RU2780410C2 - Variants of acid alpha-glucosidase and their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically to therapeutic expression vectors; it can be used in medicine for the treatment of Pompe disease (GSDII) and Cori disease (GSDIII). A recombinant vector based on an adeno-associated virus (AAV) is proposed for the expression of truncated polypeptide of alpha-glycosidase (hereinafter – GAA).

EFFECT: invention provides for expression and secretion of human GAA variants at higher levels compared to human GAA protein of a wild type, which provides improved correction of pathological accumulation of glycogen in the entire body and induction of immunological tolerance to GAA.

17 cl, 1 tbl, 10 dwg, 1 ex

Description

The present invention relates to variants of acid α-glucosidase and their use.

Pompe disease, also known as glycogen storage disease (GSD) type II and acid maltase deficiency, is an autosomal recessive metabolic myopathy caused by deficiency of the lysosomal acid α-glucosidase enzyme (GAA). GAA is an exo-1,4 and 1,6-α-glucosidase that hydrolyzes glycogen to glucose in the lysosome. GAA deficiency leads to the accumulation of glycogen in lysosomes and causes progressive damage to the respiratory, cardiac and skeletal muscles. The disease ranges from a rapidly progressive childhood course, which is usually fatal by 1–2 years of age, to a more slowly progressive and heterogeneous course, which is associated with significant disease prevalence and early mortality in children and adults. Hirschhorn RR, The Metabolic and Molecular Bases of Inherited Disease, 3: 3389-3420 (2001, McGraw-Hill); Van der Ploeg and Reuser, Lancet 372: 1342-1351 (2008).

Current human therapy for the treatment of Pompe disease involves the administration of recombinant human GAA, otherwise referred to as enzyme replacement therapy (ERT). An ERT effect has been demonstrated in severe pediatric GSDII. However, the benefit of enzyme therapy is limited by the need for frequent infusions and the development of inhibitory antibodies against recombinant hGAA (Amalfitano, A., et al. (2001) Genet. In Med. 3:132-138). In addition, ERT does not provide effective whole-body correction, likely due to a combination of poor protein biodistribution after delivery to a peripheral vein, lack of uptake from some tissues, and high immunogenicity.

As an alternative or adjunct to ERT, the feasibility of gene therapy approaches to treat GSDII has been investigated (Amalfitano, A., et al. (1999) Proc. Natl. Acad. Sci. USA 96:8861-8866, Ding, E. (2002) Mol Ther 5:436-446, Fraites, T. J., et al (2002) Mol Ther 5:571-578, Tsujino, S., et al (1998) Hum. Gene Ther 9:1609-1616). However, muscle-targeted gene transfer to correct a genetic defect has run into limitations due to the systemic nature of the disease and the fact that transgene expression in muscle tends to be more immunogenic than in other tissues.

Doerfler et al., 2016 describe the combined administration of two constructs encoding human GAA with codon optimization, one driven by a specific liver promoter and the other driven by a specific muscle promoter. Liver-specific promoter-driven GAA expression is used to promote immunological tolerance to GAA in a Gaa-/- mouse model, while muscle-specific promoter-driven GAA expression mediates the expression of the therapeutic protein in the part of tissues targeted for therapy. However, this strategy is not entirely satisfactory in that it requires the use of multiple constructs and does not result in GAA expression throughout the body.

In the past, modified GAA proteins have been proposed to improve the treatment of lysosomal storage disease. In particular, WO2004064750 and Sun et al. 2006 disclosed a chimeric GAA polypeptide containing a signal peptide operably linked to GAA as a way to enhance protein targeting into the secretory pathway.

However, the therapy available to the patient is not entirely satisfactory and improved GAA polypeptides and GAA production are still needed in the art. In particular, there is still a need for a long-term effect of GAA treatment, high levels of GAA production, improved immunological tolerance to the resulting GAA polypeptide, and improved uptake of GAA by cells and tissues in need of it. In addition, in WO2004064750 and Sun et al., 2006, tissue distribution of a chimeric GAA polypeptide is disclosed, which is not entirely satisfactory. Therefore, there is still a need for a GAA polypeptide that will be fully therapeutic, allowing for the correction of glycogen storage in most, if not all, tissues of interest.

SUMMARY OF THE INVENTION

The present invention relates to GAA variants that express and secrete at higher levels than wild-type GAA protein and which provide improved correction of pathological glycogen accumulation throughout the body and lead to the induction of immunological tolerance to GAA.

In one aspect, the invention relates to a truncated GAA polypeptide that contains a deletion of at least one amino acid from the N-terminus of the parent GAA polypeptide, where the parent polypeptide corresponds to the form of the GAA polypeptide precursor free from its signal peptide. In a particular embodiment, said truncated GAA polypeptide has at least 2, in particular at least 2, in particular at least 3, in particular at least 4, in particular at least 5, in particular at least 6, in particular at least 7, in particular at least 8 consecutive amino acids, at its N-terminus, compared to the parent GAA polypeptide. In another embodiment, said truncated GAA polypeptide has at most 75, in particular at most 70, in particular at most 60, in particular at most 55, in particular at most 50, in particular at most 47, in particular at most 46, in in particular at most 45, in particular at most 44, in particular at most 43 consecutive amino acids deleted at its N-terminus, compared to the parent GAA polypeptide. In a further specific embodiment, said truncated GAA polypeptide has at most 47, in particular at most 46, in particular at most 45, in particular at most 44, in particular at most 43 consecutive amino acids deleted at its N-terminus, compared with the parent GAA polypeptide. In another specific embodiment, said truncated GAA polypeptide has from 1 to 75, in particular from 1 to 47, in particular from 1 to 46, in particular from 1 to 45, in particular from 1 to 44, in particular, 1 to 43 consecutive amino acids deleted at its N-terminus compared to the parent GAA polypeptide. In another embodiment, said truncated GAA polypeptide has from 2 to 43, in particular from 3 to 43, in particular from 4 to 43, in particular from 5 to 43, in particular from 6 to 43, in particular from 7 to 43, in particular 8 to 43 consecutive amino acids deleted at its N-terminus, compared to the parent GAA polypeptide. In a more specific embodiment, said truncated GAA polypeptide has 6, 7, 8, 9, 10, 27, 28, 29, 30, 31, 40, 41, 42, 43, 44, 45, 46, or 47 consecutive amino acids deleted at its N-terminus, compared to the parent GAA polypeptide, in particular 7, 8, 9, 28, 29, 30, 41, 42, 43 or 44, more specifically 8, 29, 42 or 43 consecutive amino acids truncated by its N-terminus, compared to the parent GAA polypeptide. In a further specific embodiment, the parent polypeptide is human GAA (hGAA), in particular hGAA, having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1.

In a specific embodiment, the truncated GAA polypeptide of the invention has the sequence shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 34, and SEQ ID NO: 35.

In addition, the truncated GAA polypeptide of the invention may further comprise a signal peptide fused to its N-terminus, in particular a signal peptide selected from the group consisting of SEQ ID NO: 3 to 7, in particular a signal peptide from SEQ ID NO: 3.

In another aspect, the invention relates to a nucleic acid molecule encoding a truncated GAA polypeptide as described above, optionally fused to a signal peptide through its N-terminus. In some embodiments, the nucleic acid molecule has a nucleotide sequence optimized to improve expression and/or improve immunological tolerance for the truncated GAA polypeptide in vivo, particularly in a human subject.

In yet another aspect, the invention relates to a nucleic acid construct that comprises a nucleic acid molecule of the invention operably linked to one or more regulatory sequences such as a promoter, intron, polyadenylation signal, and/or enhancer (e.g., a cis-regulatory module, or CRM ). In a specific embodiment, the promoter is a specific liver promoter, preferably selected from the group consisting of the α-1-antitrypsin (hAAT) promoter, transthyretin promoter, albumin promoter, and thyroxin-binding globulin (TBG) promoter. In another specific embodiment, the promoter is a specific muscle promoter such as the Spc5-12, MCK and desmin promoters. In another embodiment, the promoter is a general purpose promoter such as the CMV, CAG and PGK promoters. The nucleic acid construct may optionally further comprise an intron, in particular an intron selected from the group consisting of a human β-globin b2 (or HBB2) intron, a FIX intron, a chicken β-globin intron, and an SV40 intron, wherein said intron is optionally a modified intron. , such as the modified HBB2 intron of SEQ ID NO: 17, the modified FIX intron of SEQ ID NO: 19, or the modified chicken β-globin intron of SEQ ID NO: 21. In a particular embodiment of the nucleic acid construct of the invention, said construct preferably contains in this order: enhancer; intron; a promoter, in particular a specific liver promoter; a nucleic acid sequence encoding a GAA protein; and a polyadenylation signal, the construct contains, preferably in this order: an ApoE control region; an HBB2 intron, in particular a modified HBB2 intron; hAAT promoter; a nucleic acid sequence encoding a truncated GAA polypeptide; and a bovine growth hormone polyadenylation signal. In specific embodiments, said nucleic acid construct more specifically comprises a nucleotide sequence from any one of SEQ ID NOs: 22 to 26.

In another aspect, the invention relates to a vector containing a nucleic acid molecule or a nucleic acid construct as disclosed herein. The vector of the invention may in particular be a viral vector, preferably a retroviral vector such as a lentiviral vector, or an AAV vector. Preferably, the vector is a single- or double-stranded self-complementary AAV vector, preferably an AAV vector with an AAV-derived capsid, e.g., a capsid from AAV1, AAV2, AAV2 variant, AAV3, AAV3 variant, AAV3B, AAV3B variant, AAV4, AAV5, AAV6, AAV6 variant , AAV7, AAV8, AAV9, AAV10 such as AAVcy10 and AAVrh10, AAVrh74, AAVdj, AAV-Anc80, AAV-LK03, AAV2i8, porcine AAV such as AAVpo4 and AAVpo6 capsid, or with a chimeric capsid. In a specific embodiment, the vector is an AAV vector with an AAV8, AAV9, AAVrh74 or AAV2i8 capsid, in particular an AAV8, AAV9 or AAVrh74 capsid, more specifically an AAV8 capsid.

In yet another aspect, the invention relates to a cell transformed with a nucleic acid molecule, a nucleic acid construct, or a vector of the invention. More specifically, the cell is a liver cell or a muscle cell.

In a particular aspect, the invention relates to a pharmaceutical composition which comprises, in a pharmaceutically acceptable carrier, a truncated GAA polypeptide, nucleic acid molecule, nucleic acid construct, vector or cell of the invention.

The invention further relates to a truncated GAA polypeptide, nucleic acid molecule, nucleic acid construct, vector or cell of the invention for use as a drug.

The invention further provides a truncated GAA polypeptide, nucleic acid molecule, nucleic acid construct, vector or cell of the invention for use in a method of treating glycogen storage disease. In a specific embodiment, the glycogen storage disease is GSDI, GSDII, GSDIII, GSDIV, GSDV, GSDVI, GSDVII, GSDVIII, or lethal congenital heart glycogen storage disease. In a more specific embodiment, the glycogen storage disease is selected from the group consisting of GSDI, GSDII and GSDIII, more specifically from the group consisting of GSDII and GSDIII. In an even more specific embodiment, the glycogen storage disease is GSDII.

DESCRIPTION OF THE DRAWINGS

Fig. 1. Deletion of parts of hGAA increases its secretion in vitro. Part A. Human hepatoma (Huh7) cells were transfected using Lipofectamine ^TM and a green fluorescent protein (GFP) expression control plasmid or plasmids expressing wild-type hGAA (hGAA) or an hGAA sequence optimized according to two different algorithms (hGAAco1 and co2, respectively). Various hGAA constructs contained a wild-type signal peptide or a human α-1-antitrypsin (sp2) signal peptide. Truncated hGAA was obtained by deleting 8 amino acids after the signal peptide (Δ8). 48 hours after transfection, hGAA activity in culture media was measured by fluorogenic enzyme assay and GAA activity was assessed from the reaction product calibration curve as described in Materials and Methods. The histogram plot shows the mean±SE for secreted hGAA levels from three different experiments. Statistical analysis was performed by paired Student's t-test, the histogram shows the resulting p-values (*=p<0.05, as indicated). Part B. Human hepatoma (Huh7) cells were transfected using lipofectamine and a GFP expression control plasmid or hGAAco1 expression plasmids with a wild-type or chymotrypsinogen B1 (sp7) signal peptide. The hGAA protein was truncated by removing 8 or 42 amino acids after the signal peptide (Δ8 and Δ42, respectively). 48 hours after transfection, hGAA activity in culture media was measured by fluorogenic enzyme assay as above. The histogram plot shows the mean±SE for secreted hGAA levels from three different experiments. Statistical analysis was performed by ANOVA (*=p<0.05 as indicated).

Fig. 2. Deletion of portions of hGAA increases its secretion into the bloodstream in a mouse model of Pompe disease. At 3 months of age, GAA −/− mice (n=4-5 mice/group) were intravenously injected with PBS or 2E12 vg/kg AAV8 vectors expressing sequence optimized hGAA (hGAAco1) under the transcriptional control of a specific liver promoter. The wild-type signal peptide from hGAA was replaced with the signal peptide of chymotrypsinogen B1 (sp7), and the hGAA sequence was used as the full-length native sequence or truncated by deleting 8 or 42 amino acids after the signal peptide (Δ8 and Δ42, respectively). One month after injection, mice were bled and hGAA activity was measured using a serum fluorogenic assay. Statistical analysis was performed by ANOVA (*=p<0.05 as indicated).

Fig. 3. Signal peptides enhance hGAA secretion. Human hepatoma (Huh7) cells were transfected with Lipofectamine ^TM and a control plasmid (GFP), wild type hGAA expression plasmid (designated as sp1), or sequence optimized Δ8 hGAA (hGAAco) expression plasmid fused with signal peptides 6-8 (sp6-8). 48 hours after transfection, hGAA activity in culture media was measured by fluorogenic enzyme assay, and GAA activity was assessed from a calibration curve for 4-methylumbelliferone. The histogram plot shows the mean±SE for secreted hGAA levels from three different experiments. Statistical analysis was performed by ANOVA (*=p<0.05 vs. mimic transfected cells).

Fig. 4. Truncated Δ8 hGAA effectively corrects glycogen storage in a mouse model of Pompe disease. At 4 months of age, wild-type (WT) and GAA-/- mice (n=4-5 mice/group) were intravenously injected with PBS or 6E11 vg/kg AAV8 vectors expressing Δ8 hGAA (hGAAco) with an optimized sequence under transcriptional control of the promoter human α-1 antitrypsin and fused to signal peptide 1, 2, 7 and 8 (sp1, 2, 7, 8). Part A. Histogram showing hGAA activity as measured by fluorogenic blood assay 3 months after vector injection. Statistical analysis was performed by ANOVA, the histogram shows the obtained p-values in comparison with GAA-/- animals treated with PBS (*=p<0.05). Part B-D. Biochemical correction of glycogen content in the heart, diaphragm and quadriceps. At 4 months of age, GAA −/− mice were treated as described above. Mice were sacrificed 3 months after injection and glycogen content was assessed. Bar graphs show glycogen content expressed as glucose released after enzymatic breakdown of glycogen measured in the heart (Part B), diaphragm (Part C), and quadriceps (Part D). Statistical analysis was performed by ANOVA (*=p<0.05 vs GAA-/- mice injected with PBS).

Fig. 5. Highly secreted hGAA reduces the humoral response in a mouse model of Pompe disease. At 4 months of age, GAA-/- mice were intravenously injected with PBS or two different doses (5E11 or 2E12 vg/kg) of AAV8 vectors containing an optimized sequence under the transcriptional control of the human α-1-antitrypsin promoter, which encode Δ8 hGAA fused to a signal peptide 1 (co), signal peptide 2 (sp2-Δ8-co), signal peptide 7 (sp7-Δ8-co), or signal peptide 8 (sp8-Δ8-co). 1 month after injection, sera were analyzed for the presence of anti-hGAA antibodies by ELISA. Quantitation was performed using purified mouse IgG as a standard. Statistical analysis was performed by ANOVA using Dunnett's post hoc test (*=p<0.01).

Fig. 6. Injection of AAV8-hAAT-sp7-Δ8-hGAAco1 leads to efficient secretion of hGAA in blood and uptake in muscle by NHP. Two Macaca fascicularis monkeys on day 0 were injected with 2E12 vg/kg AAV8-hAAT-sp7-Δ8-hGAAco1. Part A. Western blotting of hGAA was performed in serum from two monkeys, which was obtained 12 days before and 30 days after the introduction of the vector. Shown on the left are the positions of the molecular weight marker bands running parallel to the samples. Part B 3 months after vector injection, monkeys were sacrificed and tissues were harvested for biochemical evaluation of hGAA uptake. Western blotting of hGAA was performed on tissue extracts obtained from biceps and diaphragm. An anti-tubulin antibody was used as a loading control. Shown on the left are the positions of the molecular weight marker bands running parallel to the samples.

Fig. 7. Biochemical correction of liver glycogen content in GDE-/- animals injected with an hGAA-expressing vector. At 3 months of age, wild-type (WT) or GDE-/- mice were intravenously injected with PBS or AAV8 vectors expressing codon-optimized hGAA under transcriptional control of the human α-1-antitrypsin promoter and fused to signal peptide 7 (AAV8-hAAT-sp7 -Δ8-hGAAco1) at a dose of 1E11 or 1E12 vg/mouse. The histogram plot shows the glycogen content expressed as glucose released after the enzymatic breakdown of glycogen as measured in the liver. Statistical analysis was performed by ANOVA (*=p<0.05 vs GDE −/− mice injected with PBS, =p<0.05 versus WT animals injected with PBS).

Fig. 8. GAA activity in media of cells transfected with plasmids encoding various GAA variants. GAA activity was measured in HuH7 cell media 24 (part A) and 48 (part B) hours after transfection with plasmids containing optimized sequences encoding native GAA combined with native GAA sp1 signal peptide (co) or encoding engineered GAA containing native GAA combined with a heterologous sp7 signal peptide (sp7-co). The effect of various deletions in the GAA coding sequence after the sp7 signal peptide (sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co, sp7-Δ47-co, sp7-Δ62-co) was evaluated. An eGFP encoding plasmid was used as a negative control. Statistical analysis was performed by one-way ANOVA with Tukey's post hoc test. The sharp sign (#) in the columns denotes statistically significant differences compared to co; the symbol tau (τ) denotes statistically significant differences compared to sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co. Data are mean±SD of two independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, except for the use of different characters.

Fig. 9. Intracellular GAA activity of various GAA variants. GAA activity was measured in lysates of HuH7 cells 48 hours after transfection with plasmids containing optimized sequences encoding native GAA combined with native signal peptide sp1 GAA (co) or encoding engineered GAA containing native GAA combined with heterologous signal peptide sp7 (sp7 -co). The effect of various deletions in the GAA coding sequence after the signal peptide (sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co, sp7-Δ47-co, sp7-Δ62-co) was evaluated. An eGFP encoding plasmid was used as a negative control. Statistical analysis was performed by one-way ANOVA with Tukey's post hoc test. The symbol tau (τ) denotes statistically significant differences compared to sp7-co, sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co. Data are mean±SD of two independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, except for the use of different characters.

Fig. 10. Increased GAA activity in cell media using Δ8 deletion combined with sp6 or sp8 signal peptides. GAA activity was measured in media (part A) and lysates (part B) of HuH7 cells 48 hours after transfection with plasmids containing optimized sequences encoding native GAA combined with native GAA sp1 signal peptide (co) or encoding engineered GAA containing native GAA combined with a heterologous sp6 or sp8 signal peptide (sp6-co or sp8-co). The effect of deletion of 8 amino acids in the GAA coding sequence after the signal peptide (sp6-Δ8-co, sp8-Δ8-co) was evaluated. An eGFP encoding plasmid was used as a negative control. Statistical analysis was performed by one-way ANOVA with Tukey's post hoc test. Asterisks in columns denote statistically significant differences compared to co. Data are mean±SD of two independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, except for the use of different characters.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a truncated GAA polypeptide, to a nucleic acid molecule encoding such a truncated GAA polypeptide, to a nucleic acid construct containing said nucleic acid, to a vector containing said nucleic acid construct, to a cell containing said nucleic acid molecule or construct or vector , and to a pharmaceutical composition containing a polypeptide, nucleic acid molecule, nucleic acid construct, vector or cell in accordance with the invention. The inventors have surprisingly shown that the truncated form of GAA according to the invention significantly improves the secretion of GAA while reducing its immunogenicity.

Lysosomal acid α-glucosidase or "GAA" (E.C. 3.2.1.20) (1,4-α-D-glucanglucohydrolase) is an exo-1,4-α-D-glucosidase that hydrolyzes both α-1,4 and and α-1,6 bonds in oligosaccharides in order to release glucose. GAA deficiency leads to glycogen storage disease type II (GSDII), also referred to as Pompe disease (despite the term formally referring to the childhood-onset form of the disease). It catalyzes the complete destruction of glycogen with a slowdown at the branch points. human acid α-glucosidase gene in 28 t. on chromosome 17 encodes a 3.6 kb mRNA that produces a 951 amino acid polypeptide (Hoefsloot et al., (1988) EMBO J. 7: 1697; Martiniuk et al., (1990) DNA and Cell Biology 9: 85). The enzyme allows co-translational N-linked glycosylation in the endoplasmic reticulum. It is synthesized as a 110 kD precursor form that matures by extensive modification of glycosylation, phosphorylation, and by proteolytic processing through an approximately 90 kD endosomal intermediate to final 76 and 67 kD lysosomal forms (Hoefsloot, (1988) EMBO J. 7: 1697; Hoefsloot et al., (1990) Biochem J. 272: 485; Wisselaar et al., (1993) J. Biol. Chem. 268: 2223; Hermans et al., (1993) Biochem. J. 289: 681).

In patients with GSDII, acid α-glucosidase deficiency causes a massive accumulation of glycogen in lysosomes, impairing cellular function (Hirschhorn, R. and Reuser, A. J. (2001), The Metabolic and Molecular Basis for Inherited Disease, (ed. Scriver, C. R. et al. ), pp. 3389-3419 (McGraw-Hill, New York).In the most common childhood form, patients show progressive muscle degeneration and cardiomyopathy and die before age 2. Severe malnutrition occurs in the juvenile and adult forms.

In addition, patients with other GSDs may benefit from the administration of an optimized form of GAA. For example, it has been shown (Sun et al. (2013) Mol Genet Metab 108(2): 145; WO2010/005565) that GAA administration reduces glycogen in primary myoblasts in patients with type III glycogen storage disease (GSDIII).

In particular, in the context of the present invention, a "GAA precursor form" is a form of a GAA polypeptide that contains its natural signal peptide. For example, the sequence from SEQ ID NO: 2 is the precursor form of human GAA (hGAA). In SEQ ID NO: 2, amino acid residues 1-27 correspond to the signal peptide from the hGAA polypeptide. This hGAA signal peptide sequence is also shown in SEQ ID NO: 4.

In the context of the present invention, the truncated GAA polypeptide of the invention is derived from the parent GAA polypeptide. In accordance with the present invention, a "parental GAA polypeptide" is a functional GAA precursor sequence as defined above, but free of its signal peptide. For example, with reference to a typical human wild-type GAA polypeptide, the complete wild-type GAA polypeptide (i.e., the precursor form of GAA) is shown in SEQ ID NO: 2 or SEQ ID NO: 30 and has a signal peptide (corresponding to amino acids 1-27 of SEQ ID NO: 2 or SEQ ID NO: 30), while the parental GAA polypeptide serving as the basis for the truncated GAA forms of these wild-type human GAA polypeptides are shown in SEQ ID NO: 1 and SEQ ID NO: 33, respectively , and do not have a signal peptide. In this example, the latter, corresponding to amino acids 28-952 of SEQ ID NO: 2 and amino acids 28-952 of SEQ ID NO: 30, is referred to as the parental GAA polypeptide.

According to the invention, the truncated GAA polypeptide of the invention is a functional GAA polypeptide, ie it has the functionality of a wild-type GAA polypeptide. As defined above, the functionality of wild-type GAA is to hydrolyze both the α-1,4 and α-1,6 bonds of oligosaccharides and polysaccharides, more specifically glycogen, to release glucose. The functional GAA protein encoded by the nucleic acid of the invention may have a glycogen hydrolytic activity of at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or at least 100% relative to the polypeptide Wild-type GAA of SEQ ID NO: 1 or SEQ ID NO: 33. The activity of the GAA protein encoded by the nucleic acid of the invention may even be greater than 100%, such as greater than 110%, 120%, 130%, 140%, or even more than 150% of the activity of the wild-type GAA protein from SEQ ID NO: 1 or from SEQ ID NO: 33.

The amino acid sequence of the parent GAA polypeptide or its coding sequence may be from any source, including avian and mammalian species. The term "birds" as used herein includes, but is not limited to, chicken, duck, goose, quail, turkey, and pheasant. The term "mammal" as used herein includes, but is not limited to, human, simians and other non-human primates, cow, sheep, goat, horse, cat, dog, lagomorphs, etc. In embodiments of the invention , the parental GAA polypeptide is a human, mouse or quail, in particular human, GAA polypeptide.

In addition, the parent GAA polypeptide may be a functional variant of the GAA polypeptide that contains one or more amino acid modifications, such as an amino acid insertion, deletion and/or substitution, compared to the GAA polypeptide. For example, the parent polypeptide may be a functional derivative of a human GAA polypeptide, such as the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1 having at least 80, 85, 90, 95, 96 , 97, 98 or at least 99% sequence identity with this human GAA polypeptide. For example, in addition to the truncation defined above, a functional GAA polypeptide variant may have between 0 and 50, between 0 and 30, between 0 and 20, between 0 and 15, between 0 and 10, or between 0 and 5 amino acid changes in the parent a GAA polypeptide, such as the parent GAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1. In particular, the parent GAA polypeptide may consist of a human GAA polypeptide having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1.

The term "identical" and its derivatives refer to sequence identity between two nucleic acid molecules. When the position in both of the two compared sequences is occupied by the same base, for example, if the position in each of the two DNA molecules is occupied by adenine, then the molecules are identical at that position. The % identity between two sequences is a function of the number of matching positions that the two sequences have divided by the number of positions compared, x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences are 60% identical. In general, comparison is performed when two sequences are aligned in order to give maximum identity. Various bioinformatic tools known to the person skilled in the art can be used to align nucleic acid sequences, such as BLAST or FASTA.

The parent GAA polypeptide can also be a GAA variant such as GAA II as described by Kunita et al., (1997) Biochemica et Biophysica Acta 1362: 269; polymorphisms and SNPs in GAA are described by Hirschhorn, R. and Reuser, A. J. (2001), The Metabolic and Molecular Basis for Inherited Disease (eds. Scriver, C. R., Beaudet, A. L., Sly, W. S. and Valle, D.), page 3389 -3419. McGraw-Hill, New York, see pp. 3403-3405. Any GAA polypeptide variant known in the art can be used as a basis for determining the parent GAA polypeptide. Exemplary GAA polypeptide variants include SEQ ID NO: 2 (NCBI reference sequence NP_000143.2); SEQ ID NO: 29 (GenBank AAA52506.1); SEQ ID NO: 30 (GenBank CAA68763.1); SEQ ID NO: 31 (GenBank: EAW89583.1) and SEQ ID NO: 32 (GenBank ABI53718.1). Other effective options include those described in Hoefsloot et al., (1988) EMBO J. 7: 1697; and Van Hove et al., (1996) Proc. Natl. Acad. sci. USA 93: 65 (human), and Genbank accession number NM_008064 (mouse). Other GAA polypeptide variants include those described in WO2012/145644, WO00/34451 and US6858425. In a specific embodiment, the parental GAA polypeptide is derived from the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 30.

A truncated form of GAA according to the invention is an N-terminally truncated form of a parent GAA polypeptide in which at least one amino acid is removed from the N-terminus of said parent GAA polypeptide.

By "truncated form" is meant a GAA polypeptide that contains one or more consecutive amino acids removed from the N-terminal portion of the parent GAA polypeptide. For example, a GAA fragment may have from 1 to 75 consecutive amino acids, or more than 75 consecutive amino acids truncated from its N-terminus, compared to the parent GAA polypeptide. In particular, a truncated GAA polypeptide may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75 consecutive amino acids truncated from its N-terminus compared to the parent GAA protein (in particular, the truncated form of the parent hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1). Using alternative nomenclature, a GAA polypeptide resulting from a 1 amino acid truncation in the parent GAA polypeptide is referred to as the Δ1 truncated form of GAA, a GAA polypeptide resulting from a truncation of 2 consecutive amino acids from the N-terminus is referred to as the Δ2 truncated form of GAA, a GAA polypeptide resulting from a truncated 3 consecutive amino acids in parent GAA polypeptide is referred to as Δ3 truncated form GAA), etc. In a specific embodiment, the truncated GAA polypeptide of the invention is Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, δ14, δ15, δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, ON, Δ25, ONA, Δ27, Δ28, ONA, PA, δ31, δ32, ON, ON, ONA, δ36, δ37, δ37, ONA, δ37, δ37, ONA, δ37, ONA, δ37, δ37, ONAS. Δ38, δ39, δ40, δ41, Δ42, δ43, δ44, Δ45, Δ46, Δ47, Δ48, δ49, PA, Δ51, δ52, Δ53, Δ54, PA, δ56, δ57, δ58, ON, Δ60, δ61, Δ62, Δ62, Δ63, Δ64, Δ65, Δ66, Δ67, Δ68, Δ69, Δ70, Δ71, Δ72, Δ73, Δ74, or Δ75 a truncated form of GAA (in particular, a truncated form of the hGAA parent protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1).

In another specific embodiment, the truncated GAA polypeptide of the invention is Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, ONA, Δ29, Δ30, Δ31, ON32, PA, Δ34, Δ35, Δ36, ON37, ONA, δ39, Δ40, ONA, Δ42, ON43, ONA, Δ44, ONA, ONA, Δ44, ONA, δ4, ONA, PA. Δ45, Δ46 or Δ47 truncated form of GAA (particularly the truncated form of the parental hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1).

In another specific embodiment, the truncated GAA polypeptide of the invention is Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, δ27, ONA, Δ29, Δ30, δ31, ON32, ON33, PA, Δ35, Δ36, ON37, ONA, Δ39, Δ40, ONA, ONA, δ43, δ44, ONA, Δ44, ONA, δ4, ONA, δ4, ONA, δ4, IT Δ45 or Δ46 truncated form of GAA (particularly a truncated form of the parental hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1).

In another specific embodiment, the truncated GAA polypeptide of the invention is Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, δ27, ONA, Δ29, Δ30, Δ31, ON32, ON, Δ34, Δ35, ON, Δ37, PAR, δ39, Δ40, ONA, ONA, δ43, δ44 or Δ44, or ONA, or Δ44, or ONA, Δ44, or Δ45 truncated form of GAA (particularly the truncated form of the parent hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1).

In a further specific embodiment, the truncated GAA polypeptide of the invention is Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19 , Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42, Δ43 или Δ44 a truncated form of GAA (particularly a truncated form of the parent hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1).

In a further specific embodiment, the truncated GAA polypeptide of the invention is Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19 , Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 или Δ43 усеченную форму GAA (particularly a truncated form of the parent hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1).

In a further specific embodiment, the truncated GAA polypeptide of the invention is Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19 , Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ4G20 (truncated form in particular the truncated form of the parent hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1).

In a further specific embodiment, the truncated GAA polypeptide of the invention is Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20 , Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ423 in particular the truncated form of the parent hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1).

In a further specific embodiment, the truncated GAA polypeptide of the invention is Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21 . a truncated form of the parental hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1).

In a further specific embodiment, the truncated GAA polypeptide of the invention is Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22 . the parental hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1).

In a further specific embodiment, the truncated GAA polypeptide of the invention is Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23 , Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42, or Δ43 the truncated form of the GAA protein hGAA listed in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1).

In a further specific embodiment, the truncated GAA polypeptide of the invention is Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24 , Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42, or shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1).

In a further specific embodiment, the truncated GAA polypeptide of the invention is Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25 , Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42, or Δ43 the truncated form of GAA (in particular the truncated form of the parent protein in hGAA SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1).

In a further specific embodiment, the truncated GAA polypeptide of the invention is Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ226 , Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42, or Δ43 a truncated form of GAA (in particular, a truncated form of the parent hGAA protein given in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1).

In a further specific embodiment, the truncated GAA polypeptide of the invention is a Δ6, Δ7, Δ8, Δ9, or Δ10 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, specifically in SEQ ID NO: 1), in particular the Δ7, Δ8 or Δ9 truncated form of GAA (in particular the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1), more specifically the Δ8 truncated form GAA (in particular the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1).

In a further specific embodiment, the truncated GAA polypeptide of the invention is a Δ27, Δ28, Δ29, Δ30, or Δ31 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, specifically in SEQ ID NO: : 1), in particular the Δ28, Δ29 or Δ30 truncated form of GAA (in particular the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1), more specifically the Δ29 truncated form of GAA (in particular the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, specifically in SEQ ID NO: 1).

In another specific embodiment, the truncated GAA polypeptide of the invention is a Δ40, Δ41, Δ42, Δ43, or Δ44 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, specifically in SEQ ID NO : 1), in particular the Δ41, Δ42 or Δ43 truncated form of GAA (in particular the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1), more specifically the Δ42 truncated form of GAA (in particular the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, specifically in SEQ ID NO: 1).

In a further specific embodiment, the truncated GAA polypeptide of the invention is a Δ41, Δ42, Δ43, Δ44 or Δ45 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO: 1), in particular a Δ42, Δ43 or Δ44 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1), more specifically the Δ43 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO: 1) .

In another embodiment, the truncated GAA polypeptide of the invention is a Δ6, Δ7, Δ8, Δ9, Δ10, Δ27, Δ28, Δ29, Δ30, Δ31, Δ40, Δ41, Δ42, Δ43, Δ44, Δ45, Δ46, or Δ47 truncated form of GAA ( in particular the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, specifically in SEQ ID NO: 1).

In another embodiment, the truncated GAA polypeptide of the invention is a Δ7, Δ8, Δ9, Δ28, Δ29, Δ30, Δ41, Δ42, Δ43, or Δ44 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO : 33, specifically in SEQ ID NO: 1).

In another embodiment, the truncated GAA polypeptide of the invention is Δ6, Δ7, Δ8, Δ9, Δ10, Δ40, Δ41, Δ42, Δ43, or Δ44, a truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, specifically in SEQ ID NO: 1).

In another embodiment, the truncated GAA polypeptide of the invention is a Δ8, Δ29, Δ42, Δ43, or Δ47 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, specifically in SEQ ID NO: 1 ).

In another embodiment, the truncated GAA polypeptide of the invention is a Δ8, Δ29, Δ42, or Δ43 truncated form of GAA (specifically the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, specifically in SEQ ID NO: 1).

In another embodiment, the truncated GAA polypeptide of the invention is a Δ8 or Δ42 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO: 1 or SEQ ID NO: 33, specifically in SEQ ID NO: 1).

In a specific embodiment of the invention, the truncated GAA polypeptide of the invention is a truncated form of a functional human GAA polypeptide. In a further specific embodiment, the parent hGAA polypeptide is an hGAA polypeptide as set forth in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1. In an embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ1Δ9, Δ20, Δ21, Δ23, Δ22, Δ22 , Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42, Δ43, Δ44, Δ45, Δ46, Δ47, Δ48, Δ49, Δ50 , Δ51, Δ52, Δ53, Δ54, Δ55, Δ56, Δ57, Δ58, Δ59, Δ60, Δ61, Δ62, Δ63, Δ64, Δ65, Δ66, Δ67, Δ68, Δ69, Δ70, Δ71, Δ72, Δ73, Δ74 или Δ75 a truncated GAA form of an hGAA polypeptide, and more specifically an hGAA polypeptide as shown in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, and having less at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33, in particular SEQ ID NO: 1.

In a variant of this embodiment, the truncated GAA polypeptide of the invention is Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, δ20, Δ21, Δ22, Δ23, δ24, Δ25, Δ26, Δ27, ONA28, Δ29, PA, Δ31, Δ32, δ33, Δ34, ON, ON36, ONA, Δ38, Δ39, ON, ONA, δ42, δ43, PA, δ43, δ43, δ43, δ43, PAS, Δ43, Δ43, Δ43, δ43, ILS Δ44, Δ45, Δ46, or Δ47 a truncated GAA form of an hGAA polypeptide, and more particularly an hGAA polypeptide as set forth in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically SEQ ID NO: 1, or a functional variant thereof containing substitutions amino acids in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, and having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96 , 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33, in particular SEQ ID NO: 1.

In a variant of this embodiment, the truncated GAA polypeptide of the invention is Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, δ20, Δ21, Δ22, Δ23, δ24, Δ25, Δ26, Δ27, ONA28, Δ29, PA, Δ31, Δ32, δ33, Δ34, ON, ON36, ONA, Δ38, Δ39, ON, ONA, δ42, δ43, PA, δ43, δ43, δ43, δ43, PAS, Δ43, Δ43, Δ43, δ43, ILS Δ44, Δ45 or Δ46 a truncated form of the GAA of an hGAA polypeptide, and more specifically the hGAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, and having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 , 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33, in particular SEQ ID NO: 1.

In a variant of this embodiment, the truncated GAA polypeptide of the invention is Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, δ20, Δ21, Δ22, Δ23, δ24, Δ25, Δ26, Δ27, ONA28, Δ29, PA, Δ31, Δ32, δ33, Δ34, ON, ON36, ONA, Δ38, Δ39, ON, ONA, δ42, δ43, PA, δ43, δ43, δ43, δ43, PAS, Δ43, Δ43, Δ43, δ43, ILS The Δ44 or Δ45 truncated GAA form of the hGAA polypeptide, and more particularly the hGAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence, listed in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, and having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18 , Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42, Δ43 or the Δ44 truncated GAA form of an hGAA polypeptide, and more specifically the hGAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence given in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, and having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18 , Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 или Δ43 a truncated GAA form of an hGAA polypeptide, and more specifically an hGAA polypeptide as shown in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, and having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18 , Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41 или Δ42 усеченную форму The GAA of an hGAA polypeptide, and more specifically the hGAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO : 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, and having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19 , Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ4ΔA2 polypeptide or truncated form hGAA, and more specifically the hGAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, and having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20 , Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ4A truncated polypeptide GAA1 and more specifically the hGAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, and having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21 , Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, ΔGA2 or more truncated polypeptide, GAA2 specifically the hGAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO : 33, in particular SEQ ID NO: 1, and having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22 , Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41 or Δ42 a truncated form of the h hGAA listed in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33 , in particular SEQ ID NO: 1, and having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO : 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23 , Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41 or Δ42 a truncated form of the GAA polypeptide, hGAA, and more specifically hGAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in in particular SEQ ID NO: 1, and having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33 , in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24 , Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, or Δ42 a truncated GAA form of the hGAA polypeptide, and more specifically, given in hGAA polypeptide SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, and having at least 75%, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33, in in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25 , Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, or Δ42 the GAA truncated form of the hGAA polypeptide, and more specifically the hGAA polypeptide listed in the SEQID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO : 1, and having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19 , Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 или Δ43 усеченную форму The GAA of an hGAA polypeptide, and more specifically the hGAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO : 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, and having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20 , Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, hGAA, and more specifically the hGAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, and having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21 , Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ4GA3 polypeptide, or a truncated form of GAA3 polypeptide and more specifically the hGAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, and having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22 , Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42 or more truncated polypeptide GAA3 specifically the hGAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO : 33, in particular SEQ ID NO: 1, and having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23 , Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42, or Δ43 a truncated form of the polypeptide h hGAA as shown in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33 , in particular SEQ ID NO: 1, and having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO : 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24 , Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42, or Δ43 a truncated form of the GAA polypeptide, hGAA, and more specifically hGAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in in particular SEQ ID NO: 1, and having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33 , in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25 , Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42, or Δ43 a truncated GAA form of the hGAA polypeptide, and more specifically, the hGAA polypeptide is given in SEQ ID NO: 1 or SEQ ID NO: 33, even more specifically in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, and having at least 75%, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 SEQ ID NO: 33, in in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ6, Δ7, Δ8, Δ9, or Δ10, in particular Δ7, Δ8 or Δ9, more specifically the Δ8 truncated form of an hGAA polypeptide, and more specifically the hGAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1, and having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ27, Δ28, Δ29, Δ30 or Δ31, in particular Δ28, Δ29 or Δ30, more specifically Δ29 a truncated form of an hGAA polypeptide, and more specifically an hGAA polypeptide as shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1, and having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ40, Δ41, Δ42, Δ43 or Δ44, in particular Δ41, Δ42 or Δ43, more specifically Δ42 the truncated form of an hGAA polypeptide, and more specifically the hGAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1, and having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ41, Δ42, Δ43, Δ44 or Δ45, in particular Δ42, Δ43 or Δ44, more specifically Δ43 a truncated form of an hGAA polypeptide, and more specifically an hGAA polypeptide as shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1, and having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is Δ6, Δ7, Δ8, Δ9, Δ10, Δ27, Δ28, Δ29, Δ30, Δ31, Δ40, Δ41, Δ42, Δ43, Δ44, or Δ45, in particular Δ7 , Δ8, Δ9, Δ28, Δ29, Δ30, Δ41, Δ42, Δ43 or Δ44, in particular Δ8, Δ29, Δ42 or Δ43 is a truncated form of the hGAA polypeptide, and more specifically the hGAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO : 33, in particular in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1, and having at least at least 80, 85, 90, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is a Δ6, Δ7, Δ8, Δ9, Δ10, Δ40, Δ41, Δ42, Δ43 or Δ44, in particular a Δ8 or Δ42 truncated form of an hGAA polypeptide, and more specifically an hGAA polypeptide , shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1, or its functional variant containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in in particular in SEQ ID NO: 1, and having at least 80, 85, 90, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: one.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is a Δ8, Δ29, Δ42, Δ43, or Δ47 truncated form of an hGAA polypeptide, and more particularly an hGAA polypeptide as shown in SEQ ID NO: 1 or SEQ ID NO: 33, in in particular in SEQ ID NO: 1, or a functional variant thereof containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1, and having at least 80, 85 , 90, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is a Δ8, Δ29, Δ42, or Δ43 truncated form of an hGAA polypeptide, and more particularly an hGAA polypeptide as set forth in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1, or a functional variant thereof, containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1, and having at least 80, 85, 90 , 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1.

In another embodiment of this embodiment, the truncated GAA polypeptide of the invention is a Δ8 or Δ42 truncated form of an hGAA polypeptide, and more specifically the hGAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1, or a functional variant thereof, containing amino acid substitutions in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular in SEQ ID NO: 1, and having at least 80, 85, 90, 95, 96 , 97, 98 or 99% identity with SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1.

In a specific embodiment, the truncated hGAA polypeptide of the invention has an amino acid sequence consisting of the sequence shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36, or its functional variant containing from 1 to 5, in particular from 1 to 4, in particular from 1 to 3, more specifically from 1 to 2, in particular 1 amino acid substitution, compared to the sequence shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36. In another specific embodiment, the truncated hGAA polypeptide of the invention has an amino acid sequence consisting of the sequence shown in SEQ ID NO: 27 , SEQ ID NO: 28, SEQ ID NO: 34 or SEQ ID NO: 35, or a functional variant thereof containing 1 to 5 amino acid substitutions compared to the sequence shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 34 or SEQ ID NO: 35. In a specific embodiment, the truncated floor The hGAA ipeptide according to the invention has an amino acid sequence consisting of the sequence shown in SEQ ID NO: 27 or SEQ ID NO: 28, or a functional variant thereof, containing from 1 to 5, in particular from 1 to 4, in particular from 1 to 3 , more specifically 1 to 2, in particular 1 amino acid substitution, compared to the sequence shown in SEQ ID NO: 27 or SEQ ID NO: 28.

The truncated GAA polypeptide according to the invention may further comprise a signal peptide, such as a naturally occurring GAA signal peptide or an alternative signal peptide, derived from another secreted protein. Non-limiting examples of such signal peptides include those shown in SEQ ID NO: 3 to 7. The inventors have surprisingly shown that fusing a truncated GAA polypeptide of the invention with an alternative signal peptide even further enhances its secretion. The invention thus provides a chimeric GAA polypeptide comprising a signal fragment and a truncated GAA polypeptide fragment, the truncated GAA polypeptide fragment is a truncated GAA polypeptide as defined above. In a particular embodiment, the signal peptide is a naturally occurring GAA signal peptide, such as the hGAA signal peptide shown in SEQ ID NO: 4. In another embodiment, the signal peptide is an exogenous (or alternative) signal peptide derived from a protein other than GAA . In a specific embodiment, the alternative signal peptide is selected from the group consisting of SEQ ID NOs: 3, 5, 6 and 7, or a functional derivative thereof, as defined below.

The inventors have shown that an exogenous signal peptide fused to the rest of the GAA protein increases the secretion of the resulting chimeric GAA polypeptide compared to the corresponding GAA polypeptide containing its natural signal peptide. In addition, the truncated GAA polypeptide fragment also increases the secretion of the chimeric GAA polypeptide (including both the signal peptide and the truncated GAA polypeptide) compared to a chimeric GAA polypeptide containing the same signal peptide fused to the parental GAA polypeptide.

Specific exogenous signal peptides useful in the present invention include amino acids 1-20 from chymotrypsinogen B2 (SEQ ID NO: 3), human α-1-antitrypsin signal peptide (SEQ ID NO: 5), amino acids 1-25 from iduronate-2 α-sulfatase (SEQ ID NO: 6), and amino acids 1-23 from a C1 protease inhibitor (SEQ ID NO: 7). The signal peptides from SEQ ID NO: 3 and SEQ ID NO: 5 to 7 allow higher secretion of the chimeric GAA protein both in vitro and in vivo compared to GAA containing its native signal peptide. In a specific embodiment, the signal peptide has the sequence shown in SEQ ID NO: 3 to 7, or is a functional derivative thereof, i.e. a sequence containing 1 to 5, in particular 1 to 4, in particular 1 to 3, more specifically 1 to 2, in particular 1 amino acid deletion, insertion or substitution, compared to the sequences shown in SEQ ID NOs: 3 to 7, as long as the resulting sequence corresponds to a functional signal peptide, i.e. e. a signal peptide that allows the secretion of the GAA protein. In a specific embodiment, the sequence of the signal peptide fragment consists of a sequence selected from the group consisting of SEQ ID NOs: 3 to 7.

In specific embodiments, the GAA polypeptide of the invention is selected from:

- combinations of SEQ ID NO: 3 with an Δ8 truncated form of GAA, such as the Δ8 truncated form of hGAA shown in SEQ ID NO: 27;

- combinations of SEQ ID NO: 4 with an Δ8 truncated form of GAA, such as the Δ8 truncated form of hGAA shown in SEQ ID NO: 27;

- combinations of SEQ ID NO: 5 with an Δ8 truncated form of GAA, such as the Δ8 truncated form of hGAA shown in SEQ ID NO: 27;

- combinations of SEQ ID NO: 6 with an Δ8 truncated form of GAA, such as the Δ8 truncated form of hGAA shown in SEQ ID NO: 27;

- combinations of SEQ ID NO: 7 with an Δ8 truncated form of GAA, such as the Δ8 truncated form of hGAA shown in SEQ ID NO: 27;

- combinations of SEQ ID NO: 3 with an Δ29 truncated form of GAA, such as the Δ29 truncated form of hGAA shown in SEQ ID NO: 34;

- combinations of SEQ ID NO: 4 with an Δ29 truncated form of GAA, such as the Δ29 truncated form of hGAA shown in SEQ ID NO: 34;

- combinations of SEQ ID NO: 5 with an Δ29 truncated form of GAA, such as the Δ29 truncated form of hGAA shown in SEQ ID NO: 34;

a combination of SEQ ID NO: 6 with an Δ29 truncated form of GAA, such as the Δ29 truncated form of hGAA shown in SEQ ID NO: 34;

- combinations of SEQ ID NO: 7 with an Δ29 truncated form of GAA, such as the Δ29 truncated form of hGAA shown in SEQ ID NO: 34;

- combinations of SEQ ID NO: 3 with an Δ42 truncated form of GAA, such as the Δ42 truncated form of hGAA shown in SEQ ID NO: 28;

- combinations of SEQ ID NO: 4 with an Δ42 truncated form of GAA, such as the Δ42 truncated form of hGAA shown in SEQ ID NO: 28;

- combinations of SEQ ID NO: 5 with an Δ42 truncated form of GAA, such as the Δ42 truncated form of hGAA shown in SEQ ID NO: 28;

- combinations of SEQ ID NO: 6 with an Δ42 truncated form of GAA, such as the Δ42 truncated form of hGAA shown in SEQ ID NO: 28;

- combinations of SEQ ID NO: 7 with an Δ42 truncated form of GAA, such as the Δ42 truncated form of hGAA shown in SEQ ID NO: 28;

- combinations of SEQ ID NO: 3 with an Δ43 truncated form of GAA, such as the Δ43 truncated form of hGAA shown in SEQ ID NO: 35;

- combinations of SEQ ID NO: 4 with an Δ43 truncated form of GAA, such as the Δ43 truncated form of hGAA shown in SEQ ID NO: 35;

- combinations of SEQ ID NO: 5 with an Δ43 truncated form of GAA, such as the Δ43 truncated form of hGAA shown in SEQ ID NO: 35;

- combinations of SEQ ID NO: 6 with an Δ43 truncated form of GAA, such as the Δ43 truncated form of hGAA shown in SEQ ID NO: 35; and

- combinations of SEQ ID NO: 7 with an Δ43 truncated form of GAA, such as the Δ43 truncated form of hGAA shown in SEQ ID NO: 35;

- combinations of SEQ ID NO: 3 with an Δ47 truncated form of GAA, such as the Δ47 truncated form of hGAA shown in SEQ ID NO: 36;

- combinations of SEQ ID NO: 4 with an Δ47 truncated form of GAA, such as the Δ47 truncated form of hGAA shown in SEQ ID NO: 36;

- combinations of SEQ ID NO: 5 with an Δ47 truncated form of GAA, such as the Δ47 truncated form of hGAA shown in SEQ ID NO: 36;

- combinations of SEQ ID NO: 6 with an Δ47 truncated form of GAA, such as the Δ47 truncated form of hGAA shown in SEQ ID NO: 36; and

- combinations of SEQ ID NO: 7 with an Δ47 truncated form of GAA, such as the Δ47 truncated form of hGAA shown in SEQ ID NO: 36;

or it is a functional derivative thereof having at least 90% identity, in particular at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with resulting combination of sequences. In these embodiments, as indicated above, the signal peptide fragment may be a sequence containing 1 to 5, in particular 1 to 4, in particular 1 to 3, more in particular 1 to 2, in particular 1 amino acid deletion, insertion or substitution, compared to the sequences shown in SEQ ID NO: 3 to 7, as long as the resulting sequence corresponds to a functional signal peptide, i.e., a signal peptide that allows the secretion of the resulting chimeric truncated GAA protein.

The relative proportion of newly synthesized GAA that a cell secretes can be routinely determined using methods known in the art and as described in the examples. Secreted proteins can be detected by direct measurement of the protein itself (e.g. Western blot) or by analysis of protein activity (e.g. enzymatic assay) in cell culture media, serum, milk, etc.

Those skilled in the art will further appreciate that a truncated GAA polypeptide or a chimeric GAA polypeptide may contain additional amino acids, for example, as a result of nucleic acid design manipulations, such as the addition of a restriction site, as long as those additional amino acids do not signal peptide or polypeptide GAA non-functional. Additional amino acids may be cleaved off or may be retained in the mature polypeptide as long as retention does not result in a non-functional polypeptide.

In another aspect, the invention provides a nucleic acid molecule encoding a truncated GAA polypeptide of the invention or a chimeric GAA polypeptide of the invention.

The sequence of a nucleic acid molecule of the invention encoding a truncated GAA is optimized for in vivo expression of a GAA polypeptide. Sequence optimization can include a variety of changes in the nucleic acid sequence, including codon optimization, increasing GC content, reducing the number of CpG islands, reducing the number of alternative open reading frames (ARFs), and reducing the number of splicing donor and splicing acceptor sites. Due to the degeneracy of the genetic code, different nucleic acid molecules can code for the same protein. It is also well known that the genetic codes of different organisms are often biased towards using one of several codons that code for the same amino acid relative to others. Through codon optimization, changes are introduced into the nucleotide sequence that take advantage of the codon preference that exists in a given cellular context, so that the resulting codon-optimized nucleotide sequence is more likely to be expressed in that given cellular context at a relatively high level compared to a sequence without codon-optimized. In a preferred embodiment of the invention, such a sequence-optimized nucleotide sequence encoding a truncated GAA protein has a codon optimization to improve its expression in human cells compared to non-codon-optimized nucleotide sequences encoding the same truncated GAA protein, for example, taking advantage of preference for specific codons in humans.

In a specific embodiment, the optimized GAA coding sequence has optimized codons and/or has an increased GC content and/or has a reduced number of alternative open reading frames and/or has a reduced number of splice donor and/or splice acceptor sites compared to nucleotides 82-2859 of wild-type hGAA encoding sequence from SEQ ID NO: 8. For example, a nucleic acid sequence of the invention results in at least a 2%, 3%, 4%, 5%, or 10% increase in GC content in a GAA sequence compared to a wild-type GAA sequence sequence. In a specific embodiment, the nucleic acid sequence of the invention results in a 2, 3, 4 or more specifically 5% or 10% (in particular 5%) increase in GC content in the GAA sequence compared to the wild-type GAA nucleotide sequence sequence. In a specific embodiment, the nucleic acid sequence of the invention encoding a functional GAA polypeptide is "substantially identical", that is, about 70% identical, more preferably about 80% identical, even more preferably about 90% identical, even more preferably about 95% identical, even more preferably about 97%, 98% or even 99% identical to nucleotides 82-2859 of the sequence shown in SEQ ID NO: 8. As above, in addition to GC content and/or number of ARFs , sequence optimization may also include reducing the number of CpG islands in the sequence and/or reducing the number of splicing donor and splicing acceptor sites. Of course, it is well known to those skilled in the art that sequence optimization is a balance between all of these parameters, which means that a sequence can be considered optimized if at least one of the above parameters is improved while one or more of the other parameters are not, until as long as the optimized sequence leads to an improvement in the transgene, such as improved expression and/or reduced immune response to the transgene in vivo.

In addition, the adaptability of a nucleotide sequence encoding a functional GAA to human cell codon usage can be expressed as a codon adaptation coefficient (CAI). Codon adaptation coefficient is defined herein as a measure of the relative adaptability to codon usage of a gene relative to the codon usage of highly expressed human genes. The relative adaptability (w) of each codon is the ratio of each codon usage to that of the most common codon for the same amino acid. CAI is defined as the geometric mean of these relative adaptability values. Non-synonymous codons and termination codons (depending on the genetic code) are excluded. CAI values range from 0 to 1, with higher values indicating a higher proportion of the most common codons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295; also see: Kim et al, Gen. 1997, 199:293-301; zur Megede et al, Journal of Virology, 2000, 74: 2628-2635). Preferably, the nucleic acid molecule encoding GAA has a CAI of at least 0.75 (particularly 0.77), 0.8, 0.85, 0.90, 0.92, or 0.94.

The term "nucleic acid sequence" (or nucleic acid molecule) refers to a DNA or RNA molecule in single or double stranded form, in particular DNA encoding a GAA protein according to the invention.

The inventors have found that the truncated GAA polypeptide described above, when expressed from the nucleic acid molecule encoding it, provides surprisingly high levels of functional GAA protein expression both in vitro and in vivo compared to wild-type GAA cDNA. In addition, as also shown by the inventors, the truncated GAA protein produced by liver and muscle cells expressing the nucleic acid molecule of the invention does not induce an immune response. This means that this nucleic acid molecule can be used to obtain high levels of GAA protein, and it provides therapeutic effects such as the ability to avoid immunosuppressive treatment, the ability to immunosuppressive treatment at low doses, and the ability to re-administer the nucleic acid molecule of the invention to a subject in need of it. Therefore, the truncated GAA polypeptide of the invention and the nucleic acid molecule of the invention are of particular interest in the context of deficient expression and/or activity of GAA or where high levels of GAA expression may improve a disease, such as glycogen storage disease. Specifically, the glycogen storage disease can be GSDI (Girke's disease), GSDII (Pompe's disease), GSDIII (Measles disease), GSDIV, GSDV, GSDVI, GSDVII, GSDVIII, or lethal congenital heart glycogen storage disease. More specifically, glycogen storage disease is selected from the group consisting of GSDI, GSDII and GSDIII, even more specifically from the group consisting of GSDII and GSDIII. In an even more specific embodiment, the glycogen storage disease is GSDII. In particular, the nucleic acid molecules of the invention can be used in gene therapy to treat GAA-deficient conditions or other conditions associated with glycogen storage, such as GSDI (Girke's disease), GSDII (Pompe's disease), GSDIII (Measles disease) , GSDIV, GSDV, GSDVI, GSDVII, GSDVIII and lethal congenital heart glycogen storage disease, more specifically GSDI, GSDII or GSDIII, even more specifically GSDII and GSDIII. In an even more specific embodiment, the nucleic acid molecules of the invention can be used in gene therapy in order to treat GSDII.

In another embodiment of the invention, the portion of the nucleic acid molecule of the invention encoding a truncated GAA polypeptide fragment has at least 75% (e.g., 77.7%) or at least 80%, or at least 82% (e.g., 83, 1%) identity with the corresponding part of the nucleotide sequence encoding SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, which are signal peptide-free hGAA polypeptide sequences.

A truncated GAA fragment of a nucleic acid molecule according to the invention preferably has at least 85%, more preferably at least 90% and even more preferably at least 92% identity, in particular at least 95% identity, for example at least 98 , 99 or 100% identity with the nucleotide sequence of SEQ ID NO: 10 or 11, which are sequence-optimized sequences.

The term "identical" and its derivatives refer to sequence identity between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base (for example, if the position in each of the two DNA molecules is occupied by adenine), then the molecules are identical at that position. The % identity between two sequences is a function of the number of matching positions that the two sequences have, divided by the number of positions compared, × 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences are 60% identical. In general, a comparison is made when two sequences are aligned to give maximum identity. Various bioinformatic tools known to the person skilled in the art can be used to align nucleic acid sequences, such as BLAST or FASTA.

In addition, the nucleic acid molecule of the invention encodes a functional GAA protein, ie it encodes a human GAA protein which, when expressed, has the functionality of a wild-type GAA protein. As defined above, the functionality of wild-type GAA is to hydrolyze both the α-1,4 and α-1,6 bonds of oligosaccharides and polysaccharides, more specifically glycogen, to release glucose. A functional GAA protein encoded by a nucleic acid of the invention may have a glycogen hydrolytic activity of at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or at least 100% relative to the protein Wild-type GAA of SEQ ID NO: 1, 2, 30, or 33. The activity of the GAA protein encoded by the nucleic acid of the invention may even be greater than 100%, such as greater than 110%, 120%, 130%, 140%, or even more than 150% of the wild-type GAA protein activity of SEQ ID NO: 1, 2, 30 or 33.

One skilled in the art will readily be able to determine whether a nucleic acid according to the invention expresses a functional GAA protein. Suitable methods will be apparent to those skilled in the art. For example, one suitable in vitro method includes inserting the nucleic acid into a vector such as a plasmid or viral vector, transfecting or transducing host cells such as 293T or HeLa cells or other cells such as Huh7 using the vector, and assaying the activity GAA. Alternatively, a suitable in vivo method comprises transducing the vector containing the nucleic acid in a mouse model of Pompe disease or other disorder of glycogen storage and assaying functional GAA in mouse plasma and the presence of GAA in tissues. Suitable methods are described in more detail later in the experimental part.

In a specific embodiment, the nucleic acid molecule according to the invention contains the sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13 encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 27; the sequence shown in SEQ ID NO: 48 or SEQ ID NO: 49 encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 28; the sequence shown in SEQ ID NO: 50 or SEQ ID NO: 51 encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 35; or the sequence shown in SEQ ID NO: 52 or SEQ ID NO: 53 encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 36. In a further embodiment, the nucleic acid molecule of the invention comprises the sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13 encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 27; the sequence shown in SEQ ID NO: 48 or SEQ ID NO: 49 encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 28; or the sequence shown in SEQ ID NO: 50 or SEQ ID NO: 51 encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 35. In a particular embodiment, the nucleic acid molecule of the invention comprises the sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13 encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 27.

The invention also relates to a nucleic acid construct containing the nucleic acid molecule of the invention. The nucleic acid construct may correspond to an expression cassette containing the nucleic acid sequence of the invention operably linked to one or more expression control sequences and/or other sequences that enhance the expression of the transgene and/or sequences that enhance the secretion of the encoded protein and/or sequences that enhance uptake of the encoded protein. As used herein, the term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or other transcriptional control sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Expression control sequences such as promoters, enhancers (such as cis regulatory modules (CRMs)), introns, polyA signals, etc. are known in the art.

In particular, the expression cassette may contain a promoter. The promoter may be a general purpose or tissue-specific promoter, in particular the promoter is capable of promoting expression in cells or tissues in which GAA expression is desired, for example in cells or tissues in which GAA expression is desired in GAA deficient patients. In a specific embodiment, the promoter is a specific liver promoter such as the α-1-antitrypsin (hAAT) promoter (SEQ ID NO: 14), transthyretin promoter, albumin promoter, thyroxine-binding globulin (TBG) promoter, LSP promoter (containing the globulin promoter sequence binding thyroid hormone, two copies of the α1-microglobulin/bicunin enhancer sequence and a leader sequence - 34. Ill, C. R., et al.(1997). Fibrinol. 8: S23-S30.), etc. Other effective specific liver promoters are known in the art, such as those listed in the Liver Specific Gene Promoter Database compiled by Cold Spring Harbor Laboratory (http://rulai. cshl.edu/LSPD/). The preferred promoter in the context of the invention is the hAAT promoter. In another embodiment, the promoter is a promoter that drives expression in one tissue or cell of interest (eg, muscle cells) and liver cells. For example, to a certain extent, promoters with muscle cell specificity, such as the desmin, Spc5-12, and MCK promoters, may confer some leakage of expression into liver cells, which may be beneficial in inducing immunological tolerance in a subject to a GAA polypeptide expressed from a nucleic acid. acids according to the invention.

Other tissue-specific or non-tissue-specific promoters may be used in the practice of the invention. For example, the expression cassette may contain a tissue-specific promoter that is a promoter other than the specific liver promoter. For example, a promoter may be muscle specific, such as a desmin promoter (and a desmin promoter variant such as a desmin promoter containing natural or artificial enhancers), the SPc5-12 promoter, or MCK. In another embodiment, the promoter is a promoter with specificity for another cell lineage, such as the erythropoietin promoter, for expression of the GAA polypeptide in erythroid lineage cells.

In another embodiment, the promoter is a universal promoter. Representative universal promoters include cytomegalovirus enhancer/chicken β-actin (CAG) promoter, cytomegalovirus (CMV) enhancer/promoter, PGK promoter, SV40 early promoter, etc.

In addition, the promoter may also be an endogenous promoter such as an albumin promoter or a GAA promoter.

In a specific embodiment, the promoter is associated with an enhancer sequence, such as cis regulatory modules (CRMs) or an artificial enhancer sequence. For example, a promoter may be associated with an enhancer sequence such as a human ApoE control region (or human apolipoprotein E/C-I gene locus, HCR-1 hepatic control region - GenBank accession number U32510, shown in SEQ ID NO: 15). In a particular embodiment, an enhancer sequence, such as an ApoE sequence, is associated with a specific liver promoter, such as those listed above, and in particular such as the hAAT promoter. Other CRMs useful in the practice of the present invention include those described in Rincon et al., Mol Ther. 2015 Jan;23(1):43-52, Chuah et al., Mol Ther. 2014 Sep;22(9):1605-13 or Nair et al., Blood. 2014 May 15;123(20):3195-9.

In another specific embodiment, the nucleic acid construct contains an intron, in particular an intron located between the promoter and the GAA coding sequence. An intron can be introduced to increase mRNA stability and protein production. In a further embodiment, the nucleic acid construct comprises a human β-globin b2 (or HBB2) intron, a coagulation factor IX (FIX) intron, an SV40 intron, or a chicken β-globin intron. In another further embodiment, the nucleic acid construct of the invention comprises a modified intron (particularly a modified HBB2 or FIX intron) designed to reduce, or even eliminate completely, alternative open reading frames (ARFs) found in said intron. Preferably, ARFs that are longer than 50 bp are removed. and which have a stop codon in the reading frame of the start codon. ARF can be removed by modifying the intron sequence. For example, the modification may be by nucleotide substitution, insertion or deletion, preferably by nucleotide substitution. By way of illustration, one or more nucleotides, in particular one nucleotide, in an ATG or GTG start codon present in an intron sequence of interest can be changed to lead to a non-start codon. For example, ATG or GTG can be replaced with CTG, which is not the start codon, in the sequence of the intron of interest.

The classic HBB2 intron used in nucleic acid constructs is shown in SEQ ID NO: 16. For example, this HBB2 intron can be modified by eliminating start codons (ATG and GTG codons) in said intron. In a specific embodiment, the modified HBB2 intron contained in the construct has the sequence shown in SEQ ID NO: 17. The classic FIX intron used in nucleic acid constructs is derived from the first human FIX intron and is shown in SEQ ID NO: 18. FIX intron can be modified by eliminating start codons (ATG and GTG codons) in said intron. In a specific embodiment, the modified FIX intron contained in the construct of the invention has the sequence shown in SEQ ID NO: 19. The classic chicken β-globin intron used in nucleic acid constructs is shown in SEQ ID NO: 20. β-globin intron chicken can be modified by eliminating the initiation codons (ATG and GTG codons) in the specified intron. In a specific embodiment, the modified chicken β-globin intron contained in the construct of the invention has the sequence shown in SEQ ID NO: 21.

The inventors have previously shown in WO2015/162302 that such a modified intron, in particular a modified HBB2 or FIX intron, has favorable properties and can significantly improve transgene expression.

In a particular embodiment, the nucleic acid construct of the invention is an expression cassette containing, in a 5' to 3' orientation, a promoter optionally preceded by an enhancer, a coding sequence of the invention (i.e., an optimized GAA coding sequence of the invention, a chimeric coding a GAA sequence of the invention or a chimeric and optimized GAA coding sequence of the invention) and a polyadenylation signal (such as a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal, or other naturally occurring or artificial polyadenylation signal). In a specific embodiment, the nucleic acid construct of the invention is an expression cassette comprising, in a 5' to 3' orientation, a promoter optionally preceded by an enhancer (such as an ApoE control region), an intron (specifically an intron as defined above) , the coding sequence of the invention and the polyadenylation signal. In a further specific embodiment, the nucleic acid construct of the invention is an expression cassette comprising, in a 5' to 3' orientation, an enhancer such as an ApoE control region, a promoter, an intron (particularly an intron as defined above), a coding sequence according to the invention and a polyadenylation signal. In a further specific embodiment, the expression cassette comprises, in a 5' to 3' orientation, an ApoE control region, a specific liver hAAT promoter, an HBB2 intron (specifically a modified HBB2 intron as defined above), a coding sequence of the invention, and a bovine polyadenylation signal. growth hormone, for example, the construct presented in:

- SEQ ID NO: 22 containing a non-optimized nucleotide sequence encoding an Δ8 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 and encoding a signal peptide from SEQ ID NO: 5;

- SEQ ID NO: 23 containing an optimized sequence encoding an Δ8 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide from SEQ ID NO: 5;

- SEQ ID NO: 24 containing another optimized sequence encoding the Δ8 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding the signal peptide from SEQ ID NO: 5;

- SEQ ID NO: 25 containing the optimized sequence encoding the Δ8 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and the signal peptide from SEQ ID NO : 3;

- SEQ ID NO: 26 containing the optimized sequence encoding the Δ42 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and the signal peptide from SEQ ID NO : 3;

- SEQ ID NO: 37 containing the non-optimized sequence encoding the Δ29 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the non-optimized sequence of SEQ ID NO: 9) and the signal peptide from SEQ ID NO: 3;

- SEQ ID NO: 38 containing the optimized sequence encoding the Δ29 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and the signal peptide from SEQ ID NO : 3;

- SEQ ID NO: 39 containing another optimized sequence encoding the Δ29 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and a signal peptide from SEQ ID NO: 3;

- SEQ ID NO: 40: contains the non-optimized sequence encoding the Δ42 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the non-optimized sequence of SEQ ID NO: 9) and the signal peptide from SEQ ID NO: 3;

- SEQ ID NO: 41 containing another optimized sequence encoding the Δ42 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and the signal peptide from SEQ ID NO: 3;

- SEQ ID NO: 42 containing the non-optimized sequence encoding the Δ43 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the non-optimized sequence of SEQ ID NO: 9) and the signal peptide from SEQ ID NO: 3;

- SEQ ID NO: 43 containing the optimized sequence encoding the Δ43 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and the signal peptide from SEQ ID NO : 3; and

- SEQ ID NO: 44 containing another optimized sequence encoding the Δ43 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and a signal peptide from SEQ ID NO: 3;

- SEQ ID NO: 45 containing the non-optimized sequence encoding the Δ47 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the non-optimized sequence of SEQ ID NO: 9) and the signal peptide from SEQ ID NO: 3;

- SEQ ID NO: 46 containing the optimized sequence encoding the Δ47 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and the signal peptide from SEQ ID NO : 3; and

- SEQ ID NO: 47 containing another optimized sequence encoding the Δ47 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and the signal peptide from SEQ ID NO: 3.

Other expression cassettes of the invention may contain the following nucleic acid sequences:

- a non-optimized nucleotide sequence encoding an Δ8 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 and encoding a signal peptide of SEQ ID NO: 4, 6 or 7;

- a non-optimized nucleotide sequence encoding the Δ29 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 and encoding the signal peptide of SEQ ID NO: 4, 6 or 7;

- a non-optimized nucleotide sequence encoding an Δ42 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 and encoding a signal peptide of SEQ ID NO: 4, 6 or 7;

- a non-optimized nucleotide sequence encoding the Δ43 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 and encoding the signal peptide of SEQ ID NO: 4, 6 or 7;

- a non-optimized nucleotide sequence encoding the Δ47 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 and encoding the signal peptide of SEQ ID NO: 4, 6 or 7;

- an optimized nucleotide sequence encoding an Δ8 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7;

- an optimized nucleotide sequence encoding an Δ8 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7;

- an optimized nucleotide sequence encoding the ∆29 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7;

- an optimized nucleotide sequence encoding the Δ29 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7;

- an optimized nucleotide sequence encoding an Δ42 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7;

- an optimized nucleotide sequence encoding an Δ42 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7

- an optimized nucleotide sequence encoding the Δ43 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7;

- an optimized nucleotide sequence encoding the Δ43 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7

- an optimized nucleotide sequence encoding the ∆47 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7;

- an optimized nucleotide sequence encoding the ∆47 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7.

In alternative embodiments of these specific constructs, the sequence encoding SEQ ID NO: 1 is replaced with the sequence encoding SEQ ID NO: 33.

In a particular embodiment, the expression cassette contains an ApoE control region, a specific liver hAAT promoter, a codon optimized HBB2 intron, a coding sequence of the invention, and a bovine growth hormone polyadenylation signal.

In developing a nucleic acid construct of the invention, one skilled in the art will consider limiting the size of the vector used to deliver said construct to a cell or organ. In particular, one of skill in the art is aware that the main limitation of an AAV vector is its usable capacity, which may vary from one AAV serotype to another, but is presumably limited by the approximate size of the parent viral genome. For example, 5 tons. This is the maximum size usually planned to be packaged in an AAV8 capsid (Wu Z. et al., Mol Ther., 2010, 18(1): 80-86; Lai Y. et al., Mol Ther., 2010, 18(1 ): 75-79; Wang Y. et al., Hum Gene Ther Methods, 2012, 23(4): 225-33). Accordingly, those skilled in the art, in the practice of the present invention, will take care to select the components of the nucleic acid construct of the invention so that the resulting nucleic acid sequence, including sequences encoding the AAV 5'- and 3'-ITR, preferably does not exceed 110% of the useful capacity of the introduced AAV vector, in particular, preferably does not exceed 5.5 t.

The invention also relates to a vector containing a nucleic acid molecule or construct, as disclosed in the present description. In particular, the vector of the invention is a vector suitable for protein expression, preferably for use in gene therapy. In one embodiment, the vector is a plasmid vector. In another embodiment, the vector is a nanoparticle containing a nucleic acid molecule of the invention, in particular a messenger RNA encoding a GAA polypeptide of the invention. In another embodiment, the vector is a transposon based system that allows the insertion of a nucleic acid molecule or construct of the invention into the genome of a target cell, such as the Sleeping Beauty hyperactive transposon system (SB100X) (Mates et al. 2009). In another embodiment, the vector is a viral vector suitable for gene therapy that targets any cell of interest such as liver tissue or cells, muscle cell, CNS cells (such as brain cells), or hematopoietic stem cells such as erythroid lineage cells (such as erythrocytes). In this case, the nucleic acid construct of the invention also contains sequences suitable for producing an effective viral vector, as is well known in the art. In a specific embodiment, the viral vector is derived from an integrating virus. In particular, the viral vector may be derived from a retrovirus or a lentivirus. In a further specific embodiment, the viral vector is an AAV vector, such as an AAV vector suitable for liver tissue or cell transduction, more specifically AAV-1, -2, and AAV-2 variants (such as an optimized AAV-2 with a quadruple capsid mutation containing engineered capsid with Y44+500+730F+T491V changes as disclosed in Ling et al., 2016 Jul 18, Hum Gene Ther Methods [preprint electronic publication]), -3 and AAV-3 variants (such as AAV3 variant -ST containing an engineered AAV3 capsid with two amino acid changes, S663V+T492V, as disclosed in Vercauteren et al., 2016, Mol Ther vol 24(6), page 1042), -3B and variants of AAV-3B, - 4, -5, -6 and AAV-6 variants (such as the AAV6 variant containing the Y731F/Y705F/T492V triple mutation AAV6 capsid form as disclosed in Rosario et al., 2016, Mol Ther Methods Clin Dev. 3, p. . 16026), -7, -8, -9, -10 e.g. -cy10 and -rh10, -rh74, -dj, Anc80, LK03, AAV2i8, swine AAV serotypes such as A AVpo4 and AAVpo6, etc., vector or retroviral vector such as lentiviral vector and alfaretrovirus. As is known in the art, depending on the particular viral vector being considered for use, additional suitable sequences are introduced into the nucleic acid construct of the invention in order to obtain a functional viral vector. Suitable sequences include AAV ITR for AAV vector or LTR for lentiviral vectors. As such, the invention also relates to an expression cassette as described above, flanked with an ITR or LTR on each side.

The advantages of viral vectors are discussed in the next part of this disclosure. Viral vectors are preferred for delivery of a nucleic acid molecule or construct of the invention, such as a retroviral vector, eg a lentiviral vector, or a non-pathogenic parvovirus, more preferably an AAV vector. Human parvovirus adeno-associated virus (AAV) is a dependovirus having a natural replication defect that is able to integrate into the genome of an infected cell in order to establish a latent infection. The latter property appears to be unique among mammalian viruses because the insertion occurs at a specific site in the human genome called AAVS1, which is located on chromosome 19 (19q13.3-qter).

Therefore, AAV vectors are of considerable interest as potential vectors for human gene therapy. Among the favorable properties of the virus are its lack of association with any human disease, its ability to infect dividing and non-dividing cells, and the wide range of cell lines derived from various tissues that can be infected.

Among AAV serotypes isolated from humans or non-human primates (NHP) and well characterized, human serotype 2 represents the first AAV that has been developed as a gene transfer vector. Other currently used AAV serotypes include AAV-1, AAV-2 variants (such as an optimized AAV-2 with a quadruple capsid mutation containing an engineered capsid with Y44+500+730F+T491V changes as disclosed in Ling et al., 2016 Jul 18, Hum Gene Ther Methods [preprint electronic publication]), -3 and AAV-3 variants (such as the AAV3-ST variant containing an engineered AAV3 capsid with two amino acid changes, S663V+T492V, as disclosed in Vercauteren et al. , 2016, Mol. Ther. vol. 24(6), p. triple mutation Y731F/Y705F/T492V as disclosed in Rosario et al., 2016, Mol Ther Methods Clin Dev. 3, page 16026), -7, -8, -9, -10, e.g. cy10 and -rh10, -rh74, -dj, Anc80, LK03, AAV2i8, porcine AAV serotypes such as AAVpo4 and AAVpo6, and tyrosine, lysine and serine mutant capsids of AAV serotypes, etc. In addition, other non-naturally engineered bath variants and chimeric AAVs may also be effective.

AAV viruses can be engineered using standard molecular biology techniques that allow these particles to be optimized for delivery of nucleic acid sequences to specific cells, to minimize immunogenicity, to adjust for particle stability and lifetime, for efficient disruption, for precise delivery to the nucleus.

Desirable AAV fragments for assembly into vectors include cap proteins, including vp1, vp2, vp3 and hypervariable regions, rep proteins, including rep 78, rep 68, rep 52 and rep 40, and sequences encoding these proteins. These fragments can easily be used in various vector systems and host cells.

Recombinant AAV-based vectors lacking the Rep protein integrate with low efficiency into the host genome and are predominantly presented as stable round episomes that can persist for years in target cells.

As an alternative to using naturally occurring AAV serotypes, artificial AAV serotypes, including but not limited to AAV with a non-naturally occurring capsid protein, can be used in the context of the present invention. Such an artificial capsid can be generated by any suitable technique using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with heterologous sequences that can be derived from another selected AAV serotype, unrelated portions of an AAV of the same serotype, from a non-AAV viral source or from a non-viral source. The artificial AAV serotype can be, without limitation, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid.

Accordingly, the present invention relates to an AAV vector containing the nucleic acid molecule or construct of the invention. In the context of the present invention, an AAV vector contains an AAV capsid capable of transducing target cells of interest, in particular hepatocytes. According to a specific embodiment, the AAV vector refers to AAV-1, -2, AAV-2 variants (e.g., an optimized AAV-2 with a quadruple capsid mutation containing an engineered capsid with Y44+500+730F+T491V changes as disclosed in Ling et al., 2016 Jul 18, Hum Gene Ther Methods [e-published ahead of print]), -3 and AAV-3 variants (e.g. AAV3-ST variant containing an engineered AAV3 capsid with two amino acid changes, S663V+T492V, as disclosed in Vercauteren et al., 2016, Mol Ther vol 24(6), page 1042), -3B and variants AAV-3B, -4, -5, -6 and variants AAV-6 (e.g. containing the AAV6 capsid form with the Y731F/Y705F/T492V triple mutation as disclosed in Rosario et al., 2016, Mol Ther Methods Clin Dev. 3, page 16026), -7, -8, -9, -10, e.g. , -cy10 and -rh10, -rh74, -dj, Anc80, LK03, AAV2i8, porcine AAV such as AAVpo4 and AAVpo6, and tyrosine, lysine and serine capsid mutants of AAV serotypes, etc., serotype. In a specific embodiment, the AAV vector is of serotype AAV8, AAV9, AAVrh74, or AAV2i8 (i.e., the AAV vector has a capsid of serotype AAV8, AAV9, AAVrh74, or AAV2i8). In a further specific embodiment, the AAV vector is a pseudotyped vector, ie its genome and capsid are derived from AAVs of different serotypes. For example, a pseudotyped AAV vector may be a vector whose genome is derived from one of the above AAV serotypes and whose capsid is derived from another serotype. For example, the genome of a pseudotyped vector may have a capsid derived from the serotype AAV8, AAV9, AAVrh74, or AAV2i8 and its genome may be derived from another serotype. In a specific embodiment, the AAV vector has a capsid of the AAV8, AAV9 or AAVrh74 serotype, in particular the AAV8 or AAV9 serotype, more specifically the AAV8 serotype.

In a specific embodiment in which the vector is for use in delivering a transgene to muscle cells, the AAV vector may be selected from the group consisting of AAV8, AAV9 and AAVrh74, among others.

In another specific embodiment, in which the vector is for use in delivering a transgene to liver cells, the AAV vector may be selected from the group consisting of AAV5, AAV8, AAV9, AAV-LK03, AAV-Anc80, and AAV3B, among others.

In another embodiment, the capsid is a modified capsid. In the context of the present invention, a "modified capsid" can be a chimeric capsid or a capsid containing one or more variants of VP capsid proteins derived from one or more wild-type AAV VP capsid proteins.

In a specific embodiment, the AAV vector is a chimeric vector, i.e., its capsid contains VP capsid proteins derived from at least two different AAV serotypes, or contains at least one chimeric VP protein combining regions or domains of VP proteins derived from from at least two AAV serotypes. Examples of such chimeric AAV vectors effective for transducing liver cells are described in Shen et al., Molecular Therapy, 2007 and Tenney et al., Virology, 2014. For example, a chimeric AAV vector can be derived from a combination of an AAV8 capsid sequence with an AAV serotype sequence. , other than the AAV8 serotype, such as any of those specifically mentioned above. In another embodiment, the AAV vector capsid contains one or more VP capsid protein variants such as those described in WO2015013313, in particular RHM4-1, RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15 capsid variants. -4 and RHM15-6, which give a high tropism to the liver.

In another embodiment, the modified capsid may also be derived from capsid modifications inserted by error-prone PCR and/or peptide insertion (eg, as described in Bartel et al., 2011). In addition, capsid variants may contain single amino acid changes such as tyrosine mutants (eg, as described in Zhong et al., 2008)

In addition, the AAV vector genome can be a single stranded or self complementary double stranded genome (McCarty et al., Gene Therapy, 2003). Self-complementary double-stranded AAV vectors are generated by deletion of the terminal clearance site (trs) from one of the AAV terminal repeats. These modified vectors, whose replicating genome is half the length of the wild-type AAV genome, tend to package DNA dimers. In a preferred embodiment, the AAV vector used in the practice of the present invention has a single stranded genome and further preferably contains an AAV8, AAV9, AAVrh74 or AAV2i8 capsid, in particular an AAV8, AAV9 or AAVrh74 capsid, such as an AAV8 or AAV9 capsid, more specifically an AAV8 capsid .

In a particularly preferred embodiment, the invention relates to an AAV vector comprising, in a single-stranded or double-stranded self-complementary genome (eg, single-stranded genome), the nucleic acid construct of the invention. In one embodiment, the AAV vector comprises an AAV8, AAV9, AAVrh74 or AAV2i8 capsid, in particular an AAV8, AAV9 or AAVrh74 capsid, such as an AAV8 or AAV9 capsid, more specifically an AAV8 capsid. In a further specific embodiment, said nucleic acid is operably linked to a promoter, in particular a universal or specific liver promoter. According to a particular embodiment, the promoter is a universal promoter such as a cytomegalovirus enhancer/chicken β-actin (CAG) promoter, a cytomegalovirus (CMV) enhancer/promoter, a PGK promoter, and an early SV40 promoter. In a specific embodiment, the universal promoter is a CAG promoter. Alternatively, the promoter is a specific liver promoter such as the α-1-antitrypsin (hAAT) promoter, transthyretin promoter, albumin promoter, and thyroxine-binding globulin (TBG) promoter. In a specific embodiment, the specific liver promoter is the specific liver hAAT promoter of SEQ ID NO: 14. In a further specific embodiment, the nucleic acid construct contained in the genome of the AAV vector of the invention further comprises an intron as described above, such as an intron, placed between the promoter and a nucleic acid sequence encoding a GAA coding sequence (i.e., an optimized GAA coding sequence of the invention, a chimeric GAA coding sequence of the invention, or a chimeric and optimized GAA coding sequence of the invention). Representative introns that can be included in a nucleic acid construct introduced into the genome of an AAV vector include, without limitation, the human b2 (or HBB2) β-globin intron, the FIX intron, and the chicken β-globin intron. Said intron in the genome of an AAV vector may be a classical (or unmodified) intron or a modified intron designed to reduce, or even eliminate, alternative open reading frames (ARFs) in said intron. Modified and unmodified introns that can be used in the practice of this embodiment, where the nucleic acid of the invention is introduced into an AAV vector, are described in detail above. In a specific embodiment, an AAV vector, in particular an AAV vector containing an AAV8, AAV9, AAVrh74 or AAV2i8 capsid, in particular an AAV8, AAV9 or AAVrh74 capsid, for example an AAV8 or AAV9 capsid, more specifically an AAV8 capsid, according to the invention contains in its genome a modified (or optimized) intron, such as the modified HBB2 intron of SEQ ID NO: 17, the modified FIX intron of SEQ ID NO: 19, and the modified chicken β-globin intron of SEQ ID NO: 21. In a further specific embodiment, the vector of the invention is an AAV vector containing an AAV8, AAV9, AAVrh74 or AAV2i8 capsid, in particular an AAV8, AAV9 or AAVrh74 capsid, for example an AAV8 or AAV9 capsid, more specifically an AAV8 capsid, which contains a genome containing, in the orientation from 5' to 3' : AAV 5'-ITR (e.g. AAV2 5'-ITR); an ApoE control area; liver hAAT specific promoter; an HBB2 intron (particularly a modified HBB2 intron as defined above); the GAA coding sequence of the invention; bovine growth hormone polyadenylation signal; and AAV 3'-ITR (e.g. AAV2 3'-ITR) such as a genome containing the nucleic acid construct shown in SEQ ID NO: 22 to 26 and SEQ ID NO: 37 to 47 flanked with AAV 5 '-ITR (eg AAV2 5'-ITR) and AAV 3'-ITR (eg AAV2 3'-ITR). Other nucleic acid constructs useful in the practice of the present invention include those described above, including:

- a non-optimized nucleotide sequence encoding an Δ8 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 and encoding a signal peptide of SEQ ID NO: 4, 6 or 7;

- a non-optimized nucleotide sequence encoding the Δ29 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 and encoding the signal peptide of SEQ ID NO: 4, 6 or 7;

- a non-optimized nucleotide sequence encoding an Δ42 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 and encoding a signal peptide of SEQ ID NO: 4, 6 or 7;

- a non-optimized nucleotide sequence encoding the Δ43 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 and encoding the signal peptide of SEQ ID NO: 4, 6 or 7;

- non-optimized nucleotide sequence encoding the Δ47 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 and encoding the signal peptide of SEQ ID NO: 4, 6 or 7

- an optimized nucleotide sequence encoding an Δ8 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7;

- an optimized nucleotide sequence encoding an Δ8 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7;

- an optimized nucleotide sequence encoding the Δ29 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7;

- an optimized nucleotide sequence encoding the ∆29 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7;

- an optimized nucleotide sequence encoding an Δ42 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide from SEQ ID NO: 4 or 6;

- an optimized nucleotide sequence encoding an Δ42 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7;

- an optimized nucleotide sequence encoding the Δ43 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7;

- an optimized nucleotide sequence encoding the Δ43 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7;

- an optimized nucleotide sequence encoding the ∆47 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7;

- an optimized nucleotide sequence encoding the Δ47 truncated form of GAA derived from the parental hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide from SEQ ID NO: 4, 6, or 7.

In alternative embodiments of these specific constructs, the sequence encoding SEQ ID NO: 1 is replaced with the sequence encoding SEQ ID NO: 33.

In a particular embodiment of the invention, the nucleic acid construct of the invention comprises a specific liver promoter as described above and the vector is a viral vector capable of transducing liver tissue or cells as described above. The inventors further provide data showing that the protolerogenic and metabolic properties of the liver are beneficially realized by this embodiment to develop highly efficient and optimized vectors for expressing highly secreted forms of GAA in hepatocytes and inducing immunological tolerance to the protein.

Furthermore, in a further specific embodiment, the invention relates to a combination of two vectors, eg two viral vectors, in particular two AAV vectors, to improve gene delivery and treatment effect in cells of interest. For example, two vectors may carry a nucleic acid molecule of the invention encoding a GAA protein of the invention under the control of a single promoter that is different in each of the two vectors. In a particular embodiment, one vector contains a promoter that is a liver specific promoter (as one described above) and the other vector contains a promoter that has specificity for another tissue of interest for treating glycogen storage disorder, such as a muscle specific promoter. eg the desmin promoter. In a particular embodiment of this embodiment, this combination of vectors corresponds to multiple co-packaged AAV vectors obtained as described in WO2015196179.

The invention also relates to a cell, for example a liver cell, which is transformed with a nucleic acid molecule or construct according to the invention, as in the case of ex vivo gene therapy. The cells of the invention can be delivered to a subject in need, such as a GAA deficient patient, by any suitable route of administration, for example, by injection into the liver or into the blood stream of said subject. In a specific embodiment, the invention includes introducing a nucleic acid molecule, a nucleic acid construct or a vector, in particular a lentiviral vector, according to the invention into liver cells, in particular liver cells of a subject to be treated, and introducing said transformed liver cells into which the nucleic acid was introduced , subject. Advantageously, this embodiment can be used to secrete GAA from said cells. In a specific embodiment, the liver cells are liver cells from a patient to be treated or liver stem cells that are further transformed and differentiated in vitro into liver cells for subsequent administration to the patient.

The present invention further relates to a transgenic non-human animal containing in its genome a nucleic acid molecule or construct encoding a GAA polypeptide in accordance with the invention. In a particular embodiment, the animal is a mouse.

In addition to the specific delivery systems carried out in the examples below, various delivery systems are known that can be used to introduce a nucleic acid molecule or construct according to the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the coding sequence of the invention , receptor-mediated endocytosis, therapeutic nucleic acid construct as part of a retroviral or other vector, etc.

According to one embodiment, it may be desirable to introduce a GAA polypeptide, nucleic acid molecule, nucleic acid construct, or cell of the invention into the liver of a subject via any suitable route. In addition, deproteinized DNA such as minicircles and transposons can be used for delivery or lentiviral vectors. Additionally, gene editing technologies such as zinc finger nucleases, meganucleases, TALEN and CRISPR can also be used to deliver the coding sequence of the invention.

The present invention also relates to pharmaceutical compositions containing a nucleic acid molecule, a nucleic acid construct, a vector, a GAA polypeptide, or a cell of the invention. Such compositions contain a therapeutically effective amount of a therapeutic agent (nucleic acid molecule, nucleic acid construct, vector, GAA polypeptide or cell of the invention) and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approval by a state or regional government regulatory body or inclusion in the USP or European Pharmacopoeia or other generally recognized pharmacopoeias for use in animals and humans. The term "carrier" refers to a diluent, adjuvant, excipient or carrier with which a therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids such as water and oils, including those derived from petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, in particular for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene glycol, water, ethanol, and the like.

The composition, if desired, may also contain minor amounts of wetting or emulsifying agents or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The oral formulation may contain conventional carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose , pharmaceutical grade magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, E. W. Martin. Such compositions should contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with a suitable amount of carrier so as to provide a form for appropriate administration to the subject. In a specific embodiment, the nucleic acid, vector or cell of the invention is formulated in a composition containing phosphate-buffered saline supplemented with 0.25% human serum albumin. In another specific embodiment, the nucleic acid, vector or cell of the invention is formulated in a composition containing Ringer's lactate and a non-ionic surfactant such as pluronic F68 at a final concentration of 0.01-0.0001%, for example at a concentration of 0.001% , by weight of the total composition. The composition may additionally contain serum albumin, in particular human serum albumin, such as human serum albumin 0.25%. Other suitable formulations for storage or administration are known in the art, in particular from WO 2005/118792 or Allay et al., 2011.

In a preferred embodiment, the composition is formulated according to standard procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also contain a solubilizing agent and a local anesthetic such as lignocaine to relieve pain at the injection site.

In one embodiment, the nucleic acid molecule, nucleic acid construct, vector, GAA polypeptide or cell of the invention can be delivered in a vesicle, in particular a liposome. In yet another embodiment, a nucleic acid molecule, nucleic acid construct, vector, GAA polypeptide, or cell of the invention can be delivered in a controlled release system.

Methods for administering a nucleic acid molecule, nucleic acid construct, vector, GAA polypeptide, or cell of the invention include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. In a specific embodiment, administration is via the intravenous or intramuscular route. The nucleic acid molecule, nucleic acid construct, vector, GAA polypeptide, or cells of the invention, whether in vector form or not, may be administered by any convenient route, e.g., by infusion or bolus injection, by absorption through epithelial or mucocutaneous membranes (e.g., , oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. The introduction can be systemic or local.

In a particular embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment, such as the liver. This can be achieved, for example, by means of an implant, said implant of a porous, non-porous or gel-like material, including membranes such as "sialastic" membranes or fibers.

The amount of a therapeutic agent (i.e., nucleic acid molecule, nucleic acid construct, vector, GAA polypeptide, or cell of the invention) of the invention that will be effective in treating glycogen storage disease can be determined using standard clinical procedures. Additionally, in vivo and/or in vitro assays may optionally be used to help predict optimal dosage ranges. The precise dosage used in the formulation also depends on the route of administration and the severity of the disease, and should be selected according to the judgment of the practitioner and the circumstances of each patient. The dosage of a nucleic acid molecule, nucleic acid construct, vector, GAA polypeptide, or cell of the invention administered to a subject in need thereof will vary depending on several factors, including, but not limited to, the route of administration, the particular disease being treated, the age of the subject, or the level expression necessary to achieve a therapeutic effect. The person skilled in the art can readily determine, based on his knowledge of the art, the required dosage range based on these and other factors. In the case of treatment involving the introduction of a viral vector, such as an AAV vector, to a subject, typical doses of the vector are at least 1×10 ⁸ vector genomes per kilogram of body weight (wg/kg), for example, at least 1×10 ⁹ vg/ kg, at least 1×10 ¹⁰ vg/kg, at least 1×10 ¹¹ vg/kg, at least 1×10 ¹² vg/kg at least 1×10 ¹³ vg/kg or at least 1× 10 ¹⁴ vg/kg.

The invention also relates to a method for treating glycogen storage disease which comprises the step of delivering a therapeutically effective amount of a nucleic acid, vector, GAA polypeptide, pharmaceutical composition or cell of the invention to a subject in need thereof.

The invention also relates to a method for treating glycogen storage disease, said method does not induce an immune response to a transgene (i.e., to a GAA polypeptide of the invention) or induces a reduced immune response to the transgene, which includes the step of delivering a therapeutically effective amount of a nucleic acid molecule, a nucleic acid construct acid, vector, pharmaceutical composition or cell of the invention to a subject in need. The invention also relates to a method for treating glycogen storage disease, said method comprising re-administering a therapeutically effective amount of a nucleic acid molecule, nucleic acid construct, vector, pharmaceutical composition or cell of the invention to a subject in need thereof. In this aspect, the nucleic acid molecule or nucleic acid construct of the invention contains a promoter that is functional in liver cells, thereby allowing immunological tolerance to an expressed GAA polypeptide produced by them. Also in this aspect, the pharmaceutical composition used in this aspect contains a nucleic acid molecule or a nucleic acid construct containing a promoter that is functional in liver cells. In the case of delivery of liver cells, said cells may be cells previously taken from a subject in need of treatment and constructed by introducing into them the nucleic acid molecule or nucleic acid construct of the invention, thereby making them capable of producing the GAA polypeptide of the invention. According to one embodiment, in an aspect involving repeated administration, said administration may be repeated at least once or more, and may even be considered to be performed according to a periodic pattern, such as once a week, a month, or a year. . The periodic schedule may also include administration once every 2, 3, 4, 5, 6, 7, 8, 9 or 10 years or more than 10 years. In another specific embodiment, the introduction of each introduction of the viral vector of the invention is performed using a different virus for each subsequent introduction, thereby avoiding a decrease in effect due to a possible immune response to the previously introduced viral vector. For example, a first introduction of a viral vector containing an AAV8 capsid may be carried out, followed by the introduction of a vector containing an AAV9 capsid, or even the introduction of a non-AAV related virus such as a retroviral or lentiviral vector.

In accordance with the present invention, the treatment may include curative, alleviating or prophylactic effects. Accordingly, therapeutic and prophylactic treatment includes improving the symptoms of a particular glycogen storage disease or preventing or otherwise reducing the risk of developing a particular glycogen storage disease. The term "prophylactic" can be thought of as reducing the severity or onset of a particular condition. "Prophylactic" also includes preventing a recurrence of a particular condition in a patient previously diagnosed with the condition. "Therapeutic" can also reduce the severity of an existing condition. The term "treatment" is used herein to refer to any regimen that can benefit an animal, in particular a mammal, more specifically a human subject.

The invention also relates to an ex vivo gene therapy method for treating glycogen storage disease, which comprises introducing a nucleic acid molecule or nucleic acid construct of the invention into an isolated cell of a patient in need thereof, such as an isolated hematopoietic stem cell, and administering said cell to said patient, in need of it. In a specific embodiment of this aspect, the nucleic acid molecule or construct is introduced into a cell using a vector as defined above. In a specific embodiment, the vector is an integrating viral vector. In a further specific embodiment, the viral vector is a retroviral vector, such as a lentiviral vector. For example, a lentiviral vector, as disclosed in van Til et al., 2010, Blood, 115(26), page 5329, can be used in the practice of the method of the present invention.

The invention also relates to a nucleic acid molecule, nucleic acid construct, vector, GAA polypeptide or cell of the invention for use as a drug.

The invention also relates to a nucleic acid molecule, nucleic acid construct, vector, GAA polypeptide or cell of the invention for use in a method for treating a disease caused by a mutation in the GAA gene, in particular a method for treating Pompe disease. The invention further relates to a nucleic acid molecule, nucleic acid construct, vector, GAA polypeptide or cell of the invention for use in a method of treating a glycogen storage disease such as GSDI (Girke's disease), GSDII (Pompe's disease), GSDIII (Corey's disease), GSDIV , GSDV, GSDVI, GSDVII, GSDVIII and lethal congenital heart glycogen storage disease, more specifically GSDI, GSDII or GSDIII, even more specifically GSDII and GSDIII and most specifically GSDII. A truncated GAA polypeptide of the invention can be administered to a patient in need thereof for use in enzyme replacement therapy (ERT), e.g. for use in enzyme replacement therapy for glycogen storage disease such as GSDI (Girke's disease), GSDII (Pompe disease), GSDIII (Measles disease) , GSDIV, GSDV, GSDVI, GSDVII, GSDVIII and lethal congenital heart glycogen storage disease, more specifically GSDI, GSDII or GSDIII, even more specifically GSDII and GSDIII and most specifically GSDII.

The invention further relates to the use of a nucleic acid molecule, a nucleic acid construct, a vector, a GAA polypeptide, or a cell of the invention in the manufacture of a medicament effective for the treatment of a glycogen storage disease such as GSDI (Girke's disease), GSDII (Pompe disease), GSDIII (Pompe disease). Corey), GSDIV, GSDV, GSDVI, GSDVII, GSDVIII and lethal congenital heart glycogen storage disease, more specifically GSDI, GSDII or GSDIII, even more specifically GSDII and GSDIII and most specifically GSDII.

EXAMPLES

The invention is further described in detail with reference to the following Experimental Examples and the attached FIGS. These examples are provided for illustrative purposes only and are not intended to be limiting.

MATERIALS AND METHODS

GAA Activity

GAA activity was measured after homogenization of frozen tissue samples in distilled water. 50-100 mg tissue was weighed and homogenized, then centrifuged for 20 minutes at 10000×g. The reaction was carried out using 10 μl of the supernatant and 20 μl of the substrate - 4MUα-D-glucoside, in a 96-well plate. The reaction mixture was incubated at 37°C for one hour and then stopped by adding 150 μl of buffer with sodium carbonate pH 10.5. A calibration curve (4MU 0-2500 pmol/µl) was used to measure the fluorescent 4MU released from the individual reaction mixture using an EnSpire α plate reader (Perkin-Elmer) at 449 nm (emission) and 360 nm (excitation). The protein concentration of the clarified supernatant was quantified using BCA (Thermo Fisher Scientific). In order to calculate GAA activity, the concentration of 4MU released was divided by the protein concentration in the sample and the activity was reported as nmol/h/mg of protein.

Mouse studies

A GAA-/- mouse was created by targeted disruption of exon 6 and maintained in a C57BL/6J/129X1/SvJ background (Raben N. et al 1998). The vectors were delivered via the tail vein in a volume of 0.2 ml. Serum samples were taken monthly in order to monitor secreted hGAA levels. Affected animals injected with PBS and wild-type littermates were used as controls.

NHP study

Male cynomolgus monkeys were kept in stainless steel cages and maintained on a 12 hour light/dark cycle. All macaques had neutralizing antibody titers < 1:5 before the start of the study, a dose of 2E12 vg/kg AAV8-hAAT-sp7-Δ8-hGAAco1 was infused via the saphenous vein. Blood samples were taken 12 days before and 30 days after injection via the femoral vein. Whole blood was collected into tubes containing EDTA and centrifuged to separate serum. 3 months after the introduction of the vector, all macaques were sacrificed. Animals were first anesthetized with a mixture of ketamine/dexmedetomidine and then euthanized using an IV injection of sodium pentobarbital. Tissues were harvested directly and frozen in liquid nitrogen.

Western blot analysis

Complete homogenates of frozen muscles were obtained. Protein concentration was determined in the extracts using the Pierce BCA Protein Assay (Thermo Fisher Scientific) following the manufacturer's instructions. Western blotting was performed using an anti-hGAA antibody (Abcam). An anti-tubulin antibody (Sigma Aldrich) was used as a loading control.

RESULTS

In order to develop new forms of GAA with improved secretion and reduced immunogenicity, the inventors decided to obtain truncated forms of GAA, optionally combining them with alternative signal peptides.

The human GAA shown in SEQ ID NO: 2 served as the basis for the development of these new forms. SEQ ID NO: 1 corresponds to the sequence from SEQ ID NO: 2 free of the corresponding natural GAA signal peptide (amino acids 1-27 from SEQ ID NO: 2). Nucleic acid constructs were designed to encode GAA polypeptides derived from SEQ ID NO: 1 truncated at their N-terminus. The inventors started by designing a nucleic acid sequence based on the wild-type hGAA coding sequence (SEQ ID NO: 9, corresponding to nucleotides 82-2859 of SEQ ID NO: 8, which is the wild-type hGAA coding sequence containing the signal peptide coding sequence) with deletion of codons corresponding to the first 8 amino acids of SEQ ID NO: 1 (Δ8). In addition to the wild-type hGAA coding sequence, the inventors designed an optimized nucleic acid sequence encoding the Δ8 truncated hGAA polypeptide (SEQ ID NO: 10 and SEQ ID NO: 11 correspond to the optimized hGAAco1 and hGAAco2 coding sequence, respectively) to eliminate the possible effect of a specific sequences.

Subsequence wt co1 co2 CAI ^a 0.84 0.94 0.77 GC content ^b 64.7 61.9 54.4 aORF 5'→ ^3'c 2 3 0 aORF 3'→ ^5'd 5 four 0 SA ^e 3 0 one SD ^f 3 0 0 % identity compared to wt ^g 83.1 77.7 % identity compared to co1 ^h 80.8 CpG ⁱ islands four 5 one

Table 1. Description of optimized sequences. The table shows the performance of the two optimized hGAA sequences compared to the wild-type sequence. a) codon adaptation coefficient; and b) GC content was calculated using a rare codon analysis tool (http://www.genscript.com). c) and d) respectively represent alternative open reading frames calculated on 5'-3' (aORF 5'→3') and 3'-5' (aORF 3'→5') strands. e) and f) respectively are the splicing acceptor (SA) and donor (SD) regions calculated using the online splicing site prediction tool (http://www.fruitfly.org/seq_tools/splice.html). g) and h) respectively represent the percent identity calculated in comparison with the sequence of the wild type (wt) and optimized sequence co1. i) CpG islands were calculated using the MethDB online tool (http://www.methdb.de/links.html). CpG islands are sequences longer than 100 bp, with a GC content of >60% and an observed/expected ratio of >0.6.

Amino acids 1-27 from the hGAA sequences (corresponding to the natural hGAA signal peptide, here defined as sp1; their sequence is given in SEQ ID NO: 4) were replaced with amino acids 1-24 from the sequence of human α-1-antitrypsin (NP_000286.3), here defined as sp2 (the sequence is given in SEQ ID NO: 5). The inventors transfected human hepatoma (Huh-7) cells with truncated hGAA-encoding constructs in parallel with their full-length versions, and the inventors measured the amount of hGAA released into the medium after 48 hours of FIG. 1A). Deletion of Δ8 in the hGAA sequences resulted in a significant 50% increase in secretion levels for both the wild-type (hGAA) and codon-optimized (hGAAco2) sequences. The same truncation performed on another codon-optimized sequence (hGAAco1) also improved hGAA secretion to the same extent.

In order to confirm that a change in the sequence of the following signal peptide can improve hGAA secretion, the inventors further truncated the hGAA polypeptide. The inventors removed the codons corresponding to the first 42 amino acids of hGAA from the hGAAco1 construct (Δ42) and the inventors replaced them with a signal peptide derived from chymotrypsinogen B1 (sp7; the sequence is given in SEQ ID NO: 3). The inventors then compared the secretion effect obtained using this new deletion construct with its Δ8 version fused to the sp7 signal peptide and full-length hGAAco1 to either sp1 or sp7. The inventors transfected Huh-7 cells with these constructs and the inventors measured the hGAA activity in the medium after 48 hours. As expected, the inventors could measure hGAA activity after transfection with full-length hGAAco1 (p=0.055 vs. GFP) and its secretion increased two-fold when the wild-type signal peptide was replaced by sp7 (p=0.006 vs. hGAAco1). Surprisingly, both Δ8 and Δ42 hGAA sequences fused to the sp7 signal peptide showed a 2-fold increase in secreted hGAA compared to the full-length sequence (p=0.0002 and 0.0003, respectively, compared to sp7-hGAAco1, Fig. 1B) .

Taken together, these data demonstrate that truncation of the hGAA sequence coupled to an effective signal peptide allows increased protein secretion in vitro. Additionally, truncation has one important advantage over native sequence mutagenesis in that it does not generate major neoantigens, which is an advantage in therapeutic product design.

The inventors then verified these findings in vivo, in a mouse model of Pompe disease. The inventors injected GAA-/- mice (Raben et al J. Bio. Chem. 1998) with AAV8 vectors expressing full-length hGAAco1, Δ8 or Δ42 fused to the sp7 signal peptide under transcriptional control of a highly active liver-specific promoter derived from the fusion of the apolipoprotein B enhancer and the human α-1-antitrypsin (hAAT) promoter. One month after injection of 2E12 vg/kg of the vectors described above, mice were bled and serum hGAA activity was measured. Treatment of mice with vectors expressing full length hGAAco1 fused to sp7 showed elevated circulating levels of hGAA (p=0.115 vs. PBS). Surprisingly, both truncated hGAA, Δ8 and Δ42, led to a significant increase in serum hGAA levels (p=0.014 and 0.013, respectively).

These data show that the deletion of the first amino acids of hGAA led to a significant improvement in the level of hGAA secreted into the bloodstream.

In addition, another signal peptide was fused to the Δ8 truncated form of hGAA corresponding to amino acids 1-25 from iduronate-2-sulfatase (sp6; SEQ ID NO: 6). The inventors transfected hepatoma (Huh-7) cells with plasmids expressing GFP or wild-type hGAA (hGAA; parental polypeptide corresponding to amino acid residues 28-952 of SEQ ID NO: 30) in parallel with plasmids expressing optimized hGAA (hGAAco1) fused with sp1, sp2, sp6, sp7 or sp8. 48 hours after transfection, the growth medium was analyzed for the presence of hGAA. Of note, these constructs resulted in significantly higher levels of hGAA secretion than were observed in the negative control represented by GFP-transfected cells (FIG. 3).

The inventors then assessed the glycogen content in the heart, diaphragm and quadriceps of GAA-/- mice treated as described above with Δ8-hGAA. Of note, the inventors observed high levels of hGAA in tissues following treatment with Δ8-hGAAco expression vectors (data not shown), which correlated with a significant decrease in glycogen content in all tissues considered (Fig. 4B-D). In particular, in the heart (Fig. 4B), the glycogen level measured after treatment with vectors carrying the highly effective signal peptides sp7 and 8 was indistinguishable from that observed in unaffected animals (p=0.983 and 0.996 compared to WT, respectively) . Importantly, the level observed after treatment with the sp7 and sp8 vectors was significantly reduced compared to GAA-/- animals that received PBS injections or treatment with a vector expressing hGAAco fused to the sp1 signal peptide.

The inventors also tested whether liver transduction using the vectors of the inventors induced a humoral response to the transgene. Mice were intravenously injected with AAV8 vectors expressing hGAAco1 with native signal peptide sp1 (co) or Δ8-hGAAco1 fused to sp2, sp7 or sp8 under the transcriptional control of a specific liver promoter. The results are shown in FIG. 5. Gaa-/- received an intramuscular injection of AAV expressing Δ8-hGAAco1 under the transcriptional control of a constitutive promoter, which showed a very high level of total IgG (~150 μg/ml), while in general the vector expressing the same protein in the liver showed lower humoral response. Interestingly, mice injected with the hGAAco1 (co) sp1 expression vector exhibited detectable levels of antibodies at both doses, while mice injected with high secretion engineered vectors had undetectable IgG levels. These data indicate that hepatic expression of the transgene is necessary to induce peripheral tolerance, and they also provide indications that high circulating hGAA levels, achieved through fusion with an effective signal peptide, induce a reduction in the humoral response to the protein itself.

The most potent vector selected in the mouse study was injected with two non-human primates (NHP, Macaca fascicularis sp.) in order to verify the effect of our vector secretion and muscle uptake. The inventors injected two monkeys with 2E12 vg/kg AAV8-hAAT-sp7-Δ8-hGAAco1. One month after injection, the inventors measured the serum levels of hGAA in two animals by Western blotting using a specific antibody against hGAA. In two monkeys, the inventors observed a clear band with a size consistent with that of hGAA. This band was absent in serum samples obtained 12 days before vector injection, thus confirming the specificity of the method of detection of the inventors (Fig. 6A). 3 months after the injection, the inventors sacrificed the animals and the inventors obtained tissues in order to verify if the hGAA secreted by the liver into the bloodstream was effectively taken up by the muscle. The inventors performed a Western blot using an antibody specific for hGAA on total lysates obtained from the biceps and diaphragm of two monkeys. Interestingly, the inventors could observe a clear band in animal #2, which also showed the highest circulating levels of hGAA (FIG. 6B). Also in Animal No. 1, the inventors could observe a faint band with a molecular weight corresponding to that of hGAA in both analyzed muscles. These data show that the AAV8-hAAT-sp7-Δ8-hGAAco1 vector efficiently transduces the liver in NHP. They also demonstrate that the protein secreted into the bloodstream is efficiently taken up by the muscle and that this uptake correlates with the level of hGAA measured in the blood.

The inventors also determined the effect of the most potent vector selected in the mouse study (AAV8-hAAT-sp7-Δ8-hGAAco1) in a GSDIII mouse model. The inventors developed a knockout mouse model for glycogen debranching enzyme (GDE). This model replicates the disease phenotype seen in humans affected by glycogen storage disease type III (GSDIII). In particular, GDE -/- mice, which completely lack GDE activity, have a weakening of muscle strength and accumulation of glycogen in various tissues. It is also interesting that they accumulate glycogen in the liver, which is also observed in humans. Here, the inventors tested whether overexpression of sp7-Δ8-hGAA in the liver prevents the accumulation of glycogen observed in GDE -/- mice. The inventors injected GDE-/- mice 1E11 or 1E12 vg/mouse AAV8-hAAT-sp7-Δ8-hGAAco1. As controls, we injected PBS into wild-type (WT) and GDE-/- mice in parallel. 3 months after the introduction of the vector, the mice were sacrificed and the level of glycogen in the liver was quantified. The results are shown in FIG. 7. As already reported (Pagliarani et al and the inventors' model), GDE -/- mice showed a significant increase in hepatic glycogen accumulation (p=1.3E-7) - 5 times more glycogen compared to wild-type animals. Surprisingly, treatment with 1E11 and 1E12 vg/mouse vector AAV8-hAAT-sp7-Δ8-hGAAco1 caused a statistically significant decrease in glycogen content (p=4.5E-5 and 1.4E-6, respectively). Importantly, glycogen levels measured in the liver of mice injected with the AAV8-hAAT-sp7-Δ8-hGAAco1 vector did not differ from those measured in wild-type animals, in particular at the highest dose (p=0.053 for the 1E11 dose cohort and 0.244 for the 1E12 dose cohort).

The inventors analyzed GAA activity in media and lysates of HuH7 cells transfected with different versions of GAA (all with codon optimization): 1. native GAA containing the native signal peptide sp1 GAA (co), 2. engineered GAA containing the heterologous signal peptide sp7 ( sp7-co), and 3. an engineered GAA containing a heterologous sp7 signal peptide followed by a variable amino acid deletion (sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co, sp7 -Δ47-co and sp7-Δ62-co, in which the 8, 29, 42, 47 and 62 first N-terminal amino acids from SEQ ID NO: 1 are removed, respectively. The analysis showed (FIG. 8) significantly higher GAA activity in the media of cells transfected with the Δ8, Δ29, Δ42 and Δ43 versions of GAA compared to both engineered GAA without deletions (sp7-co) and native GAA (co) . Conversely, significantly lower GAA activity was observed in the media of cells transfected with the Δ47 and Δ62 versions of GAA compared to other engineered versions of GAA [with deletions (sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7 -Δ43-co) and without deletions (sp7-co)]. Interestingly, (Fig. 9) intracellular GAA activity did not differ between productive deletions (sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co) and non-deleted versions (sp7-co) , which indicates that all of them are efficiently produced and processed in the cell. Conversely, intracellular GAA activity was very low for sp7-Δ47-co and sp7-Δ62-co versions and significantly lower compared to all other engineered versions [with deletions (sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42- co, sp7-Δ43-co) and without deletions (sp7-co)].

The inventors also performed an analysis of GAA activity in media and lysates of HuH7 cells transfected with different versions of GAA (all with optimized codons): 1. native GAA containing the native signal peptide sp1 GAA (co), 2. engineered GAA containing the heterologous signal peptide sp6 or sp8 (sp6-co, sp8-co), and 3. engineered GAA containing a heterologous sp6 or sp8 signal peptide followed by an 8 amino acid deletion (sp6-Δ8-co, sp8-Δ8-co). The analysis showed (FIG. 10) significantly higher GAA activity in the media of cells transfected with Δ8 versions compared to: i. their respective engineered versions of GAA without deletions (sp6-co or sp8-co); and ii. native GAA (co). Interestingly, the intracellular activity of GAA did not differ among all engineered versions of GAA (both with and without deletions), indicating that they are all efficiently produced and processed in the cell (part of cell lysates). Conversely, intracellular GAA activity was significantly higher with native GAA (co) compared to the engineered versions, indicating that native GAA is preferentially retained in the cell.

--->

SEQUENCE LIST

<110> GENETHON et al.

<120> ACID ALPHA-GLUCOSIDASE VARIANTS AND THEIR USES

<130> B2299PC00

<160> 53

<170> PatentIn version 3.3

<210> 1

<211> 925

<212> Protein

<213> artificial

<220>

<223> hGAAwt without sp

<400> 1

Gly His Ile Leu Leu His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser

1 5 10 15

Gly Ser Ser Pro Val Leu Glu Glu Thr His Pro Ala His Gln Gln Gly

20 25 30

Ala Ser Arg Pro Gly Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro

35 40 45

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

50 55 60

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

65 70 75 80

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

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Gln Pro Trp Cys Phe Phe Pro Ser Tyr Pro Ser Tyr Lys Leu Glu

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Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr

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

130 135 140

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

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Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr Pro His Val His Ser Arg

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Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly

180 185 190

Val Ile Val Arg Arg Gln Leu Asp Gly Arg Val Leu Leu Asn Thr

195 200 205

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

210 215 220

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

225 230 235 240

Met Leu Ser Thr Ser Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu

245 250 255

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

260 265 270

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

275 280 285

Asn Ala Met Asp Val Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg

290 295 300

Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro

305 310 315 320

Lys Ser Val Val Gln Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met

325 330 335

Pro Pro Tyr Trp Gly Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser

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Ser Thr Ala Ile Thr Arg Gln Val Val Glu Asn Met Thr Arg Ala His

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Phe Pro Leu Asp Val Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg

370 375 380

Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met

385 390 395 400

Val Gln Glu Leu His Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp

405 410 415

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

420 425 430

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

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Leu Ile Gly Lys Val Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr

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Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp Met Val Ala Glu Phe His

465 470 475 480

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

485 490 495

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

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Asn Pro Pro Tyr Val Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala

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

530 535 540

His Asn Leu Tyr Gly Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu

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

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

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

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Leu Leu Gly Val Pro Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly

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Asn Thr Ser Glu Glu Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe

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

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Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu

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

675 680 685

Ala His Val Ala Gly Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe

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Pro Lys Asp Ser Ser Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly

705 710 715 720

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

725 730 735

Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro

740 745 750

Val Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu

755 760 765

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

770 775 780

Asp Thr Ile Asn Val His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln

785 790 795 800

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

805 810 815

Ala Val Ala Leu Thr Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp

820 825 830

Asp Asp Gly Glu Ser Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln

835 840 845

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

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

865 870 875 880

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

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

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

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Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys

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Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu

20 25 30

His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val

35 40 45

Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly

50 55 60

Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr

65 70 75 80

Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys

85 90 95

Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro

100 105 110

Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe

115 120 125

Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser

130 135 140

Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe

145 150 155 160

Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu

165 170 175

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

180 185 190

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

195 200 205

Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val Arg Arg

210 215 220

Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe

225 230 235 240

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

245 250 255

Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser

260 265 270

Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly

275 280 285

Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly

290 295 300

Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val

305 310 315 320

Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile

325 330 335

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

340 345 350

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

355 360 365

Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr

370 375 380

Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val

385 390 395 400

Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe

405 410 415

Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His

420 425 430

Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser

435 440 445

Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg

450 455 460

Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val

465 470 475 480

Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu

485 490 495

Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe

500 505 510

Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly

515 520 525

Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val

530 535 540

Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser

545 550 555 560

Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu

645 650 655

Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg

660 665 670

Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser

675 680 685

Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala

690 695 700

Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly

705 710 715 720

Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser

725 730 735

Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile

740 745 750

Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro

755 760 765

Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Val Glu Ala Leu Gly

770 775 780

Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser

785 790 795 800

Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val

805 810 815

His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr

820 825 830

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

835 840 845

Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser

850 855 860

Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala

865 870 875 880

Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly

885 890 895

Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala

900 905 910

Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr

915 920 925

Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly

930 935 940

Glu Gln Phe Leu Val Ser Trp Cys

945 950

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<220>

<223> sp7

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Met Ala Phe Leu Trp Leu Leu Ser Cys Trp Ala Leu Leu Gly Thr Thr

1 5 10 15

Phe Gly

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<220>

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Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys

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Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu

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<213> artificial

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Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly Leu Cys

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Cys Leu Val Pro Val Ser Leu Ala

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Met Pro Pro Pro Arg Thr Gly Arg Gly Leu Leu Trp Leu Gly Leu Val

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Leu Ser Ser Val Cys Val Ala Leu Gly

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<210> 7

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Met Ala Ser Arg Leu Thr Leu Leu Thr Leu Leu Leu Leu Leu Leu Ala

1 5 10 15

Gly Asp Arg Ala Ser Ser

twenty

<210> 8

<211> 2859

<212> DNA

<213> homo sapiens

<400> 8

atgggagtga ggcacccgcc ctgctcccac cggctcctgg ccgtctgcgc cctcgtgtcc 60

ttggcaaccg cagcgctcct ggggcacatc ctactccatg atttcctgct ggttccccga 120

gagctgagtg gctcctcccc agtcctggag gagactcacc cagctcacca gcagggagcc 180

agcagaccag ggccccggga tgcccaggca caccccggggc ggccgcgagc agtgcccaca 240

cagtgcgacg tcccccccaa cagccgcttc gattgcgccc ctgacaaggc catcacccag 300

gaacagtgcg aggcccgcgg ctgttgctac atccctgcaa agcaggggct gcagggagcc 360

cagatggggc agccctggtg cttcttccca cccagctacc ccagctacaa gctggagaac 420

ctgagctcct ctgaaatggg ctacacggcc accctgaccc gtaccacccc caccttcttc 480

cccaaggaca tcctgaccct gcggctggac gtgatgatgg agactgagaa ccgcctccac 540

ttcacgatca aagatccagc taacaggcgc tacgaggtgc ccttggagac cccgcatgtc 600

cacagccggg caccgtcccc actctacagc gtggagttct ccgaggagcc cttcggggtg 660

atcgtgcgcc ggcagctgga cggccgcgtg ctgctgaaca cgacggtggc gcccctgttc 720

tttgcggacc agttccttca gctgtccacc tcgctgccct cgcagtatat cacaggcctc 780

gccgagcacc tcagtcccct gatgctcagc accagctgga ccaggatcac cctgtggaac 840

cgggaccttg cgccccgcc cggtgcgaac ctctacgggt ctcacccttt ctacctggcg 900

ctggaggacg gcgggtcggc acacggggtg ttcctgctaa acagcaatgc catggatgtg 960

gtcctgcagc cgagccctgc ccttagctgg aggtcgacag gtgggatcct ggatgtctac 1020

atcttcctgg gccgagcc caagagcgtg gtgcagcagt acctggacgt tgtgggatac 1080

ccgttcatgc cgccatactg gggcctgggc ttccacctgt gccgctgggg ctactcctcc 1140

accgctatca cccgccaggt ggtggagaac atgaccaggg cccacttccc cctggacgtc 1200

cagtggaacg acctggacta catggactcc cggagggact tcacgttcaa caaggatggc 1260

ttccgggact tcccggccat ggtgcaggag ctgcaccagg gcggccggcg ctacatgatg 1320

atcgtggatc ctgccatcag cagctcgggc cctgccggga gctacaggcc ctacgacgag 1380

ggtctgcgga ggggggtttt catcaccaac gagaccggcc agccgctgat tgggaaggta 1440

tggcccgggt ccactgcctt ccccgacttc accaacccca cagccctggc ctggtgggag 1500

gacatggtgg ctgagttcca tgaccaggtg cccttcgacg gcatgtggat tgacatgaac 1560

gagccttcca acttcatcag gggctctgag gacggctgcc ccaacaatga gctggagaac 1620

ccaccctacg tgcctggggt ggttgggggg accctccagg cggccaccat ctgtgcctcc 1680

agccaccagt ttctctccac acactacaac ctgcacaacc tctacggcct gaccgaagcc 1740

atcgcctccc acagggcgct ggtgaaggct cgggggacac gcccatttgt gatctcccgc 1800

tcgacctttg ctggcacgg ccgatacgcc ggccactgga cggggggacgt gtggagctcc 1860

tgggagcagc tcgcctcctc cgtgccagaa atcctgcagt ttaacctgct gggggtgcct 1920

ctggtcgggg ccgacgtctg cggcttcctg ggcaacacct cagaggagct gtgtgtgcgc 1980

tggacccagc tgggggcctt ctaccccttc atgcggaacc acaacagcct gctcagtctg 2040

ccccaggagc cgtacagctt cagcgagccg gcccagcagg ccatgaggaa ggccctcacc 2100

ctgcgctacg cactcctccc ccacctctac acactgttcc accaggccca cgtcgcgggg 2160

gagaccgtgg cccggcccct cttcctggag ttccccaagg actctagcac ctggactgtg 2220

gaccaccagc tcctgtgggg ggaggccctg ctcatcaccc cagtgctcca ggccgggaag 2280

gccgaagtga ctggctactt ccccttggggc acatggtacg acctgcagac ggtgccagta 2340

gaggcccttg gcagcctccc acccccacct gcagctcccc gtgagccagc catccacagc 2400

gaggggcagt gggtgacgct gccggccccc ctggacacca tcaacgtcca cctccgggct 2460

gggtacatca tccccctgca gggccctggc ctcacaacca cagagtcccg ccagcagccc 2520

atggccctgg ctgtggccct gaccaagggt ggggaggccc gaggggagct gttctgggac 2580

gatggagaga gcctggaagt gctggagcga ggggcctaca cacaggtcat cttcctggcc 2640

aggaataaca cgatcgtgaa tgagctggta cgtgtgacca gtgagggagc tggcctgcag 2700

ctgcagaagg tgactgtcct gggcgtggcc acggcgcccc agcaggtcct ctccaacggt 2760

gtccctgtct ccaacttcac ctacagcccc gacaccaagg tcctggacat ctgtgtctcg 2820

ctgttgatgg gagagcagtt tctcgtcagc tggtgttag 2859

<210> 9

<211> 2778

<212> DNA

<213> artificial

<220>

<223> hGAAwt without sp

<400> 9

gggcacatcc tactccatga tttcctgctg gttccccgag agctgagtgg ctcctcccca 60

gtcctggagg agactcaccc agctcaccag cagggagcca gcagaccagg gccccgggat 120

gcccaggcac accccgggcg gccgcgagca gtgcccacac agtgcgacgt cccccccaac 180

agccgcttcg attgcgcccc tgacaaggcc atcacccagg aacagtgcga ggcccgcggc 240

tgttgctaca tccctgcaaa gcaggggctg cagggagccc agatggggca gccctggtgc 300

ttcttcccac ccagctaccc cagctacaag ctggagaacc tgagctcctc tgaaatggggc 360

tacacggcca ccctgacccg taccaccccc accttcttcc ccaaggacat cctgaccctg 420

cggctggacg tgatgatgga gactgagaac cgcctccact tcacgatcaa agatccagct 480

aacaggcgct acgaggtgcc cttggagacc ccgcatgtcc acagccggggc accgtcccca 540

ctctacagcg tggagttctc cgaggagccc ttcggggtga tcgtgcgccg gcagctggac 600

ggccgcgtgc tgctgaacac gacggtggcg cccctgttct ttgcggacca gttccttcag 660

ctgtccacct cgctgccctc gcagtatatc acaggcctcg ccgagcacct cagtcccctg 720

atgctcagca ccagctggac caggatcacc ctgtggaacc gggaccttgc gcccacgccc 780

ggtgcgaacc tctacgggtc tcaccctttc tacctggcgc tggaggacgg cgggtcggca 840

cacggggtgt tcctgctaaa cagcaatgcc atggatgtgg tcctgcagcc gagccctgcc 900

cttagctgga ggtcgacagg tgggatcctg gatgtctaca tcttcctgggg cccagagccc 960

aagagcgtgg tgcagcagta cctggacgtt gtgggatacc cgttcatgcc gccatactgg 1020

ggcctgggct tccacctgtg ccgctggggc tactcctcca ccgctatcac ccgccaggtg 1080

gtggagaaca tgaccagggc ccacttcccc ctggacgtcc agtggaacga cctggactac 1140

atggactccc ggagggactt cacgttcaac aaggatggct tccgggactt cccggccatg 1200

gtgcaggagc tgcaccaggg cggccggcgc tacatgatga tcgtggatcc tgccatcagc 1260

agctcgggcc ctgccgggag ctacaggccc tacgacgagg gtctgcggag gggggttttc 1320

atcaccaacg agaccggcca gccgctgatt gggaaggtat ggcccgggtc cactgccttc 1380

ccgacttca ccaaccccac agccctggcc tggtgggagg acatggtggc tgagttccat 1440

gaccaggtgc ccttcgacgg catgtggatt gacatgaacg agccttccaa cttcatcagg 1500

ggctctgagg acggctgccc caacaatgag ctggagaacc caccctacgt gcctggggtg 1560

gttgggggga ccctccaggc ggccaccatc tgtgcctcca gccaccagtt tctctccaca 1620

cactacaacc tgcacaacct ctacggcctg accgaagcca tcgcctccca cagggcgctg 1680

gtgaaggctc gggggacacg cccatttgtg atctcccgct cgacctttgc tggccacggc 1740

cgatacgccg gccactggac gggggacgtg tggagctcct gggagcagct cgcctcctcc 1800

gtgccagaaa tcctgcagtt taacctgctg ggggtgcctc tggtcggggc cgacgtctgc 1860

ggcttcctgg gcaacacctc agaggagctg tgtgtgcgct ggacccagct gggggccttc 1920

taccccttca tgcggaacca caacagcctg ctcagtctgc cccaggagcc gtacagcttc 1980

agcgagccgg cccagcaggc catgaggaag gccctcaccc tgcgctacgc actcctcccc 2040

cacctctaca cactgttcca ccaggcccac gtcgcgggggg agaccgtggc ccggcccctc 2100

ttcctggagt tccccaagga ctctagcacc tggactgtgg accaccagct cctgtggggg 2160

gaggccctgc tcatcacccc agtgctccag gccgggaagg ccgaagtgac tggctacttc 2220

cccttgggca catggtacga cctgcagacg gtgccagtag aggcccttgg cagcctccca 2280

cccccacctg cagctccccg tgagccagcc atccacagcg aggggcagtg ggtgacgctg 2340

ccggcccccc tggacaccat caacgtccac ctccgggctg ggtacatcat ccccctgcag 2400

ggccctggcc tcacaaccac agagtcccgc cagcagccca tggccctggc tgtggccctg 2460

accaagggtg gggaggcccg aggggagctg ttctgggacg atggagagag cctggaagtg 2520

ctggagcgag gggcctacac acaggtcatc ttcctggcca ggaataacac gatcgtgaat 2580

gagctggtac gtgtgaccag tgagggagct ggcctgcagc tgcagaaggt gactgtcctg 2640

ggcgtggcca cggcgcccca gcaggtcctc tccaacggtg tccctgtctc caacttcacc 2700

tacagccccg acaccaaggt cctggacatc tgtgtctcgc tgttgatggg agagcagttt 2760

ctcgtcagct ggtgttag 2778

<210> 10

<211> 2778

<212> DNA

<213> artificial

<220>

<223> hGAAco1 no sp

<400> 10

ggccatatcc tgctgcacga ctttctacta gtgccgag agctgagcgg cagctctccc 60

gtgctggaag aaacacaccc tgcccatcag cagggcgcct ctagacctgg acctagagat 120

gcccaggccc accccggcag acctagagct gtgcctaccc agtgtgacgt gccccccaac 180

agcagattcg actgcgcccc tgacaaggcc atcacccagg aacagtgcga ggccagaggc 240

tgctgctaca tccctgccaa gcagggactg cagggcgctc agatgggaca gccctggtgc 300

ttcttcccac cctcctaccc cagctacaag ctggaaaacc tgagcagcag cgagatgggc 360

tacaccgcca ccctgaccag aaccaccccc acattcttcc caaaggacat cctgaccctg 420

cggctggacg tgatgatgga aaccgagaac cggctgcact tcaccatcaa ggaccccgcc 480

aatcggagat acgaggtgcc ccctggaaacc ccccacgtgc actctagagc ccccagccct 540

ctgtacagcg tggaattcag cgaggaaccc ttcggcgtga tcgtgcggag acagctggat 600

ggcagagtgc tgctgaacac caccgtggcc cctctgttct tcgccgacca gttcctgcag 660

ctgagcacca gcctgcccag ccagtacatc acaggactgg ccgagcacct gagccccctg 720

atgctgagca catcctggac ccggatcacc ctgtggaaca gggatctggc ccctacccct 780

ggcgccaatc tgtacggcag ccaccctttc tacctggccc tggaagatgg cggatctgcc 840

cacggagtgt ttctgctgaa ctccaacgcc atggacgtgg tgctgcagcc tagccctgcc 900

ctgtcttgga gaagcacagg cggcatcctg gatgtgtaca tctttctggg ccccgagccc 960

aagagcgtgg tgcagcagta tctggatgtc gtgggctacc ccttcatgcc cccttactgg 1020

ggcctgggat tccacctgtg cagatggggc tactccagca ccgccatcac cagacaggtg 1080

gtggaaaaca tgaccagagc ccacttccca ctggatgtgc agtggaacga cctggactac 1140

atggacagca gacgggactt caccttcaac aaggacggct tccgggactt ccccgccatg 1200

gtgcaggaac tgcatcaggg cggcagacgg tacatgatga tcgtggatcc cgccatcagc 1260

tcctctggcc ctgccggctc ttacagaccc tacgacgagg gcctgcggag aggcgtgttc 1320

atcaccaacg agacaggcca gcccctgatc ggcaaagtgt ggcctggcag cacagccttc 1380

cccgacttca ccaatcctac cgccctggct tggtgggagg acatggtggc cgagttccac 1440

gaccaggtgc ccttcgacgg catgtggatc gacatgaacg agcccagcaa cttcatccgg 1500

ggcagcgagg atggctgccc caacaacgaa ctggaaaatc ccccttacgt gcccggcgtc 1560

gtgggcggaa cactgcaggc cgctacaatc tgtgccagca gccaccagtt tctgagcacc 1620

cactacaacc tgcacaacct gtacggcctg accgaggcca ttgccagcca ccgcgctctc 1680

gtgaaagcca gaggcacacg gcccttcgtg atcagcagaa gcacctttgc cggccacggc 1740

agatacgccg gacattggac tggcgacgtg tggtcctctt gggagcagct ggcctctagc 1800

gtgcccgaga tcctgcagtt caatctgctg ggcgtgccac tcgtgggcgc cgatgtgtgt 1860

ggcttcctgg gcaacacctc cgaggaactg tgtgtgcggt ggacacagct gggcgccttc 1920

1980

agcgagcctg cacagcaggc catgcggaag gccctgacac tgagatacgc tctgctgccc 2040

cacctgtaca ccctgtttca ccaggcccat gtggccggcg agacagtggc cagacctctg 2100

tttctggaat tccccaagga cagcagcacc tggaccgtgg accatcagct gctgtgggga 2160

gaggctctgc tgattacccc agtgctgcag gcaggcaagg ccgaagtgac cggctacttt 2220

cccctgggca cttggtacga cctgcagacc gtgcctgtgg aagccctggg atctctgcct 2280

ccacctcctg ccgctcctag agagcctgcc attcactctg agggccagtg ggtcacactg 2340

cctgcccccc tggataccat caacgtgcac ctgaggggccg gctacatcat accactgcag 2400

ggacctggcc tgaccaccac cgagtctaga cagcagccaa tggccctggc cgtggccctg 2460

accaaaggcg gagaagctag gggcgagctg ttctgggacg atggcgagag cctggaagtg 2520

ctggaaagag gcgcctatac ccaagtgatc ttcctggccc ggaacaacac catcgtgaac 2580

gagctggtgc gcgtgacctc tgaaggcgct ggactgcagc tgcagaaagt gaccgtgctg 2640

ggagtggcca cagcccctca gcaggtgctg tctaatggcg tgcccgtgtc caacttcacc 2700

2760

ctggtgtcct ggtgctga 2778

<210> 11

<211> 2778

<212> DNA

<213> artificial

<220>

<223> hGAAco2 without sp

<400> 11

ggacacatcc tgctgcacga cttcctgttg gtgcctagag agctgagcgg atcatcccca 60

gtgctggagg agactcatcc tgctcaccaa cagggagctt ccagaccagg accgagagac 120

gcccaagcc atcctggtag accaaggct gtgcctaccc aatgcgacgt gccacccaac 180

tcccgattcg actgcgcgcc agataaggct attacccaag agcagtgtga agccagaggt 240

tgctgctaca tcccagcgaa gcaaggattg caaggcgccc aaatgggaca accttggtgt 300

ttcttccccc cttcgtaccc atcatataaa ctcgaaaacc tgtcctcttc ggaaatgggt 360

tatactgcca ccctcaccag aactactcct actttcttcc cgaaagacat cttgaccttg 420

aggctggacg tgatgatgga gactgaaaac cggctgcatt tcactatcaa agatcctgcc 480

aatcggcgat acgaggtccc tctggaaacc cctcacgtgc actcacggggc tccttctccg 540

ctttactccg tcgaattctc tgaggaaccc ttcggagtga tcgttagacg ccagctggat 600

ggtagagtgc tgttgaacac tactgtggcc ccacttttct tcgctgacca gtttctgcaa 660

ctgtccactt ccctgccatc ccagtacatt actggactcg ccgaacacct gtcgccactg 720

atgctctcga cctcttggac tagaatcact ttgtggaaca gagacttggc ccctactccg 780

ggagcaaatc tgtacggaag ccaccctttt tacctggcgc tcgaagatgg cggatccgct 840

cacggagtgt tcctgctgaa tagcaacgca atggacgtgg tgctgcaacc ttcccctgca 900

ctcagttgga gaagtaccgg gggtattctg gacgtgtaca tcttcctcgg accagaaccc 960

aagagcgtgg tgcagcaata tctggacgtg gtcggatacc cttttatgcc tccttactgg 1020

ggactgggat tccacctttg ccgttggggc tactcatcca ccgccattac cagacaggtg 1080

gtggagaata tgaccagagc ccacttccct ctcgacgtgc agtggaacga tctggactat 1140

1200

1260

agcagcggac ccgctggatc ttatagacct tacgacgaag gccttaggag aggagtgttc 1320

1380

cctgacttta ccaatcccac tgccttggct tggtgggagg acatggtggc cgaattccac 1440

gaccaagtcc cctttgatgg aatgtggatc gatatgaacg aaccaagcaa ttttatcaga 1500

ggttccgaag acggttgccc caacaacgaa ctggaaaacc ctccttatgt gcccggagtc 1560

gtgggcggaa cattacaggc cgcgactatt tgcgccagca gccaccaatt cctgtccact 1620

cactacaacc tccacaacct ttatggatta accgaagcta ttgcaagtca cagggctctg 1680

gtgaaggcta gagggactag gccctttgtg atctcccgat ccacctttgc cggacacggg 1740

agatacgccg gtcactggac tggtgacgtg tggagctcat gggaacaact ggcctcctcc 1800

1860

gggtttcttg gtaacacctc cgaggaactg tgtgtgcgct ggactcaact gggtgcattc 1920

tacccattca tgagaaacca caactccttg ctgtccctgc cacaagagcc ctactcgttc 1980

agcgagcctg cacaacaggc tatgcggaag gcactgaccc tgagatacgc cctgcttcca 2040

cacttataca ctctcttcca tcaagcgcat gtggcaggag aaaccgttgc aaggcctctt 2100

ttccttgaat tccccaagga ttcctcgact tggacggtgg atcatcagct gctgtgggga 2160

gaagctctgc tgattactcc agtgttgcaa gccggaaaag ctgaggtgac cggatacttt 2220

ccgctgggaa cctggtacga ccctccagact gtccctgttg aagcccttgg atcactgcct 2280

ccgcctccgg cagctccacg cgaaccagct atacattccg agggacagtg ggttacatta 2340

ccagctcctc tggacaat caacgtccac ttaagagctg gctacattat ccctctgcaa 2400

ggaccaggac tgactacgac cgagagcaga cagcagccaa tggcactggc tgtggctctg 2460

accaagggag gggaagctag aggagaactc ttctgggatg atggggagtc ccttgaagtg 2520

ctggaaagag gcgcttacac tcaagtcatt ttccttgcac ggaacaacac cattgtgaac 2580

gaattggtgc gagtgaccag cgaaggagct ggacttcaac tgcagaaggt cactgtgctc 2640

ggagtggcta ccgctcctca gcaagtgctg tcgaatggag tccccgtgtc aaactttacc 2700

tactcccctg acactaaggt gctcgacatt tgcgtgtccc tcctgatggg agagcagttc 2760

cttgtgtcct ggtgttga 2778

<210> 12

<211> 2754

<212> DNA

<213> artificial

<220>

<223> hGAAco1-delta-8 no sp

<400> 12

ctactagtgc ccagagagct gagcggcagc tctcccgtgc tggaagaaac acaccctgcc 60

catcagcagg gcgcctctag acctggacct agagatgccc aggcccaccc cggcagacct 120

agagctgtgc ctacccagtg tgacgtgccc cccaacagca gattcgactg cgcccctgac 180

aaggccatca ccgggaaca gtgcgaggcc agaggctgct gctacatccc tgccaagcag 240

ggactgcagg gcgctcagat gggacagccc tggtgcttct tcccaccctc ctaccccagc 300

tacaagctgg aaaacctgag cagcagcgag atgggctaca ccgccaccct gaccagaacc 360

acccccacat tcttcccaaa ggacatcctg accctgcggc tggacgtgat gatggaaacc 420

gagaaccggc tgcacttcac catcaaggac cccgccaatc ggagatacga ggtgcccctg 480

gaaacccccc acgtgcactc tagagccccc agccctctgt acagcgtgga attcagcgag 540

gaacccttcg gcgtgatcgt gcggagacag ctggatggca gagtgctgct gaacaccacc 600

gtggcccctc tgttcttcgc cgaccagttc ctgcagctga gcaccagcct gcccagccag 660

tacatcacag gactggccga gcacctgagc cccctgatgc tgagcacatc ctggacccgg 720

atcaccctgt ggaacagggga tctggcccct acccctggcg ccaatctgta cggcagccac 780

cctttctacc tggccctgga agatggcggga tctgcccacg gagtgtttct gctgaactcc 840

aacgccatgg acgtggtgct gcagcctagc cctgccctgt cttggagaag cacaggcggc 900

atcctggatg tgtacatctt tctgggcccc gagcccaaga gcgtggtgca gcagtatctg 960

gatgtcgtgg gctacccctt catgccccct tactggggcc tgggattcca cctgtgcaga 1020

tggggctact ccagcaccgc catcaccaga caggtggtgg aaaacatgac cagagcccac 1080

ttcccactgg atgtgcagtg gaacgacctg gactacatgg acagcagacg ggacttcacc 1140

ttcaacaagg acggcttccg ggacttcccc gccatggtgc aggaactgca tcagggcggc 1200

agacggtaca tgatgatcgt ggatcccgcc atcagctcct ctggccctgc cggctcttac 1260

agaccctacg acgaggggcct gcggagaggc gtgttcatca ccaacgagac aggccagccc 1320

ctgatcggca aagtgtggcc tggcagcaca gccttccccg acttcaccaa tcctaccgcc 1380

ctggcttggt gggaggacat ggtggccgag ttccacgacc aggtgccctt cgacggcatg 1440

tggatcgaca tgaacgagcc cagcaacttc atccggggca gcgaggatgg ctgccccaac 1500

aacgaactgg aaaatccccc ttacgtgccc ggcgtcgtgg gcggaacact gcaggccgct 1560

1620

ggcctgaccg aggccattgc cagccaccgc gctctcgtga aagccagagg cacacggccc 1680

ttcgtgatca gcagaagcac ctttgccggc cacggcagat acgccggaca ttggactggc 1740

gacgtgtggt cctcttggga gcagctggcc tctagcgtgc ccgagatcct gcagttcaat 1800

ctgctgggcg tgccactcgt gggcgccgat gtgtgtggct tcctgggcaa cacctccgag 1860

gaactgtgtg tgcggtggac acagctgggc gccttctacc ctttcatgag aaaccacaac 1920

agcctgctga gcctgcccca ggaaccctac agctttagcg agcctgcaca gcaggccatg 1980

cggaaggccc tgacactgag atacgctctg ctgccccacc tgtacaccct gtttcaccag 2040

gcccatgtgg ccggcgagac agtggccaga cctctgtttc tggaattccc caaggacagc 2100

agcacctgga ccgtggacca tcagctgctg tggggagagg ctctgctgat taccccagtg 2160

ctgcaggcag gcaaggccga agtgaccggc tactttcccc tgggcacttg gtacgacctg 2220

cagaccgtgc ctgtggaagc cctgggatct ctgcctccac ctcctgccgc tcctagagag 2280

cctgccattc actctgagg ccagtgggtc acactgcctg cccccctgga taccatcaac 2340

gtgcacctga gggccggcta catcatacca ctgcagggac ctggcctgac caccaccgag 2400

tctagacagc agccaatggc cctggccgtg gccctgacca aaggcggaga agctagggggc 2460

gagctgttct gggacgatgg cgagagcctg gaagtgctgg aaagaggcgc ctatacccaa 2520

gtgatcttcc tggcccggaa caacaccatc gtgaacgagc tggtgcgcgt gaccctctgaa 2580

ggcgctggac tgcagctgca gaaagtgacc gtgctgggag tggccacagc ccctcagcag 2640

gtgctgtcta atggcgtgcc cgtgtccaac ttcacctaca gccccgacac caaggtgctg 2700

gacatctgcg tgtcactgct gatgggagag cagtttctgg tgtcctggtg ctga 2754

<210> 13

<211> 2754

<212> DNA

<213> artificial

<220>

<223> hGAAco2-delta8 without sp

<400> 13

ctgttggtgc ctagagagct gagcggatca tccccagtgc tggaggagac tcatcctgct 60

caccaacagg gagcttccag accaggaccg agagacgccc aagcccatcc tggtagacca 120

agagctgtgc ctacccaatg cgacgtgcca cccaactccc gattcgactg cgcgccagat 180

aaggctatta cccaaagca gtgtgaagcc agaggttgct gctacatccc agcgaagcaa 240

ggattgcaag gcgcccaaat gggacaacct tggtgtttct tccccccttc gtacccatca 300

tataaactcg aaaacctgtc ctcttcggaa atgggttata ctgccaccct caccagaact 360

actcctactt tcttcccgaa agacatcttg accttgaggc tggacgtgat gatggagact 420

gaaaaccggc tgcatttcac tatcaaagat cctgccaatc ggcgatacga ggtccctctg 480

gaaacccctc acgtgcactc acgggctcct tctccgcttt actccgtcga attctctgag 540

gaacccttcg gagtgatcgt tagacgccag ctggatggta gagtgctgtt gaacactact 600

gtggccccac ttttcttcgc tgaccagttt ctgcaactgt ccacttccct gccatcccag 660

tacattactg gactcgccga acacctgtcg ccactgatgc tctcgacctc ttggactaga 720

atcactttgt ggaacagaga cttggcccct actccgggag caaatctgta cggaagccac 780

cctttttacc tggcgctcga agatggcgga tccgctcacg gagtgttcct gctgaatagc 840

aacgcaatgg acgtggtgct gcaaccttcc cctgcactca gttggagaag taccgggggt 900

attctggacg tgtacatctt cctcggacca gaacccaaga gcgtggtgca gcaatatctg 960

gacgtggtcg gatacccttt tatgcctcct tactggggac tgggattcca cctttgccgt 1020

tggggctact catccaccgc cattaccaga caggtggtgg agaatatgac cagagcccac 1080

ttccctctcg acgtgcagtg gaacgatctg gactatatgg actcccggag agatttcacc 1140

ttcaacaagg acgggttccg cgattttccc gcgatggttc aagagctcca ccagggtggt 1200

cgaagatata tgatgatcgt cgacccagcc atttcgagca gcggacccgc tggatcttat 1260

agaccttacg acgaaggcct taggagagga gtgttcatca caaacgagac tggacagcct 1320

ttgatcggta aagtgtggcc tggatcaacc gcctttcctg actttaccaa tcccactgcc 1380

ttggcttggt gggaggacat ggtggccgaa ttccacgacc aagtcccctt tgatggaatg 1440

tggatcgata tgaacgaacc aagcaatttt atcagaggtt ccgaagacgg ttgccccaac 1500

aacgaactgg aaaaccctcc ttatgtgccc ggagtcgtgg gcggaacatt acaggccgcg 1560

actatttgcg ccagcagcca ccaattcctg tccactcact acaacctcca caacctttat 1620

ggattaaccg aagctattgc aagtcacagg gctctggtga aggctagagg gactaggccc 1680

tttgtgatct cccgatccac ctttgccgga cacgggagat acgccggtca ctggactggt 1740

gacgtgtgga gctcatggga acaactggcc tcctccgtgc cggaaatctt acagttcaac 1800

cttctgggtg tccctcttgt cggagcagac gtgtgtgggt ttcttggtaa cacctccgag 1860

gaactgtgtg tgcgctggac tcaactgggt gcattctacc cattcatgag aaaccacaac 1920

tccttgctgt ccctgccaca agagccctac tcgttcagcg agcctgcaca acaggctatg 1980

cggaaggcac tgaccctgag atacgccctg cttccacact tatacactct cttccatcaa 2040

gcgcatgtgg caggagaaac cgttgcaagg cctcttttcc ttgaattccc caaggattcc 2100

tcgacttgga cggtggatca tcagctgctg tggggagaag ctctgctgat tactccagtg 2160

ttgcaagccg gaaaagctga ggtgaccgga tactttccgc tgggaacctg gtacgacctc 2220

cagactgtcc ctgttgaagc ccttggatca ctgcctccgc ctccggcagc tccacgcgaa 2280

ccagctatac attccgagg acagtgggtt acattaccag ctcctctgga cacaatcaac 2340

gtccacttaa gagctggcta cattatccct ctgcaaggac caggactgac tacgaccgag 2400

agcagacagc agccaatggc actggctgtg gctctgacca agggagggga agctagagga 2460

gaactcttct gggatgatgg ggagtccctt gaagtgctgg aaagaggcgc ttacactcaa 2520

gtcattttcc ttgcacggaa caacaccatt gtgaacgaat tggtgcgagt gaccagcgaa 2580

ggagctggac ttcaactgca gaaggtcact gtgctcggag tggctaccgc tcctcagcaa 2640

gtgctgtcga atggagtccc cgtgtcaaac tttacctact cccctgacac taaggtgctc 2700

gacatttgcg tgtccctcct gatgggagag cagttccttg tgtcctggtg ttga 2754

<210> 14

<211> 397

<212> DNA

<213> artificial

<220>

<223> hAAT promoter

<400> 14

gatcttgcta ccagtggaac agccactaag gattctgcag tgagagcaga gggccagcta 60

agtggtactc tcccagagac tgtctgactc acgccacccc ctccaccttg gacacaggac 120

gctgtggttt ctgagccagg tacaatgact cctttcggta agtgcagtgg aagctgtaca 180

ctgcccaggc aaagcgtccg ggcagcgtag gcgggcgact cagatcccag ccagtggact 240

tagcccctgt ttgctcctcc gataactggg gtgaccttgg ttaatattca ccagcagcct 300

cccccgttgc ccctctggat ccactgctta aatacggacg aggacagggc cctgtctcct 360

cagcttcagg caccaccact gacctgggac agtgaat 397

<210> 15

<211> 321

<212> DNA

<213> artificial

<220>

<223> ApoE control area

<400> 15

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctggggc ccatgccacc 240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300

ggtttaggta gtgtgagagg g 321

<210> 16

<211> 441

<212> DNA

<213> artificial

<220>

<223> HBB2 intron

<400> 16

gtacacatat tgaccaaatc agggtaattt tgcatttgta attttaaaaa atgctttctt 60

cttttaatat acttttttgt ttatcttatt tctaatactt tccctaatct ctttctttca 120

gggcaataat gtacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata 180

atttctgggt taaggcaata gcaatatttc tgcatataaa tatttctgca tataaattgt 240

aactgatgta agaggtttca tattgctaat agcagctaca atccagctac cattctgctt 300

ttatttttatg gttgggataa ggctggatta ttctgagtcc aagctaggcc cttttgctaa 360

tcatgttcat acctcttatc ttcctcccac agctcctggg caacgtgctg gtctgtgtgc 420

tggcccatca ctttggcaaa g 441

<210> 17

<211> 441

<212> DNA

<213> artificial

<220>

<223> modified HBB2 intron

<400> 17

gtacacatat tgaccaaatc agggtaattt tgcatttgta attttaaaaa atgctttctt 60

cttttaatat acttttttgt ttatcttatt tctaatactt tccctaatct ctttctttca 120

gggcaataat gtacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata 180

atttctgggt taaggcaata gcaatatttc tgcatataaa tatttctgca tataaattgt 240

aactgatgta agaggtttca tattgctaat agcagctaca atccagctac cattctgctt 300

ttatttttctg gttgggataa ggctggatta ttctgagtcc aagctaggcc cttttgctaa 360

tcttgttcat acctcttatc ttcctcccac agctcctggg caacctgctg gtctctctgc 420

tggcccatca ctttggcaaa g 441

<210> 18

<211> 1438

<212> DNA

<213> artificial

<220>

<223> intron FIX

<400> 18

ggtttgtttc cttttttaaa atacattgag tatgcttgcc tttttagatat agaaatatct 60

gatgctgtct tcttcactaa attttgatta catgatttga cagcaatatt gaagagtcta 120

acagccagca cgcaggttgg taagtactgg ttctttgtta gctaggtttt cttcttcttc 180

atttttaaaa ctaaatagat cgacaatgct tatgatgcat ttatgtttaa taaacactgt 240

tcagttcatg atttggtcat gtaattcctg ttagaaaaca ttcatctcct tggtttaaaa 300

360

tttttgtttg gacttaccac tttgaaatca aaatgggaaa caaaagcaca aacaatggcc 420

ttatttacac aaaaagtctg attttaagat atatgacatt tcaaggtttc agaagtatgt 480

aatgaggtgt gtctctaatt ttttaaatta tatatcttca atttaaagtt ttagttaaaa 540

cataaagatt aacctttcat tagcaagctg ttagttatca ccaacgcttt tcatggatta 600

660

tactcagttg agttccctag gggagaaaag cacgcttaag aattgacata aagagtagga 720

agttagctaa tgcaacatat atcactttgt tttttcacaa ctacagtgac tttatgtatt 780

tccgagga aggcatacag ggaagaaatt atcccatttg gacaaacagc atgttctcac 840

aggaagcatt tatcacactt acttgtcaac tttctagaat caaatctagt agctgacagt 900

accaggatca ggggtgccaa ccctaagcac ccccagaaag ctgactggcc ctgtggttcc 960

cactccagac atgatgtcag ctgtgaaatc gacgtcgctg gaccataatt aggcttctgt 1020

tcttcaggag acatttgttc aaagtcattt gggcaaccat attctgaaaa cagcccagcc 1080

agggtgatgg atcactttgc aaagatcctc aatgagctat tttcaagtga tgacaaagtg 1140

tgaagttaac cgctcatttg agaactttct ttttcatcca aagtaaattc aaatatgatt 1200

agaaatctga ccttttatta ctggaattct cttgactaaa agtaaaattg aattttaatt 1260

cctaaatctc catgtgtata cagtactgtg ggaacatcac agattttggc tccatgccct 1320

1380

aaattttcat gatgttttct tttttgctaa aactaaagaa ttattctttt acatttca 1438

<210> 19

<211> 1438

<212> DNA

<213> artificial

<220>

<223> modified FIX intron

<400> 19

ggtttgtttc cttttttaaa atacattgag tatgcttgcc tttttagatat agaaatatct 60

gatgctgtct tcttcactaa attttgatta catgatttga cagcaatatt gaagagtcta 120

acagccagca cgcaggttgg taagtactgg ttctttgtta gctaggtttt cttcttcttc 180

atttttaaaa ctaaatagat cgacattgct tttgttgcat ttatgtttaa taaacactgt 240

tcagttcatg atttggtcat gtaattcctg ttagaaaaca ttcatctcct tggtttaaaa 300

360

tttttgtttg gacttaccac tttgaaatca aattgggaaa caaaagcaca aacaatggcc 420

ttatttacac aaaaagtctg attttaagat atatgacatt tcaaggtttc agaagtatgt 480

aatgaggtgt gtctctaatt ttttaaatta tatatcttca atttaaagtt ttagttaaaa 540

cataaagatt aacctttcat tagcaagctg ttagttatca ccaacgcttt tcatggatta 600

660

tactcagttg agttccctag gggagaaaag cacgcttaag aattgacata aagagtagga 720

agttagctat tgcaacatat atcactttgt tttttcacaa ctacagtgac tttttgtatt 780

tccgagga aggcatacag ggaagaaatt atcccatttg gacaaacagc ttgttctcac 840

aggaagcatt tatcacactt acttgtcaac tttctagaat caaatctagt agctgacagt 900

accaggatca ggggtgccaa ccctaagcac ccccagaaag ctgactggcc ctgtggttcc 960

cactccagac atgatgtcag ctgtgaaatc gacgtcgctg gaccataatt aggcttctgt 1020

tcttcaggag acatttgttc aaagtcattt gggcaaccat attctgaaaa cagcccagcc 1080

agggtgttgg atcactttgc aaagatcctc attgagctat tttcaagtgt tgacaaagtg 1140

tgaagttaac cgctcatttg agaactttct ttttcatcca aagtaaattc aaatatgatt 1200

agaaatctga ccttttatta ctggaattct cttgactaaa agtaaaattg aattttaatt 1260

cctaaatctc catgtgtata cagtactgtg ggaacatcac agattttggc tccatgccct 1320

1380

1438

<210> 20

<211> 881

<212> DNA

<213> artificial

<220>

<223> chicken beta-globin intron

<400> 20

gcgggagtcg ctgcgttgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc 60

gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc 120

180

aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg 240

cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg 300

ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg 360

gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt 420

gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca 480

cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg 540

tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc 600

ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg 660

cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagaggggc 720

gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac 780

cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga 840

gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct c 881

<210> 21

<211> 881

<212> DNA

<213> artificial

<220>

<223> modified chicken beta-globin intron

<400> 21

gcgggagtcg ctgcgttgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc 60

gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc 120

180

aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg 240

cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg 300

ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg 360

gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt 420

gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca 480

cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg 540

tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc 600

ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg 660

cggctgtcga ggcgcggcga gccgcagcca ttgccttttt tggtaatcgt gcgagaggggc 720

gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac 780

cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaat tgggcgggga 840

gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct c 881

<210> 22

<211> 4318

<212> DNA

<213> artificial

<220>

<223> construct: sp2+hGAAwt-delta-8

<400> 22

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctggggc ccatgccacc 240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300

ggtttaggta gtgtgagagg ggtacccggg gatcttgcta ccagtggaac agccactaag 360

gattctgcag tgagagcaga gggccagcta agtggtactc tcccagagac tgtctgactc 420

acgccacccc ctccaccttg gacacaggac gctgtggttt ctgagccagg tacaatgact 480

cctttcggta agtgcagtgg aagctgtaca ctgcccaggc aaagcgtccg ggcagcgtag 540

gcgggcgact cagatcccag ccagtggact tagcccctgt ttgctcctcc gataactggg 600

660

aatacggacg aggacagggc cctgtctcct cagcttcagg caccaccact gacctgggac 720

agtgaataga tcctgagaac ttcagggtga gtctatggga cccttgatgt tttctttccc 780

cttcttttct atggttaagt tcatgtcata ggaaggggag aagtaacagg gtacacatat 840

tgaccaaatc agggtaattt tgcatttgta attttaaaaa atgctttctt cttttaatat 900

acttttttgt ttatcttatt tctaatactt tccctaatct ctttctttca gggcaataat 960

gatacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata atttctgggt 1020

taaggcaata gcaatatttc tgcatataaa tatttctgca tataaattgt aactgatgta 1080

1140

gttgggataa ggctggatta ttctgagtcc aagctaggcc cttttgctaa tcttgttcat 1200

acctcttatc ttcctcccac agctcctggg caacctgctg gtctctctgc tggcccatca 1260

ctttggcaaa gcacgcgtgc caccatgccg tcttctgtct cgtggggcat cctcctgctg 1320

gcaggcctgt gctgcctggt ccctgtctcc ctggctctgc tggttccccg agagctgagt 1380

ggctcctccc cagtcctgga ggagactcac ccagctcacc agcagggagc cagcagacca 1440

gggccccggg atgcccaggc acaccccggg cggccgcgag cagtgcccac acagtgcgac 1500

gtccccccca acagccgctt cgattgcgcc cctgacaagg ccatcaccca ggaacagtgc 1560

gaggcccgcg gctgttgcta catccctgca aagcaggggc tgcagggagc ccagatgggg 1620

cagccctggt gcttcttccc acccagctac cccagctaca agctggagaa cctgagctcc 1680

tctgaaatgg gctacacggc caccctgacc cgtaccaccc ccaccttctt ccccaaggac 1740

atcctgaccc tgcggctgga cgtgatgatg gagactgaga accgcctcca cttcacgatc 1800

1860

gcaccgtccc cactctacag cgtggagttc tccgaggagc ccttcggggt gatcgtgcgc 1920

cggcagctgg acggccgcgt gctgctgaac acgacggtgg cgcccctgtt ctttgcggac 1980

cagttccttc agctgtccac ctcgctgccc tcgcagtata tcacaggcct cgccgagcac 2040

ctcagtcccc tgatgctcag caccagctgg accaggatca ccctgtggaa ccgggacctt 2100

gcgccccgc ccggtgcgaa cctctacggg tctcaccctt tctacctggc gctggaggac 2160

ggcgggtcgg cacacggggt gttcctgcta aacagcaatg ccatggatgt ggtcctgcag 2220

ccgagccctg cccttagctg gaggtcgaca ggtgggatcc tggatgtcta catcttcctg 2280

ggccgagc ccaagagcgt ggtgcagcag tacctggacg ttgtgggata cccgttcatg 2340

ccgccatact ggggcctggg cttccacctg tgccgctggg gctactcctc caccgctatc 2400

acccgccagg tggtggagaa catgaccagg gcccacttcc ccctggacgt ccagtggaac 2460

gacctggact acatggactc ccggagggac ttcacgttca acaaggatgg cttccgggac 2520

ttcccggcca tggtgcagga gctgcaccag ggcggccggc gctacatgat gatcgtggat 2580

cctgccatca gcagctcggg ccctgccggg agctacaggc cctacgacga gggtctgcgg 2640

aggggggttt tcatcaccaa cgagaccggc cagccgctga ttgggaaggt atggcccggg 2700

tccactgcct tccccgactt caccaacccc acagccctgg cctggtggga ggacatggtg 2760

gctgagttcc atgaccaggt gcccttcgac ggcatgtgga ttgacatgaa cgagccttcc 2820

aacttcatca ggggctctga ggacggctgc cccaacaatg agctggagaa cccaccctac 2880

gtgcctgggg tggttggggg gaccctccag gcggccacca tctgtgcctc cagccaccag 2940

tttctctcca cacactacaa cctgcacaac ctctacggcc tgaccgaagc catcgcctcc 3000

3060

gctggccacg gccgatacgc cggccactgg acggggggacg tgtggagctc ctgggagcag 3120

ctcgcctcct ccgtgccaga aatcctgcag tttaacctgc tgggggtgcc tctggtcggg 3180

gccgacgtct gcggcttcct gggcaacacc tcagaggagc tgtgtgtgcg ctggacccag 3240

ctgggggcct tctacccctt catgcggaac cacaacagcc tgctcagtct gccccaggag 3300

ccgtacagct tcagcgagcc ggcccagcag gccatgagga aggccctcac cctgcgctac 3360

gcactcctcc cccacctcta cacactgttc caccaggccc acgtcgcggg ggagaccgtg 3420

gcccggcccc tcttcctgga gttccccaag gactctagca cctggactgt ggaccaccag 3480

ctcctgtggggggaggccct gctcatcacc ccagtgctcc aggccgggaa ggccgaagtg 3540

actggctact tccccttggg cacatggtac gacctgcaga cggtgccagt agaggccctt 3600

ggcagcctcc cacccccacc tgcagctccc cgtgagccag ccatccacag cgaggggcag 3660

tgggtgacgc tgccggcccc cctggacacc atcaacgtcc acctccggggc tgggtacatc 3720

atccccctgc agggccctgg cctcacaacc acagagtccc gccagcagcc catggccctg 3780

gctgtggccc tgaccaaggg tggggaggcc cgaggggagc tgttctggga cgatggagag 3840

agcctggaag tgctggagcg aggggcctac acacaggtca tcttcctggc caggaataac 3900

acgatcgtga atgagctggt acgtgtgacc agtgagggag ctggcctgca gctgcagaag 3960

gtgactgtcc tgggcgtggc cacggcgcc cagcaggtcc tctccaacgg tgtccctgtc 4020

tccaacttca cctacagccc cgacaccaag gtcctggaca tctgtgtctc gctgttgatg 4080

ggagagcagt ttctcgtcag ctggtgttag ctcgagagat ctaccggtga attcaccgcg 4140

ggtttaaact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 4200

cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 4260

gcattgtctg agtaggtgtc attctattct ggggggtggg gtgggggcta gctctaga 4318

<210> 23

<211> 4318

<212> DNA

<213> artificial

<220>

<223> construct: sp2+hGAAco1-delta-8

<400> 23

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctggggc ccatgccacc 240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300

ggtttaggta gtgtgagagg ggtacccggg gatcttgcta ccagtggaac agccactaag 360

gattctgcag tgagagcaga gggccagcta agtggtactc tcccagagac tgtctgactc 420

acgccacccc ctccaccttg gacacaggac gctgtggttt ctgagccagg tacaatgact 480

cctttcggta agtgcagtgg aagctgtaca ctgcccaggc aaagcgtccg ggcagcgtag 540

gcgggcgact cagatcccag ccagtggact tagcccctgt ttgctcctcc gataactggg 600

660

aatacggacg aggacagggc cctgtctcct cagcttcagg caccaccact gacctgggac 720

agtgaataga tcctgagaac ttcagggtga gtctatggga cccttgatgt tttctttccc 780

cttcttttct atggttaagt tcatgtcata ggaaggggag aagtaacagg gtacacatat 840

tgaccaaatc agggtaattt tgcatttgta attttaaaaa atgctttctt cttttaatat 900

acttttttgt ttatcttatt tctaatactt tccctaatct ctttctttca gggcaataat 960

gatacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata atttctgggt 1020

taaggcaata gcaatatttc tgcatataaa tatttctgca tataaattgt aactgatgta 1080

1140

gttgggataa ggctggatta ttctgagtcc aagctaggcc cttttgctaa tcttgttcat 1200

acctcttatc ttcctcccac agctcctggg caacctgctg gtctctctgc tggcccatca 1260

ctttggcaaa gcacgcgtgc caccatgcct agctctgtgt cctggggcat tctgctgctg 1320

gccggcctgt gttgtctggt gcctgtgtct ctggccctac tagtgcccag agagctgagc 1380

ggcagctctc ccgtgctgga agaaacacac cctgcccatc agcaggggcgc ctctagacct 1440

ggacctagag atgcccaggc ccaccccggc agacctagag ctgtgcctac ccagtgtgac 1500

gtgcccccca acagcagatt cgactgcgcc cctgacaagg ccatcaccca ggaacagtgc 1560

gaggccagag gctgctgcta catccctgcc aagcagggac tgcagggcgc tcagatggga 1620

cagccctggt gcttcttccc accctcctac cccagctaca agctggaaaa cctgagcagc 1680

agcgagatgg gctacaccgc caccctgacc agaaccaccc ccacattctt cccaaaggac 1740

atcctgaccc tgcggctgga cgtgatgatg gaaaccgaga accggctgca cttcaccatc 1800

aaggaccccg ccaatcggag atacgaggtg cccctggaaa cccccccgt gcactctaga 1860

gcccccagcc ctctgtacag cgtggaattc agcgaggaac ccttcggcgt gatcgtgcgg 1920

agacagctgg atggcagagt gctgctgaac accaccgtgg cccctctgtt cttcgccgac 1980

cagttcctgc agctgagcac cagcctgccc agccagtaca tcacaggact ggccgagcac 2040

ctgagccccc tgatgctgag cacatcctgg acccggatca ccctgtggaa cagggatctg 2100

gcccctaccc ctggcgccaa tctgtacggc agccaccctt tctacctggc cctggaagat 2160

ggcggatctg cccacggagt gtttctgctg aactccaacg ccatggacgt ggtgctgcag 2220

cctagccctg ccctgtcttg gagaagcaca ggcggcatcc tggatgtgta catctttctg 2280

ggccccgagc ccaagagcgt ggtgcagcag tatctggatg tcgtgggcta ccccttcatg 2340

cccccttact ggggcctggg attccacctg tgcagatggg gctactccag caccgccatc 2400

accagacagg tggtggaaaa catgaccaga gcccacttcc cactggatgt gcagtggaac 2460

gacctggact acatggacag cagacgggac ttcaccttca acaaggacgg cttccgggac 2520

ttccccgcca tggtgcagga actgcatcag ggcggcagac ggtacatgat gatcgtggat 2580

cccgccatca gctcctctgg ccctgccggc tcttacagac cctacgacga gggcctgcgg 2640

agaggcgtgt tcatcaccaa cgagacaggc cagcccctga tcggcaaagt gtggcctggc 2700

agcacagcct tccccgactt caccaatcct accgccctgg cttggtggga ggacatggtg 2760

gccgagttcc acgaccaggt gcccttcgac ggcatgtgga tcgacatgaa cgagcccagc 2820

aacttcatcc ggggcagcga ggatggctgc cccaacaacg aactggaaaa tcccccttac 2880

gtgcccggcg tcgtgggcgg aacactgcag gccgctacaa tctgtgccag cagccaccag 2940

tttctgagca cccactacaa cctgcacaac ctgtacggcc tgaccgaggc cattgccagc 3000

caccgcgctc tcgtgaaagc cagaggcaca cggcccttcg tgatcagcag aagcaccttt 3060

gccggccacg gcagatacgc cggacattgg actggcgacg tgtggtcctc ttgggagcag 3120

ctggcctcta gcgtgcccga gatcctgcag ttcaatctgc tgggcgtgcc actcgtgggc 3180

gccgatgtgt gtggcttcct gggcaacacc tccgaggaac tgtgtgtgcg gtggacacag 3240

ctgggcgcct tctacccttt catgagaaac cacaacagcc tgctgagcct gccccaggaa 3300

ccctacagct ttagcgagcc tgcacagcag gccatgcgga aggccctgac actgagatac 3360

gctctgctgc cccacctgta caccctgttt caccaggccc atgtggccgg cgagacagtg 3420

gccagacctc tgtttctgga attccccaag gacagcagca cctggaccgt ggaccatcag 3480

ctgctgtggg gagaggctct gctgattacc ccagtgctgc aggcaggcaa ggccgaagtg 3540

accggctact ttcccctggg cacttggtac gacctgcaga ccgtgcctgt ggaagccctg 3600

ggatctctgc ctccacctcc tgccgctcct agagagcctg ccattcactc tgagggccag 3660

tgggtcacac tgcctgcccc cctggatacc atcaacgtgc acctgagggc cggctacatc 3720

ataccactgc agggacctgg cctgaccacc accgagtcta gacagcagcc aatggccctg 3780

gccgtggccc tgaccaaagg cggagaagct aggggcgagc tgttctggga cgatggcgag 3840

agcctggaag tgctggaaag aggcgcctat acccaagtga tcttcctggc ccggaacaac 3900

accatcgtga acgagctggt gcgcgtgacc tctgaaggcg ctggactgca gctgcagaaa 3960

gtgaccgtgc tgggagtggc cacagcccct cagcaggtgc tgtctaatgg cgtgcccgtg 4020

tccaacttca cctacagccc cgacaccaag gtgctggaca tctgcgtgtc actgctgatg 4080

ggagagcagt ttctggtgtc ctggtgctga ctcgagagat ctaccggtga attcaccgcg 4140

ggtttaaact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 4200

cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 4260

gcattgtctg agtaggtgtc attctattct ggggggtggg gtgggggcta gctctaga 4318

<210> 24

<211> 4318

<212> DNA

<213> artificial

<220>

<223> construct: sp2+hGAAco2-delta-8

<400> 24

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctggggc ccatgccacc 240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300

ggtttaggta gtgtgagagg ggtacccggg gatcttgcta ccagtggaac agccactaag 360

gattctgcag tgagagcaga gggccagcta agtggtactc tcccagagac tgtctgactc 420

acgccacccc ctccaccttg gacacaggac gctgtggttt ctgagccagg tacaatgact 480

cctttcggta agtgcagtgg aagctgtaca ctgcccaggc aaagcgtccg ggcagcgtag 540

gcgggcgact cagatcccag ccagtggact tagcccctgt ttgctcctcc gataactggg 600

660

aatacggacg aggacagggc cctgtctcct cagcttcagg caccaccact gacctgggac 720

agtgaataga tcctgagaac ttcagggtga gtctatggga cccttgatgt tttctttccc 780

cttcttttct atggttaagt tcatgtcata ggaaggggag aagtaacagg gtacacatat 840

tgaccaaatc agggtaattt tgcatttgta attttaaaaa atgctttctt cttttaatat 900

acttttttgt ttatcttatt tctaatactt tccctaatct ctttctttca gggcaataat 960

gatacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata atttctgggt 1020

taaggcaata gcaatatttc tgcatataaa tatttctgca tataaattgt aactgatgta 1080

1140

gttgggataa ggctggatta ttctgagtcc aagctaggcc cttttgctaa tcttgttcat 1200

acctcttatc ttcctcccac agctcctggg caacctgctg gtctctctgc tggcccatca 1260

ctttggcaaa gcacgcgtgc caccatgcca tcgtcagtgt cttggggcat tcttctgctc 1320

gccggattgt gttgcctggt gcctgtctca ttggccctgt tggtgcctag agagctgagc 1380

ggatcatccc cagtgctgga ggagactcat cctgctcacc aacagggagc ttccagacca 1440

ggaccgagag acgcccaagc ccatcctggt agaccaagag ctgtgcctac ccaatgcgac 1500

1560

gaagccagag gttgctgcta catcccagcg aagcaaggat tgcaaggcgc ccaaatggga 1620

caaccttggt gtttcttccc cccttcgtac ccatcatata aactcgaaaa cctgtcctct 1680

tcggaaatgg gttatactgc caccctcacc agaactactc ctactttctt cccgaaagac 1740

atcttgacct tgaggctgga cgtgatgatg gagactgaaa accggctgca tttcactatc 1800

aaagatcctg ccaatcggcg atacgaggtc cctctggaaa cccctcacgt gcactcacgg 1860

gctccttctc cgctttactc cgtcgaattc tctgaggaac ccttcggagt gatcgttaga 1920

cgccagctgg atggtagagt gctgttgaac actactgtgg ccccactttt cttcgctgac 1980

cagtttctgc aactgtccac ttccctgcca tcccagtaca ttactggact cgccgaacac 2040

ctgtcgccac tgatgctctc gacctcttgg actagaatca ctttgtggaa cagagacttg 2100

gcccctactc cgggagcaaa tctgtacgga agccaccctt tttacctggc gctcgaagat 2160

ggcggatccg ctcacggagt gttcctgctg aatagcaacg caatggacgt ggtgctgcaa 2220

ccttcccctg cactcagttg gagaagtacc gggggtattc tggacgtgta catcttcctc 2280

ggaccagaac ccaagagcgt ggtgcagcaa tatctggacg tggtcggata cccttttatg 2340

cctccttact ggggactggg attccacctt tgccgttggg gctactcatc caccgccatt 2400

accagacagg tggtggagaa tatgaccaga gcccacttcc ctctcgacgt gcagtggaac 2460

gatctggact atatggactc ccggagagat ttcaccttca acaaggacgg gttccgcgat 2520

tttcccgcga tggttcaaga gctccaccag ggtggtcgaa gatatatgat gatcgtcgac 2580

ccagccattt cgagcagcgg acccgctggga tcttatagac cttacgacga aggccttagg 2640

agaggagtgt tcatcacaaa cgagactgga cagcctttga tcggtaaagt gtggcctgga 2700

tcaaccgcct ttcctgactt taccaatccc actgccttgg cttggtggga ggacatggtg 2760

gccgaattcc acgaccaagt cccctttgat ggaatgtgga tcgatatgaa cgaaccaagc 2820

2880

gtgcccggag tcgtgggcgg aacattacag gccgcgacta tttgcgccag cagccaccaa 2940

ttcctgtcca ctcactacaa cctccacaac ctttatggat taaccgaagc tattgcaagt 3000

3060

gccggacacg ggagatacgc cggtcactgg actggtgacg tgtggagctc atgggaacaa 3120

ctggcctcct ccgtgccgga aatcttacag ttcaaccttc tgggtgtccc tcttgtcgga 3180

3240

ctgggtgcat tctacccatt catgagaaac cacaactcct tgctgtccct gccacaagag 3300

ccctactcgt tcagcgagcc tgcacaacag gctatgcgga aggcactgac cctgagatac 3360

gccctgcttc cacacttata cactctcttc catcaagcgc atgtggcagg agaaaccgtt 3420

gcaaggcctc ttttccttga attccccaag gattcctcga cttggacggt ggatcatcag 3480

ctgctgtggg gagaagctct gctgattact ccagtgttgc aagccggaaa agctgaggtg 3540

accggatact ttccgctggg aacctggtac gacctccaga ctgtccctgt tgaagccctt 3600

ggatcactgc ctccgcctcc ggcagctcca cgcgaaccag ctatacattc cgagggacag 3660

3720

atccctctgc aaggaccagg actgactacg accgagagca gacagcagcc aatggcactg 3780

gctgtggctc tgaccaaggg aggggaagct agaggagaac tcttctggga tgatggggag 3840

tcccttgaag tgctggaaag aggcgcttac actcaagtca ttttccttgc acggaacaac 3900

accattgtga acgaattggt gcgagtgacc agcgaaggag ctggacttca actgcagaag 3960

gtcactgtgc tcggagtggc taccgctcct cagcaagtgc tgtcgaatgg agtccccgtg 4020

4080

ggagagcagt tccttgtgtc ctggtgttga ctcgagagat ctaccggtga attcaccgcg 4140

ggtttaaact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 4200

cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 4260

gcattgtctg agtaggtgtc attctattct ggggggtggg gtgggggcta gctctaga 4318

<210> 25

<211> 4300

<212> DNA

<213> artificial

<220>

<223> construct: sp7+hGAAco1-delta-8

<400> 25

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctggggc ccatgccacc 240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300

ggtttaggta gtgtgagagg ggtacccggg gatcttgcta ccagtggaac agccactaag 360

gattctgcag tgagagcaga gggccagcta agtggtactc tcccagagac tgtctgactc 420

acgccacccc ctccaccttg gacacaggac gctgtggttt ctgagccagg tacaatgact 480

cctttcggta agtgcagtgg aagctgtaca ctgcccaggc aaagcgtccg ggcagcgtag 540

gcgggcgact cagatcccag ccagtggact tagcccctgt ttgctcctcc gataactggg 600

660

aatacggacg aggacagggc cctgtctcct cagcttcagg caccaccact gacctgggac 720

agtgaataga tcctgagaac ttcagggtga gtctatggga cccttgatgt tttctttccc 780

cttcttttct atggttaagt tcatgtcata ggaaggggag aagtaacagg gtacacatat 840

tgaccaaatc agggtaattt tgcatttgta attttaaaaa atgctttctt cttttaatat 900

acttttttgt ttatcttatt tctaatactt tccctaatct ctttctttca gggcaataat 960

gatacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata atttctgggt 1020

taaggcaata gcaatatttc tgcatataaa tatttctgca tataaattgt aactgatgta 1080

1140

gttgggataa ggctggatta ttctgagtcc aagctaggcc cttttgctaa tcttgttcat 1200

acctcttatc ttcctcccac agctcctggg caacctgctg gtctctctgc tggcccatca 1260

1320

ctgggcacca ccttcggcct actagtgccc agagagctga gcggcagctc tcccgtgctg 1380

gaagaaacac accctgccca tcagcagggc gcctctagac ctggacctag agatgcccag 1440

gcccaccccg gcagacctag agctgtgcct acccagtgtg acgtgccccc caacagcaga 1500

ttcgactgcg cccctgacaa ggccatcacc caggaacagt gcgaggccag aggctgctgc 1560

tacatccctg ccaagcaggg actgcaggggc gctcagatgg gacagccctg gtgcttcttc 1620

ccaccctcct accccagcta caagctggaa aacctgagca gcagcgagat gggctacacc 1680

gccaccctga ccagaaccac ccccacattc ttcccaaagg acatcctgac cctgcggctg 1740

gacgtgatga tggaaaccga gaaccggctg cacttcacca tcaaggaccc cgccaatcgg 1800

agatacgagg tgcccctgga aaccccccac gtgcactcta gagcccccag ccctctgtac 1860

agcgtggaat tcagcgagga acccttcggc gtgatcgtgc ggagacagct ggatggcaga 1920

gtgctgctga acaccaccgt ggcccctctg ttcttcgccg accagttcct gcagctgagc 1980

accagcctgc ccagccagta catcacagga ctggccgagc acctgagccc cctgatgctg 2040

agcacatcct ggacccggat caccctgtgg aacagggatc tggcccctac ccctggcgcc 2100

aatctgtacg gcagccaccc tttctacctg gccctggaag atggcggatc tgccccggga 2160

gtgtttctgc tgaactccaa cgccatggac gtggtgctgc agcctagccc tgccctgtct 2220

tggagaagca caggcggcat cctggatgtg tacatctttc tgggccccga gcccaagagc 2280

gtggtgcagc agtatctgga tgtcgtgggc taccccttca tgccccctta ctggggcctg 2340

ggattccacc tgtgcagatg gggctactcc agcaccgcca tcaccagaca ggtggtggaa 2400

aacatgacca gagcccactt cccactggat gtgcagtgga acgacctgga ctacatggac 2460

agcagacggg acttcacctt caacaaggac ggcttccggg acttccccgc catggtgcag 2520

gaactgcatc agggcggcag acggtacatg atgatcgtgg atcccgccat cagctcctct 2580

ggccctgccg gctcttacag accctacgac gagggcctgc ggagaggcgt gttcatcacc 2640

aacgagacag gccagcccct gatcggcaaa gtgtggcctg gcagcacagc cttccccgac 2700

ttcaccaatc ctaccgccct ggcttggtgg gaggacatgg tggccgagtt ccacgaccag 2760

gtgcccttcg acggcatgtg gatcgacatg aacgagccca gcaacttcat ccggggcagc 2820

gaggatggct gccccaacaa cgaactggaa aatccccctt acgtgcccgg cgtcgtggggc 2880

ggaacactgc aggccgctac aatctgtgcc agcagccacc agtttctgag cacccactac 2940

aacctgcaca acctgtacgg cctgaccgag gccattgcca gccaccgcgc tctcgtgaaa 3000

gccagaggca cacggccctt cgtgatcagc agaagcacct ttgccggcca cggcagatac 3060

gccggacatt ggactggcga cgtgtggtcc tcttgggagc agctggcctc tagcgtgccc 3120

gagatcctgc agttcaatct gctgggcgtg ccactcgtgg gcgccgatgt gtgtggcttc 3180

ctgggcaaca cctccgagga actgtgtgtg cggtggacac agctgggcgc cttctaccct 3240

ttcatgagaa accacaacag ctgctgagc ctgccccagg aaccctacag ctttagcgag 3300

ccctgcacagc aggccatgcg gaaggccctg acactgagat acgctctgct gccccacctg 3360

3420

gaattcccca aggacagcag cacctggacc gtggaccatc agctgctgtg gggagaggct 3480

ctgctgatta ccccagtgct gcaggcaggc aaggccgaag tgaccggcta ctttcccctg 3540

ggcacttggt acgacctgca gaccgtgcct gtggaagccc tgggatctct gcctccacct 3600

cctgccgctc ctagagagcc tgccattcac tctgaggggcc agtgggtcac actgcctgcc 3660

cccctggata ccatcaacgt gcacctgagg gccggctaca tcataccact gcagggacct 3720

ggcctgacca ccaccgagtc tagacagcag ccaatggccc tggccgtggc cctgaccaaa 3780

ggcggagaag ctaggggcga gctgttctgg gacgatggcg agagcctgga agtgctggaa 3840

agaggcgcct atacccaagt gatcttcctg gccgggaaca acaccatcgt gaacgagctg 3900

gtgcgcgtga cctctgaagg cgctggactg cagctgcaga aagtgaccgt gctgggagtg 3960

gccacagccc ctcagcaggt gctgtctaat ggcgtgcccg tgtccaactt cacctacagc 4020

cccgacacca aggtgctgga catctgcgtg tcactgctga tgggagagca gtttctggtg 4080

tcctggtgct gactcgagag atctaccggt gaattcaccg cgggtttaaa ctgtgccttc 4140

tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc 4200

cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg 4260

tcattctatt ctggggggtg gggtggggggc tagctctaga 4300

<210> 26

<211> 4198

<212> DNA

<213> artificial

<220>

<223> sp7+hGAAco1-delta-42

<400> 26

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctggggc ccatgccacc 240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300

ggtttaggta gtgtgagagg ggtacccggg gatcttgcta ccagtggaac agccactaag 360

gattctgcag tgagagcaga gggccagcta agtggtactc tcccagagac tgtctgactc 420

acgccacccc ctccaccttg gacacaggac gctgtggttt ctgagccagg tacaatgact 480

cctttcggta agtgcagtgg aagctgtaca ctgcccaggc aaagcgtccg ggcagcgtag 540

gcgggcgact cagatcccag ccagtggact tagcccctgt ttgctcctcc gataactggg 600

660

aatacggacg aggacagggc cctgtctcct cagcttcagg caccaccact gacctgggac 720

agtgaataga tcctgagaac ttcagggtga gtctatggga cccttgatgt tttctttccc 780

cttcttttct atggttaagt tcatgtcata ggaaggggag aagtaacagg gtacacatat 840

tgaccaaatc agggtaattt tgcatttgta attttaaaaa atgctttctt cttttaatat 900

acttttttgt ttatcttatt tctaatactt tccctaatct ctttctttca gggcaataat 960

gatacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata atttctgggt 1020

taaggcaata gcaatatttc tgcatataaa tatttctgca tataaattgt aactgatgta 1080

1140

gttgggataa ggctggatta ttctgagtcc aagctaggcc cttttgctaa tcttgttcat 1200

acctcttatc ttcctcccac agctcctggg caacctgctg gtctctctgc tggcccatca 1260

1320

ctgggcacca ccttcggcgc ccaccccggc agacctagag ctgtgcctac ccagtgtgac 1380

gtgcccccca acagcagatt cgactgcgcc cctgacaagg ccatcaccca ggaacagtgc 1440

gaggccagag gctgctgcta catccctgcc aagcagggac tgcagggcgc tcagatggga 1500

cagccctggt gcttcttccc accctcctac cccagctaca agctggaaaa cctgagcagc 1560

agcgagatgg gctacaccgc caccctgacc agaaccaccc ccacattctt cccaaaggac 1620

atcctgaccc tgcggctgga cgtgatgatg gaaaccgaga accggctgca cttcaccatc 1680

aaggaccccg ccaatcggag atacgaggtg cccctggaaa cccccccgt gcactctaga 1740

gcccccagcc ctctgtacag cgtggaattc agcgaggaac ccttcggcgt gatcgtgcgg 1800

agacagctgg atggcagagt gctgctgaac accaccgtgg cccctctgtt cttcgccgac 1860

cagttcctgc agctgagcac cagcctgccc agccagtaca tcacaggact ggccgagcac 1920

ctgagcccccc tgatgctgag cacatcctgg acccggatca ccctgtggaa cagggatctg 1980

gcccctaccc ctggcgccaa tctgtacggc agccaccctt tctacctggc cctggaagat 2040

ggcggatctg cccacggagt gtttctgctg aactccaacg ccatggacgt ggtgctgcag 2100

cctagccctg ccctgtcttg gagaagcaca ggcggcatcc tggatgtgta catctttctg 2160

ggccccgagc ccaagagcgt ggtgcagcag tatctggatg tcgtgggcta ccccttcatg 2220

cccccttact ggggcctggg attccacctg tgcagatggg gctactccag caccgccatc 2280

accagacagg tggtggaaaa catgaccaga gcccacttcc cactggatgt gcagtggaac 2340

gacctggact acatggacag cagacgggac ttcaccttca acaaggacgg cttccgggac 2400

ttccccgcca tggtgcagga actgcatcag ggcggcagac ggtacatgat gatcgtggat 2460

cccgccatca gctcctctgg ccctgccggc tcttacagac cctacgacga gggcctgcgg 2520

agaggcgtgt tcatcaccaa cgagacaggc cagcccctga tcggcaaagt gtggcctggc 2580

agcacagcct tccccgactt caccaatcct accgccctgg cttggtggga ggacatggtg 2640

gccgagttcc acgaccaggt gcccttcgac ggcatgtgga tcgacatgaa cgagcccagc 2700

aacttcatcc ggggcagcga ggatggctgc cccaacaacg aactggaaaa tcccccttac 2760

gtgcccggcg tcgtgggcgg aacactgcag gccgctacaa tctgtgccag cagccaccag 2820

tttctgagca cccactacaa cctgcacaac ctgtacggcc tgaccgaggc cattgccagc 2880

caccgcgctc tcgtgaaagc cagaggcaca cggcccttcg tgatcagcag aagcaccttt 2940

gccggccacg gcagatacgc cggacattgg actggcgacg tgtggtcctc ttgggagcag 3000

ctggcctcta gcgtgcccga gatcctgcag ttcaatctgc tgggcgtgcc actcgtgggc 3060

gccgatgtgt gtggcttcct gggcaacacc tccgaggaac tgtgtgtgcg gtggacacag 3120

ctgggcgcct tctacccttt catgagaaac cacaacagcc tgctgagcct gccccaggaa 3180

ccctacagct ttagcgagcc tgcacagcag gccatgcgga aggccctgac actgagatac 3240

gctctgctgc cccacctgta caccctgttt caccaggccc atgtggccgg cgagacagtg 3300

gccagacctc tgtttctgga attccccaag gacagcagca cctggaccgt ggaccatcag 3360

ctgctgtggg gagaggctct gctgattacc ccagtgctgc aggcaggcaa ggccgaagtg 3420

accggctact ttcccctggg cacttggtac gacctgcaga ccgtgcctgt ggaagccctg 3480

ggatctctgc ctccacctcc tgccgctcct agagagcctg ccattcactc tgagggccag 3540

tgggtcacac tgcctgcccc cctggatacc atcaacgtgc acctgagggc cggctacatc 3600

ataccactgc agggacctgg cctgaccacc accgagtcta gacagcagcc aatggccctg 3660

gccgtggccc tgaccaaagg cggagaagct aggggcgagc tgttctggga cgatggcgag 3720

agcctggaag tgctggaaag aggcgcctat acccaagtga tcttcctggc ccggaacaac 3780

accatcgtga acgagctggt gcgcgtgacc tctgaaggcg ctggactgca gctgcagaaa 3840

gtgaccgtgc tgggagtggc cacagcccct cagcaggtgc tgtctaatgg cgtgcccgtg 3900

tccaacttca cctacagccc cgacaccaag gtgctggaca tctgcgtgtc actgctgatg 3960

ggagagcagt ttctggtgtc ctggtgctga ctcgagagat ctaccggtga attcaccgcg 4020

ggtttaaact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 4080

cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 4140

gcattgtctg agtaggtgtc attctattct ggggggtggg gtgggggcta gctctaga 4198

<210> 27

<211> 917

<212> Protein

<213> artificial

<220>

<223> hGAA-delta-8

<400> 27

Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val Leu Glu Glu

1 5 10 15

Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly Pro Arg Asp

20 25 30

Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr Gln Cys Asp

35 40 45

Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr

50 55 60

Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro Ala Lys Gln

65 70 75 80

Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe Phe Pro Pro

85 90 95

Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser Glu Met Gly

100 105 110

Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp

115 120 125

Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu Asn Arg Leu

130 135 140

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

145 150 155 160

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

165 170 175

Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val Arg Arg Gln Leu Asp

180 185 190

Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe Phe Ala Asp

195 200 205

Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr Ile Thr Gly

210 215 220

Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser Trp Thr Arg

225 230 235 240

Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly Ala Asn Leu

245 250 255

Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser Ala

260 265 270

His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val Val Leu Gln

275 280 285

Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile Leu Asp Val

290 295 300

Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln Gln Tyr Leu

305 310 315 320

Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe

325 330 335

His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr Arg Gln Val

340 345 350

Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val Gln Trp Asn

355 360 365

Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp

370 375 380

Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His Gln Gly Gly

385 390 395 400

Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser Ser Gly Pro

405 410 415

Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg Gly Val Phe

420 425 430

Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val Trp Pro Gly

435 440 445

Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp

450 455 460

Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe Asp Gly Met

465 470 475 480

Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly Ser Glu Asp

485 490 495

Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val Pro Gly Val

500 505 510

Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser Ser His Gln

515 520 525

Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu

530 535 540

Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly Thr Arg Pro

545 550 555 560

Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly

565 570 575

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

580 585 590

Val Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro Leu Val Gly

595 600 605

Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu Glu Leu Cys Val

610 615 620

Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn

625 630 635 640

Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser Glu Pro Ala

645 650 655

Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro

660 665 670

His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly Glu Thr Val

675 680 685

Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr

690 695 700

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

705 710 715 720

Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly Thr

725 730 735

Trp Tyr Asp Leu Gln Thr Val Pro Val Glu Ala Leu Gly Ser Leu Pro

740 745 750

Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser Glu Gly Gln

755 760 765

Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val His Leu Arg

770 775 780

Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr Thr Thr Glu

785 790 795 800

Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr Lys Gly Gly

805 810 815

Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Val

820 825 830

Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala Arg Asn Asn

835 840 845

Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly Ala Gly Leu

850 855 860

Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala Pro Gln Gln

865 870 875 880

Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp

885 890 895

Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly Glu Gln Phe

900 905 910

Leu Val Ser Trp Cys

915

<210> 28

<211> 883

<212> Protein

<213> artificial

<220>

<223> hGAA-delta-42

<400> 28

Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr Gln Cys Asp Val Pro

1 5 10 15

Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr Gln Glu

20 25 30

Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro Ala Lys Gln Gly Leu

35 40 45

Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe Phe Pro Pro Ser Tyr

50 55 60

Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr

65 70 75 80

Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp Ile Leu

85 90 95

Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu Asn Arg Leu His Phe

100 105 110

Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr

115 120 125

Pro His Val His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe

130 135 140

Ser Glu Glu Pro Phe Gly Val Ile Val Arg Arg Gln Leu Asp Gly Arg

145 150 155 160

Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe Phe Ala Asp Gln Phe

165 170 175

Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr Ile Thr Gly Leu Ala

180 185 190

Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser Trp Thr Arg Ile Thr

195 200 205

Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly

210 215 220

Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly

225 230 235 240

Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val Val Leu Gln Pro Ser

245 250 255

Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile

260 265 270

Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln Gln Tyr Leu Asp Val

275 280 285

Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe His Leu

290 295 300

Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr Arg Gln Val Val Glu

305 310 315 320

Asn Met Thr Arg Ala His Phe Pro Leu Asp Val Gln Trp Asn Asp Leu

325 330 335

Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe

340 345 350

Arg Asp Phe Pro Ala Met Val Gln Glu Leu His Gln Gly Gly Arg Arg

355 360 365

Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly

370 375 380

Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg Gly Val Phe Ile Thr

385 390 395 400

Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val Trp Pro Gly Ser Thr

405 410 415

Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp

420 425 430

Met Val Ala Glu Phe His Asp Gln Val Pro Phe Asp Gly Met Trp Ile

435 440 445

Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly Ser Glu Asp Gly Cys

450 455 460

Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val Pro Gly Val Val Gly

465 470 475 480

Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser Ser His Gln Phe Leu

485 490 495

Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu Ala Ile

500 505 510

Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly Thr Arg Pro Phe Val

515 520 525

Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp

530 535 540

Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu Ala Ser Ser Val Pro

545 550 555 560

Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro Leu Val Gly Ala Asp

565 570 575

Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu Leu Cys Val Arg Trp

580 585 590

Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn Ser Leu

595 600 605

Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln

610 615 620

Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro His Leu

625 630 635 640

Tyr Thr Leu Phe His Gln Ala His Val Ala Gly Glu Thr Val Ala Arg

645 650 655

Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr Val Asp

660 665 670

His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile Thr Pro Val Leu Gln

675 680 685

Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr

690 695 700

Asp Leu Gln Thr Val Pro Val Glu Ala Leu Gly Ser Leu Pro Pro Pro

705 710 715 720

Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser Glu Gly Gln Trp Val

725 730 735

Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val His Leu Arg Ala Gly

740 745 750

Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg

755 760 765

Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr Lys Gly Gly Glu Ala

770 775 780

Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Val Leu Glu

785 790 795 800

Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala Arg Asn Asn Thr Ile

805 810 815

Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly Ala Gly Leu Gln Leu

820 825 830

Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala Pro Gln Gln Val Leu

835 840 845

Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys

850 855 860

Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly Glu Gln Phe Leu Val

865 870 875 880

Ser Trp Cys

<210> 29

<211> 952

<212> Protein

<213> homo sapiens

<400> 29

Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys

1 5 10 15

Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu

20 25 30

His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val

35 40 45

Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly

50 55 60

Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr

65 70 75 80

Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys

85 90 95

Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro

100 105 110

Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe

115 120 125

Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser

130 135 140

Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe

145 150 155 160

Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu

165 170 175

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

180 185 190

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

195 200 205

Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val Arg Arg

210 215 220

Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe

225 230 235 240

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

245 250 255

Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser

260 265 270

Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly

275 280 285

Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly

290 295 300

Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val

305 310 315 320

Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile

325 330 335

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

340 345 350

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

355 360 365

Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr

370 375 380

Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val

385 390 395 400

Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe

405 410 415

Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His

420 425 430

Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser

435 440 445

Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg

450 455 460

Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val

465 470 475 480

Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu

485 490 495

Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe

500 505 510

Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly

515 520 525

Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val

530 535 540

Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser

545 550 555 560

Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu

645 650 655

Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg

660 665 670

Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser

675 680 685

Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala

690 695 700

Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly

705 710 715 720

Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser

725 730 735

Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile

740 745 750

Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro

755 760 765

Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly

770 775 780

Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser

785 790 795 800

Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val

805 810 815

His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr

820 825 830

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

835 840 845

Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser

850 855 860

Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala

865 870 875 880

Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly

885 890 895

Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala

900 905 910

Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr

915 920 925

Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly

930 935 940

Glu Gln Phe Leu Val Ser Trp Cys

945 950

<210> 30

<211> 952

<212> Protein

<213> homo sapiens

<400> 30

Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys

1 5 10 15

Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu

20 25 30

His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val

35 40 45

Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly

50 55 60

Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr

65 70 75 80

Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys

85 90 95

Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro

100 105 110

Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe

115 120 125

Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser

130 135 140

Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe

145 150 155 160

Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu

165 170 175

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

180 185 190

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

195 200 205

Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val His Arg

210 215 220

Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe

225 230 235 240

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

245 250 255

Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser

260 265 270

Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly

275 280 285

Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly

290 295 300

Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val

305 310 315 320

Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile

325 330 335

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

340 345 350

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

355 360 365

Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr

370 375 380

Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val

385 390 395 400

Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe

405 410 415

Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His

420 425 430

Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser

435 440 445

Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg

450 455 460

Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val

465 470 475 480

Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu

485 490 495

Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe

500 505 510

Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly

515 520 525

Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val

530 535 540

Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser

545 550 555 560

Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu

645 650 655

Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg

660 665 670

Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser

675 680 685

Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala

690 695 700

Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly

705 710 715 720

Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser

725 730 735

Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile

740 745 750

Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro

755 760 765

Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly

770 775 780

Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser

785 790 795 800

Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val

805 810 815

His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr

820 825 830

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

835 840 845

Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser

850 855 860

Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala

865 870 875 880

Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly

885 890 895

Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala

900 905 910

Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr

915 920 925

Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly

930 935 940

Glu Gln Phe Leu Val Ser Trp Cys

945 950

<210> 31

<211> 957

<212> Protein

<213> homo sapiens

<400> 31

Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys

1 5 10 15

Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu

20 25 30

His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val

35 40 45

Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly

50 55 60

Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr

65 70 75 80

Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys

85 90 95

Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro

100 105 110

Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe

115 120 125

Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser

130 135 140

Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe

145 150 155 160

Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu

165 170 175

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

180 185 190

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

195 200 205

Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val Arg Arg

210 215 220

Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe

225 230 235 240

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

245 250 255

Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser

260 265 270

Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly

275 280 285

Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly

290 295 300

Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val

305 310 315 320

Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile

325 330 335

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

340 345 350

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

355 360 365

Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr

370 375 380

Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val

385 390 395 400

Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe

405 410 415

Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His

420 425 430

Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser

435 440 445

Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg

450 455 460

Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val

465 470 475 480

Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu

485 490 495

Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe

500 505 510

Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly

515 520 525

Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val

530 535 540

Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser

545 550 555 560

Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu

645 650 655

Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg

660 665 670

Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser

675 680 685

Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala

690 695 700

Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly

705 710 715 720

Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser

725 730 735

Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile

740 745 750

Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro

755 760 765

Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly

770 775 780

Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser

785 790 795 800

Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val

805 810 815

His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr

820 825 830

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

835 840 845

Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser

850 855 860

Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala

865 870 875 880

Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly

885 890 895

Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala

900 905 910

Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr

915 920 925

Ser Pro Asp Thr Lys Ala Arg Gly Pro Arg Val Leu Asp Ile Cys Val

930 935 940

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

945 950 955

<210> 32

<211> 952

<212> Protein

<213> homo sapiens

<400> 32

Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys

1 5 10 15

Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu

20 25 30

His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val

35 40 45

Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly

50 55 60

Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr

65 70 75 80

Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys

85 90 95

Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro

100 105 110

Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe

115 120 125

Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser

130 135 140

Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe

145 150 155 160

Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu

165 170 175

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

180 185 190

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

195 200 205

Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val His Arg

210 215 220

Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe

225 230 235 240

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

245 250 255

Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser

260 265 270

Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly

275 280 285

Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly

290 295 300

Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val

305 310 315 320

Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile

325 330 335

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

340 345 350

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

355 360 365

Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr

370 375 380

Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val

385 390 395 400

Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe

405 410 415

Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His

420 425 430

Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser

435 440 445

Ser Gly Pro Ala Gly Ser Tyr Arg Leu Tyr Asp Glu Gly Leu Arg Arg

450 455 460

Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val

465 470 475 480

Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu

485 490 495

Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe

500 505 510

Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly

515 520 525

Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val

530 535 540

Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser

545 550 555 560

Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu

645 650 655

Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg

660 665 670

Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser

675 680 685

Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala

690 695 700

Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly

705 710 715 720

Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser

725 730 735

Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile

740 745 750

Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro

755 760 765

Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly

770 775 780

Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser

785 790 795 800

Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val

805 810 815

His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr

820 825 830

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

835 840 845

Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser

850 855 860

Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala

865 870 875 880

Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly

885 890 895

Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala

900 905 910

Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr

915 920 925

Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly

930 935 940

Glu Gln Phe Leu Val Ser Trp Cys

945 950

<210> 33

<211> 925

<212> Protein

<213> artificial

<220>

<223> hGAAwt variant without sp

<400> 33

Gly His Ile Leu Leu His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser

1 5 10 15

Gly Ser Ser Pro Val Leu Glu Glu Thr His Pro Ala His Gln Gln Gly

20 25 30

Ala Ser Arg Pro Gly Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gln Pro Trp Cys Phe Phe Pro Ser Tyr Pro Ser Tyr Lys Leu Glu

100 105 110

Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr

115 120 125

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

130 135 140

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

145 150 155 160

Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr Pro Arg Val His Ser Arg

165 170 175

Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly

180 185 190

Val Ile Val His Arg Gln Leu Asp Gly Arg Val Leu Leu Asn Thr

195 200 205

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

210 215 220

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

225 230 235 240

Met Leu Ser Thr Ser Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu

245 250 255

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

260 265 270

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

275 280 285

Asn Ala Met Asp Val Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg

290 295 300

Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro

305 310 315 320

Lys Ser Val Val Gln Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met

325 330 335

Pro Pro Tyr Trp Gly Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser

340 345 350

Ser Thr Ala Ile Thr Arg Gln Val Val Glu Asn Met Thr Arg Ala His

355 360 365

Phe Pro Leu Asp Val Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg

370 375 380

Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met

385 390 395 400

Val Gln Glu Leu His Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp

405 410 415

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

420 425 430

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

435 440 445

Leu Ile Gly Lys Val Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr

450 455 460

Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp Met Val Ala Glu Phe His

465 470 475 480

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

485 490 495

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

500 505 510

Asn Pro Pro Tyr Val Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala

515 520 525

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

530 535 540

His Asn Leu Tyr Gly Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

Leu Leu Gly Val Pro Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly

610 615 620

Asn Thr Ser Glu Glu Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe

625 630 635 640

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

645 650 655

Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu

660 665 670

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

675 680 685

Ala His Val Ala Gly Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe

690 695 700

Pro Lys Asp Ser Ser Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly

705 710 715 720

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

725 730 735

Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro

740 745 750

Ile Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu

755 760 765

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

770 775 780

Asp Thr Ile Asn Val His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln

785 790 795 800

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

805 810 815

Ala Val Ala Leu Thr Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp

820 825 830

Asp Asp Gly Glu Ser Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

<210> 34

<211> 896

<212> Protein

<213> artificial

<220>

<223> hGAA-delta-29

<400> 34

Gln Gln Gly Ala Ser Arg Pro Gly Pro Arg Asp Ala Gln Ala His Pro

1 5 10 15

Gly Arg Pro Arg Ala Val Pro Thr Gln Cys Asp Val Pro Pro Asn Ser

20 25 30

Arg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr Gln Glu Gln Cys Glu

35 40 45

Ala Arg Gly Cys Cys Tyr Ile Pro Ala Lys Gln Gly Leu Gln Gly Ala

50 55 60

Gln Met Gly Gln Pro Trp Cys Phe Phe Pro Pro Ser Tyr Pro Ser Tyr

65 70 75 80

Lys Leu Glu Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu

85 90 95

Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp Ile Leu Thr Leu Arg

100 105 110

Leu Asp Val Met Met Glu Thr Glu Asn Arg Leu His Phe Thr Ile Lys

115 120 125

Asp Pro Ala Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr Pro His Val

130 135 140

His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe Ser Glu Glu

145 150 155 160

Pro Phe Gly Val Ile Val Arg Arg Gln Leu Asp Gly Arg Val Leu Leu

165 170 175

Asn Thr Thr Val Ala Pro Leu Phe Phe Ala Asp Gln Phe Leu Gln Leu

180 185 190

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

195 200 205

Ser Pro Leu Met Leu Ser Thr Ser Trp Thr Arg Ile Thr Leu Trp Asn

210 215 220

Arg Asp Leu Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly Ser His Pro

225 230 235 240

Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly Val Phe Leu

245 250 255

Leu Asn Ser Asn Ala Met Asp Val Val Leu Gln Pro Ser Pro Ala Leu

260 265 270

Ser Trp Arg Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile Phe Leu Gly

275 280 285

Pro Glu Pro Lys Ser Val Val Gln Gln Tyr Leu Asp Val Val Gly Tyr

290 295 300

Pro Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe His Leu Cys Arg Trp

305 310 315 320

Gly Tyr Ser Ser Thr Ala Ile Thr Arg Gln Val Val Glu Asn Met Thr

325 330 335

Arg Ala His Phe Pro Leu Asp Val Gln Trp Asn Asp Leu Asp Tyr Met

340 345 350

Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg Asp Phe

355 360 365

Pro Ala Met Val Gln Glu Leu His Gln Gly Gly Arg Arg Tyr Met Met

370 375 380

Ile Val Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly Ser Tyr Arg

385 390 395 400

Pro Tyr Asp Glu Gly Leu Arg Arg Gly Val Phe Ile Thr Asn Glu Thr

405 410 415

Gly Gln Pro Leu Ile Gly Lys Val Trp Pro Gly Ser Thr Ala Phe Pro

420 425 430

Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp Met Val Ala

435 440 445

Glu Phe His Asp Gln Val Pro Phe Asp Gly Met Trp Ile Asp Met Asn

450 455 460

Glu Pro Ser Asn Phe Ile Arg Gly Ser Glu Asp Gly Cys Pro Asn Asn

465 470 475 480

Glu Leu Glu Asn Pro Pro Tyr Val Pro Gly Val Val Gly Gly Thr Leu

485 490 495

Gln Ala Ala Thr Ile Cys Ala Ser Ser His Gln Phe Leu Ser Thr His

500 505 510

Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu Ala Ile Ala Ser His

515 520 525

Arg Ala Leu Val Lys Ala Arg Gly Thr Arg Pro Phe Val Ile Ser Arg

530 535 540

Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp Thr Gly Asp

545 550 555 560

Val Trp Ser Ser Trp Glu Gln Leu Ala Ser Ser Val Pro Glu Ile Leu

565 570 575

Gln Phe Asn Leu Leu Gly Val Pro Leu Val Gly Ala Asp Val Cys Gly

580 585 590

Phe Leu Gly Asn Thr Ser Glu Glu Leu Cys Val Arg Trp Thr Gln Leu

595 600 605

Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn Ser Leu Leu Ser Leu

610 615 620

Pro Gln Glu Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln Ala Met Arg

625 630 635 640

Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro His Leu Tyr Thr Leu

645 650 655

Phe His Gln Ala His Val Ala Gly Glu Thr Val Ala Arg Pro Leu Phe

660 665 670

Leu Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr Val Asp His Gln Leu

675 680 685

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

690 695 700

Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu Gln

705 710 715 720

Thr Val Pro Val Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro Ala Ala

725 730 735

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

740 745 750

Ala Pro Leu Asp Thr Ile Asn Val His Leu Arg Ala Gly Tyr Ile Ile

755 760 765

Pro Leu Gln Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg Gln Gln Pro

770 775 780

Met Ala Leu Ala Val Ala Leu Thr Lys Gly Gly Glu Ala Arg Gly Glu

785 790 795 800

Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Val Leu Glu Arg Gly Ala

805 810 815

Tyr Thr Gln Val Ile Phe Leu Ala Arg Asn Asn Thr Ile Val Asn Glu

820 825 830

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

835 840 845

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

850 855 860

Val Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys Val Leu Asp

865 870 875 880

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

885 890 895

<210> 35

<211> 882

<212> Protein

<213> artificial

<220>

<223> hGAA-delta-43

<400> 35

His Pro Gly Arg Pro Arg Ala Val Pro Thr Gln Cys Asp Val Pro Pro

1 5 10 15

Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr Gln Glu Gln

20 25 30

Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro Ala Lys Gln Gly Leu Gln

35 40 45

Gly Ala Gln Met Gly Gln Pro Trp Cys Phe Phe Pro Pro Ser Tyr Pro

50 55 60

Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr Ala

65 70 75 80

Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp Ile Leu Thr

85 90 95

Leu Arg Leu Asp Val Met Met Glu Thr Glu Asn Arg Leu His Phe Thr

100 105 110

Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr Pro

115 120 125

His Val His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe Ser

130 135 140

Glu Glu Pro Phe Gly Val Ile Val Arg Arg Gln Leu Asp Gly Arg Val

145 150 155 160

Leu Leu Asn Thr Thr Val Ala Pro Leu Phe Phe Ala Asp Gln Phe Leu

165 170 175

Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr Ile Thr Gly Leu Ala Glu

180 185 190

His Leu Ser Pro Leu Met Leu Ser Thr Ser Trp Thr Arg Ile Thr Leu

195 200 205

Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly Ser

210 215 220

His Pro Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly Val

225 230 235 240

Phe Leu Leu Asn Ser Asn Ala Met Asp Val Val Leu Gln Pro Ser Pro

245 250 255

Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile Phe

260 265 270

Leu Gly Pro Glu Pro Lys Ser Val Val Gln Gln Tyr Leu Asp Val Val

275 280 285

Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe His Leu Cys

290 295 300

Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr Arg Gln Val Val Glu Asn

305 310 315 320

Met Thr Arg Ala His Phe Pro Leu Asp Val Gln Trp Asn Asp Leu Asp

325 330 335

Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg

340 345 350

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

355 360 365

Met Met Ile Val Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly Ser

370 375 380

Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg Gly Val Phe Ile Thr Asn

385 390 395 400

Glu Thr Gly Gln Pro Leu Ile Gly Lys Val Trp Pro Gly Ser Thr Ala

405 410 415

Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp Met

420 425 430

Val Ala Glu Phe His Asp Gln Val Pro Phe Asp Gly Met Trp Ile Asp

435 440 445

Met Asn Glu Pro Ser Asn Phe Ile Arg Gly Ser Glu Asp Gly Cys Pro

450 455 460

Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val Pro Gly Val Val Gly Gly

465 470 475 480

Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser Ser His Gln Phe Leu Ser

485 490 495

Thr His Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu Ala Ile Ala

500 505 510

Ser His Arg Ala Leu Val Lys Ala Arg Gly Thr Arg Pro Phe Val Ile

515 520 525

Ser Arg Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp Thr

530 535 540

Gly Asp Val Trp Ser Ser Trp Glu Gln Leu Ala Ser Ser Val Pro Glu

545 550 555 560

Ile Leu Gln Phe Asn Leu Leu Gly Val Pro Leu Val Gly Ala Asp Val

565 570 575

Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu Leu Cys Val Arg Trp Thr

580 585 590

Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn Ser Leu Leu

595 600 605

Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln Ala

610 615 620

Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro His Leu Tyr

625 630 635 640

Thr Leu Phe His Gln Ala His Val Ala Gly Glu Thr Val Ala Arg Pro

645 650 655

Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr Val Asp His

660 665 670

Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile Thr Pro Val Leu Gln Ala

675 680 685

Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp

690 695 700

Leu Gln Thr Val Pro Val Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro

705 710 715 720

Ala Ala Pro Arg Glu Pro Ala Ile His Ser Glu Gly Gln Trp Val Thr

725 730 735

Leu Pro Ala Pro Leu Asp Thr Ile Asn Val His Leu Arg Ala Gly Tyr

740 745 750

Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg Gln

755 760 765

Gln Pro Met Ala Leu Ala Val Ala Leu Thr Lys Gly Gly Glu Ala Arg

770 775 780

Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Val Leu Glu Arg

785 790 795 800

Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala Arg Asn Asn Thr Ile Val

805 810 815

Asn Glu Leu Val Arg Val Thr Ser Glu Gly Ala Gly Leu Gln Leu Gln

820 825 830

Lys Val Thr Val Leu Gly Val Ala Thr Ala Pro Gln Gln Val Leu Ser

835 840 845

Asn Gly Val Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys Val

850 855 860

Leu Asp Ile Cys Val Ser Leu Leu Met Gly Glu Gln Phe Leu Val Ser

865 870 875 880

Trp Cys

<210> 36

<211> 878

<212> Protein

<213> artificial

<220>

<223> hGAA-delta-47

<400> 36

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Gln Pro Trp Cys Phe Phe Pro Ser Tyr Pro Ser Tyr Lys Leu

50 55 60

Glu Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg

65 70 75 80

Thr Thr Pro Thr Phe Phe Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp

85 90 95

Val Met Met Glu Thr Glu Asn Arg Leu His Phe Thr Ile Lys Asp Pro

100 105 110

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

115 120 125

Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe

130 135 140

Gly Val Ile Val Arg Arg Gln Leu Asp Gly Arg Val Leu Leu Asn Thr

145 150 155 160

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

165 170 175

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

180 185 190

Leu Met Leu Ser Thr Ser Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp

195 200 205

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

210 215 220

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

225 230 235 240

Ser Asn Ala Met Asp Val Val Leu Gln Pro Ser Pro Ala Leu Ser Trp

245 250 255

Arg Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu

260 265 270

Pro Lys Ser Val Val Gln Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe

275 280 285

Met Pro Pro Tyr Trp Gly Leu Gly Phe His Leu Cys Arg Trp Gly Tyr

290 295 300

Ser Ser Thr Ala Ile Thr Arg Gln Val Val Glu Asn Met Thr Arg Ala

305 310 315 320

His Phe Pro Leu Asp Val Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser

325 330 335

Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala

340 345 350

Met Val Gln Glu Leu His Gln Gly Gly Arg Arg Tyr Met Met Ile Val

355 360 365

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

370 375 380

Asp Glu Gly Leu Arg Arg Gly Val Phe Ile Thr Asn Glu Thr Gly Gln

385 390 395 400

Pro Leu Ile Gly Lys Val Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe

405 410 415

Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp Met Val Ala Glu Phe

420 425 430

His Asp Gln Val Pro Phe Asp Gly Met Trp Ile Asp Met Asn Glu Pro

435 440 445

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

450 455 460

Glu Asn Pro Pro Tyr Val Pro Gly Val Val Gly Gly Thr Leu Gln Ala

465 470 475 480

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

485 490 495

Leu His Asn Leu Tyr Gly Leu Thr Glu Ala Ile Ala Ser His Arg Ala

500 505 510

Leu Val Lys Ala Arg Gly Thr Arg Pro Phe Val Ile Ser Arg Ser Thr

515 520 525

Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp Thr Gly Asp Val Trp

530 535 540

Ser Ser Trp Glu Gln Leu Ala Ser Ser Val Pro Glu Ile Leu Gln Phe

545 550 555 560

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

565 570 575

Gly Asn Thr Ser Glu Glu Leu Cys Val Arg Trp Thr Gln Leu Gly Ala

580 585 590

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

595 600 605

Glu Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln Ala Met Arg Lys Ala

610 615 620

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

625 630 635 640

Gln Ala His Val Ala Gly Glu Thr Val Ala Arg Pro Leu Phe Leu Glu

645 650 655

Phe Pro Lys Asp Ser Ser Thr Trp Thr Val Asp His Gln Leu Leu Trp

660 665 670

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

675 680 685

Val Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val

690 695 700

Pro Val Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg

705 710 715 720

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

725 730 735

Leu Asp Thr Ile Asn Val His Leu Arg Ala Gly Tyr Ile Ile Pro Leu

740 745 750

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

755 760 765

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

770 775 780

Trp Asp Asp Gly Glu Ser Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875

<210> 37

<211> 2745

<212> DNA

<213> artificial

<220>

<223> sp7+hGAAwt-delta-29

<400> 37

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggccagcag 60

ggagccagca gaccaggggcc ccgggatgcc caggcacacc ccgggcggcc gcgagcagtg 120

cccacacagt gcgacgtccc ccccaacagc cgcttcgatt gcgcccctga caaggccatc 180

acccaggaac agtgcgaggc ccgcggctgt tgctacatcc ctgcaaagca ggggctgcag 240

ggagcccaga tggggcagcc ctggtgcttc ttcccaccca gctaccccag ctacaagctg 300

gagaacctga gctcctctga aatgggctac acggccaccc tgacccgtac cacccccacc 360

ttcttcccca aggacatcct gaccctgcgg ctggacgtga tgatggagac tgagaaccgc 420

ctccacttca cgatcaaaga tccagctaac aggcgctacg aggtgccctt ggagaccccg 480

catgtccaca gccgggcacc gtccccactc tacagcgtgg agttctccga ggagcccttc 540

ggggtgatcg tgcgccggca gctggacggc cgcgtgctgc tgaacacgac ggtggcgccc 600

ctgttctttg cggaccagtt ccttcagctg tccacctcgc tgccctcgca gtatatcaca 660

ggcctcgccg agcacctcag tcccctgatg ctcagcacca gctggaccag gatcaccctg 720

tggaaccggg accttgcgcc cacgcccggt gcgaacctct acgggtctca ccctttctac 780

ctggcgctgg aggacggcgg gtcggcacac ggggtgttcc tgctaaacag caatgccatg 840

gatgtggtcc tgcagccgag ccctgccctt agctggaggt cgacaggtgg gatcctggat 900

gtctacatct tcctgggccc agagcccaag agcgtggtgc agcagtacct ggacgttgtg 960

ggatacccgt tcatgccgcc atactggggc ctgggcttcc acctgtgccg ctggggctac 1020

1080

1140

gatggcttcc gggacttccc ggccatggtg caggagctgc accagggcgg ccggcgctac 1200

atgatgatcg tggatcctgc catcagcagc tcgggccctg ccgggagcta caggccctac 1260

gacgagggtc tgcggagggg ggttttcatc accaacgaga ccggccagcc gctgattggg 1320

aaggtatggc ccgggtccac tgccttcccc gacttcacca accccacagc cctggcctgg 1380

tgggaggaca tggtggctga gttccatgac caggtgccct tcgacggcat gtggattgac 1440

atgaacgagc cttccaactt catcaggggc tctgaggacg gctgccccaa caatgagctg 1500

gagaacccac cctacgtgcc tggggtggtt ggggggaccc tccaggcggc caccatctgt 1560

gcctccagcc accagtttct ctccacacac tacaacctgc acaacctcta cggcctgacc 1620

gaagccatcg cctcccacag ggcgctggtg aaggctcggg ggacacgccc atttgtgatc 1680

1740

agctcctggg agcagctcgc ctcctccgtg ccagaaatcc tgcagtttaa cctgctgggg 1800

gtgcctctgg tcggggccga cgtctgcggc ttcctgggca acacctcaga ggagctgtgt 1860

gtgcgctgga cccagctggg ggccttctac cccttcatgc ggaaccacaa cagcctgctc 1920

agtctgcccc aggagccgta cagcttcagc gagccggccc agcaggccat gaggaaggcc 1980

ctcaccctgc gctacgcact cctcccccac ctctacacac tgttccacca ggccccgtc 2040

gcggggggaga ccgtggcccg gcccctcttc ctggagttcc ccaaggactc tagcacctgg 2100

actgtggacc accagctcct gtggggggag gccctgctca tcaccccagt gctccaggcc 2160

gggaaggccg aagtgactgg ctacttcccc ttgggcacat ggtacgacct gcagacggtg 2220

ccagtagagg cccttggcag ccctcccaccc ccacctgcag ctccccgtga gccagccatc 2280

cacagcgagg ggcagtgggt gacgctgccg gcccccctgg acaccatcaa cgtccacctc 2340

cgggctgggt acatcatccc cctgcagggc cctggcctca caaccacaga gtcccgccag 2400

cagcccatgg ccctggctgt ggccctgacc aagggtgggg aggcccgagg ggagctgttc 2460

tgggacgatg gagagagcct ggaagtgctg gagcgagggg cctacacaca ggtcatcttc 2520

ctggccagga ataacacgat cgtgaatgag ctggtacgtg tgaccagtga gggagctggc 2580

ctgcagctgc agaaggtgac tgtcctgggc gtggccacgg cgccccagca ggtcctctcc 2640

aacggtgtcc ctgtctccaa cttcacctac agccccgaca ccaaggtcct ggacatctgt 2700

gtctcgctgt tgatgggaga gcagtttctc gtcagctggt gttag 2745

<210> 38

<211> 2745

<212> DNA

<213> artificial

<220>

<223> sp7+hGAAco1-delta-29

<400> 38

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggccagcag 60

ggcgcctcta gacctggacc tagagatgcc caggcccacc ccggcagacc tagagctgtg 120

cctacccagt gtgacgtgcc ccccaacagc agattcgact gcgcccctga caaggccatc 180

acccaggaac agtgcgaggc cagaggctgc tgctacatcc ctgccaagca gggactgcag 240

ggcgctcaga tgggacagcc ctggtgcttc ttcccaccct cctaccccag ctacaagctg 300

gaaaacctga gcagcagcga gatgggctac accgccaccc tgaccagaac cacccccaca 360

ttcttcccaa aggacatcct gaccctgcgg ctggacgtga tgatggaaac cgagaaccgg 420

ctgcacttca ccatcaagga ccccgccaat cggagatacg aggtgcccct ggaaaccccc 480

cacgtgcact ctagagcccc cagccctctg tacagcgtgg aattcagcga ggaacccttc 540

ggcgtgatcg tgcggagaca gctggatggc agagtgctgc tgaacaccac cgtggcccct 600

ctgttcttcg ccgaccagtt cctgcagctg agcaccagcc tgcccagcca gtacatcaca 660

ggactggccg agcacctgag ccccctgatg ctgagcacat cctggacccg gatcaccctg 720

tggaacaggg atctggcccc tacccctggc gccaatctgt acggcagcca ccctttctac 780

ctggccctgg aagatggcgg atctgcccac ggagtgtttc tgctgaactc caacgccatg 840

gacgtggtgc tgcagcctag ccctgccctg tcttggagaa gcacaggcgg catcctggat 900

gtgtacatct ttctgggccc cgagcccaag agcgtggtgc agcagtatct ggatgtcgtg 960

ggctacccct tcatgccccc ttactggggc ctgggattcc acctgtgcag atggggctac 1020

tccagcaccg ccatcaccag acaggtggtg gaaaacatga cccagccca cttcccactg 1080

gatgtgcagt ggaacgacct ggactacatg gacagcagac gggacttcac cttcaacaag 1140

gacggcttcc gggacttccc cgccatggtg caggaactgc atcagggcgg cagacggtac 1200

atgatgatcg tggatcccgc catcagctcc tctggccctg ccggctctta cagaccctac 1260

gacgaggggcc tgcggagagg cgtgttcatc accaacgaga caggccagcc cctgatcggc 1320

aaagtgtggc ctggcagcac agccttcccc gacttcacca atcctaccgc cctggcttgg 1380

tgggaggaca tggtggccga gttccacgac caggtgccct tcgacggcat gtggatcgac 1440

atgaacgagc ccagcaactt catccggggc agcgaggatg gctgccccaa caacgaactg 1500

gaaaatcccc cttacgtgcc cggcgtcgtg ggcggaacac tgcaggccgc tacaatctgt 1560

gccagcagcc accagtttct gagcacccac tacaacctgc acaacctgta cggcctgacc 1620

gaggccattg ccagccaccg cgctctcgtg aaagccagag gcacacggcc cttcgtgatc 1680

agcagaagca cctttgccgg ccacggcaga tacgccggac attggactgg cgacgtgtgg 1740

tcctcttggg agcagctggc ctctagcgtg cccgagatcc tgcagttcaa tctgctgggc 1800

gtgccactcg tgggcgccga tgtgtgtggc ttcctgggca acacctccga ggaactgtgt 1860

gtgcggtgga cacagctggg cgccttctac cctttcatga gaaaccacaa cagcctgctg 1920

agcctgcccc aggaacccta cagctttagc gagcctgcac agcaggccat gcggaaggcc 1980

ctgacactga gatacgctct gctgccccac ctgtacaccc tgtttcacca ggcccatgtg 2040

gccggcgaga cagtggccag acctctgttt ctggaattcc ccaaggacag cagcacctgg 2100

accgtggacc atcagctgct gtggggagag gctctgctga ttaccccagt gctgcaggca 2160

ggcaaggccg aagtgaccgg ctactttccc ctgggcactt ggtacgacct gcagaccgtg 2220

cctgtggaag ccctgggatc tctgcctcca cctcctgccg ctcctagaga gcctgccatt 2280

cactctgagg gccagtgggt cacactgcct gcccccctgg ataccatcaa cgtgcacctg 2340

agggccggct acatcatacc actgcaggga cctggcctga ccaccaccga gtctagacag 2400

cagccaatgg ccctggccgt ggccctgacc aaaggcggag aagctaggggg cgagctgttc 2460

tgggacgatg gcgagagcct ggaagtgctg gaaagaggcg cctataccca agtgatcttc 2520

ctggcccgga acaacaccat cgtgaacgag ctggtgcgcg tgacctctga aggcgctgga 2580

ctgcagctgc agaaagtgac cgtgctggga gtggccacag cccctcagca ggtgctgtct 2640

aatggcgtgc ccgtgtccaa cttcacctac agccccgaca ccaaggtgct ggacatctgc 2700

gtgtcactgc tgatgggaga gcagtttctg gtgtcctggt gctga 2745

<210> 39

<211> 2745

<212> DNA

<213> artificial

<220>

<223> sp7+hGAAco2-delta-29

<400> 39

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggccaacag 60

ggagcttcca gaccaggacc gagagacgcc caagcccatc ctggtagacc aagagctgtg 120

cctacccaat gcgacgtgcc acccaactcc cgattcgact gcgcgccaga taaggctatt 180

acccaagagc agtgtgaagc cagaggttgc tgctacatcc cagcgaagca aggattgcaa 240

ggcgcccaaa tgggacaacc ttggtgtttc ttcccccctt cgtacccatc atataaactc 300

gaaaacctgt cctcttcgga aatgggttat actgccaccc tcaccagaac tactcctact 360

ttcttcccga aagacatctt gaccttgagg ctggacgtga tgatggagac tgaaaaccgg 420

ctgcatttca ctatcaaaga tcctgccaat cggcgatacg aggtccctct ggaaacccct 480

cacgtgcact cacgggctcc ttctccgctt tactccgtcg aattctctga ggaacccttc 540

ggagtgatcg ttagacgcca gctggatggt agagtgctgt tgaacactac tgtggcccca 600

cttttcttcg ctgaccagtt tctgcaactg tccacttccc tgccatccca gtacattact 660

ggactcgccg aacacctgtc gccactgatg ctctcgacct cttggactag aatcactttg 720

tggaacagag acttggcccc tactccggga gcaaatctgt acggaagcca ccctttttac 780

ctggcgctcg aagatggcgg atccgctcac ggagtgttcc tgctgaatag caacgcaatg 840

gacgtggtgc tgcaaccttc ccctgcactc agttggagaa gtaccggggg tattctggac 900

gtgtacatct tcctcggacc agaacccaag agcgtggtgc agcaatatct ggacgtggtc 960

ggataccctt ttatgcctcc ttactgggga ctgggattcc acctttgccg ttggggctac 1020

tcatccaccg ccattaccag acaggtggtg gagaatatga ccagagccca cttccctctc 1080

1140

gacgggttcc gcgattttcc cgcgatggtt caagagctcc accagggtgg tcgaagatat 1200

atgatgatcg tcgacccagc catttcgagc agcggacccg ctggatctta tagaccttac 1260

1320

aaagtgtggc ctggatcaac cgcctttcct gactttacca atcccactgc cttggcttgg 1380

tgggaggaca tggtggccga attccacgac caagtcccct ttgatggaat gtggatcgat 1440

atgaacgaac caagcaattt tatcagaggt tccgaagacg gttgccccaa caacgaactg 1500

gaaaaccctc cttatgtgcc cggagtcgtg ggcggaacat tacaggccgc gactatttgc 1560

gccagcagcc accaattcct gtccactcac tacaacctcc acaaccttta tggattaacc 1620

gaagctattg caagtcacag ggctctggtg aaggctagag ggactaggcc ctttgtgatc 1680

tcccgatcca cctttgccgg acacggggaga tacgccggtc actggactgg tgacgtgtgg 1740

1800

gtccctcttg tcggagcaga cgtgtgtggg tttcttggta acacctccga ggaactgtgt 1860

gtgcgctgga ctcaactggg tgcattctac ccattcatga gaaaccacaa ctccttgctg 1920

tccctgccac aagagccta ctcgttcagc gagcctgcac aacaggctat gcggaaggca 1980

ctgaccctga gatacgccct gcttccacac ttatacactc tcttccatca agcgcatgtg 2040

gcaggagaaa ccgttgcaag gcctcttttc cttgaattcc ccaaggattc ctcgacttgg 2100

acggtggatc atcagctgct gtggggagaa gctctgctga ttactccagt gttgcaagcc 2160

ggaaaagctg aggtgaccgg atactttccg ctgggaacct ggtacgacct cggactgtc 2220

ccctgttgaag cccttggatc actgcctccg ccctccggcag ctccacgcga accagctata 2280

cattccgagg gacagtgggt tacattacca gctcctctgg acacaatcaa cgtccactta 2340

agagctggct acattatccc tctgcaagga ccaggactga ctacgaccga gagcagacag 2400

cagccaatgg cactggctgt ggctctgacc aagggagggg aagctagagg agaactcttc 2460

tgggatgatg gggagtccct tgaagtgctg gaaagaggcg cttacactca agtcattttc 2520

cttgcacgga acaacaccat tgtgaacgaa ttggtgcgag tgaccagcga aggagctgga 2580

cttcaactgc agaaggtcac tgtgctcgga gtggctaccg ctcctcagca agtgctgtcg 2640

aatggagtcc ccgtgtcaaa ctttacctac tcccctgaca ctaaggtgct cgacatttgc 2700

gtgtccctcc tgatgggaga gcagttcctt gtgtcctggt gttga 2745

<210> 40

<211> 2706

<212> DNA

<213> artificial

<220>

<223> sp7+hGAAwt-delta-42

<400> 40

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggcgcacac 60

cccgggcggc cgcgagcagt gcccacacag tgcgacgtcc cccccaacag ccgcttcgat 120

tgcgcccctg acaaggccat cacccaggaa cagtgcgagg cccgcggctg ttgctacatc 180

cctgcaaagc aggggctgca gggagcccag atggggcagc cctggtgctt cttcccaccc 240

agctacccca gctacaagct ggagaacctg agctcctctg aaatgggcta cacggccacc 300

ctgacccgta ccacccccac cttcttcccc aaggacatcc tgaccctgcg gctggacgtg 360

atgatggaga ctgagaaccg ccctccacttc acgatcaaag atccagctaa caggcgctac 420

gaggtgccct tggagacccc gcatgtccac agccgggcac cgtccccact ctacagcgtg 480

gagttctccg aggagccctt cggggtgatc gtgcgccggc agctggacgg ccgcgtgctg 540

ctgaacacga cggtggcgcc cctgttcttt gcggaccagt tccttcagct gtccacctcg 600

ctgccctcgc agtatatcac aggcctcgcc gagcacctca gtcccctgat gctcagcacc 660

agctggacca ggatcaccct gtggaaccgg gaccttgcgc ccacgcccgg tgcgaacctc 720

tacgggtctc accctttcta cctggcgctg gaggacggcg ggtcggcaca cggggtgttc 780

ctgctaaaca gcaatgccat ggatgtggtc ctgcagccga gccctgccct tagctggagg 840

tcgacaggtg ggatcctgga tgtctacatc ttcctgggcc cagagcccaa gagcgtggtg 900

cagcagtacc tggacgttgt gggatacccg ttcatgccgc catactgggg cctgggcttc 960

cacctgtgcc gctggggcta ctcctccacc gctatcaccc gccaggtggt ggagaacatg 1020

accagggccc acttccccct ggacgtccag tggaacgacc tggactacat ggactcccgg 1080

agggacttca cgttcaacaa ggatggcttc cgggacttcc cggccatggt gcaggagctg 1140

caccagggcg gccggcgcta catgatgatc gtggatcctg ccatcagcag ctcgggccct 1200

gccgggggct acaggcccta cgacgagggt ctgcggaggg gggttttcat caccaacgag 1260

accggccagc cgctgattgg gaaggtatgg cccgggtcca ctgccttccc cgacttcacc 1320

aaccccacag ccctggcctg gtgggaggac atggtggctg agttccatga ccaggtgccc 1380

ttcgacggca tgtggattga catgaacgag ccttccaact tcatcagggg ctctgaggac 1440

ggctgcccca acaatgagct ggagaaccca ccctacgtgc ctggggtggt tggggggacc 1500

ctccaggcgg ccaccatctg tgcctccagc caccagtttc tctccacaca ctacaacctg 1560

cacaacctct acggcctgac cgaagccatc gcctcccaca gggcgctggt gaaggctcgg 1620

gggacacgcc catttgtgat ctcccgctcg acctttgctg gccacggccg atacgccggc 1680

cactggacgg gggacgtgtg gagctcctgg gagcagctcg cctcctccgt gccagaaatc 1740

ctgcagttta acctgctggg ggtgcctctg gtcggggccg acgtctgcgg cttcctgggc 1800

aacacctcag aggagctgtg tgtgcgctgg acccagctgg gggccttcta ccccttcatg 1860

cggaaccaca acagcctgct cagtctgccc caggagccgt acagcttcag cgagccggcc 1920

cagcaggcca tgaggaaggc cctcaccctg cgctacgcac tcctccccca cctctacaca 1980

ctgttccacc aggcccacgt cgcgggggag accgtggccc ggcccctctt cctggagttc 2040

cccaaggact ctagcacctg gactgtggac caccagctcc tgtgggggga ggccctgctc 2100

atcaccccag tgctccaggc cgggaaggcc gaagtgactg gctacttccc cttgggcaca 2160

tggtacgacc tgcagacggt gccagtagag gcccttggca gcctcccacc cccacctgca 2220

gctccccgtg agccagccat ccacagcgag gggcagtggg tgacgctgcc ggcccccctg 2280

gacaccacct acgtccacct ccgggctggg tacatcatcc ccctgcaggg ccctggcctc 2340

acaaccacag agtcccgcca gcagcccatg gccctggctg tggccctgac caaggggtggg 2400

gaggcccgag gggagctgtt ctgggacgat ggagagagcc tggaagtgct ggagcgaggg 2460

gcctacacac aggtcatctt cctggccagg aataacacga tcgtgaatga gctggtacgt 2520

gtgaccagtg agggagctgg cctgcagctg cagaaggtga ctgtcctggg cgtggccacg 2580

gcgccccagc aggtcctctc caacggtgtc cctgtctcca acttcaccta cagccccgac 2640

accaaggtcc tggacatctg tgtctcgctg ttgatgggag agcagtttct cgtcagctgg 2700

tgttag 2706

<210> 41

<211> 2706

<212> DNA

<213> artificial

<220>

<223> sp7-hGAAco2-delta-42

<400> 41

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggcgcccat 60

cctggtagac caagagctgt gcctacccaa tgcgacgtgc cacccaactc ccgattcgac 120

tgcgcgccag ataaggctat tacccaagag cagtgtgaag ccagaggttg ctgctacatc 180

ccagcgaagc aaggattgca aggcgcccaa atgggacaac cttggtgttt cttcccccct 240

tcgtacccat catataaact cgaaaacctg tcctcttcgg aaatgggtta tactgccacc 300

ctcaccagaa ctactcctac tttcttcccg aaagacatct tgaccttgag gctggacgtg 360

atgatggaga ctgaaaaccg gctgcatttc actatcaaag atcctgccaa tcggcgatac 420

gaggtccctc tggaaacccc tcacgtgcac tcacgggctc cttctccgct ttactccgtc 480

gaattctctg aggaaccctt cggagtgatc gttagacgcc agctggatgg tagagtgctg 540

ttgaacacta ctgtggcccc acttttcttc gctgaccagt ttctgcaact gtccacttcc 600

ctgccatccc agtacattac tggactcgcc gaacacctgt cgccactgat gctctcgacc 660

tcttggacta gaatcacttt gtggaacaga gacttggccc ctactccggg agcaaatctg 720

tacggaagcc acccttttta cctggcgctc gaagatggcg gatccgctca cggagtgttc 780

ctgctgaata gcaacgcaat ggacgtggtg ctgcaacctt cccctgcact cagttggaga 840

agtaccgggg gtattctgga cgtgtacatc ttcctcggac cagaacccaa gagcgtggtg 900

cagcaatatc tggacgtggt cggataccct tttatgcctc cttactgggg actgggattc 960

cacctttgcc gttggggcta ctcatccacc gccattacca gacaggtggt ggagaatatg 1020

accagagccc acttccctct cgacgtgcag tggaacgatc tggactatat ggactcccgg 1080

agagatttca ccttcaacaa ggacgggttc cgcgattttc ccgcgatggt tcaagagctc 1140

caccagggtg gtcgaagata tatgatgatc gtcgacccag ccatttcgag cagcggaccc 1200

gctggatctt atagacctta cgacgaaggc cttaggagag gagtgttcat cacaaacgag 1260

1320

1380

tttgatggaa tgtggatcga tatgaacgaa ccaagcaatt ttatcagagg ttccgaagac 1440

ggttgcccca acaacgaact ggaaaaccct ccttatgtgc ccggagtcgt gggcggaaca 1500

ttacaggccg cgactatttg cgccagcagc caccaattcc tgtccactca ctacaacctc 1560

cacaaccttt atggattaac cgaagctatt gcaagtcaca gggctctggt gaaggctaga 1620

gggactaggc cctttgtgat ctcccgatcc acctttgccg gacacgggag atacgccggt 1680

cactggactg gtgacgtgtg gagctcatgg gaacaactgg cctcctccgt gccggaaatc 1740

ttacagttca accttctggg tgtccctctt gtcggagcag acgtgtgtgg gtttcttggt 1800

aacacctccg aggaactgtg tgtgcgctgg actcaactgg gtgcattcta cccattcatg 1860

agaaaccaca actccttgct gtccctgcca caagagccct actcgttcag cgagcctgca 1920

caacaggcta tgcggaaggc actgaccctg agatacgccc tgcttccaca cttatacact 1980

ctcttccatc aagcgcatgt ggcaggagaa accgttgcaa ggcctctttt ccttgaattc 2040

cccaaggatt cctcgacttg gacggtggat catcagctgc tgtggggaga agctctgctg 2100

attactccag tgttgcaagc cggaaaagct gaggtgaccg gatactttcc gctgggaacc 2160

tggtacgacc tccagactgt ccctgttgaa gcccttggat cactgcctcc gcctccggca 2220

gctccacgcg aaccagctat acattccgag ggacagtggg ttacattacc agctcctctg 2280

gacacaatca acgtccactt aagagctggc tacattatcc ctctgcaagg accaggactg 2340

actacgaccg agagcagaca gcagccaatg gcactggctg tggctctgac caagggaggg 2400

gaagctagag gagaactctt ctgggatgat ggggagtccc ttgaagtgct ggaaagaggc 2460

gcttacactc aagtcatttt ccttgcacgg aacaacacca ttgtgaacga attggtgcga 2520

gtgaccagcg aaggagctgg acttcaactg cagaaggtca ctgtgctcgg agtggctacc 2580

gctcctcagc aagtgctgtc gaatggagtc cccgtgtcaa actttaccta ctcccctgac 2640

actaaggtgc tcgacatttg cgtgtccctc ctgatgggag agcagttcct tgtgtcctgg 2700

tgttga 2706

<210> 42

<211> 2703

<212> DNA

<213> artificial

<220>

<223> sp7-hGAAwt-delta-43

<400> 42

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggccacccc 60

gggcggccgc gagcagtgcc cacacagtgc gacgtccccc ccaacagccg cttcgattgc 120

gcccctgaca aggccatcac cggaacag tgcgaggccc gcggctgttg ctacatccct 180

gcaaagcagg ggctgcaggg agccgatg gggcagccct ggtgcttctt cccacccagc 240

taccccagct acaagctgga gaacctgagc tcctctgaaa tgggctacac ggccaccctg 300

acccgtacca cccccacctt cttccccaag gacatcctga ccctgcggct ggacgtgatg 360

atggagactg agaaccgcct ccacttcacg atcaaagatc cagctaacag gcgctacgag 420

gtgcccttgg agaccccgca tgtccacagc cgggcaccgt ccccactcta cagcgtggag 480

ttctccgagg agcccttcgg ggtgatcgtg cgccggcagc tggacggccg cgtgctgctg 540

aacacgacgg tggcgcccct gttctttgcg gaccagttcc ttcagctgtc cacctcgctg 600

ccctcgcagt atatcacagg cctcgccgag cacctcagtc ccctgatgct cagcaccagc 660

tggaccagga tcaccctgtg gaaccgggac cttgcgccca cgcccggtgc gaacctctac 720

gggtctcacc ctttctacct ggcgctggag gacggcgggt cggcacacgg ggtgttcctg 780

ctaaacagca atgccatgga tgtggtcctg cagccgagcc ctgcccttag ctggaggtcg 840

acaggtggga tcctggatgt ctacatcttc ctgggcccag agcccaagag cgtggtgcag 900

cagtacctgg acgttgtggg atacccgttc atgccgccat actggggcct gggcttccac 960

ctgtgccgct ggggctactc ctccaccgct atcacccgcc aggtggtgga gaacatgacc 1020

agggcccact tccccctgga cgtccagtgg aacgacctgg actacatgga ctcccgggagg 1080

gacttcacgt tcaacaagga tggcttccgg gacttcccgg ccatggtgca ggagctgcac 1140

cagggcggcc ggcgctacat gatgatcgtg gatcctgcca tcagcagctc gggccctgcc 1200

gggagctaca ggccctacga cgagggtctg cggagggggg ttttcatcac caacgagacc 1260

ggccagccgc tgattgggaa ggtatggccc gggtccactg ccttccccga cttcaccaac 1320

cccacagccc tggcctggtg ggaggacatg gtggctgagt tccatgacca ggtgcccttc 1380

gacggcatgt ggattgacat gaacgagcct tccaacttca tcaggggctc tgaggacggc 1440

tgccccaaca atgagctgga gaacccaccc tacgtgcctg gggtggttgg ggggaccctc 1500

caggcggcca ccatctgtgc ctccagccac cagtttctct ccacacacta caacctgcac 1560

aacctctacg gcctgaccga agccatcgcc tcccacaggg cgctggtgaa ggctcggggg 1620

acacgcccat ttgtgatctc ccgctcgacc tttgctggcc acggccgata cgccggccac 1680

tggacggggg acgtgtggag ctcctgggag cagctcgcct cctccgtgcc agaaatcctg 1740

cagtttaacc tgctgggggt gcctctggtc ggggccgacg tctgcggctt cctgggcaac 1800

acctcagagg agctgtgtgt gcgctggacc cagctggggg ccttctaccc cttcatgcgg 1860

aaccacaaca gcctgctcag tctgccccag gagccgtaca gcttcagcga gccggcccag 1920

caggccatga ggaaggccct caccctgcgc tacgcactcc tcccccacct ctacacactg 1980

ttccaccagg cccacgtcgc gggggagacc gtggcccggc ccctcttcct ggagttcccc 2040

aaggactcta gcacctggac tgtggaccac cagctcctgt ggggggaggc cctgctcatc 2100

accccagtgc tccaggccgg gaaggccgaa gtgactggct acttcccctt gggcacatgg 2160

tacgacctgc agacggtgcc agtagaggcc cttggcagcc tcccaccccc acctgcagct 2220

ccccgtgagc cagccatcca cagcgagggg cagtgggtga cgctgccggc ccccctggac 2280

accatcaacg tccacctccg ggctgggtac atcatccccc tgcagggccc tggcctcaca 2340

accacagagt cccgccagca gcccatggcc ctggctgtgg ccctgaccaa gggtggggag 2400

gcccgagggg agctgttctg ggacgatgga gagagcctgg aagtgctgga gcgaggggcc 2460

tacacacagg tcatcttcct ggccaggaat aacacgatcg tgaatgagct ggtacgtgtg 2520

accagtgagg gagctggcct gcagctgcag aaggtgactg tcctgggcgt ggccacggcg 2580

ccccagcagg tcctctccaa cggtgtccct gtctccaact tcacctacag ccccgacacc 2640

aaggtcctgg acatctgtgt ctcgctgttg atgggagagc agtttctcgt cagctggtgt 2700

tag 2703

<210> 43

<211> 2703

<212> DNA

<213> artificial

<220>

<223> sp7+hGAAco1-delta-43

<400> 43

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggccacccc 60

ggcagaccta gagctgtgcc tacccagtgt gacgtgcccc ccaacagcag attcgactgc 120

gcccctgaca aggccatcac cggaacag tgcgaggcca gaggctgctg ctacatccct 180

gccaagcagg gactgcaggg cgctcagatg ggacagccct ggtgcttctt cccaccctcc 240

taccccagct acaagctgga aaacctgagc agcagcgaga tgggctacac cgccaccctg 300

accagaacca cccccacatt cttcccaaag gacatcctga ccctgcggct ggacgtgatg 360

atggaaaccg agaaccggct gcacttcacc atcaaggacc ccgccaatcg gagatacgag 420

gtgcccctgg aaaccccca cgtgcactct agagccccca gccctctgta cagcgtggaa 480

ttcagcgagg aacccttcgg cgtgatcgtg cggagacagc tggatggcag agtgctgctg 540

aacaccaccg tggcccctct gttcttcgcc gaccagttcc tgcagctgag caccagcctg 600

cccagccagt acatcacagg actggccgag cacctgagcc ccctgatgct gagcacatcc 660

tggacccgga tcaccctgtg gaacagggat ctggccccta cccctggcgc caatctgtac 720

ggcagccacc ctttctacct ggccctggaa gatggcggat ctgccccgg agtgtttctg 780

ctgaactcca acgccatgga cgtggtgctg cagcctagcc ctgccctgtc ttggagaagc 840

acaggcggca tcctggatgt gtacatcttt ctgggccccg agcccaagag cgtggtgcag 900

cagtatctgg atgtcgtggg ctaccccttc atgccccctt actggggcct gggattccac 960

ctgtgcagat ggggctactc cagcaccgcc atcaccagac aggtggtgga aaacatgacc 1020

agagcccact tcccactgga tgtgcagtgg aacgacctgg actacatgga cagcagacgg 1080

gacttcacct tcaacaagga cggcttccgg gacttccccg ccatggtgca ggaactgcat 1140

cagggcggca gacggtacat gatgatcgtg gatcccgcca tcagctcctc tggccctgcc 1200

ggctcttaca gaccctacga cgaggggcctg cggagaggcg tgttcatcac caacgagaca 1260

ggccagcccc tgatcggcaa agtgtggcct ggcagcacag ccttccccga cttcaccaat 1320

cctaccgccc tggcttggtg ggaggacatg gtggccgagt tccacgacca ggtgcccttc 1380

gacggcatgt ggatcgacat gaacgagccc agcaacttca tccggggcag cgaggatggc 1440

tgccccaaca acgaactgga aaatccccct tacgtgcccg gcgtcgtggg cggaacactg 1500

caggccgcta caatctgtgc cagcagccac cagtttctga gcacccacta caacctgcac 1560

aacctgtacg gcctgaccga ggccattgcc agccaccgcg ctctcgtgaa agccagaggc 1620

acacggccct tcgtgatcag cagaagcacc tttgccggcc acggcagata cgccggacat 1680

tggactggcg acgtgtggtc ctcttgggag cagctggcct ctagcgtgcc cgagatcctg 1740

cagttcaatc tgctgggcgt gccactcgtg ggcgccgatg tgtgtggctt cctgggcaac 1800

acctccgagg aactgtgtgt gcggtggaca cagctgggcg ccttctaccc tttcatgaga 1860

aaccacaaca gcctgctgag cctgccccag gaaccctaca gctttagcga gcctgcacag 1920

caggccatgc ggaaggccct gacactgaga tacgctctgc tgccccacct gtacaccctg 1980

tttcaccagg cccatgtggc cggcgagaca gtggccagac ctctgtttct ggaattcccc 2040

aaggacagca gcacctggac cgtggaccat cagctgctgt ggggagaggc tctgctgatt 2100

accccagtgc tgcaggcagg caaggccgaa gtgaccggct actttcccct gggcacttgg 2160

tacgacctgc agaccgtgcc tgtggaagcc ctgggatctc tgcctccacc tcctgccgct 2220

cctagagc ctgccattca ctctgagggc cagtgggtca cactgcctgc ccccctggat 2280

accatcaacg tgcacctgag ggccggctac atcataccac tgcagggacc tggcctgacc 2340

accaccgagt ctagacagca gccaatggcc ctggccgtgg ccctgaccaa aggcggagaa 2400

gctagggggcg agctgttctg ggacgatggc gagagcctgg aagtgctgga aagaggcgcc 2460

tatacccaag tgatcttcct ggccgggaac aacaccatcg tgaacgagct ggtgcgcgtg 2520

acctctgaag gcgctggact gcagctgcag aaagtgaccg tgctgggagt ggccacagcc 2580

ccctcagcagg tgctgtctaa tggcgtgccc gtgtccaact tcacctacag ccccgacacc 2640

aaggtgctgg acatctgcgt gtcactgctg atgggagagc agtttctggt gtcctggtgc 2700

tga 2703

<210> 44

<211> 2703

<212> DNA

<213> artificial

<220>

<223> sp7+hGAAco2-delta-43

<400> 44

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggccatcct 60

ggtagaccaa gagctgtgcc tacccaatgc gacgtgccac ccaactcccg attcgactgc 120

gcgccagata aggctattac ccaagagcag tgtgaagcca gaggttgctg ctacatccca 180

gcgaagcaag gattgcaagg cgcccaaatg ggacaacctt ggtgtttctt ccccccttcg 240

tacccatcat ataaactcga aaacctgtcc tcttcggaaa tgggttatac tgccaccctc 300

accagaacta ctcctacttt cttcccgaaa gacatcttga ccttgaggct ggacgtgatg 360

atggagactg aaaaccggct gcatttcact atcaaagatc ctgccaatcg gcgatacgag 420

gtccctctgg aaacccctca cgtgcactca cgggctcctt ctccgcttta ctccgtcgaa 480

ttctctgagg aacccttcgg agtgatcgtt agacgccagc tggatggtag agtgctgttg 540

aacactactg tggccccact tttcttcgct gaccagtttc tgcaactgtc cacttccctg 600

ccatcccagt acattactgg actcgccgaa cacctgtcgc cactgatgct ctcgacctct 660

tggactagaa tcactttgtg gaacagagac ttggccccta ctccgggagc aaatctgtac 720

ggaagccacc ctttttacct ggcgctcgaa gatggcggat ccgctcacgg agtgttcctg 780

ctgaatagca acgcaatgga cgtggtgctg caaccttccc ctgcactcag ttggagaagt 840

accgggggta ttctggacgt gtacatcttc ctcggaccag aacccaagag cgtggtgcag 900

caatatctgg acgtggtcgg ataccctttt atgcctcctt actggggact gggattccac 960

ctttgccgtt ggggctactc atccaccgcc attaccagac aggtggtgga gaatatgacc 1020

agagcccact tccctctcga cgtgcagtgg aacgatctgg actatatgga ctcccggaga 1080

gatttcacct tcaacaagga cgggttccgc gattttcccg cgatggttca agagctccac 1140

cagggtggtc gaagatatat gatgatcgtc gacccagcca tttcgagcag cggacccgct 1200

ggatcttata gaccttacga cgaaggcctt aggagaggag tgttcatcac aaacgagact 1260

ggacagcctt tgatcggtaa agtgtggcct ggatcaaccg ccttttcctga ctttaccaat 1320

1380

gatggaatgt ggatcgatat gaacgaacca agcaatttta tcagaggttc cgaagacggt 1440

tgccccaaca acgaactgga aaaccctcct tatgtgcccg gagtcgtggg cggaacatta 1500

caggccgcga ctatttgcgc cagcagccac caattcctgt ccactcacta caacctccac 1560

1620

actaggccct ttgtgatctc ccgatccacc tttgccggac acgggagata cgccggtcac 1680

tggactggtg acgtgtggag ctcatgggaa caactggcct cctccgtgcc ggaaatctta 1740

cagttcaacc ttctgggtgt ccctcttgtc ggagcagacg tgtgtgggtt tcttggtaac 1800

acctccgagg aactgtgtgt gcgctggact caactgggtg cattctaccc attcatgaga 1860

aaccacaact ccttgctgtc cctgccacaa gagccctact cgttcagcga gcctgcacaa 1920

caggctatgc ggaaggcact gaccctgaga tacgccctgc ttccacactt atacactctc 1980

ttccatcaag cgcatgtggc aggagaaacc gttgcaaggc ctcttttcct tgaattcccc 2040

aaggattcct cgacttggac ggtggatcat cagctgctgt ggggagaagc tctgctgatt 2100

actccagtgt tgcaagccgg aaaagctgag gtgaccggat actttccgct gggaacctgg 2160

tacgacctcc agactgtccc tgttgaagcc cttggatcac tgcctccgcc tccggcagct 2220

ccacgcgaac cagctataca ttccgaggga cagtgggtta cattaccagc tcctctggac 2280

acaatcaacg tccacttaag agctggctac attatccctc tgcaaggacc aggactgact 2340

acgaccgaga gcagacagca gccaatggca ctggctgtgg ctctgaccaa gggaggggaa 2400

gctagaggag aactcttctg ggatgatggg gagtcccttg aagtgctgga aagaggcgct 2460

2520

accagcgaag gagctggact tcaactgcag aaggtcactg tgctcggagt ggctaccgct 2580

ccctcagcaag tgctgtcgaa tggagtcccc gtgtcaaact ttacctactc ccctgacact 2640

aaggtgctcg acatttgcgt gtccctcctg atgggagagc agttccttgt gtcctggtgt 2700

tga 2703

<210> 45

<211> 2691

<212> DNA

<213> artificial

<220>

<223> sp7+hGAAwt-delta-47

<400> 45

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggcccgcga 60

gcagtgccca cacagtgcga cgtccccccc aacagccgct tcgattgcgc ccctgacaag 120

gccatcaccc aggaacagtg cgaggcccgc ggctgttgct acatccctgc aaagcagggg 180

ctgcagggag cccagatggg gcagccctgg tgcttcttcc cacccagcta ccccagctac 240

aagctggaga acctgagctc ctctgaaatg ggctacacgg ccaccctgac ccgtaccacc 300

cccaccttct tccccaagga catcctgacc ctgcggctgg acgtgatgat ggagactgag 360

aaccgcctcc acttcacgat caaagatcca gctaacaggc gctacgaggt gcccttggag 420

accccgcatg tccacagccg ggcaccgtcc ccactctaca gcgtggagtt ctccgaggag 480

cccttcgggg tgatcgtgcg ccggcagctg gacggccgcg tgctgctgaa cacgacggtg 540

gcgcccctgt tctttgcgga ccagttcctt cagctgtcca cctcgctgcc ctcgcagtat 600

atcacaggcc tcgccgagca cctcagtccc ctgatgctca gcaccagctg gaccaggatc 660

accctgtgga accgggacct tgcgccccg cccggtgcga acctctacgg gtctcaccct 720

ttctacctgg cgctggagga cggcgggtcg gcacacgggg tgttcctgct aaacagcaat 780

gccatggatg tggtcctgca gccgagccct gcccttagct ggaggtcgac aggtgggatc 840

ctggatgtct acatcttcct gggccgag cccaagagcg tggtgcagca gtacctggac 900

gttgtgggat acccgttcat gccgccatac tggggcctgg gcttccacct gtgccgctgg 960

ggctactcct ccaccgctat cacccgccag gtggtggaga acatgaccag ggcccacttc 1020

cccctggacg tccagtggaa cgacctggac tacatggact cccggaggga cttcacgttc 1080

aacaaggatg gcttccggga cttcccggcc atggtgcagg agctgcacca gggcggccgg 1140

cgctacatga tgatcgtgga tcctgccatc agcagctcgg gccctgccgg gagctacagg 1200

ccctacgacg agggtctgcg gaggggggtt ttcatcacca acgagaccgg ccagccgctg 1260

attgggaagg tatggcccgg gtccactgcc ttccccgact tcaccaaccc cacagccctg 1320

gcctggtggg aggacatggt ggctgagttc catgaccagg tgcccttcga cggcatgtgg 1380

attgacatga acgagccttc caacttcatc aggggctctg aggacggctg ccccaacaat 1440

gagctggaga acccacccta cgtgcctggg gtggttgggg ggaccctcca ggcggccacc 1500

atctgtgcct ccagccacca gtttctctcc acacactaca acctgcacaa cctctacggc 1560

ctgaccgaag cccatcgcctc ccacaggggcg ctggtgaagg ctcgggggac acgcccattt 1620

gtgatctccc gctcgacctt tgctggccac ggccgatacg ccggccactg gacgggggac 1680

gtgtggagct cctgggagca gctcgcctcc tccgtgccag aaatcctgca gtttaacctg 1740

ctgggggtgc ctctggtcgg ggccgacgtc tgcggcttcc tgggcaacac ctcagaggag 1800

ctgtgtgtgc gctggaccca gctgggggcc ttctacccct tcatgcggaa ccacaacagc 1860

ctgctcagtc tgccccagga gccgtacagc ttcagcgagc cggcccagca ggccatgagg 1920

aaggccctca ccctgcgcta cgcactcctc ccccacctct acacactgtt ccaccaggcc 1980

cacgtcgcgg gggagaccgt ggcccggccc ctcttcctgg agttccccaa ggactctagc 2040

acctggactg tggaccacca gctcctgtgg ggggaggccc tgctcatcac cccagtgctc 2100

caggccggga aggccgaagt gactggctac ttccccttgg gcacatggta cgacctgcag 2160

acggtgccag tagaggccct tggcagcctc ccacccccac ctgcagctcc ccgtgagcca 2220

gccatccaca gcgaggggca gtgggtgacg ctgccggccc ccctggacac catcaacgtc 2280

cacctccggg ctgggtacat catccccctg cagggccctg gcctcacaac cacagagtcc 2340

cgccagcagc ccatggccct ggctgtggcc ctgaccaagg gtgggggaggc ccgaggggag 2400

ctgttctggg acgatggaga gagcctggaa gtgctggagc gaggggccta cacacaggtc 2460

atcttcctgg cgggaataa cacgatcgtg aatgagctgg tacgtgtgac cagtgaggga 2520

2580

ctctccaacg gtgtccctgt ctccaacttc acctacagcc ccgacaccaa ggtcctggac 2640

atctgtgtct cgctgttgat gggagagcag tttctcgtca gctggtgtta g 2691

<210> 46

<211> 2691

<212> DNA

<213> artificial

<220>

<223> sp7+hGAAco1-delta-47

<400> 46

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggccctaga 60

gctgtgccta cccagtgtga cgtgcccccc aacagcagat tcgactgcgc ccctgacaag 120

gccatcaccc aggaacagtg cgaggccaga ggctgctgct acatccctgc caagcaggga 180

ctgcagggcg ctcagatggg acagccctgg tgcttcttcc caccctccta ccccagctac 240

aagctggaaa acctgagcag cagcgagatg ggctacaccg ccaccctgac cagaaccacc 300

cccacattct tcccaaagga catcctgacc ctgcggctgg acgtgatgat ggaaaccgag 360

aaccggctgc acttcaccat caaggacccc gccaatcgga gatacgaggt gcccctggaa 420

accccccg tgcactctag agcccccagc cctctgtaca gcgtggaatt cagcgaggaa 480

cccttcggcg tgatcgtgcg gagacagctg gatggcagag tgctgctgaa caccaccgtg 540

gcccctctgt tcttcgccga ccagttcctg cagctgagca ccagcctgcc cagccagtac 600

atcacaggac tggccgagca cctgagcccc ctgatgctga gcacatcctg gacccggatc 660

accctgtgga acagggatct ggcccctacc cctggcgcca atctgtacgg cagccaccct 720

ttctacctgg ccctggaaga tggcggatct gcccacggag tgtttctgct gaactccaac 780

gccatggacg tggtgctgca gcctagccct gccctgtctt ggagaagcac aggcggcatc 840

ctggatgtgt acatctttct gggccccgag cccaagagcg tggtgcagca gtatctggat 900

gtcgtgggct accccttcat gcccccttac tggggcctgg gattccacct gtgcagatgg 960

ggctactcca gcaccgccat caccagacag gtggtggaaa acatgaccag agcccacttc 1020

ccactggatg tgcagtggaa cgacctggac tacatggaca gcagacggga cttcaccttc 1080

aacaaggacg gcttccggga cttccccgcc atggtgcagg aactgcatca gggcggcaga 1140

cggtacatga tgatcgtgga tcccgccatc agctcctctg gccctgccgg ctcttacaga 1200

ccctacgacg agggcctgcg gagaggcgtg ttcatcacca acgagacagg ccagcccctg 1260

atcggcaaag tgtggcctgg cagcacagcc ttccccgact tcaccaatcc taccgccctg 1320

gcttggtggg aggacatggt ggccgagttc cacgaccagg tgcccttcga cggcatgtgg 1380

atcgacatga acgagcccag caacttcatc cggggcagcg aggatggctg ccccaacaac 1440

gaactggaaa atccccctta cgtgcccggc gtcgtgggcg gaacactgca ggccgctaca 1500

atctgtgcca gcagccacca gtttctgagc acccactaca acctgcacaa cctgtacggc 1560

ctgaccgagg ccattgccag ccaccgcgct ctcgtgaaag ccagaggcac acggcccttc 1620

gtgatcagca gaagcacctt tgccggccac ggcagatacg ccggacattg gactggcgac 1680

gtgtggtcct cttgggagca gctggcctct agcgtgcccg agatcctgca gttcaatctg 1740

ctgggcgtgc cactcgtggg cgccgatgtg tgtggcttcc tgggcaacac ctccgaggaa 1800

ctgtgtgtgc ggtggacaca gctgggcgcc ttctaccctt tcatgagaaa ccacaacagc 1860

ctgctgagcc tgccccagga accctacagc tttagcgagc ctgcacagca ggccatgcgg 1920

aaggccctga cactgagata cgctctgctg ccccacctgt acaccctgtt tcaccaggcc 1980

catgtggccg gcgagacagt ggccagacct ctgtttctgg aattccccaa ggacagcagc 2040

acctggaccg tggaccatca gctgctgtgg ggagaggctc tgctgattac cccagtgctg 2100

caggcaggca aggccgaagt gaccggctac tttcccctgg gcacttggta cgacctgcag 2160

accgtgcctg tggaagccct gggatctctg cctccacctc ctgccgctcc tagagagcct 2220

gccattcact ctgagggcca gtgggtcaca ctgcctgccc ccctggatac catcaacgtg 2280

cacctgaggg ccggctacat cataccactg cagggacctg gcctgaccac caccgagtct 2340

agacagcagc caatggccct ggccgtggcc ctgaccaaag gcggagaagc taggggcgag 2400

ctgttctggg acgatggcga gagcctggaa gtgctggaaa gaggcgccta tacccaagtg 2460

atcttcctgg ccgggaacaa caccatcgtg aacgagctgg tgcgcgtgac ctctgaaggc 2520

gctggactgc agctgcagaa agtgaccgtg ctgggagtgg ccacagcccc tcagcaggtg 2580

ctgtctaatg gcgtgcccgt gtccaacttc acctacagcc ccgacaccaa ggtgctggac 2640

atctgcgtgt cactgctgat gggagagcag tttctggtgt cctggtgctg a 2691

<210> 47

<211> 2691

<212> DNA

<213> artificial

<220>

<223> sp7+hGAAco2-delta-47

<400> 47

atggcctttc tgtggctgct gagctgttgg gccctgctgg gcaccacctt cggcccaaga 60

gctgtgccta cccaatgcga cgtgccaccc aactcccgat tcgactgcgc gccagataag 120

gctattaccc aagagcagtg tgaagccaga ggttgctgct acatcccagc gaagcaagga 180

ttgcaaggcg cccaaatggg acaaccttgg tgtttcttcc ccccttcgta cccatcatat 240

aaactcgaaa acctgtcctc ttcggaaatg ggttatactg ccaccctcac cagaactact 300

cctactttct tcccgaaaga catcttgacc ttgaggctgg acgtgatgat ggagactgaa 360

aaccggctgc atttcactat caaagatcct gccaatcggc gatacgaggt ccctctggaa 420

acccctcacg tgcactcacg ggctccttct ccgctttact ccgtcgaatt ctctgaggaa 480

cccttcggag tgatcgttag acgccagctg gatggtagag tgctgttgaa cactactgtg 540

gccccacttt tcttcgctga ccagtttctg caactgtcca cttccctgcc atcccagtac 600

attactggac tcgccgaaca cctgtcgcca ctgatgctct cgacctcttg gactagaatc 660

actttgtgga acagagactt ggcccctact ccgggagcaa atctgtacgg aagccaccct 720

ttttacctgg cgctcgaaga tggcggatcc gctcacggag tgttcctgct gaatagcaac 780

gcaatggacg tggtgctgca accttcccct gcactcagtt ggagaagtac cgggggtatt 840

ctggacgtgt acatcttcct cggaccagaa cccaagagcg tggtgcagca atatctggac 900

gtggtcggat acccttttat gcctccttac tggggactgg gattccacct ttgccgttgg 960

ggctactcat ccaccgccat taccagacag gtggtggaga atatgaccag agcccacttc 1020

cctctcgacg tgcagtggaa cgatctggac tatatggact cccggagaga tttcaccttc 1080

1140

agatatatga tgatcgtcga cccagccatt tcgagcagcg gacccgctgg atcttataga 1200

ccttacgacg aaggccttag gagaggagtg ttcatcacaa acgagactgg acagcctttg 1260

atcggtaaag tgtggcctgg atcaaccgcc tttcctgact ttaccaatcc cactgccttg 1320

1380

atcgatatga acgaaccaag caattttatc agaggttccg aagacggttg ccccaacaac 1440

gaactggaaa accctcctta tgtgccggga gtcgtgggcg gaacattaca ggccgcgact 1500

atttgcgcca gcagccacca attcctgtcc actcactaca acctccacaa cctttatgga 1560

ttaaccgaag ctattgcaag tcacaggggct ctggtgaagg ctagagggac taggcccttt 1620

gtgatctccc gatccacctt tgccggacac gggagatacg ccggtcactg gactggtgac 1680

gtgtggagct catgggaaca actggcctcc tccgtgccgg aaatcttaca gttcaacctt 1740

ctgggtgtcc ctcttgtcgg agcagacgtg tgtgggtttc ttggtaacac ctccgaggaa 1800

ctgtgtgtgc gctggactca actgggtgca ttctacccat tcatgagaaa ccacaactcc 1860

ttgctgtccc tgccacaaga gccctactcg ttcagcgagc ctgcacaaca ggctatgcgg 1920

aaggcactga ccctgagata cgccctgctt ccacacttat acactctctt ccatcaagcg 1980

catgtggcag gagaaaccgt tgcaaggcct cttttccttg aattccccaa ggattcctcg 2040

acttggacgg tggatcatca gctgctgtgg ggagaagctc tgctgattac tccagtgttg 2100

caagccggaa aagctgaggt gaccggatac tttccgctgg gaacctggta cgacctccag 2160

actgtccctg ttgaagccct tggatcactg cctccgcctc cggcagctcc acgcgaacca 2220

gctatacatt ccgagggaca gtgggttaca ttaccagctc ctctggacac aatcaacgtc 2280

cacttaagag ctggctacat tatccctctg caaggaccag gactgactac gaccgagagc 2340

agacagcagc caatggcact ggctgtggct ctgaccaagg gaggggaagc tagaggagaa 2400

ctcttctggg atgatgggga gtcccttgaa gtgctggaaa gaggcgctta cactcaagtc 2460

attttccttg cacggaacaa caccattgtg aacgaattgg tgcgagtgac cagcgaagga 2520

gctggacttc aactgcagaa ggtcactgtg ctcggagtgg ctaccgctcc tcagcaagtg 2580

ctgtcgaatg gagtccccgt gtcaaacttt acctactccc ctgacactaa ggtgctcgac 2640

atttgcgtgt ccctcctgat gggagagcag ttccttgtgt cctggtgttg a 2691

<210> 48

<211> 2652

<212> DNA

<213> artificial

<220>

<223> hGAAco1-delta-42

<400> 48

gcccaccccg gcagacctag agctgtgcct acccagtgtg acgtgccccc caacagcaga 60

ttcgactgcg cccctgacaa ggccatcacc caggaacagt gcgaggccag aggctgctgc 120

tacatccctg ccaagcaggg actgcaggggc gctcagatgg gacagccctg gtgcttcttc 180

ccaccctcct accccagcta caagctggaa aacctgagca gcagcgagat gggctacacc 240

gccaccctga ccagaaccac ccccacattc ttcccaaagg acatcctgac cctgcggctg 300

gacgtgatga tggaaaccga gaaccggctg cacttcacca tcaaggaccc cgccaatcgg 360

agatacgagg tgcccctgga aaccccccac gtgcactcta gagcccccag ccctctgtac 420

agcgtggaat tcagcgagga acccttcggc gtgatcgtgc ggagacagct ggatggcaga 480

gtgctgctga acaccaccgt ggcccctctg ttcttcgccg accagttcct gcagctgagc 540

accagcctgc ccagccagta catcacagga ctggccgagc acctgagccc cctgatgctg 600

agcacatcct ggacccggat caccctgtgg aacagggatc tggcccctac ccctggcgcc 660

aatctgtacg gcagccaccc tttctacctg gccctggaag atggcggatc tgccccggga 720

gtgtttctgc tgaactccaa cgccatggac gtggtgctgc agcctagccc tgccctgtct 780

tggagaagca caggcggcat cctggatgtg tacatctttc tgggccccga gcccaagagc 840

gtggtgcagc agtatctgga tgtcgtgggc taccccttca tgccccctta ctggggcctg 900

ggattccacc tgtgcagatg gggctactcc agcaccgcca tcaccagaca ggtggtggaa 960

aacatgacca gagcccactt cccactggat gtgcagtgga acgacctgga ctacatggac 1020

agcagacggg acttcacctt caacaaggac ggcttccggg acttccccgc catggtgcag 1080

gaactgcatc agggcggcag acggtacatg atgatcgtgg atcccgccat cagctcctct 1140

ggccctgccg gctcttacag accctacgac gagggcctgc ggagaggcgt gttcatcacc 1200

aacgagacag gccagcccct gatcggcaaa gtgtggcctg gcagcacagc cttccccgac 1260

ttcaccaatc ctaccgccct ggcttggtgg gaggacatgg tggccgagtt ccacgaccag 1320

gtgcccttcg acggcatgtg gatcgacatg aacgagccca gcaacttcat ccggggcagc 1380

gaggatggct gccccaacaa cgaactggaa aatccccctt acgtgcccgg cgtcgtggggc 1440

ggaacactgc aggccgctac aatctgtgcc agcagccacc agtttctgag cacccactac 1500

aacctgcaca acctgtacgg cctgaccgag gccattgcca gccaccgcgc tctcgtgaaa 1560

gccagaggca cacggccctt cgtgatcagc agaagcacct ttgccggcca cggcagatac 1620

gccggacatt ggactggcga cgtgtggtcc tcttgggagc agctggcctc tagcgtgccc 1680

gagatcctgc agttcaatct gctgggcgtg ccactcgtgg gcgccgatgt gtgtggcttc 1740

ctgggcaaca cctccgagga actgtgtgtg cggtggacac agctgggcgc cttctaccct 1800

ttcatgagaa accacaacag ctgctgagc ctgccccagg aaccctacag ctttagcgag 1860

ccctgcacagc aggccatgcg gaaggccctg acactgagat acgctctgct gccccacctg 1920

tacaccctgt ttcaccaggc ccatgtggcc ggcgagacag tggccagacc tctgtttctg 1980

gaattcccca aggacagcag cacctggacc gtggaccatc agctgctgtg gggagaggct 2040

ctgctgatta ccccagtgct gcaggcaggc aaggccgaag tgaccggcta ctttcccctg 2100

ggcacttggt acgacctgca gaccgtgcct gtggaagccc tgggatctct gcctccacct 2160

cctgccgctc ctagagagcc tgccattcac tctgaggggcc agtgggtcac actgcctgcc 2220

cccctggata ccatcaacgt gcacctgagg gccggctaca tcataccact gcagggacct 2280

ggcctgacca ccaccgagtc tagacagcag ccaatggccc tggccgtggc cctgaccaaa 2340

ggcggagaag ctaggggcga gctgttctgg gacgatggcg agagcctgga agtgctggaa 2400

agaggcgcct atacccaagt gatcttcctg gccgggaaca acaccatcgt gaacgagctg 2460

gtgcgcgtga cctctgaagg cgctggactg cagctgcaga aagtgaccgt gctgggagtg 2520

gccacagccc ctcagcaggt gctgtctaat ggcgtgcccg tgtccaactt cacctacagc 2580

cccgacacca aggtgctgga catctgcgtg tcactgctga tgggagagca gtttctggtg 2640

tcctggtgct ga 2652

<210> 49

<211> 2652

<212> DNA

<213> artificial

<220>

<223> hGAAco2-delta-42

<400> 49

gcccatcctg gtagaccaag agctgtgcct acccaatgcg acgtgccacc caactcccga 60

ttcgactgcg cgccagataa ggctattacc caagagcagt gtgaagccag aggttgctgc 120

tacatcccag cgaagcaagg attgcaaggc gcccaaatgg gacaaccttg gtgtttcttc 180

cccccttcgt acccatcata taaactcgaa aacctgtcct cttcggaaat gggttatact 240

gccaccctca ccagaactac tcctactttc ttcccgaaag acatcttgac cttgaggctg 300

gacgtgatga tggagactga aaaccggctg catttcacta tcaaagatcc tgccaatcgg 360

cgatacgagg tccctctgga aacccctcac gtgcactcac gggctccttc tccgctttac 420

tccgtcgaat tctctgagga acccttcgga gtgatcgtta gacgccagct ggatggtaga 480

gtgctgttga accactactgt ggccccactt ttcttcgctg accagtttct gcaactgtcc 540

acttccctgc catcccagta cattactgga ctcgccgaac acctgtcgcc actgatgctc 600

tcgacctctt ggactagaat cactttgtgg aacagagact tggcccctac tccgggagca 660

aatctgtacg gaagccaccc tttttacctg gcgctcgaag atggcggatc cgctcacgga 720

gtgttcctgc tgaatagcaa cgcaatggac gtggtgctgc aaccttcccc tgcactcagt 780

tggagaagta ccggggggtat tctggacgtg tacatcttcc tcggaccaga acccaagagc 840

gtggtgcagc aatatctgga cgtggtcgga taccctttta tgcctcctta ctggggactg 900

ggattccacc tttgccgttg gggctactca tccaccgcca ttaccagaca ggtggtggag 960

aatatgacca gagcccactt ccctctcgac gtgcagtgga acgatctgga ctatatggac 1020

tcccggagag atttcacctt caacaaggac gggttccgcg attttcccgc gatggttcaa 1080

gagctccacc agggtggtcg aagatatatg atgatcgtcg acccagccat ttcgagcagc 1140

ggacccgctg gatcttatag accttacgac gaaggcctta ggagaggagt gttcatcaca 1200

aacgagactg gacagccttt gatcggtaaa gtgtggcctg gatcaaccgc ctttcctgac 1260

tttaccaatc ccactgcctt ggcttggtgg gaggacatgg tggccgaatt ccacgaccaa 1320

gtcccctttg atggaatgtg gatcgatatg aacgaaccaa gcaattttat cagaggttcc 1380

gaagacggtt gccccaacaa cgaactggaa aaccctcctt atgtgcccgg agtcgtggggc 1440

ggaacattac aggccgcgac tatttgcgcc agcagccacc aattcctgtc cactcactac 1500

aacctccaca acctttatgg attaaccgaa gctattgcaa gtcacagggc tctggtgaag 1560

gctagaggga ctaggccctt tgtgatctcc cgatccacct ttgccggaca cgggagatac 1620

gccggtcact ggactggtga cgtgtggagc tcatgggaac aactggcctc ctccgtgccg 1680

gaaatcttac agttcaacct tctgggtgtc cctcttgtcg gagcagacgt gtgtgggttt 1740

cttggtaaca cctccgagga actgtgtgtg cgctggactc aactgggtgc attctaccca 1800

ttcatgagaa accacaactc cttgctgtcc ctgccacaag agccctactc gttcagcgag 1860

cctgcacaac aggctatgcg gaaggcactg accctgagat acgccctgct tccacactta 1920

tacactctct tccatcaagc gcatgtggca ggagaaaccg ttgcaaggcc tcttttcctt 1980

gaattcccca aggattcctc gacttggacg gtggatcatc agctgctgtg gggagaagct 2040

ctgctgatta ctccagtgtt gcaagccgga aaagctgagg tgaccggata ctttccgctg 2100

ggaacctggt acgacctcca gactgtccct gttgaagccc ttggatcact gcctccgcct 2160

ccggcagctc cacgcgaacc agctatacat tccgagggac agtgggttac attaccagct 2220

cctctggaca caatcaacgt ccacttaaga gctggctaca ttatccctct gcaaggacca 2280

ggactgacta cgaccgagag cagacagcag ccaatggcac tggctgtggc tctgaccaag 2340

ggaggggaag ctagaggaga actcttctgg gatgatgggg agtcccttga agtgctggaa 2400

agaggcgctt acactcaagt cattttcctt gcacggaaca acaccattgt gaacgaattg 2460

gtgcgagtga ccagcgaagg agctggactt caactgcaga aggtcactgt gctcggagtg 2520

gctaccgctc ctcagcaagt gctgtcgaat ggagtccccg tgtcaaactt tacctactcc 2580

cctgacacta aggtgctcga catttgcgtg tccctcctga tgggagagca gttccttgtg 2640

tcctggtgtt ga 2652

<210> 50

<211> 2649

<212> DNA

<213> artificial

<220>

<223> hGAAco1-delta-43

<400> 50

caccccggca gacctagagc tgtgcctacc cagtgtgacg tgccccccaa cagcagattc 60

gactgcgccc ctgacaaggc catcacccag gaacagtgcg aggccagagg ctgctgctac 120

atccctgcca agcagggact gcagggcgct cagatgggac agccctggtg cttcttccca 180

ccctcctacc ccagctacaa gctggaaaac ctgagcagca gcgagatgggg ctacaccgcc 240

accctgacca gaaccacccc cacattcttc ccaaaggaca tcctgaccct gcggctggac 300

gtgatgatgg aaaccgagaa ccggctgcac ttcaccatca aggaccccgc caatcggaga 360

tacgaggtgc ccctggaaac cccccacgtg cactctagag cccccagccc tctgtacagc 420

gtggaattca gcgaggaacc cttcggcgtg atcgtgcgga gacagctgga tggcagagtg 480

ctgctgaaca ccaccgtggc ccctctgttc ttcgccgacc agttcctgca gctgagcacc 540

agcctgccca gccagtacat cacaggactg gccgagcacc tgagccccct gatgctgagc 600

acatcctgga cccggatcac cctgtggaac agggatctgg cccctacccc tggcgccaat 660

ctgtacggca gccacccttt ctacctggcc ctggaagatg gcggatctgc ccacggagtg 720

tttctgctga actccaacgc catggacgtg gtgctgcagc ctagccctgc cctgtcttgg 780

agaagcacag gcggcatcct ggatgtgtac atctttctgg gccccgagcc caagagcgtg 840

gtgcagcagt atctggatgt cgtgggctac cccttcatgc ccccttactg gggcctggga 900

ttccacctgt gcagatgggg ctactccagc accgccatca ccagacaggt ggtggaaaac 960

atgaccagag cccacttccc actggatgtg cagtggaacg acctggacta catggacagc 1020

agacgggact tcaccttcaa caaggacggc ttccgggact tccccgccat ggtgcaggaa 1080

ctgcatcagg gcggcagacg gtacatgatg atcgtggatc ccgccatcag ctcctctggc 1140

cctgccggct cttacagacc ctacgacgag ggcctgcgga gaggcgtgtt catcaccaac 1200

gagacaggcc agcccctgat cggcaaagtg tggcctggca gcacagcctt ccccgacttc 1260

accaatccta ccgccctggc ttggtgggag gacatggtgg ccgagttcca cgaccaggtg 1320

cccttcgacg gcatgtggat cgacatgaac gagcccagca acttcatccg gggcagcgag 1380

gatggctgcc ccaacaacga actggaaaat cccccttacg tgcccggcgt cgtgggcgga 1440

acactgcagg ccgctacaat ctgtgccagc agccaccagt ttctgagcac ccactacaac 1500

ctgcacaacc tgtacggcct gaccgaggcc attgccagcc accgcgctct cgtgaaagcc 1560

agaggcacac ggcccttcgt gatcagcaga agcacctttg ccggccacgg cagatacgcc 1620

ggacattgga ctggcgacgt gtggtcctct tgggagcagc tggcctctag cgtgcccgag 1680

atcctgcagt tcaatctgct gggcgtgcca ctcgtgggcg ccgatgtgtg tggcttcctg 1740

ggcaacacct ccgaggaact gtgtgtgcgg tggacacagc tgggcgcctt ctaccctttc 1800

atgagaaacc acaacagcct gctgagcctg ccccaggaac cctacagctt tagcgagcct 1860

gcacagcagg ccatgcggaa ggccctgaca ctgagatacg ctctgctgcc ccacctgtac 1920

accctgtttc accaggccca tgtggccggc gagacagtgg ccagacctct gtttctggaa 1980

ttccccaagg acagcagcac ctggaccgtg gaccatcagc tgctgtgggg agaggctctg 2040

ctgattaccc cagtgctgca ggcaggcaag gccgaagtga ccggctactt tcccctgggc 2100

acttggtacg acctgcagac cgtgcctgtg gaagccctgg gatctctgcc tccacctcct 2160

gccgctccta gagagcctgc cattcactct gagggccagt gggtcacact gcctgccccc 2220

ctggatacca tcaacgtgca cctgagggcc ggctacatca taccactgca gggacctggc 2280

ctgaccacca ccgagtctag acagcagcca atggccctgg ccgtggccct gaccaaaggc 2340

ggagaagcta ggggcgagct gttctgggac gatggcgaga gcctggaagt gctggaaaga 2400

ggcgcctata cccaagtgat cttcctggcc cggaacaaca ccatcgtgaa cgagctggtg 2460

cgcgtgacct ctgaaggcgc tggactgcag ctgcagaaag tgaccgtgct gggagtggcc 2520

acagcccctc agcaggtgct gtctaatggc gtgcccgtgt ccaacttcac ctacagcccc 2580

gacaccaagg tgctggacat ctgcgtgtca ctgctgatgg gagagcagtt tctggtgtcc 2640

tggtgctga 2649

<210> 51

<211> 2649

<212> DNA

<213> artificial

<220>

<223> hGAAco2-delta-43

<400> 51

catcctggta gaccaagagc tgtgcctacc caatgcgacg tgccacccaa ctcccgattc 60

gactgcgcgc cagataaggc tattacccaa gagcagtgtg aagccagagg ttgctgctac 120

atcccagcga agcaaggatt gcaaggcgcc caaatgggac aaccttggtg tttcttcccc 180

ccttcgtacc catcatataa actcgaaaac ctgtcctctt cggaaatgggg ttatactgcc 240

accctcacca gaactactcc tactttcttc ccgaaagaca tcttgacctt gaggctggac 300

gtgatgatgg agactgaaaa ccggctgcat ttcactatca aagatcctgc caatcggcga 360

tacgaggtcc ctctggaaac ccctcacgtg cactcacggg ctccttctcc gctttactcc 420

gtcgaattct ctgaggaacc cttcggagtg atcgttagac gccagctgga tggtagagtg 480

ctgttgaaca ctactgtggc cccacttttc ttcgctgacc agtttctgca actgtccact 540

tccctgccat cccagtacat tactggactc gccgaacacc tgtcgccact gatgctctcg 600

acctcttgga ctagaatcac tttgtggaac agagacttgg cccctactcc gggagcaaat 660

ctgtacggaa gccacccttt ttacctggcg ctcgaagatg gcggatccgc tcacggagtg 720

ttcctgctga atagcaacgc aatggacgtg gtgctgcaac cttcccctgc actcagttgg 780

agaagtaccg ggggtattct ggacgtgtac atcttcctcg gaccagaacc caagagcgtg 840

gtgcagcaat atctggacgt ggtcggatac ccttttatgc ctccttactg gggactggga 900

ttccaccttt gccgttgggg ctactcatcc accgccatta ccagacaggt ggtggagaat 960

atgaccagag cccacttccc tctcgacgtg cagtggaacg atctggacta tatggactcc 1020

1080

ctccaccagg gtggtcgaag atatatgatg atcgtcgacc cagccatttc gagcagcgga 1140

cccgctggat cttatagacc ttacgacgaa ggccttagga gaggagtgtt catcacaaac 1200

gagactggac agcctttgat cggtaaagtg tggcctggat caaccgcctt tcctgacttt 1260

accaatccca ctgccttggc ttggtgggag gacatggtgg ccgaattcca cgaccaagtc 1320

ccctttgatg gaatgtggat cgatatgaac gaaccaagca attttatcag aggttccgaa 1380

1440

acattacagg ccgcgactat ttgcgccagc agccaccaat tcctgtccac tcactacaac 1500

ctccacaacc tttatggatt aaccgaagct attgcaagtc acagggctct ggtgaaggct 1560

agagggacta ggccctttgt gatctcccga tccacctttg ccggacacgg gagatacgcc 1620

ggtcactgga ctggtgacgt gtggagctca tgggaacaac tggcctcctc cgtgccggaa 1680

atcttacagt tcaaccttct gggtgtccct cttgtcggag cagacgtgtg tgggtttctt 1740

ggtaacacct ccgaggaact gtgtgtgcgc tggactcaac tgggtgcatt ctacccattc 1800

atgagaaacc acaactcctt gctgtccctg ccacaagagc cctactcgtt cagcgagcct 1860

gcacaacagg ctatgcggaa ggcactgacc ctgagatacg ccctgcttcc acacttatac 1920

actctcttcc atcaagcgca tgtggcagga gaaaccgttg caaggcctct tttccttgaa 1980

ttccccaagg attcctcgac ttggacggtg gatcatcagc tgctgtgggg agaagctctg 2040

ctgattactc cagtgttgca agccggaaaa gctgaggtga ccggatactt tccgctggga 2100

acctggtacg acctccagac tgtccctgtt gaagcccttg gatcactgcc tccgcctccg 2160

gcagctccac gcgaaccagc tatacattcc gagggacagt gggttacatt accagctcct 2220

ctggacacaa tcaacgtcca cttaaagct ggctacatta tccctctgca aggacgga 2280

ctgactacga ccgagagcag acagcagcca atggcactgg ctgtggctct gaccaaggga 2340

ggggaagcta gaggagaact cttctgggat gatggggagt cccttgaagt gctggaaaga 2400

ggcgcttaca ctcaagtcat ttttccttgca cggaacaaca ccattgtgaa cgaattggtg 2460

cgagtgacca gcgaaggagc tggacttcaa ctgcagaagg tcactgtgct cggagtggct 2520

accgctcctc agcaagtgct gtcgaatgga gtccccgtgt caaactttac ctactcccct 2580

gacactaagg tgctcgacat ttgcgtgtcc ctcctgatgg gagagcagtt ccttgtgtcc 2640

tggtgttga 2649

<210> 52

<211> 2637

<212> DNA

<213> artificial

<220>

<223> hGAAco1-delta-47

<400> 52

cctagagctg tgcctaccca gtgtgacgtg ccccccaaca gcagattcga ctgcgcccct 60

gacaaggcca tcaccagga acagtgcgag gccagaggct gctgctacat ccctgccaag 120

cagggactgc agggcgctca gatgggacag ccctggtgct tcttcccacc ctcctacccc 180

agctacaagc tggaaaacct gagcagcagc gagatgggct acaccgccac cctgaccaga 240

accaccccca cattcttccc aaaggacatc ctgaccctgc ggctggacgt gatgatggaa 300

accgagaacc ggctgcactt caccatcaag gaccccgcca atcggagata cgaggtgccc 360

ctggaaaccc cccacgtgca ctctagagcc cccagccctc tgtacagcgt ggaattcagc 420

gaggaaccct tcggcgtgat cgtgcggaga cagctggatg gcagagtgct gctgaacacc 480

accgtggccc ctctgttctt cgccgaccag ttcctgcagc tgagcaccag cctgcccagc 540

cagtacatca caggactggc cgagcacctg agccccctga tgctgagcac atcctggacc 600

cggatcaccc tgtggaacag ggatctggcc cctacccctg gcgccaatct gtacggcagc 660

caccctttct acctggccct ggaagatggc ggatctgccc acggagtgtt tctgctgaac 720

tccaacgcca tggacgtggt gctgcagcct agccctgccc tgtcttggag aagcacaggc 780

ggcatcctgg atgtgtacat ctttctgggc cccgagccca agagcgtggt gcagcagtat 840

ctggatgtcg tgggctaccc cttcatgccc ccttactggg gcctgggatt ccacctgtgc 900

agatggggct actccagcac cgccatcacc agacaggtgg tggaaaacat gacgagcc 960

cacttcccac tggatgtgca gtggaacgac ctggactaca tggacagcag acgggacttc 1020

accttcaaca aggacggctt ccgggacttc cccgccatgg tgcaggaact gcatcagggc 1080

ggcagacggt acatgatgat cgtggatccc gccatcagct cctctggccc tgccggctct 1140

tacagaccct acgacgagggg cctgcggaga ggcgtgttca tcaccaacga gacaggccag 1200

cccctgatcg gcaaagtgtg gcctggcagc acagccttcc ccgacttcac caatcctacc 1260

gccctggctt ggtgggagga catggtggcc gagttccacg accaggtgcc cttcgacggc 1320

atgtggatcg acatgaacga gccagcaac ttcatccggg gcagcgagga tggctgcccc 1380

aacaacgaac tggaaaatcc cccttacgtg cccggcgtcg tgggcggaac actgcaggcc 1440

gctacaatct gtgccagcag ccaccagttt ctgagcaccc actacaacct gcacaacctg 1500

tacggcctga ccgaggccat tgccagccac cgcgctctcg tgaaagccag aggcacacgg 1560

cccttcgtga tcagcagaag cacctttgcc ggccacggca gatacgccgg acattggact 1620

ggcgacgtgt ggtcctcttg ggagcagctg gcctctagcg tgcccgagat cctgcagttc 1680

aatctgctgg gcgtgccact cgtgggcgcc gatgtgtgtg gcttcctggg caacacctcc 1740

gaggaactgt gtgtgcggtg gacacagctg ggcgccttct accctttcat gagaaaccac 1800

aacagcctgc tgagcctgcc cggaaccc tacagcttta gcgagcctgc acagcaggcc 1860

atgcggaagg ccctgacact gagatacgct ctgctgcccc acctgtacac cctgtttcac 1920

caggcccatg tggccggcga gacagtggcc agacctctgt ttctggaatt ccccaaggac 1980

agcagcacct ggaccgtgga ccatcagctg ctgtggggag aggctctgct gattacccca 2040

gtgctgcagg caggcaaggc cgaagtgacc ggctactttc ccctgggcac ttggtacgac 2100

ctgcagaccg tgcctgtgga agccctggga tctctgcctc cacctcctgc cgctcctaga 2160

gagcctgcca ttcactctga gggccagtgg gtcacactgc ctgcccccct ggataccatc 2220

aacgtgcacc tgagggccgg ctacatcata ccactgcagg gacctggcct gaccaccacc 2280

gagtctagac agcagccaat ggccctggcc gtggccctga ccaaaggcgg agaagctagg 2340

ggcgagctgt tctgggacga tggcgagagc ctggaagtgc tggaaagagg cgcctatacc 2400

caagtgatct tcctggcccg gaacaacacc atcgtgaacg agctggtgcg cgtgacctct 2460

gaaggcgctg gactgcagct gcagaaagtg accgtgctgg gagtggccac agcccctcag 2520

caggtgctgt ctaatggcgt gcccgtgtcc aacttcacct acagccccga caccaaggtg 2580

ctggacatct gcgtgtcact gctgatggga gagcagtttc tggtgtcctg gtgctga 2637

<210> 53

<211> 2637

<212> DNA

<213> artificial

<220>

<223> hGAAco2-delta-47

<400> 53

ccaagagctg tgcctaccca atgcgacgtg ccacccaact cccgattcga ctgcgcgcca 60

gataaggcta ttacccaaga gcagtgtgaa gccagaggtt gctgctacat cccagcgaag 120

caaggattgc aaggcgccca aatgggacaa ccttggtgtt tcttcccccc ttcgtaccca 180

tcatataaac tcgaaaacct gtcctcttcg gaaatgggtt atactgccac cctcaccaga 240

actactccta ctttcttccc gaaagacatc ttgaccttga ggctggacgt gatgatggag 300

actgaaaacc ggctgcattt cactatcaaa gatcctgcca atcggcgata cgaggtccct 360

ctggaaaccc ctcacgtgca ctcacgggct ccttctccgc tttactccgt cgaattctct 420

gaggaaccct tcggagtgat cgttagacgc cagctggatg gtagagtgct gttgaacact 480

actgtggccc cacttttctt cgctgaccag tttctgcaac tgtccacttc cctgccatcc 540

cagtacatta ctggactcgc cgaacacctg tcgccactga tgctctcgac ctcttggact 600

agaatcactt tgtggaacag agacttggcc cctactccgg gagcaaatct gtacggaagc 660

cacccttttt acctggcgct cgaagatggc ggatccgctc acggagtgtt cctgctgaat 720

agcaacgcaa tggacgtggt gctgcaacct tcccctgcac tcagttggag aagtaccgggg 780

ggtattctgg acgtgtacat cttcctcgga ccagaaccca agagcgtggt gcagcaatat 840

ctggacgtgg tcggataccc ttttatgcct ccttactgggg gactgggatt ccacctttgc 900

cgttggggct actcatccac cgccattacc agacaggtgg tggagaatat gaccagagcc 960

cacttccctc tcgacgtgca gtggaacgat ctggactata tggactcccg gagagatttc 1020

1080

ggtcgaagat atatgatgat cgtcgaccca gccatttcga gcagcggacc cgctggatct 1140

tatagacctt acgacgaagg ccttaggaga ggagtgttca tcacaaacga gactggacag 1200

cctttgatcg gtaaagtgtg gcctggatca accgcctttc ctgactttac caatcccact 1260

gccttggctt ggtgggagga catggtggcc gaattccacg accaagtccc ctttgatgga 1320

atgtggatcg atatgaacga accaagcaat tttatcagag gttccgaaga cggttgcccc 1380

aacaacgaac tggaaaaccc tccttatgtg cccggagtcg tgggcggaac attacaggcc 1440

gcgactattt gcgccagcag ccaccaattc ctgtccactc actacaacct ccacaacctt 1500

tgcaagtcac agggctctgg tgaaggctag agggactagg 1560

ccctttgtga tctcccgatc cacctttgcc ggacacggga gatacgccgg tcactggact 1620

ggtgacgtgt ggagctcatg ggaacaactg gcctcctccg tgccggaaat cttacagttc 1680

aaccttctgg gtgtccctct tgtcggagca gacgtgtgtg ggtttcttgg taacacctcc 1740

gaggaactgt gtgtgcgctg gactcaactg ggtgcattct acccattcat gagaaaccac 1800

aactccttgc tgtccctgcc acaagagccc tactcgttca gcgagcctgc acaacaggct 1860

atgcggaagg cactgaccct gagatacgcc ctgcttccac acttatacac tctcttccat 1920

caagcgcatg tggcaggaga aaccgttgca aggcctcttt tccttgaatt ccccaaggat 1980

tcctcgactt ggacggtgga tcatcagctg ctgtggggag aagctctgct gattactcca 2040

gtgttgcaag ccggaaaagc tgaggtgacc ggatactttc cgctgggaac ctggtacgac 2100

ctccagactg tccctgttga agcccttgga tcactgcctc cgcctccggc agctccacgc 2160

gaaccagcta tacattccga gggacagtgg gttacattac cagctcctct ggacacaatc 2220

aacgtccact taagagctgg ctacattatc cctctgcaag gaccaggact gactacgacc 2280

gagagcagac agcagccaat ggcactggct gtggctctga ccaagggagg ggaagctaga 2340

ggagaactct tctgggatga tggggagtcc cttgaagtgc tggaaagagg cgcttacact 2400

caagtcattt tccttgcacg gaacaacacc attgtgaacg aattggtgcg agtgaccagc 2460

gaaggagctg gacttcaact gcagaaggtc actgtgctcg gagtggctac cgctcctcag 2520

caagtgctgt cgaatggagt ccccgtgtca aactttacct actcccctga cactaaggtg 2580

ctcgacattt gcgtgtccct cctgatggga gagcagttcc ttgtgtcctg gtgttga 2637

<---

Claims (19)

1. A recombinant adeno-associated virus (AAV) vector for expression of a truncated alpha-glucosidase (GAA) polypeptide containing an expression cassette for expression of a GAA polypeptide, wherein the expression cassette contains a nucleic acid molecule encoding a truncated GAA polypeptide containing a deletion of 1-43 consecutive amino acids at its N-terminus compared to the parental GAA polypeptide, wherein said parental polypeptide corresponds to the precursor form of the GAA polypeptide free of its signal peptide, wherein said truncated GAA polypeptide further comprises a signal peptide fused to its N-terminus, and wherein said parental GAA polypeptide is a human GAA polypeptide having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33. 2. The recombinant AAV vector of claim 1, wherein said truncated GAA polypeptide has 6, 7, 8, 9, 10, 40, 41, 42, or 43 consecutive amino acids deleted at its N-terminus compared to said parental GAA polypeptide . 3. The recombinant AAV vector of claim 2, wherein said truncated GAA polypeptide has 8, 42, or 43 consecutive amino acids deleted at its N-terminus compared to said parental GAA polypeptide. 4. Recombinant AAV vector GAA according to any one of paragraphs. 1-3, wherein the truncated GAA polypeptide has the amino acid sequence shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 34, or SEQ ID NO: 35. 5. Recombinant AAV vector according to any one of paragraphs. 1-4, wherein said fused signal peptide is selected from the group consisting of SEQ ID NO: 3-7, in particular the signal peptide of SEQ ID NO: 3. 6. Recombinant AAV vector according to any one of paragraphs. 1-5, wherein said nucleic acid molecule encoding a truncated GAA polypeptide is a nucleotide sequence optimized to improve expression and/or immunological tolerance for said truncated GAA polypeptide in vivo, said nucleic acid molecule comprising the nucleic acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51. 7. Recombinant AAV vector according to any one of paragraphs. 1-6, wherein said nucleic acid molecule is operably linked to a promoter, in particular a promoter which is a specific liver promoter, preferably selected from the group consisting of an α-1-antitrypsin (hAAT) promoter, a transthyretin promoter, an albumin promoter and a thyroxin-binding promoter. globulin (TBG), wherein said expression cassette further optionally comprises an intron, in particular an intron selected from the group consisting of human β-globin b2 intron (HBB2), FIX intron, chicken β-globin intron, and SV40 intron, wherein said intron optionally is a modified intron such as the modified HBB2 intron of SEQ ID NO: 17, the modified FIX intron of SEQ ID NO: 19, or the modified chicken β-globin intron of SEQ ID NO: 21. 8. The recombinant AAV vector according to claim 7, which preferably contains in this order: enhancer; intron; a promoter, in particular a specific liver promoter; said nucleic acid molecule encoding said truncated GAA polypeptide; and a polyadenylation signal, wherein the expression cassette contains preferably in this order: an ApoE control region; an HBB2 intron, in particular a modified HBB2 intron; hAAT promoter; said nucleic acid molecule encoding said truncated GAA polypeptide; and a bovine growth hormone polyadenylation signal, wherein said expression cassette more specifically comprises a nucleotide sequence of any of SEQ ID NOs: 22-26. 9. Recombinant AAV vector according to any one of paragraphs. 1-8 which is a single-stranded or double-stranded self-complementary AAV vector, preferably an AAV vector with an AAV-derived capsid, such as the capsid of AAV1, AAV2, AAV2 variant, AAV3, AAV3 variant, AAV3B, AAV3B variant, AAV4, AAV5, AAV6, variant AAV6, AAV7, AAV8, AAV9, AAV10, such as AAVcy10 and AAVrh10, AAVrh74, AAVdj, AAV-Anc80, AAV-LK03, AAV2i8 and AAV swine, such as AAVpo4 and AAVpo6, or with a chimeric capsid. 10. The recombinant AAV vector according to claim 9, wherein the AAV vector has an AAV8, AAV9, AAVrh74 or AAV2i8 capsid, in particular an AAV8, AAV9 or AAVrh74 capsid, more specifically an AAV8 capsid. 11. The recombinant AAV vector of claim 1, wherein said nucleic acid molecule encoding said truncated GAA polypeptide is about 70 percent, about 80 percent, about 90 percent, about 95 percent, about 97 percent, about 98 percent, or about 99 percent identity with nucleotides 82-2859 SEQ ID NO: 8; or has at least 75 percent, at least 80 percent, or at least 82 percent sequence identity with the corresponding portion of the nucleotide sequence encoding SEQ ID NO: 1 or SEQ ID NO: 33; or has at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 98 percent, at least 99 percent, or 100 percent identity with the corresponding portion of the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11. 12. The recombinant AAV vector according to claim 1, wherein said truncated GAA polypeptide has the amino acid sequence of SEQ ID NO: 27, where said signal peptide has the amino acid sequence of SEQ ID NO: 3, and where said recombinant AAV vector further comprises one or more protein variants VP capsid derived from AAVrh74 or selected from the group consisting of RHM4-1, RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4 and RHM15-6. 13. A cell transformed with a recombinant AAV vector according to any one of paragraphs. 1-12 for expressing a truncated GAA polypeptide, wherein said cell is particularly a liver cell or a muscle cell. 14. The use of a recombinant AAV vector according to any one of paragraphs. 1-12 or cells according to claim 13 in an effective amount in a pharmaceutical composition with a pharmaceutically acceptable carrier for the treatment of GSDII (Pompe's disease). 15. The use of a recombinant AAV vector according to any one of paragraphs. 1-12 or cells according to claim 13 in an effective amount in a pharmaceutical composition with a pharmaceutically acceptable carrier for the treatment of GSDIII (Measles disease). 16. The use of a recombinant AAV vector according to any one of paragraphs. 1-12 or cells according to claim 13 for the manufacture of a medicament for the treatment of GSDII (Pompe's disease). 17. The use of a recombinant AAV vector according to any one of paragraphs. 1-12 or cells according to claim 13 for the manufacture of a medicament for the treatment of GSDIII (Measles disease).

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