealing buffer (the D-PBS solution of 10% heat inactivation horse serum (Invitrogen)) sealing.Microscope slide is hatched with first Mus monoclonal anti-GAA antibody 3A6-1F2 (1: 5,000, in the sealing buffer), then with the secondary rabbit anti--Mus IgG AF594 binding antibody (Invitrogen A1 1032,1: 200, in the sealing buffer) hatches.Then, hatch FITC-in conjunction with rat anti-mice LAMP-1 (BDPharmingen 553793,1: 50, the sealing buffer in).Microscope slide usefulness contains (Invitrogen) sealing of sealing solution (mounting solution) of DAPI, and observes with the NikonEclipse 80i microscope (Chroma Technology) that is equipped with Fluorescein isothiocyanate, texas Red (texas red) and DAPI filter.Adopt photometer cascade camera captures images by MetaMorph software (UniversalImaging) control.Image adopts Photoshop software (Adobe) to merge.Figure 11 shows the colocated by anti--GAA antibody institute detection signal, and this anti--GAA antibody has by the antibody institute detection signal at lysosome label LAMP1.Therefore, this result has confirmed that the GAA of GILT-labelling is delivered to lysosome.

Embodiment 11: the removing of glycogen in the body

The purpose of this experiment is to determine after the GAA or wt GAA of Pompe mice single IV injection GILT-labelling the speed that glycogen is removed from heart tissue.

Pompe mice (Pompe mouse model 6 ^Neo/ 6 ^Neo, in document Raben (1998) JBC.273:19086-19092, describe, its disclosure is hereby expressly incorporated by reference), inject jugular vein with the GAA (ZC-701) of unlabelled GAA of 10mg/kg (ZC-635) or GILT-labelling.1,5,10 and 15 day time is killed mice after injection then.Each data point is represented the meansigma methods from three Mus.Carry out homogenizing of heart tissue sample according to standardization program, and analyze glycogen content.Substantially adopt according to Zhu et al. (2005) Biochem J., the aspergillus niger of describing among the 389:619-628 (A.niger) amyloglucosidase and

Red Glucose mensuration test kit (Invitrogen) detects the glycogen content in these tissue homogenate mixture.Result shown in Figure 12 shows that the mouse heart tissue that the ZC-701 that uses by oneself handles demonstrates glycogen and is close to removing fully, and demonstrate glycogen content with the mice that wt GAA handles minor variations only takes place.

Embodiment 12: the pathological counter-rotating of Pompe

Verified, treatment of the present invention is more more effective in treatment than wt GAA in vivo with fusion rotein.Carried out a research so that relatively ZC-701 and wt GAA use Pompe mouse model 6 from the ability of the skeletal muscle tissue removing glycogen of Pompe mice ^Neo/ 6 ^NeoAnimal (Raben (1998) JBC 273:19086-19092).Pompe mice (5/group) is accepted weekly in two dosage wt GAA of IV injection of 4 times or ZC-701 (5mg/kg or 20mg/kg) or the carrier.Five untreated animals are with comparing, and accept 4 pump pickle solution weekly.Animal 1h before the 2nd, 3 and 4 injection accepts oral diphenhydramine (5mg/kg).In this research, when animal during age, in order to make their tolerances, is injected 25 μ gZC-701s via subcutaneous at the cervical region collare with the Pompe knock-out mice less than 48h.1 week killed these mices in the 4th injection back, and gather in the crops various tissues (diaphragm, heart, lung, liver, musculus soleus, musculus quadriceps, gastrocnemius, TA, EDL, tongue) and be used for tissue and biochemical analysis.Glycogen content in these tissue homogenate things adopts aspergillus niger amyloglucosidase and Amplex Red Glucose to measure test kit and detects.Research design is summarised in the table 4.

Table 4

Mice	Injection	Dosage	The injection back time-to-live	The # animal
					GAA skin-/-	??PBS	??0	1 week of back of injection at last	?N＝6
GAA skin-/-	??Wt?GAA(ZC-635)	??5,20mg/kg	1 week of back of injection at last	N=6/ dosage amounts to=12
					GAA skin-/-	The GAA (ZC-701/ZC-1026) that GILT-modifies	??5,20mg/kg	1 week of back of injection at last	N=6/ dosage amounts to=12

GAA enzyme level in the different tissues homogenate adopts standardization program to measure, and the result is summarised in the table 5.

GAA level in table 5 tissue

Tissue	??GAA	Unit/mg albumen	??SD
				Gastrocnemius	??635-20	??15.918	??9.659
	??701-20	??7.495	??1.435
					??701-5	??7.263	??0.859
	??635-5	??4.828	??0.251
					??PBS	??4.380	??0.193
Musculus quadriceps	??635-20	??9.164	??3.297
					??701-20	??6.715	??1.408
	??701-5	??6.158	??1.140
					??635-5	??4.363	??0.145
	??PBS	??4.018	??0.298
				Barrier film	??635-20	??42.178	??53.517
	??701-20	??24.945	??27.799
					??701-5	??7.795	??0.387
	??635-5	??6.364	??1.058
					??PBS	??5.254	??0.281
Heart	??635-20	??5.121	??2.082
					??701-20	??5.363	??0.683
	??701-5	??6.330	??1.310
					??635-5	??4.354	??0.652
	??PBS	??3.911	??0.311
				Tongue	??635-20	??10.418	??3.901
	??701-20	??5.668	??0.568
					??701-5	??4.786	??0.327
	??635-5	??4.783	??0.494
					??PBS	??4.587	??0.470
Musculus soleus	??635-20	??34.397	??22.049
					??701-20	??20.621	??6.685

	??701-5	??13.618	??2.804
					??635-5	??7.310	??1.236
	??PBS	??7.490	??3.137
				??TA	??635-20	??19.623	??13.242
	??701-20	??7.148	??1.318
					??701-5	??7.398	??0.507
	??635-5	??4.688	??0.140
					??PBS	??4.790	??0.797
??EDL	??635-20	??23.9828	??12.6501
					??701-20	??13.0170	??1.9414
	??701-5	??9.1218	??0.7590
					??635-5	??7.2197	??0.4682
	??PBS	??6.7099	??1.1557

Glycogen content in these tissues adopts aspergillus niger amyloglucosidase and AmplexRed Glucose to measure test kit (Invitrogen) substantially according to Zhu et al. (2005) Biochem J., the description of 389:619-628 detects.The glycogen data description is in Figure 13 A-H.The unmarked GAA employed as Figure 13 A-H, that GAA 5 is meant at 5mg/kg dosage; The unmarked GAA that GAA 20 is meant at 20mg/kg dosage; 701 5 are meant the GAA ZC-701 at the GILT-of 5mg/kg dosage labelling; 701 20 are meant the GAA ZC-701 at the GILT-of 20mg/kg dosage labelling.PBS is with comparing.These results show that the GAA of GILT-labelling (ZC-701) compares with unlabelled GAA (ZC-635), have remarkable advantages at it aspect the ability of various muscular tissues removing glycogens.Particularly, ZC-701 removes aspect the ability of glycogen more more effective than wtGAA significantly at it from multiple skeletal muscle tissue.Accept the glycogen levels that the Pompe mice of one of any wt GAA of two kinds of dosage had and be different from the animal glycogen levels that PBS handles.On the contrary, the animal of accepting ZC-701 shows significantly lower glycogen levels.

Embodiment 13: dosage, administration time interval and the optimization at main body age

Dosage

In experiment before, the dosage of 5mg/kg is the same effective with the dosage of 20mg/kg on the removing glycogen in many tissues.The dose titration experiment is used for determining can be enough to treat the minimum effective dose of human patients.Every group 5～7 toleration Pompe knock-out mices are injected the GAA of the GILT-labelling of various dose weekly.For example, toleration Pompe mice with 1.0,1.5,2,2.5,5,10,20mg/kg dosage injects.

The Pompe knock-out mice injected for 8 weeks, killed then.Take a sample from different tissues, and determine glycogen levels according to the description of embodiment 11 and 12.The histochemistry that glycogen and enzyme distribute is also measured according to standardization program.In addition, physiology's detection detects as muscle strength and the ventilation of response hypercapnia can be according to document Mah et al. (2007) Molecular TherapyBeing described in of (online open) uses barometrical total body plethysmography to determine in the mice.

Interval

In addition, assessed treatment time at interval and the clinical effect of maximum time determined to produce to(for) given dose at interval., dose titration adopts different treatment times to inject in for example per 2,3 or 4 weeks at interval and carry out as mentioned above.Skeletal muscle tissue for example the glycogen in musculus soleus or the musculus quadriceps remove general with acting on the indication of determining the optimum balance between dosage and treatment time are at interval in the mouse model.Aforesaid other clinical coherent detections also can be used.

For example, an experimental design is used for investigating the GAA administration time of GILT-labelling at interval in the influence of Pompe mouse model to its usefulness.The Pompe mice at 2～3 monthly ages (Raben JBC 1998 273:19086-19092) is divided into the group of a plurality of 8 animals.One winding is subjected to weekly PBS injection (matched group), one winding is subjected to weekly, and the GAA of 5mg/kg GILT-labelling injects, one winding is injected by the GAA of 10mg/kg GILT-labelling week about, one winding is injected by the GAA of per three all 15mg/kg GILT-labellings, and a winding is injected by every GAA of 20mg/kg GILT-labelling all around.Through the week after the injection of 12 weeks, all animals are all slaughtered.The tissue sample that sampling is analyzed comprises: heart, musculus soleus, gastrocnemius, EDL, TA, musculus quadriceps, psoas muscle, diaphragm, brain, tongue.

Analysis comprises: the histochemical stain of biochemical glycogen analysis, glycogen, selected structural EM, selected structural immunostaining, the antibody serum sample analysis by ELISA, detect (force-frequency measurement) at the external power on the musculus soleus-frequency, glycogen content before the Fructus Vitis viniferae tetrose analysis during lived study portion in the urine, therapeutic scheme and afterwards ¹³C NMR spectrum is determined.

Wherein the matrix of the condition of dosage and interval variation can generate to form the understanding to the relation between these parameters.

Can predict to confirm it is effectively at interval, the explanation of low burden therapeutic scheme is provided for the people who suffers from Pompe thus with the longer time between the higher dosage medication.For example,, can predict that the dosage of 5mg/kg administration weekly will produce dosed administration similar effects with per two all 10mg/kg or per three all 20mg/kg based on the glycogen data shown in Figure 13 A-H.Therefore, expect per three or once replace the GAA perfusion of every GILT-labelling biweekly all around, in people's Pompe patient, will be enough to realize curative effect.Treatment time is long more at interval to be highly favourable, because this will reduce patient's burden and inconvenience at least.

The age of main body

Determined Therapeutic Method begin in Mus age to the influence of therapeutic outcome.It is reported that in Pompe mice II type muscle fiber, the autophagy vacuole will generate in time, this can interfere normal transport pathway, comprises that exogenous enzyme sends to lysosomal.Referring to Fukuda etal (2006) Mol.Therapy, 14 (6): 831-839; Fukuda et al. (2006) Ann. Neurol., 59 (4): 700-708; Fukuda et al. (2006) Autophagy.2 (4): 318-320.Scientist is verified, neo-rhGAA, its be have up to 6 chemical couplings synthetic each contain the reorganization GAA of the oligosaccharide of 2 M6P, can be fully knock out and remove glycogen the Mus from the Pompe at 13 monthly ages.Zhu?et?al，(2005) Biochem?J.，389：619-628。This shows that if adopt the enzyme that CI-MPR is had high-affinity then the cytopathology of report such as Fukuda can be reversible.The GAA that supposes the GILT-labelling has high-affinity for CI-MPR and it is more effective than unlabelled GAA to sending of muscle cell, then it is contemplated that, the GAA enzyme of enough GILT-labellings can be delivered to lysosome, thereby removes the foundation that glycogen also reverses autophagocytosis subsequently.This directly tests in the Pompe mice at 12～13 monthly ages.These mices accept weekly 20 or 4 injections of the GAA of 40mg/kg GILT-labelling, and slaughter in 1 week after last injection.Substantially according to the description of Zhu et al. (2005), adopt aspergillus niger amyloglucosidase and AmplexRed Glucose to measure test kit (Invitrogen) and assess glycogen content.According to Lynch etal, (2005) J.Histochem.Cytochem., the description of 53:63-73 is also estimated glycogen by histochemical stain.

In addition, enforcement mensuration enters with the GAA picked-up of analyzing the GILT-labelling to be had from the independent completion muscle fiber of the phagocytotic animal of body,, with as the comparing of descriptions such as Fukuda with unlabelled GAA, the direct lysosomal ability of GAA targeting of GILT-labelling more under these conditions.It is more effective than unlabelled enzyme that expection GILT-labelling GAA targeting has the muscle fiber of autophagocytosis.

Experiment 14: human clinical research

Based on the treatment of animals of success, 6 month I/II phase dosage range researchs of GAA in department of pediatrics Pompe patient of GILT-labelling have been designed.This clinical trial is open label Proof of Concept people research, implements safety, toleration, usefulness and the pharmacokinetics of GAA in the patient who suffers from children's property sent out Pompe to estimate the GILT-labelling.In this research, overall treatment time is whenever biweekly (per two week ground) at interval.What on the one hand expectation increased in addition is that the interval of wherein treating with given dose is per 3 or 4 weeks 1 time.

The main purpose of this clinical trial comprises to be determined in suffering from the patient treatment that the children sends out the property Pompe, and the GAA of GILT-labelling is by dabbling 4 dosage levels of per two all intravenouss, promptly 2.5,5,10 and the usefulness of 20mg/kg.Secondary objective comprises that (1) estimate in suffering from the patient that the children sends out the property Pompe safety and the pharmacokinetics of GAA of GILT-labelling of pouring into 4 various dose levels of administrations by per two all intravenouss; (2) determine in suffering from the patient that the children sends out the property Pompe to pour into the pharmacokinetics of GILT-GAA of 4 dosage levels of administrations by per two all intravenouss; And (3) determine each influence that the muscle glycogen among the patient who suffers from the children and send out the property Pompe is existed of the GILT-GAA of 4 dosage levels by per two all intravenouss perfusion administrations.The detailed protocol summary of this clinical trial is as shown in table 6.

Table 6 people clinical trial

Figure 14 shows the detail flowchart of clinical research program.

In addition, expectation comprises that one tolerates the patient or the step of immunosuppressed patient.In the clinical trial of other lysosomal enzyme alternative medicine, observe the high-titer antibody of many patients' generations at GAA.For example, this phenomenon is adopting

Gaucher's disease (Gaucher) patient in observe.Under the sort of situation, that Most patients becomes toleration naturally and stop to produce antibody in response to therapeutic scheme.Do not expect to be bound by theory, think that anti--GAA antibody with interferases targeting CI-MPR, has changed the bio distribution of enzyme thus.A kind of tolerance strategy be before the GAA of the GILT-labelling treatment or during, use

(monoclonal antibody of a kind of anti-CD20) treatment Pompe patient.Used on the Pompe patient

Dosage is similar at Sperr et al. (2007) Haematologica.Jan; 92 (1): used dosage in some autoimmune diseasees of the treatment of instructing among the 66-71, the instruction content of the document is hereby expressly incorporated by reference.This chemical compound is also united other immunosuppressant such as steroid uses together.

Based on the histochemical stain of biopsy material, use the Pompe patient expection of the GAA treatment of GILT-labelling will confirm remarkable removing glycogen after 10～12 weeks.The seriality that Thurberg etc. have designed based on the ultrastructure damage is categorized into 5 stage cytopathologys with Pompe.Thurberg?et?al.(2006)Lab.Invest.，86：1208-1220。For example, early stage disease stage 1 cell contains the lysosome that a small amount of glycogen between complete sarcostyle is filled.Stages 3 cell contains the lysosome that many glycogens are filled, so wherein many glycogens leak in the Cytoplasm because lysosome membrane breaks.These stages seem that the autophagy accumulation of describing with Fukada etc. is relevant.At the beginning of the treatment beginning patient in this research is carried out cytopathology analyses, expection will indicate clinical effectiveness.The existence that the patient of response generally has low percent is the cytopathologic myocyte of severe form more.Especially, have the patient who surpasses 50% stages 2 myocyte and have better clinical effectiveness.This it is contemplated that also littler patient generally can have better clinical effectiveness, and the patient with I type meat fiber of higher percentage ratio also has better clinical effectiveness.

The factor of regulating the cytopathology severity comprises: because the remaining GAA active existence of the allelic character of patient GAA in the patient; Patient's age in the treatment beginning; And patient's immune response.For example, more serious in the CRIM-negative patient to the antibody response of the GAA of GILT-labelling.

Based on from initial zooperal result, the GAA of GILT-labelling expection will be more more effective in the human patients treatment than unlabelled GAA.Can predict, give similar dosage, have the more a high proportion of patient of given cytopathology level, will be advantageously when algucerase begins in response to the GAA therapy of GILT-labelling.For example, for

Crucial clinical trial in, among 18 patients 12 when 52 weeks in the muscle glycogen reduce and surpass 20%.Yet, only in 18 only 3 patients glycogen content occurs and reduce by 50% or higher.Kishnani?et?al.(2007) Neurology，68：99-109。Based on the work of animal experiment data and Zhu etc., the GAA of expection GILT-labelling will cause glycogen content decline 80% in Most patients.Patient's expection with the more vast scale of the GAA of GILT-labelling treatment will be presented at physiological parameter such as motor function, the Repiration aspect is improved and improve, and more patient's expection will begin back survival 1,2 and 5 year in therapy.

Have myocyte's cytopathology in late period more and begin the patient of algucerase, for example just begin the patient of therapy greater than 6 monthly ages, on the GAA of GILT-labelling algucerase, also expect and on the muscle glycogen, to take place significantly to reduce, improved aspect respiratory capacity, motor function and the better long-term efficacy and improving.

Incorporating into of list of references

Yin Shu all publications and patent documentation be all with its in full combination in this application, is attached to herein degree as the content of each single publication or patent documentation with it.

Sequence table

＜110〉Zystor Therapeutics Inc. (ZYSTOR THERAPEUTICS, INC.)

＜120〉be used for the treatment of the method for Pompe

<130>P25884BAY

<140>PCT/US2007/023881

<141>2007-11-13

<160>8

<170>PatentIn?version?3.2

<210>1

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>1

ggaattccaa?ccatgggagt?gaggcacccg?ccc?????????????????????????????????????????????33

<210>2

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>2

gctctagact?aacaccagct?gacgagaaac?tgc?????????????????????????????????????????????33

<210>3

<211>276

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used to produce the DNA box of the GAA ZC-701 of reorganization GILT labelling

<400>3

gaattcacac?caatgggaat?cccaatgggg?aagtcgatgc?tggtgcttct?caccttcttg?????60

gccttcgcct?cgtgctgcat?tgctgctctg?tgcggcgggg?agctggtgga?caccctccag????120

ttcgtctgtg?gggaccgcgg?cttctacttc?agcaggcccg?caagccgtgt?gagccgtcgc????180

agccgtggca?tcgttgagga?gtgctgtttc?cgcagctgtg?acctggccct?cctggagacg????240

tactgtgcta?cccccgccaa?gtccgagggc?gcgccg??????????????????????????????276

<210>4

<211>971

<212>PRT

＜213〉artificial sequence

<220>

＜223〉box 701 usefulness EcoRI and XbaI digestion is by handling passivation with the Ke Lunnuo archaeal dna polymerase

<400>4

Met?Gly?Ile?Pro?Met?Gly?Lys?Ser?Met?Leu?Val?Leu?Leu?Thr?Phe?Leu

1???????????????5???????????????????10??????????????????15

Ala?Phe?Ala?Ser?Cys?Cys?Ile?Ala?Ala?Leu?Cys?Gly?Gly?Glu?Leu?Val

20??????????????????25??????????????????30

Asp?Thr?Leu?Gln?Phe?Val?Cys?Gly?Asp?Arg?Gly?Phe?Tyr?Phe?Ser?Arg

35??????????????????40??????????????????45

Pro?Ala?Ser?Arg?Val?Ser?Arg?Arg?Ser?Arg?Gly?Ile?Val?Glu?Glu?Cys

50??????????????????55??????????????????60

Cys?Phe?Arg?Ser?Cys?Asp?Leu?Ala?Leu?Leu?Glu?Thr?Tyr?Cys?Ala?Thr

65??????????????????70??????????????????75??????????????????80

Pro?Ala?Lys?Ser?Glu?Gly?Ala?Pro?Ala?His?Pro?Gly?Arg?Pro?Arg?Ala

85??????????????????90??????????????????95

Val?Pro?Thr?Gln?Cys?Asp?Val?Pro?Pro?Asn?Ser?Arg?Phe?Asp?Cys?Ala

100?????????????????105?????????????????110

Pro?Asp?Lys?Ala?Ile?Thr?Gln?Glu?Gln?Cys?Glu?Ala?Arg?Gly?Cys?Cys

115?????????????????120?????????????????125

Tyr?Ile?Pro?Ala?Lys?Gln?Gly?Leu?Gln?Gly?Ala?Gln?Met?Gly?Gln?Pro

130?????????????????135?????????????????140

Trp?Cys?Phe?Phe?Pro?Pro?Ser?Tyr?Pro?Ser?Tyr?Lys?Leu?Glu?Asn?Leu

145?????????????????150?????????????????155?????????????????160

Ser?Ser?Ser?Glu?Met?Gly?Tyr?Thr?Ala?Thr?Leu?Thr?Arg?Thr?Thr?Pro

165?????????????????170?????????????????175

Thr?Phe?Phe?Pro?Lys?Asp?Ile?Leu?Thr?Leu?Arg?Leu?Asp?Val?Met?Met

180?????????????????185?????????????????190

Glu?Thr?Glu?Asn?Arg?Leu?His?Phe?Thr?Ile?Lys?Asp?Pro?Ala?Asn?Arg

195?????????????????200?????????????????205

Arg?Tyr?Glu?Val?Pro?Leu?Glu?Thr?Pro?Arg?Val?His?Ser?Arg?Ala?Pro

210?????????????????215?????????????????220

Ser?Pro?Leu?Tyr?Ser?Val?Glu?Phe?Ser?Glu?Glu?Pro?Phe?Gly?Val?Ile

225?????????????????230?????????????????235?????????????????240

Val?His?Arg?Gln?Leu?Asp?Gly?Arg?Val?Leu?Leu?Asn?Thr?Thr?Val?Ala

245?????????????????250?????????????????255

Pro?Leu?Phe?Phe?Ala?Asp?Gln?Phe?Leu?Gln?Leu?Ser?Thr?Ser?Leu?Pro

260?????????????????265?????????????????270

Ser?Gln?Tyr?Ile?Thr?Gly?Leu?Ala?Glu?His?Leu?Ser?Pro?Leu?Met?Leu

275?????????????????280?????????????????285

Ser?Thr?Ser?Trp?Thr?Arg?Ile?Thr?Leu?Trp?Asn?Arg?Asp?Leu?Ala?Pro

290?????????????????295?????????????????300

Thr?Pro?Gly?Ala?Asn?Leu?Tyr?Gly?Ser?His?Pro?Phe?Tyr?Leu?Ala?Leu

305?????????????????310?????????????????315?????????????????320

Glu?Asp?Gly?Gly?Ser?Ala?His?Gly?Val?Phe?Leu?Leu?Asn?Ser?Asn?Ala

325?????????????????330?????????????????335

Met?Asp?Val?Val?Leu?Gln?Pro?Ser?Pro?Ala?Leu?Ser?Trp?Arg?Ser?Thr

340?????????????????345?????????????????350

Gly?Gly?Ile?Leu?Asp?Val?Tyr?Ile?Phe?Leu?Gly?Pro?Glu?Pro?Lys?Ser

355?????????????????360?????????????????365

Val?Val?Gln?Gln?Tyr?Leu?Asp?Val?Val?Gly?Tyr?Pro?Phe?Met?Pro?Pro

370?????????????????375?????????????????380

Tyr?Trp?Gly?Leu?Gly?Phe?His?Leu?Cys?Arg?Trp?Gly?Tyr?Ser?Ser?Thr

385?????????????????390?????????????????395?????????????????400

Ala?Ile?Thr?Arg?Gln?Val?Val?Glu?Asn?Met?Thr?Arg?Ala?His?Phe?Pro

405?????????????????410?????????????????415

Leu?Asp?Val?Gln?Trp?Asn?Asp?Leu?Asp?Tyr?Met?Asp?Ser?Arg?Arg?Asp

420?????????????????425?????????????????430

Phe?Thr?Phe?Asn?Lys?Asp?Gly?Phe?Arg?Asp?Phe?Pro?Ala?Met?Val?Gln

435?????????????????440?????????????????445

Glu?Leu?His?Gln?Gly?Gly?Arg?Arg?Tyr?Met?Met?Ile?Val?Asp?Pro?Ala

450?????????????????455?????????????????460

Ile?Ser?Ser?Ser?Gly?Pro?Ala?Gly?Ser?Tyr?Arg?Pro?Tyr?Asp?Glu?Gly

465?????????????????470?????????????????475?????????????????480

Leu?Arg?Arg?Gly?Val?Phe?Ile?Thr?Asn?Glu?Thr?Gly?Gln?Pro?Leu?Ile

485?????????????????490?????????????????495

Gly?Lys?Val?Trp?Pro?Gly?Ser?Thr?Ala?Phe?Pro?Asp?Phe?Thr?Asn?Pro

500?????????????????505?????????????????510

Thr?Ala?Leu?Ala?Trp?Trp?Glu?Asp?Met?Val?Ala?Glu?Phe?His?Asp?Gln

515?????????????????520?????????????????525

Val?Pro?Phe?Asp?Gly?Met?Trp?Ile?Asp?Met?Asn?Glu?Pro?Ser?Asn?Phe

530?????????????????535?????????????????540

Ile?Arg?Gly?Ser?Glu?Asp?Gly?Cys?Pro?Asn?Asn?Glu?Leu?Glu?Asn?Pro

545?????????????????550?????????????????555?????????????????560

Pro?Tyr?Val?Pro?Gly?Val?Val?Gly?Gly?Thr?Leu?Gln?Ala?Ala?Thr?Ile

565?????????????????570?????????????????575

Cys?Ala?Ser?Ser?His?Gln?Phe?Leu?Ser?Thr?His?Tyr?Asn?Leu?His?Asn

580?????????????????585?????????????????590

Leu?Tyr?Gly?Leu?Thr?Glu?Ala?Ile?Ala?Ser?His?Arg?Ala?Leu?Val?Lys

595?????????????????600?????????????????605

Ala?Arg?Gly?Thr?Arg?Pro?Phe?Val?Ile?Ser?Arg?Ser?Thr?Phe?Ala?Gly

610?????????????????615?????????????????620

His?Gly?Arg?Tyr?Ala?Gly?His?Trp?Thr?Gly?Asp?Val?Trp?Ser?Ser?Trp

625?????????????????630?????????????????635?????????????????640

Glu?Gln?Leu?Ala?Ser?Ser?Val?Pro?Glu?Ile?Leu?Gln?Phe?Asn?Leu?Leu

645?????????????????650?????????????????655

Gly?Val?Pro?Leu?Val?Gly?Ala?Asp?Val?Cys?Gly?Phe?Leu?Gly?Asn?Thr

660?????????????????665?????????????????670

Ser?Glu?Glu?Leu?Cys?Val?Arg?Trp?Thr?Gln?Leu?Gly?Ala?Phe?Tyr?Pro

675?????????????????680?????????????????685

Phe?Met?Arg?Asn?His?Asn?Ser?Leu?Leu?Ser?Leu?Pro?Gln?Glu?Pro?Tyr

690?????????????????695?????????????????700

Ser?Phe?Ser?Glu?Pro?Ala?Gln?Gln?Ala?Met?Arg?Lys?Ala?Leu?Thr?Leu

705?????????????????710?????????????????715?????????????????720

Arg?Tyr?Ala?Leu?Leu?Pro?His?Leu?Tyr?Thr?Leu?Phe?His?Gln?Ala?His

725?????????????????730?????????????????735

Val?Ala?Gly?Glu?Thr?Val?Ala?Arg?Pro?Leu?Phe?Leu?Glu?Phe?Pro?Lys

740?????????????????745?????????????????750

Asp?Ser?Ser?Thr?Trp?Thr?Val?Asp?His?Gln?Leu?Leu?Trp?Gly?Glu?Ala

755?????????????????760?????????????????765

Leu?Leu?Ile?Thr?Pro?Val?Leu?Gln?Ala?Gly?Lys?Ala?Glu?Val?Thr?Gly

770?????????????????775?????????????????780

Tyr?Phe?Pro?Leu?Gly?Thr?Trp?Tyr?Asp?Leu?Gln?Thr?Val?Pro?Ile?Glu

785?????????????????790?????????????????795?????????????????800

Ala?Leu?Gly?Ser?Leu?Pro?Pro?Pro?Pro?Ala?Ala?Pro?Arg?Glu?Pro?Ala

805?????????????????810?????????????????815

Ile?His?Ser?Glu?Gly?Gln?Trp?Val?Thr?Leu?Pro?Ala?Pro?Leu?Asp?Thr

820?????????????????825?????????????????830

Ile?Asn?Val?His?Leu?Arg?Ala?Gly?Tyr?Ile?Ile?Pro?Leu?Gln?Gly?Pro

835?????????????????840?????????????????845

Gly?Leu?Thr?Thr?Thr?Glu?Ser?Arg?Gln?Gln?Pro?Met?Ala?Leu?Ala?Val

850?????????????????855?????????????????860

Ala?Leu?Thr?Lys?Gly?Gly?Glu?Ala?Arg?Gly?Glu?Leu?Phe?Trp?Asp?Asp

865?????????????????870?????????????????875?????????????????880

Gly?Glu?Ser?Leu?Glu?Val?Leu?Glu?Arg?Gly?Ala?Tyr?Thr?Gln?Val?Ile

885?????????????????890?????????????????895

Phe?Leu?Ala?Arg?Asn?Asn?Thr?Ile?Val?Asn?Glu?Leu?Val?Arg?Val?Thr

900?????????????????905?????????????????910

Ser?Glu?Gly?Ala?Gly?Leu?Gln?Leu?Gln?Lys?Val?Thr?Val?Leu?Gly?Val

915?????????????????920?????????????????925

Ala?Thr?Ala?Pro?Gln?Gln?Val?Leu?Ser?Asn?Gly?Val?Pro?Val?Ser?Asn

930?????????????????935?????????????????940

Phe?Thr?Tyr?Ser?Pro?Asp?Thr?Lys?Val?Leu?Asp?Ile?Cys?Val?Ser?Leu

945?????????????????950?????????????????955?????????????????960

Leu?Met?Gly?Glu?Gln?Phe?Leu?Val?Ser?Trp?Cys

965?????????????????970

<210>5

<211>2040

<212>DNA

＜213〉artificial sequence

<220>

＜223〉His-biotin-Ci-Mpr domain 10-13

<400>5

atgggaatcc?caatggggaa?gtcgatgctg?gtgcttctca?ccttcttggc?cttcgcctcg?????60

tgctgcattg?ctgctggcgc?gccgaccggt?caccatcacc?atcaccacgc?gccgggcctg????120

aacgacatct?tcgaggccca?gaagatcgag?tggcacgaac?ctttcgatct?gactgaatgt????180

tcattcaaag?atggggctgg?caactccttc?gacctctcgt?ccctgtcaag?gtacagtgac????240

aactgggaag?ccatcactgg?gacgggggac?ccggagcact?acctcatcaa?tgtctgcaag????300

tctctggccc?cgcaggctgg?cactgagccg?tgccctccag?aagcagccgc?gtgtctgctg????360

ggtggctcca?agcccgtgaa?cctcggcagg?gtaagggacg?gacctcagtg?gagagatggc????420

ataattgtcc?tgaaatacgt?tgatggcgac?ttatgtccag?atgggattcg?gaaaaagtca????480

accaccatcc?gattcacctg?cagcgagagc?caagtgaact?ccaggcccat?gttcatcagc????540

gccgtggagg?actgtgagta?cacctttgcc?tggcccacag?ccacagcctg?tcccatgaag????600

agcaacgagc?atgatgactg?ccaggtcacc?aacccaagca?caggacacct?gtttgatctg????660

agctccttaa?gtggcagggc?gggattcaca?gctgcttaca?gcgagaaggg?gttggtttac????720

atgagcatct?gtggggagaa?tgaaaactgc?cctcctggcg?tgggggcctg?ctttggacag????780

accaggatta?gcgtgggcaa?ggccaacaag?aggctgagat?acgtggacca?ggtcctgcag????840

ctggtgtaca?aggatgggtc?cccttgtccc?tccaaatccg?gcctgagcta?taagagtgtg????900

atcagtttcg?tgtgcaggcc?tgaggccggg?ccaaccaata?ggcccatgct?catctccctg????960

gacaagcaga?catgcactct?cttcttctcc?tggcacacgc?cgctggcctg?cgagcaagcg???1020

accgaatgtt?ccgtgaggaa?tggaagctct?attgttgact?tgtctcccct?tattcatcgc???1080

actggtggtt?atgaggctta?tgatgagagt?gaggatgatg?cctccgatac?caaccctgat???1140

ttctacatca?atatttgtca?gccactaaat?cccatgcacg?gagtgccctg?tcctgccgga???1200

gccgctgtgt?gcaaagttcc?tattgatggt?ccccccatag?atatcggccg?ggtagcagga???1260

ccaccaatac?tcaatccaat?agcaaatgag?atttacttga?attttgaaag?cagtactcct???1320

tgcttagcgg?acaagcattt?caactacacc?tcgctcatcg?cgtttcactg?taagagaggt???1380

gtgagcatgg?gaacgcctaa?gctgttaagg?accagcgagt?gcgactttgt?gttcgaatgg???1440

gagactcctg?tcgtctgtcc?tgatgaagtg?aggatggatg?gctgtaccct?gacagatgag???1500

cagctcctct?acagcttcaa?cttgtccagc?ctttccacga?gcacctttaa?ggtgactcgc???1560

gactcgcgca?cctacagcgt?tggggtgtgc?acctttgcag?tcgggccaga?acaaggaggc???1620

tgtaaggacg?gaggagtctg?tctgctctca?ggcaccaagg?gggcatcctt?tggacggctg????1680

caatcaatga?aactggatta?caggcaccag?gatgaagcgg?tcgttttaag?ttacgtgaat????1740

ggtgatcgtt?gccctccaga?aaccgatgac?ggcgtcccct?gtgtcttccc?cttcatattc????1800

aatgggaaga?gctacgagga?gtgcatcata?gagagcaggg?cgaagctgtg?gtgtagcaca????1860

actgcggact?acgacagaga?ccacgagtgg?ggcttctgca?gacactcaaa?cagctaccgg????1920

acatccagca?tcatatttaa?gtgtgatgaa?gatgaggaca?ttgggaggcc?acaagtcttc????1980

agtgaagtgc?gtgggtgtga?tgtgacattt?gagtggaaaa?caaaagttgt?ctgcccttga????2040

<210>6

<211>679

<212>PRT

＜213〉artificial sequence

<220>

＜223〉His-biotin-Ci-Mpr domain 10-13 protein sequence

<400>6

Met?Gly?Ile?Pro?Met?Gly?Lys?Ser?Met?Leu?Val?Leu?Leu?Thr?Phe?Leu

1???????????????5???????????????????10??????????????????15

Ala?Phe?Ala?Ser?Cys?Cys?Ile?Ala?Ala?Gly?Ala?Pro?Thr?Gly?His?His

20??????????????????25??????????????????30

His?His?His?His?Ala?Pro?Gly?Leu?Asn?Asp?Ile?Phe?Glu?Ala?Gln?Lys

35??????????????????40??????????????????45

Ile?Glu?Trp?His?Glu?Pro?Phe?Asp?Leu?Thr?Glu?Cys?Ser?Phe?Lys?Asp

50??????????????????55??????????????????60

Gly?Ala?Gly?Asn?Ser?Phe?Asp?Leu?Ser?Ser?Leu?Ser?Arg?Tyr?Ser?Asp

65??????????????????70??????????????????75??????????????????80

Asn?Trp?Glu?Ala?Ile?Thr?Gly?Thr?Gly?Asp?Pro?Glu?His?Tyr?Leu?Ile

85??????????????????90??????????????????95

Asn?Val?Cys?Lys?Ser?Leu?Ala?Pro?Gln?Ala?Gly?Thr?Glu?Pro?Cys?Pro

100?????????????????105?????????????????110

Pro?Glu?Ala?Ala?Ala?Cys?Leu?Leu?Gly?Gly?Ser?Lys?Pro?Val?Asn?Leu

115?????????????????120?????????????????125

Gly?Arg?Val?Arg?Asp?Gly?Pro?Gln?Trp?Arg?Asp?Gly?Ile?Ile?Val?Leu

130?????????????????135?????????????????140

Lys?Tyr?Val?Asp?Gly?Asp?Leu?Cys?Pro?Asp?Gly?Ile?Arg?Lys?Lys?Ser

145?????????????????150?????????????????155?????????????????160

Thr?Thr?Ile?Arg?Phe?Thr?Cys?Ser?Glu?Ser?Gln?Val?Asn?Ser?Arg?Pro

165?????????????????170?????????????????175

Met?Phe?Ile?Ser?Ala?Val?Glu?Asp?Cys?Glu?Tyr?Thr?Phe?Ala?Trp?Pro

180?????????????????185?????????????????190

Thr?Ala?Thr?Ala?Cys?Pro?Met?Lys?Ser?Asn?Glu?His?Asp?Asp?Cys?Gln

195?????????????????200?????????????????205

Val?Thr?Asn?Pro?Ser?Thr?Gly?His?Leu?Phe?Asp?Leu?Ser?Ser?Leu?Ser

210?????????????????215?????????????????220

Gly?Arg?Ala?Gly?Phe?Thr?Ala?Ala?Tyr?Ser?Glu?Lys?Gly?Leu?Val?Tyr

225?????????????????230?????????????????235?????????????????240

Met?Ser?Ile?Cys?Gly?Glu?Asn?Glu?Asn?Cys?Pro?Pro?Gly?Val?Gly?Ala

245?????????????????250?????????????????255

Cys?Phe?Gly?Gln?Thr?Arg?Ile?Ser?Val?Gly?Lys?Ala?Asn?Lys?Arg?Leu

260?????????????????265?????????????????270

Arg?Tyr?Val?Asp?Gln?Val?Leu?Gln?Leu?Val?Tyr?Lys?Asp?Gly?Ser?Pro

275?????????????????280?????????????????285

Cys?Pro?Ser?Lys?Ser?Gly?Leu?Ser?Tyr?Lys?Ser?Val?Ile?Ser?Phe?Val

290?????????????????295?????????????????300

Cys?Arg?Pro?Glu?Ala?Gly?Pro?Thr?Asn?Arg?Pro?Met?Leu?Ile?Ser?Leu

305?????????????????310?????????????????315?????????????????320

Asp?Lys?Gln?Thr?Cys?Thr?Leu?Phe?Phe?Ser?Trp?His?Thr?Pro?Leu?Ala

325?????????????????330?????????????????335

Cys?Glu?Gln?Ala?Thr?Glu?Cys?Ser?Val?Arg?Asn?Gly?Ser?Ser?Ile?Val

340?????????????????345?????????????????350

Asp?Leu?Ser?Pro?Leu?Ile?His?Arg?Thr?Gly?Gly?Tyr?Glu?Ala?Tyr?Asp

355?????????????????360?????????????????365

Glu?Ser?Glu?Asp?Asp?Ala?Ser?Asp?Thr?Asn?Pro?Asp?Phe?Tyr?Ile?Asn

370?????????????????375?????????????????380

Ile?Cys?Gln?Pro?Leu?Asn?Pro?Met?His?Gly?Val?Pro?Cys?Pro?Ala?Gly

385?????????????????390?????????????????395?????????????????400

Ala?Ala?Val?Cys?Lys?Val?Pro?Ile?Asp?Gly?Pro?Pro?Ile?Asp?Ile?Gly

405?????????????????410?????????????????415

Arg?Val?Ala?Gly?Pro?Pro?Ile?Leu?Asn?Pro?Ile?Ala?Asn?Glu?Ile?Tyr

420?????????????????425?????????????????430

Leu?Asn?Phe?Glu?Ser?Ser?Thr?Pro?Cys?Leu?Ala?Asp?Lys?His?Phe?Asn

435?????????????????440?????????????????445

Tyr?Thr?Ser?Leu?Ile?Ala?Phe?His?Cys?Lys?Arg?Gly?Val?Ser?Met?Gly

450?????????????????455?????????????????460

Thr?Pro?Lys?Leu?Leu?Arg?Thr?Ser?Glu?Cys?Asp?Phe?Val?Phe?Glu?Trp

465?????????????????470?????????????????475?????????????????480

Glu?Thr?Pro?Val?Val?Cys?Pro?Asp?Glu?Val?Arg?Met?Asp?Gly?Cys?Thr

485?????????????????490?????????????????495

Leu?Thr?Asp?Glu?Gln?Leu?Leu?Tyr?Ser?Phe?Asn?Leu?Ser?Ser?Leu?Ser

500?????????????????505?????????????????510

Thr?Ser?Thr?Phe?Lys?Val?Thr?Arg?Asp?Ser?Arg?Thr?Tyr?Ser?Val?Gly

515?????????????????520?????????????????525

Val?Cys?Thr?Phe?Ala?Val?Gly?Pro?Glu?Gln?Gly?Gly?Cys?Lys?Asp?Gly

530?????????????????535?????????????????540

Gly?Val?Cys?Leu?Leu?Ser?Gly?Thr?Lys?Gly?Ala?Ser?Phe?Gly?Arg?Leu

545?????????????????550?????????????????555?????????????????560

Gln?Ser?Met?Lys?Leu?Asp?Tyr?Arg?His?Gln?Asp?Glu?Ala?Val?Val?Leu

565?????????????????570?????????????????575

Ser?Tyr?Val?Asn?Gly?Asp?Arg?Cys?Pro?Pro?Glu?Thr?Asp?Asp?Gly?Val

580?????????????????585?????????????????590

Pro?Cys?Val?Phe?Pro?Phe?Ile?Phe?Asn?Gly?Lys?Ser?Tyr?Glu?Glu?Cys

595?????????????????600?????????????????605

Ile?Ile?Glu?Ser?Arg?Ala?Lys?Leu?Trp?Cys?Ser?Thr?Thr?Ala?Asp?Tyr

610?????????????????615?????????????????620

Asp?Arg?Asp?His?Glu?Trp?Gly?Phe?Cys?Arg?His?Ser?Asn?Ser?Tyr?Arg

625?????????????????630?????????????????635?????????????????640

Thr?Ser?Ser?Ile?Ile?Phe?Lys?Cys?Asp?Glu?Asp?Glu?Asp?Ile?Gly?Arg

645?????????????????650?????????????????655

Pro?Gln?Val?Phe?Ser?Glu?Val?Arg?Gly?Cys?Asp?Val?Thr?Phe?Glu?Trp

660?????????????????665?????????????????670

Lys?Thr?Lys?Val?Val?Cys?Pro

675

<210>7

<21l>2040

<212>DNA

＜213〉artificial sequence

<220>

＜223〉His-biotin-Ci-Mpr domain 10-13 I1572T.The underscore sequence variation causes point mutation I1572T and generates the silent mutation S1573 in diagnosis SpeI site.

<400>7

atgggaatcc?caatggggaa?gtcgatgctg?gtgcttctca?ccttcttggc?cttcgcctcg?????60

tgctgcattg?ctgctggcgc?gccgaccggt?caccatcacc?atcaccacgc?gccgggcctg????120

aacgacatct?tcgaggccca?gaagatcgag?tggcacgaac?ctttcgatct?gactgaatgt????180

tcattcaaag?atggggctgg?caactccttc?gacctctcgt?ccctgtcaag?gtacagtgac????240

aactgggaag?ccatcactgg?gacgggggac?ccggagcact?acctcatcaa?tgtctgcaag????300

tctctggccc?cgcaggctgg?cactgagccg?tgccctccag?aagcagccgc?gtgtctgctg????360

ggtggctcca?agcccgtgaa?cctcggcagg?gtaagggacg?gacctcagtg?gagagatggc????420

ataattgtcc?tgaaatacgt?tgatggcgac?ttatgtccag?atgggattcg?gaaaaagtca????480

accaccatcc?gattcacctg?cagcgagagc?caagtgaact?ccaggcccat?gttcatcagc????540

gccgtggagg?actgtgagta?cacctttgcc?tggcccacag?ccacagcctg?tcccatgaag????600

agcaacgagc?atgatgactg?ccaggtcacc?aacccaagca?caggacacct?gtttgatctg????660

agctccttaa?gtggcagggc?gggattcaca?gctgcttaca?gcgagaaggg?gttggtttac????720

atgagcatct?gtggggagaa?tgaaaactgc?cctcctggcg?tgggggcctg?ctttggacag????780

accaggacta?gtgtgggcaa?ggccaacaag?aggctgagat?acgtggacca?ggtcctgcag????840

ctggtgtaca?aggatgggtc?cccttgtccc?tccaaatccg?gcctgagcta?taagagtgtg????900

atcagtttcg?tgtgcaggcc?tgaggccggg?ccaaccaata?ggcccatgct?catctccctg????960

gacaagcaga?catgcactct?cttcttctcc?tggcacacgc?cgctggcctg?cgagcaagcg???1020

accgaatgtt?ccgtgaggaa?tggaagctct?attgttgact?tgtctcccct?tattcatcgc???1080

actggtggtt?atgaggctta?tgatgagagt?gaggatgatg?cctccgatac?caaccctgat???1140

ttctacatca?atatttgtca?gccactaaat?cccatgcacg?gagtgccctg?tcctgccgga???1200

gccgctgtgt?gcaaagttcc?tattgatggt?ccccccatag?atatcggccg?ggtagcagga???1260

ccaccaatac?tcaatccaat?agcaaatgag?atttacttga?attttgaaag?cagtactcct???1320

tgcttagcgg?acaagcattt?caactacacc?tcgctcatcg?cgtttcactg?taagagaggt???1380

gtgagcatgg?gaacgcctaa?gctgttaagg?accagcgagt?gcgactttgt?gttcgaatgg???1440

gagactcctg?tcgtctgtcc?tgatgaagtg?aggatggatg?gctgtaccct?gacagatgag???1500

cagctcctct?acagcttcaa?cttgtccagc?ctttccacga?gcacctttaa?ggtgactcgc???1560

gactcgcgca?cctacagcgt?tggggtgtgc?acctttgcag?tcgggccaga?acaaggaggc???1620

tgtaaggacg?gaggagtctg?tctgctctca?ggcaccaagg?gggcatcctt?tggacggctg???1680

caatcaatga?aactggatta?caggcaccag?gatgaagcgg?tcgttttaag?ttacgtgaat???1740

ggtgatcgtt?gccctccaga?aaccgatgac?ggcgtcccct?gtgtcttccc?cttcatattc???1800

aatgggaaga?gctacgagga?gtgcatcata?gagagcaggg?cgaagctgtg?gtgtagcaca???1860

actgcggact?acgacagaga?ccacgagtgg?ggcttctgca?gacactcaaa?cagctaccgg???1920

acatccagca?tcatatttaa?gtgtgatgaa?gatgaggaca?ttgggaggcc?acaagtcttc???1980

agtgaagtgc?gtgggtgtga?tgtgacattt?gagtggaaaa?caaaagttgt?ctgcccttga???2040

<210>8

<211>679

<212>PRT

＜213〉artificial sequence

<220>

＜223〉His-biotin-Ci-Mpr domain 10-13 I1572T protein sequence.The I1572T sudden change has added underscore.

<400>8

Met?Gly?Ile?Pro?Met?Gly?Lys?Ser?Met?Leu?Val?Leu?Leu?Thr?Phe?Leu

1???????????????5???????????????????10??????????????????15

Ala?Phe?Ala?Ser?Cys?Cys?Ile?Ala?Ala?Gly?Ala?Pro?Thr?Gly?His?His

20??????????????????25??????????????????30

His?His?His?His?Ala?Pro?Gly?Leu?Asn?Asp?Ile?Phe?Glu?Ala?Gln?Lys

35??????????????????40??????????????????45

Ile?Glu?Trp?His?Glu?Pro?Phe?Asp?Leu?Thr?Glu?Cys?Ser?Phe?Lys?Asp

50??????????????????55??????????????????60

Gly?Ala?Gly?Asn?Ser?Phe?Asp?Leu?Ser?Ser?Leu?Ser?Arg?Tyr?Ser?Asp

65??????????????????70??????????????????75??????????????????80

Asn?Trp?Glu?Ala?Ile?Thr?Gly?Thr?Gly?Asp?Pro?Glu?His?Tyr?Leu?Ile

85??????????????????90??????????????????95

Asn?Val?Cys?Lys?Ser?Leu?Ala?Pro?Gln?Ala?Gly?Thr?Glu?Pro?Cys?Pro

100?????????????????105?????????????????110

Pro?Glu?Ala?Ala?Ala?Cys?Leu?Leu?Gly?Gly?Ser?Lys?Pro?Val?Asn?Leu

115?????????????????120?????????????????125

Gly?Arg?Val?Arg?Asp?Gly?Pro?Gln?Trp?Arg?Asp?Gly?Ile?Ile?Val?Leu

130?????????????????135?????????????????140

Lys?Tyr?Val?Asp?Gly?Asp?Leu?Cys?Pro?Asp?Gly?Ile?Arg?Lys?Lys?Ser

145?????????????????150?????????????????155?????????????????160

Thr?Thr?Ile?Arg?Phe?Thr?Cys?Ser?Glu?Ser?Gln?Val?Asn?Ser?Arg?Pro

165?????????????????170?????????????????175

Met?Phe?Ile?Ser?Ala?Val?Glu?Asp?Cys?Glu?Tyr?Thr?Phe?Ala?Trp?Pro

180?????????????????185?????????????????190

Thr?Ala?Thr?Ala?Cys?Pro?Met?Lys?Ser?Asn?Glu?His?Asp?Asp?Cys?Gln

195?????????????????200?????????????????205

Val?Thr?Asn?Pro?Ser?Thr?Gly?His?Leu?Phe?Asp?Leu?Ser?Ser?Leu?Ser

210?????????????????215?????????????????220

Gly?Arg?Ala?Gly?Phe?Thr?Ala?Ala?Tyr?Ser?Glu?Lys?Gly?Leu?Val?Tyr

225?????????????????230?????????????????235?????????????????240

Met?Ser?Ile?Cys?Gly?Glu?Asn?Glu?Asn?Cys?Pro?Pro?Gly?Val?Gly?Ala

245?????????????????250?????????????????255

Cys?Phe?Gly?Gln?Thr?Arg?Thr?Ser?Val?Gly?Lys?Ala?Asn?Lys?Arg?Leu

260?????????????????265?????????????????270

Arg?Tyr?Val?Asp?Gln?Val?Leu?Gln?Leu?Val?Tyr?Lys?Asp?Gly?Ser?Pro

275?????????????????280?????????????????285

Cys?Pro?Ser?Lys?Ser?Gly?Leu?Ser?Tyr?Lys?Ser?Val?Ile?Ser?Phe?Val

290?????????????????295?????????????????300

Cys?Arg?Pro?Glu?Ala?Gly?Pro?Thr?Asn?Arg?Pro?Met?Leu?Ile?Ser?Leu

305?????????????????310?????????????????315?????????????????320

Asp?Lys?Gln?Thr?Cys?Thr?Leu?Phe?Phe?Ser?Trp?His?Thr?Pro?Leu?Ala

325?????????????????330?????????????????335

Cys?Glu?Gln?Ala?Thr?Glu?Cys?Ser?Val?Arg?Asn?Gly?Ser?Ser?Ile?Val

340?????????????????345?????????????????350

Asp?Leu?Ser?Pro?Leu?Ile?His?Arg?Thr?Gly?Gly?Tyr?Glu?Ala?Tyr?Asp

355?????????????????360?????????????????365

Glu?Ser?Glu?Asp?Asp?Ala?Ser?Asp?Thr?Asn?Pro?Asp?Phe?Tyr?Ile?Asn

370?????????????????375?????????????????380

Ile?Cys?Gln?Pro?Leu?Asn?Pro?Met?His?Gly?Val?Pro?Cys?Pro?Ala?Gly

385?????????????????390?????????????????395?????????????????400

Ala?Ala?Val?Cys?Lys?Val?Pro?Ile?Asp?Gly?Pro?Pro?Ile?Asp?Ile?Gly

405?????????????????410?????????????????415

Arg?Val?Ala?Gly?Pro?Pro?Ile?Leu?Asn?Pro?Ile?Ala?Asn?Glu?Ile?Tyr

420?????????????????425?????????????????430

Leu?Asn?Phe?Glu?Ser?Ser?Thr?Pro?Cys?Leu?Ala?Asp?Lys?His?Phe?Asn

435?????????????????440?????????????????445

Tyr?Thr?Ser?Leu?Ile?Ala?Phe?His?Cys?Lys?Arg?Gly?Val?Ser?Met?Gly

450?????????????????455?????????????????460

Thr?Pro?Lys?Leu?Leu?Arg?Thr?Ser?Glu?Cys?Asp?Phe?Val?Phe?Glu?Trp

465?????????????????470?????????????????475?????????????????480

Glu?Thr?Pro?Val?Val?Cys?Pro?Asp?Glu?Val?Arg?Met?Asp?Gly?Cys?Thr

485?????????????????490?????????????????495

Leu?Thr?Asp?Glu?Gln?Leu?Leu?Tyr?Ser?Phe?Asn?Leu?Ser?Ser?Leu?Ser

500?????????????????505?????????????????510

Thr?Ser?Thr?Phe?Lys?Val?Thr?Arg?Asp?Ser?Arg?Thr?Tyr?Ser?Val?Gly

515?????????????????520?????????????????525

Val?Cys?Thr?Phe?Ala?Val?Gly?Pro?Glu?Gln?Gly?Gly?Cys?Lys?Asp?Gly

530?????????????????535?????????????????540

Gly?Val?Cys?Leu?Leu?Ser?Gly?Thr?Lys?Gly?Ala?Ser?Phe?Gly?Arg?Leu

545?????????????????550?????????????????555?????????????????560

Gln?Ser?Met?Lys?Leu?Asp?Tyr?Arg?His?Gln?Asp?Glu?Ala?Val?Val?Leu

565?????????????????570?????????????????575

Ser?Tyr?Val?Asn?Gly?Asp?Arg?Cys?Pro?Pro?Glu?Thr?Asp?Asp?Gly?Val

580?????????????????585?????????????????590

Pro?Cys?Val?Phe?Pro?Phe?Ile?Phe?Asn?Gly?Lys?Ser?Tyr?Glu?Glu?Cys

595?????????????????600?????????????????605

Ile?Ile?Glu?Ser?Arg?Ala?Lys?Leu?Trp?Cys?Ser?Thr?Thr?Ala?Asp?Tyr

610?????????????????615?????????????????620

Asp?Arg?Asp?His?Glu?Trp?Gly?Phe?Cys?Arg?His?Ser?Asn?Ser?Tyr?Arg

625?????????????????630?????????????????635?????????????????640

Thr?Ser?Ser?Ile?Ile?Phe?Lys?Cys?Asp?Glu?Asp?Glu?Asp?Ile?Gly?Arg

645?????????????????650?????????????????655

Pro?Gln?Val?Phe?Ser?Glu?Val?Arg?Gly?Cys?Asp?Val?Thr?Phe?Glu?Trp

660?????????????????665?????????????????670

Lys?Thr?Lys?Val?Val?Cys?Pro

675

Claims (40)

1. method that is used for the treatment of the Pompe of main body, comprise a kind of fusion rotein for the treatment of effective dose to described main body, described fusion rotein comprises the acid alpha-Glucosidase (GAA) of people or its fragment, and lysosome targeting domain, wherein, described lysosome targeting domain in non-Man-6-P dependent form mode in conjunction with people's non-cationic dependent form Man-6-P receptor.

2. method according to claim 1, wherein, described lysosome targeting domain comprises ripe human insulin-like growth factor II (IGF-II) or its fragment or sequence variants.

3. according to the method for claim 2, wherein, described lysosome targeting domain comprises into amino acid/11 and the 8-67 of acquaintance IGF-II.

4. method according to claim 1, wherein, described fusion rotein comprises the aminoacid 70-952 of people GAA.

5. method according to claim 1 wherein, is compared with wild type people GAA, has Man-6-P (M6P) level of reduction on the described fusion rotein.

6. method according to claim 1 wherein, does not have functional M6P level on the described fusion rotein.

7. method according to claim 1, wherein, described treatment effective dose is in the scope of the described main body body weight of 2.5～20mg/kg.

8. method according to claim 1, wherein, described fusion rotein is via intravenous administration.

9. method according to claim 1, wherein, described fusion rotein with per the bimester, every month, per three weeks, per two weeks, weekly, administration every day or interval administration to change.

10. method that is used for the treatment of the Pompe of main body, comprise a kind of fusion rotein for the treatment of effective dose to described main body, described fusion rotein comprises the amino acid/11 of ripe human insulin-like growth factor II (IGF-II) and the aminoacid 70-952 of 8-67 and the acid alpha-Glucosidase of people (GAA).

11. method according to claim 10, wherein, described fusion rotein further is included in the intervening sequence Gly-Ala-Pro between the aminoacid of the aminoacid of described one-tenth acquaintance IGF-II and described people GAA.

12. method according to claim 10 wherein, is compared with wild type people GAA, has Man-6-P (M6P) level of reduction on the described fusion rotein.

13. method according to claim 10 wherein, does not have functional M6P level on the described fusion rotein.

14. method that is used to reduce glycogen levels in the body, comprise a kind of fusion rotein that gives effective dose to the main body of suffering from Pompe, described fusion rotein comprises the acid alpha-Glucosidase (GAA) of people or its fragment, and lysosome targeting domain, wherein, described lysosome targeting domain in non-Man-6-P dependent form mode in conjunction with people's non-cationic dependent form Man-6-P receptor.

15. method according to claim 14, wherein, described lysosome targeting domain comprises ripe human insulin-like growth factor II (IGF-II) or its fragment or sequence variants.

16. method according to claim 15, wherein, described lysosome targeting domain comprises into amino acid/11 and the 8-67 of acquaintance IGF-II.

17. method according to claim 14, wherein, described fusion rotein comprises the aminoacid 70-952 of people GAA.

18. method according to claim 14 wherein, is compared with wild type people GAA, has Man-6-P (M6P) level of reduction on the described fusion rotein.

19. method according to claim 14 wherein, does not have functional M6P level on the described fusion rotein.

20. method according to claim 14, wherein, described treatment effective dose is in the scope of the described main body body weight of 2.5～20mg/kg.

21. method according to claim 14, wherein, described fusion rotein is via intravenous administration.

22. method according to claim 14, wherein, described fusion rotein with per the bimester, every month, per three weeks, per two weeks, weekly, administration every day or interval administration to change.

23. method that is used for reducing the glycogen levels of mammal lysosome, comprise the described lysosome of a kind of fusion rotein targeting that makes effective dose, described fusion rotein comprises the acid alpha-Glucosidase (GAA) of people or its fragment, and lysosome targeting domain, wherein, described lysosome targeting domain in non-Man-6-P dependent form mode in conjunction with people's non-cationic dependent form Man-6-P receptor.

24. method according to claim 23, wherein, described lysosome targeting domain comprises ripe human insulin-like growth factor II (IGF-II) or its fragment or sequence variants.

25. method according to claim 23, wherein, described lysosome targeting domain comprises into amino acid/11 and the 8-67 of acquaintance IGF-II.

26. method according to claim 23, wherein, described fusion rotein comprises the aminoacid 70-952 of people GAA.

27. the method for the glycogen levels of a muscular tissue that is used for reducing the main body of suffering from Pompe, comprise a kind of fusion rotein from effective dose to described muscular tissue that send, described fusion rotein comprises the acid alpha-Glucosidase (GAA) of people or its fragment, and lysosome targeting domain, wherein, described lysosome targeting domain in non-Man-6-P dependent form mode in conjunction with people's non-cationic dependent form Man-6-P receptor.

28. method according to claim 27, wherein, described muscular tissue is skeletal muscle.

29. one kind is used for the treatment of the main body myocardiac method relevant with Pompe, comprise a kind of fusion rotein that gives effective dose to described main body, described fusion rotein comprises the acid alpha-Glucosidase (GAA) of people or its fragment, and lysosome targeting domain, wherein, described lysosome targeting domain in non-Man-6-P dependent form mode in conjunction with people's non-cationic dependent form Man-6-P receptor.

30. method that is used for the treatment of the main body myopathy relevant with Pompe, comprise a kind of fusion rotein that gives effective dose to described main body, described fusion rotein comprises the acid alpha-Glucosidase (GAA) of people or its fragment, and lysosome targeting domain, wherein, described lysosome targeting domain in non-Man-6-P dependent form mode in conjunction with people's non-cationic dependent form Man-6-P receptor.

31. one kind is used to increase the active method of main body inner acidic alpha-Glucosidase (GAA) of suffering from Pompe, comprise to described main body and give a kind of fusion rotein, described fusion rotein comprises the acid alpha-Glucosidase (GAA) of people or its fragment, and lysosome targeting domain, wherein, described lysosome targeting domain in non-Man-6-P dependent form mode in conjunction with people's non-cationic dependent form Man-6-P receptor.

32. pharmaceutical composition that is applicable to the treatment Pompe, comprise a kind of fusion rotein for the treatment of effective dose, described fusion rotein comprises the acid alpha-Glucosidase (GAA) of people or its fragment, and lysosome targeting domain, wherein, described lysosome targeting domain in non-Man-6-P dependent form mode in conjunction with people's non-cationic dependent form Man-6-P receptor.

33. pharmaceutical composition according to claim 32, wherein, described lysosome targeting domain comprises ripe human insulin-like growth factor II (IGF-II) or its fragment or sequence variants.

34. pharmaceutical composition according to claim 32, wherein, described lysosome targeting domain comprises into amino acid/11 and the 8-67 of acquaintance IGF-II.

35. pharmaceutical composition according to claim 32, wherein, described fusion rotein comprises the aminoacid 70-952 of people GAA.

36. pharmaceutical composition according to claim 32, wherein, described fusion rotein comprises the aminoacid 70-952 of people GAA and amino acid/11 and the 8-67 that becomes acquaintance IGF-II.

37. pharmaceutical composition according to claim 36, wherein, described fusion rotein is included in the intervening sequence Gly-Ala-Pro between the aminoacid of the aminoacid of described people GAA and described one-tenth acquaintance IGF-II.

38. pharmaceutical composition according to claim 32 wherein, is compared with wild type people GAA, has Man-6-P (M6P) level of reduction on the described fusion rotein.

39. pharmaceutical composition according to claim 32 wherein, does not have functional M6P level on the described fusion rotein.

40. pharmaceutical composition according to claim 32, wherein, described pharmaceutical composition further comprises pharmaceutical carrier.

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