Targeting the lysosome

Abstract

Means and methods are provided for targeting a protein of interest to a lysosome. The means and methods encompass proteinaceous substances, nucleic acid constructs, and cells transduced with these nucleic acid constructs.

Description

[0001] Targeting the lysosome

[0002] Description

[0003] The invention relates to means and methods for the delivery of proteinaceous substances (hereinafter also referred to as proteins) of interest to the lysosomes of cells in need of said protein(s) of interest. In particular, the invention relates to means and methods for replacing a (partially) absent and / or (partially) defective lysosomal protein, e.g. an enzyme, wherein said absence and / or defect leads to disease, for example a lysosomal storage disease. The invention relates to providing the (partially) absent and / or (partially) defective protein of interest to a subject in need thereof. More in particular, the invention relates to gene therapy methods to deliver genetic constructs to cells that thereupon can produce the protein(s) of interest and thereby enable delivery of said protein(s) of interest to lysosomes in need thereof. Typically, the proteins of interest are provided with a signal peptide and / or tag that enables delivery to lysosomes. The lysosomes may be in the cells producing the protein of interest themselves (in which case a signal sequence for secretion may not be required) or in other cells through expression in one cell, secretion (through the presence of a signal sequence) and uptake by the target cell and transport to the lysosome (through the presence of a tag). Upon synthesis and secretion of the protein of interest by a certain cell in a subject, the protein of interest will be contacted with the cell containing the lysosome in need of said protein of interest through the extracellular space or circulation system of the subject. Said cell typically has a specific binding partner for a counterpart in the protein of interest. A typical proteinaceous substance according to the invention thus will be produced in a first cell as a protein of interest provided with a signal sequence (for secretion) and a tag (for lysosomal uptake in a second cell). The proteinaceous substance may also comprise a means for being internalized by said second cell, in particular when the protein of interest does not have or does not sufficiently have such a means included in its structure.

[0004] Description of Figures

[0005] Figure 1 gives a schematic representation of cross correction according to the invention. The protein of interest is expressed in the cell that has been genetically modified through ex vivo gene therapy. The protein of interest in secreted from said cell. It binds to the target cell through a specific binding site associated with the surface of said cell, such as one of the receptors (in this case exemplified by M6PR). Thereceptor with the bound enzyme (protein of interest) is internalized and the receptor and enzyme are routed to the lysosome by the tag.

[0006] Figure 2: Schematics of three expression constructs. “P” denotes promoter, preferably constitutive such as MND, EFS or PGK; “sig” denotes signal peptide; ENZ; denotes protein of interest, e.g. lysosomal enzyme or a marker; “L1” and “L2” denote optional linker sequences in which L1 and L2 may be equal or different; “N-tag” denotes an N-terminal lysosomal targeting sequence, for example a sequence derived from IGF2, apolipoprotein or a combination thereof, preferably a sequence derived from IGF-2; “C-tag” denotes a C-terminal lysosomal targeting sequence, preferably amino acids 518-593 of human progranulin; “T2A” denotes a T2A sequence which is an exemplary 2A sequence used to result in a transcriptional jump by ribosomes ensuring that the translated sequences upstream and downstream of T2A produce two distinct proteins upon expression; “M” denotes a marker protein, for example zsGreen or mCherry. The constructs aim to drive expression of two separate proteins: the fusion protein (signal peptide, N- and / or C-terminal tags and protein of interest) and the marker protein (e.g. mCherry or zsGreen). In B) a plasmid map is depicted in which the expression cassettes depicted in A) are inserted to provide for plasmids expressing lentiviral vector genomes.

[0007] Figure 3: Example binding of a fusion protein. CI-M6PR: cation-independent mannose 6 phosphate receptor, IGF2: insulin-like growth factor 2-derived lysosomal targeting tag, PRGN: progranulin-derived lysosomal targeting tag, PSPN: prosaposin-derived lysosomal targeting tag.

[0008] Figure 4: Example binding of an alternative fusion protein. CI-M6PR: cationindependent mannose 6 phosphate receptor, IGF2: insulin-like growth factor 2-derived lysosomal targeting tag, PGRN: progranulin-derived lysosomal targeting tag, PSPN: prosaposin-derived lysosomal targeting tag.

[0009] Figure 5: Schematic representation of constructs coding for C-terminal tagged GAAco proteins. P: promoter. Sp: Signal Peptide. N-Tag: N-terminal lysosomal targeting sequence. GAAco: codon-optimized glucosidase acid alpha (GAA). C-Tag: C-terminal lysosomal targeting sequence. WPRE: Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element. PolyA: polyadenylation signal. GAA sp: signal peptide of GAA. hGAA: human glucosidase acid alpha. PGRN: progranulin-derived lysosomal targeting tag combining both prosaposin binding and sortilin binding domains. PSAP: progranulin-derived lysosomal targeting tag (prosaposin binding domain). SRT: sortilin-binding domain lysosomal targeting tag. RQLL: progranulin-derived tag (4 terminal amino acids of progranulin).Figure 6: Schematic representation of constructs encoding for N-terminal tagged GAA proteins. P: promoter. Sp: Signal Peptide. N-Tag: N-terminal lysosomal targeting sequence. GAAco: codon-optimized glucosidase acid alpha (GAA). C-Tag: C-terminal lysosomal targeting sequence. WPRE: Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element. PolyA: polyadenylation signal. GAA sp: signal peptide of GAA. hGAA: human glucosidase acid alpha. PGRN: progranulin-derived lysosomal targeting tag combining both prosaposin binding and sortilin binding domains. L: GAP (Glycine-Alanine-Proline) linker. PSAP: progranulin-derived lysosomal targeting tag (prosaposin binding domain). SRT: sortilin-binding domain lysosomal targeting tag. IGF2: insulin-like growth factor 2-derived lysosomal targeting tag. IGF2 sp: signal peptide of IGF2. PGRN sp: signal peptide of progranulin.

[0010] Figure 7: GAA activity in HEK293T cells upon transient transfection of plasmids encoding various C-tagged GAA variants. HEK293T cells were co-transfected with a (secreted) NanoLuc plasmid and GAA constructs. Forty-eight hours post-transfection relative luciferase unit (RLU) were measured in the supernatant. GAA activity was measured in (A) the cell lysates and (B) supernatant (n=2).

[0011] Figure 8: GAA activity in HEK293T cells upon transient transfection of various N-tagged GAA variants. HEK293T cells were co-transfected with a (secreted) NanoLuc plasmid and GAA constructs. Forty-eight hours post-transfection relative luciferase unit (RLU) were measured in the supernatant. GAA activity was measured in (A) the cell lysates and (B) supernatant (n=2).

[0012] Figure 9. Nanoluc expression detected in the supernatant of HEK293T cells transfected with various GAA constructs. Forty-eight hours post-transfection, relative luciferase units (RLU) were measured in the supernatant (n=2).

[0013] Figure 10. Western blot analyses to detect precursor and mature GAA forms in the cell lysates of the transfected HEK293T cells. GAPDH staining was used as loading control. Untr: untransfected HEK293T cells, rhGAA: recombinant human GAA was used as positive control.

[0014] Figure 11. Time course experiments to determine optimal secretion timepoint of GAA in conditioned medium. Conditioned medium was obtained by transfection of HEK293T cells with plasmids encoding GAA. GAA activity was determined for the (A) supernatant at 24, 48, and 72 hrs post-transfection and (B) cell lysates at 48 hrs posttransfection (n=1 / 2).Figure 12. Western blot analysis of expressed GAA in conditioned medium obtained from HEK293T cells transduced with lentiviral vectors encoding various GAA variants. rhGAA: recombinant human GAA was used as positive control. Samples were diluted in (A) PBS at pH 7.4 (A) and in (B) GAA activity assay buffer at pH 4.5-4.7.

[0015] Figure 13. GAA activity in GAA conditioned medium used for cross-correction of Pompe fibroblasts and HAP1 GAA (-)cells. GAA activity data refers to GAA conditioned medium conditioned production A) batch 1 and B) batch 2. Untr: untransduced HEK293T cells.

[0016] Figure 14. Expression of A) mannose 6 phosphate receptor (M6PR) (B) prosaposin, (C) sortilin (SORT1) and (was determined on various cells: healthy (GM07525) and patients’ fibroblasts (GM00244, GM20124, and GM20089) and cell lines: HAP1 GAA (+), HAP1 GAA (-), HEK293T, and HMC3 cells. Relative expression to actin B (ACTB) was determined.

[0017] Figure 15. Cross-correction / uptake experiments in HAP1 GAA deficient cells were performed using GAA conditioned medium (derived from LV transduced HEK293T cells). Negative control was untransfected HAP1 GAA (-) cells and positive control was HAP1 GAA (+) cells that express GAA (GAA+ / +; n=3 / 4) Statistical analysis: one-way ANOVA with Dunnett’s post-test. **: p<0.01; *** p<0.001.

[0018] Figure 16. Cross-correction / uptake experiments in Pompe patients’ fibroblasts using GAA conditioned medium (derived from LV transduced HEK293T cells). GAA uptake was determined in 3 Pompe fibroblast lines: A) GM20089, B) GM20124, and C) GM20244. Negative control was untransfected HAP1 GAA (-) cells and positive control was HAP1 GAA (+) cells that express GAA (n=2 / 5). Statistical analysis: one-way ANOVA with Dunnett’s post-test. **: p<0.01; *** p<0.001.

[0019] Figure 17. Western blot analysis of GAA in cell lysates of cross-corrected Pompe fibroblasts GM20089. (A) GAA protein detection, (B) GAPDH protein detection (loading control), and C) Relative GAA proteins quantification normalized for GAPDH, rhGAA: recombinant human GAA was used as running control. Myozyme was used for crosscorrection, and the cell lysate of the cross-corrected fibroblasts was used as a positive control.Figure 18. Western blot analysis of GAA in cell lysates of cross-corrected Pompe fibroblasts GM20124. (A) GAA protein detection and (B) GAPDH protein detection (loading control), and C) Relative GAA proteins quantification normalized for GAPDH. rhGAA and Myozyme were used as running controls.

[0020] Figure 19. Glycogen depletion (lysosomal fraction) was measured in cross-corrected Pompe patient’s fibroblasts A) GM20089 and B) GM20124. Untr GD: untreated fibroblasts; glucose-deprived, Myozyme: 250 ng and 500 ng was used for crosscorrection as positive controls. C) Glycogen levels over time in culture determined in healthy (GM07525) and Pompe patients’ fibroblasts (GM20089 and GM20124) in glucose depleted medium.

[0021] Figure 20. Characterisation of expressed GAA in conditioned medium by Western blot analysis. GAA with or without de-glycosylation by PNGase F (- / +). (A) C-terminally tagged GAA (A) and (B) N-terminally tagged GAA were characterised for their N-glycans content in the absence or presence of PNGase F. Myozyme was used as a positive control.

[0022] Figure 21. Western blot analysis of GAA in conditioned medium with or without Endo H. A) C-terminally tagged GAA and B) N-terminally tagged GAA were characterised for the presence of high-mannose glycans by Endo H enzyme treatment.

[0023] Figure 22. GAA uptake in the presence of receptor blocking agents. GAA activity was measured in cell lysates of HAP1 GAA (-) exposed to A) C-terminally tagged GAA and (B) N-terminally tagged GAA conditioned media (n=2 / 4) in the presence of M6P, RAP, AF38469, or Neurotensin. Statistical analysis: 2way ANOVA with Tukey’s multiple comparison test. ***: p<0.001.

[0024] Figure 23. Relative Fluorescence Units (RFU) measured in Gamillus GFP (gamillus green fluorescent protein) variants conditioned medium produced by plasmid transfection of HEK293T cells. Various Gamillus constructs encoding secreted Gamillus (SecGamillus) without or with C-or N-terminal peptide Tags were used to generate conditioned medium that is used for subsequent cross-correction studies.Figure 24. Cross-correction of tagged Gamillus Fluorescent Protein in HMC3 cells. A) Images of LysoTracker Red stained HMC3 cells, the cross-corrected HMC3 cells and the overlay pictures. B) Zoomed in images of LysoTracker Red stained HMC3 cells, the cross-corrected HMC3 cells and the overlay pictures. C) Pearson's r correlation for colocalization of tagged Gamillus proteins with Lysotracker Red. D) Ratio of Gamillus Fluorescence and LysoTracker Red double-positive area relative to the total LysoTracker Red stained area.

[0025] Lysosomal storage diseases

[0026] Lysosomal storage diseases (LSDs) are a group of over 70 rare inherited metabolic disorders that result from defects in lysosomal function.

[0027] Lysosomes are vesicles within cells that contain enzymes that digest large molecules and pass the fragments on to other parts of the cell for recycling. This process of digesting large molecules requires several critical enzymes. If one or more of these enzymes is / are defective due to a mutation, the large molecules accumulate within the cell, eventually killing it. Lysosomal storage disorders are caused by lysosomal dysfunction usually as a consequence of deficiency of a single enzyme required for the metabolism of lipids, glycoproteins (sugar-containing proteins), or mucopolysaccharides.

[0028] Individually, lysosomal storage diseases typically occur with incidences of less than 1:100,000 although incidences may be as high as around 1:20,000 births for Pompe and Gaucher disease. Moreover, as a group, the incidence of lysosomal storage diseases is about 1:5,000 - 1:10,000. Most of these disorders are autosomal recessively inherited such as Niemann-Pick disease, type C, but a few are X-linked recessively inherited, such as Fabry disease and Hunter syndrome (MPS II).

[0029] The lysosome is commonly referred to as the cell's recycling center because it processes large molecules and unwanted material into substances that the cell can use. Lysosomes break down this unwanted matter by enzymes, highly specialized proteins essential for survival. Lysosomal disorders are usually caused by a deficiency in a particular enzyme, for example when it does not function sufficiently or exists in too small an amount or is missing altogether. When this happens, the substrates of said enzyme accumulate in the cell. In other words, when the lysosome does not function normally, excess products destined for breakdown and recycling are stored in the cell. Another reason for accumulation of excess products may be adysfunction in transportation from the lysosome. If this process is managed by a proteinaceous substance located in or at the lysosome, the present invention is also suitable to treat such disorders.

[0030] Like other genetic disorders, individuals inherit lysosomal storage diseases from their parents. Although each disorder results from different mutations that translate into a deficiency in enzyme activity, they all share a common biochemical characteristic - all lysosomal disorders originate from an abnormal accumulation of substances inside the lysosome.

[0031] Lysosomal storage diseases often affect children and if left untreated they often die at a young age, many within a few months or years of birth.

[0032] Lysosomal storage diseases may be classified by the type of protein that is deficient and is causing buildup. Table 1 below provides an overview of lysosomal storage diseases and the affected genes and proteins.

[0033] Disease Gene Protein Aspartylglucosaminuria AGA Aspartylglucosaminidase CLN1 Disease CLN1 / PPT1 Palmitoyl protein thioesterase 1, PPT1 CLN10 disease CLN10 / CTSD Cathepsin D

[0034] CLN11 disease CLN11 / GRN Progranulin

[0035] CLN12 disease CLN12 / ATP13A2 ATPase type 13A2

[0036] CLN13 disease CLN13 / CTSF Cathepsin F

[0037] CLN14 disease CLN14 / KCTD7 Potassium channel tetramerization domain containing protein 7

[0038] CLN2 Disease CLN2 / TPP1 Tripeptidyl peptidase 1, TPP1

[0039] CLN3 disease CLN3 CLN3 transmembrane

[0040] protein / Battenin

[0041]

[0042] CLN4 disease CLN4 / DNAJC5 Cysteine string protein a

[0043] CLN5 disease CLN5 Ceroid-lipofuscinosis neuronal protein 5

[0044] CLN6 disease CLN6 Ceroid-lipofuscinosis neuronal protein 6

[0045] CLN7 disease CLN7 / MFSD8 Major facilitator superfamily domain-containing protein 8 CLN8 disease CLN8 Protein CLN8

[0046] Fabry disease GLA alpha-galactosidase

[0047] Fucosidosis FUCA1 Fucosidase

[0048] Galactosialidosis CTSA Cathepsin A

[0049] Gaucher disease GBA glucocerebrosidase

[0050] GM1 gangliosidosis GLB1 GM1 gangliosidosis

[0051] GM2 Activator Deficiency GM2A GM2 Activator Protein

[0052] Infantile sialic acid storage SLC17A5 Sialin

[0053] disease

[0054] Krabbe disease GALC Galactosylceramidase

[0055] Metachromatic leukodystrophy ASA Aryl Sulfatase A

[0056] MPS I (Hurler, Scheie IDUA Alpha-L-iduronidase syndrome)

[0057] MPS II (Hunter syndrome) IDS Iduronate-2-sulfatase

[0058] MPS 111 A (Sanfilippo syndrome) SGSH Heparan-N-sulfatase

[0059] MPS 111 B (Sanfilippo syndrome) NAGLU N-acetylglucosaminidase

[0060] MPS IIIC (Sanfilippo syndrome) HGSNAT Acetyl CoA glucosamine N- acetyltransferase

[0061] MPS HID (Sanfilippo syndrome) GNS N-acetyl-glucosamine-6- sulfatase

[0062]

[0063] MPS IVA (Morquio syndrome A) GALNS N-acetylgalactosamine-6- sulfate sulfatase

[0064] MPS IVB (Morquio syndrome B) GLB1 β-galactosidase

[0065] MPS IX HYAL1 Hyaluronidase

[0066] MPS VI (Maroteaux-Lamy ARSB Arylsulfatase B

[0067] syndrome)

[0068] MPS VII (Sly syndrome) GUSB β-glucuronidase

[0069] Mucolipidosis I (Sialidosis) NEU1 a-neuraminidase

[0070] Mucolipidosis ll / lll GNPTAB, UDP-N-acetylglucosamine- GNPTG 1 -phosphotransferase Mucolipidosis IV MCOLN1 Mucolipin-1

[0071] Niemann-Pick Disease type A / B SMPD1 sphingomyelinase

[0072] Acid sphingomyelinase

[0073] deficiency

[0074] Niemann-Pick disease type C1 NPC1 NPC1 Protein

[0075] Niemann-Pick disease type C2 NPC2 NPC2 Protein

[0076] Pompe disease GAA alpha-glucosidase

[0077] Salla disease SLC17A5 Sialin

[0078] Sandhoff disease HEXB Hexosaminidase B

[0079] Schindler disease NAGA a-N- acetylgalactosaminidase Tay-Sachs disease HEXA Hexosaminidase A

[0080] Wolman Disease, lysosomal LIPA Lysosomal acid lipase acid lipase deficiency

[0081] a-mannosidosis MAN2B1 a-mannosidase

[0082] β-mannosidosis MANBA β-mannosidase

[0083]

[0084] Any of the listed proteins, or therapeutically active parts / derivatives thereof, could thus be considered as part of the proteins (or proteinaceous substances) of interest for delivery to lysosomes of cells in need thereof.Lysosomal disease treatment options

[0085] The current treatment of lysosomal storage diseases typically is a form of replacement therapy, also known as enzyme replacement therapy (ERT). In ERT, patients are provided with the missing / defective protein through parenteral administration. The provided proteins must enter the cells that have unwanted lysosomal storage (typically buildup of lysosomal substrate) and enter the lysosome to exert their (enzymatic) action. The proteins administered to the patients may be foreign proteins to them if there is no enzyme naturally present (no cross-reactive immunologic material (CRIM)), and after a certain number of administrations an immune response may occur. Although it is sometimes possible to provide higher doses that cannot be neutralized by the immune system, the treatment is lifelong, invasive, requires frequent hospital visits, expensive, and often inadequate to resolve all symptoms. Moreover, the replacement proteins typically do not cross the blood brain barrier, which prohibits amelioration of the buildup of substrate in the central nervous system, leading to neurological dysfunctions. There is thus a need to provide improved proteinaceous substances that overcome the limitations of current enzyme replacement therapy.

[0086] In one embodiment, the invention provides for a proteinaceous substance comprising an amino acid sequence of interest and at least a binding domain for a membrane receptor and / or a binding domain for prosaposin. The term “amino acid sequence” as used herein refers to an order of amino acid (including naturally occurring or synthetic amino acids) residues forming a (poly)peptide or protein, whether native, recombinant or synthetic, optionally including posttranslational modifications and variants that retain the intended biological activity. Preferred amino acid sequences are polypeptides. Therefore, in a further embodiment, the invention provides for a proteinaceous substance comprising a polypeptide of interest and at least a binding domain for a membrane receptor and / or a binding domain for prosaposin. The term “proteinaceous substance” as used herein refers broadly to compounds comprising (poly)peptide chains, including naturally occurring proteins, recombinant proteins, synthetic (poly)peptides, protein fragments, and protein conjugates, as well as complexes and assemblies thereof and includes post-translational modifications such as for example glycosylation, lipidation, or phosphorylation. As used herein, a proteinaceous substance includes agents or molecules that comprise peptides or proteins or contain a significant amount of protein.The term “polypeptide” includes any chain of amino acids or protein, whether obtained from a natural source or produced by recombinant expression or chemical synthesis, and includes post-translationally modified forms such as glycosylated, phosphorylated, acetylated, amidated, lipidated, or disulfide-bonded variants. “Variants” or “protein variants” include allelic variants, isoforms, and engineered variants comprising amino acid substitutions, insertions, deletions, truncations, extensions, or domain swaps, as well as sequence variants exhibiting at least a certain amount of amino acid sequence identity, such as 70%, 80%, 90%, 95%, 98%, or 99% identity to a reference sequence and retaining the intended biological activity.

[0087] In the context of lysosomal storage diseases, a proteinaceous substance thus refers to a compound comprising therapeutic proteins, enzymes, or fragments thereof that can complement the missing or defective protein (often an enzyme) and / or circumvent, bypass or correct any barrier affecting the degradation of any aberrantly stored lysosomal materials. In one embodiment the polypeptide of interest can thus be any of the listed proteins (or a therapeutically (hyper)active fragment and / or derivative thereof) as listed in Table 1. A preferred polypeptide of interest is GAA, which plays a role in Pompe disease. Outside the context of lysosomal storage diseases, the skilled person would understand that other polypeptides of interest can also be used in the proteinaceous substance of the present invention. Interestingly, the present inventors have demonstrated that a protein such as GFP can successfully be expressed and targeted to the lysosome of recipient cells through fusion with a lysosomal targeting tag according to the invention (see the example section), demonstrating that the polypeptide of interest can indeed be varied.

[0088] In some embodiments, the proteinaceous substance may be a conjugate, a complex, or a derivative formed by covalent or noncovalent association with other molecules. Typically, the proteinaceous substance can be a combination of two or more (fragments of) proteins that are linked together through a linker, which can be a chemical linker or a peptide or protein linker and at least one of the two or more (fragments of) proteins can influence its distribution, metabolism and / or efficacy. “Fragments” include portions of a reference protein that maintain binding and / or function (in kind, not necessarily in amount), such as enzymatic function, and can refer for example to receptor binding domains or catalytic subdomains of enzymes.Linkers to combine the two or more (fragments of) proteins are well known in the art. The term “linker” means any peptide or non-peptidic spacer covalently connecting two (fragments of) proteins, including flexible glycine-serine linkers, structured linkers, protease-cleavable linkers, and glycan or PEG-based spacers. Linkers may be flexible (such as (G4S)n) or (semi-)rigid (such as (G4P)n).

[0089] When amino acid sequences of different origin are linked together through a peptide bond, or when a peptide linker is used for fusion of the two or more (fragments of) proteins, the proteinaceous substance is typically referred to as so-called fusion protein. The term “fusion protein” refers to at least one single polypeptide chain. Fusion proteins comprising a polypeptide of interest and a lysosomal targeting sequence are useful as enzyme replacement but could also be very useful for any other application that requires processing through the lysosomal route within a cell. When a chemical linker is used to link the two or more (fragments of) proteins, the proteinaceous substance is a so-called chemically linked protein. The term “chemically linked protein” is defined as understood by a person skilled in the art as a construct where two or more protein or peptide moieties are (covalently) attached to each other using chemical methods, including the use of cross-linking agents, such as bifunctional reagents, click chemistry, or enzymatic conjugation.

[0090] As used herein, the term “binding domain” or “targeting domain” or simply “tag” (all terms are used interchangeably) means a part of a protein or peptide that, by virtue of its three dimensional structure and constituent amino acid residues, specifically recognises and reversibly binds a selected molecular target (for example, a receptor, ligand, cofactor or partner protein) with measurable affinity under physiological conditions, thereby mediating the interaction required for recognition, uptake, trafficking and / or biological activity. A binding domain may be native, modified or engineered (such as a hybrid domain), may include necessary flanking residues for proper folding, and retains its binding function when combined with, a heterologous polypeptide. Preferred binding domains are lysosomal targeting domains.

[0091] According to the invention, the targeting of the polypeptides of interest is achieved / improved by providing these polypeptides with an amino acid sequence that is specifically recognized by specific binding partners associated with cells (such as receptors on the cells) that upon binding of said amino acid sequence directly or indirectly lead to endocytosis of the protein of interest and then towards the lysosomes to achieve an intended goal, for example breakdown of undesirable substrates for theproteins of interest and / or further buildup of such unwanted substrate, or at least slowing down the further buildup of such undesired substrates.

[0092] The term “membrane receptor” means a protein embedded in or associated with a cellular lipid bilayer that specifically recognises and binds extracellular compounds, and transduces this interaction into cellular responses such as endocytosis, trafficking, or signalling. A membrane receptor typically comprises an extracellular compoundbinding region, one or more transmembrane segments, and an intracellular domain mediating downstream effects. Preferred membrane receptors undergo recycling to lysosomal compartments and include for example a mannose 6 phosphate receptor, LDL-receptors (e.g. LRP1; Low Density Lipoprotein Receptor-Related Protein 1), or sortilin (SORT1). The cation-independent mannose-6 phosphate receptor (CI-M6PR) recognizes mannose residues for trafficking enzymes to lysosomes and has an additional binding site for IGF2 (IGF2R), which is beneficial because IGF2 has been used as a lysosomal targeting tag. IGF-2 thus leverages another binding site of Cl-M6PR. The LRP-1 belongs to the LDL-receptor family and the ApoE receptor binding domain (or a tandem repeat thereof) for LRP-1 has also been used as a lysosomal targeting tag. Sortilin (SORT1) belongs to the VSP10 receptor family and is known as “trash” receptor in many cells and plays a role in progranulin homeostasis, much like CI-M6PR plays a role in IGF-2 homeostasis. Progranulin binds to SORT1 leading to receptor mediated endocytosis. Interestingly, progranulin is still partially localized to lysosomes in sortilin-deficient neurons, which means there is a second lysosomal targeting mechanism for progranulin. Indeed, this is mediated by prosaposin, which is a precursor of saposin peptides (A, B, C, and D) that serve as activators of lysosomal sphingolipid metabolizing enzymes. Prosaposin bridges progranulin towards CI-M6PR and LRP1 which is dependent on Granulin E1, independent of sortilin. Granulin E has similar affinity to prosaposin as full progranulin.

[0093] Therefore, in a further embodiment, the invention provides for a proteinaceous substance comprising a polypeptide of interest and at least a binding domain for a membrane receptor selected from a mannose 6 phosphate receptor, an LRP1 and a sortilin, and / or a binding domain for prosaposin. In an alternative embodiment, the invention provides for a proteinaceous substance comprising a polypeptide of interest and at least a binding domain for prosaposin. Preferably, a binding domain of sortilin and / or a binding domain for prosaposin may be present in the proteinaceous substance. Thus, in another embodiment the invention provides for a proteinaceoussubstance comprising a polypeptide of interest and at least a binding domain for sortilin and / or a binding domain for prosaposin.

[0094] Prosaposin is a secreted and lysosomal glycoprotein precursor of the four saposin activator peptides (saposins A, B, C, and D) that facilitate the degradation of sphingolipids by lysosomal hydrolases. Prosaposin is synthesised as a single polypeptide, trafficked to lysosomes and extracellular spaces, and can bind partner proteins including progranulin as well as mediate interactions with receptors such as the cation-independent mannose-6-phosphate receptor and LRP1, thereby contributing to lysosomal targeting and lipid catabolism. Any fragment or derivative of prosaposin that has the same or similar function is therefor of use in the present invention.

[0095] It is known that sortilin plays a role in progranulin homeostasis and binding of progranulin to SORT1 results in receptor mediated endocytosis of progranulin. Interestingly, progranulin is still partially localized to lysosomes in sortilin-deficient neurons. Therefore, at least one binding domain of the proteinaceous substance of the present invention further comprises a granulin binding domain.

[0096] The binding domain of the proteinaceous substance according to the invention is preferably positioned at a terminal end of the proteinaceous substance and can thus be at the C-terminal end or the N-terminal end.

[0097] Thus, in one embodiment the proteinaceous substance is configured with the lysosomal targeting tag at the C-terminus of the polypeptide of interest. A C-terminal tag may be coupled to the polypeptide of interest through flexible linkers to avoid conformational constraints, to accommodate posttranslational modifications of the enzyme (including glycosylation and maturation), and to maintain high affinity binding to target receptors.

[0098] For some proteins, including for example GAA, C-terminal tagging has historically proven to be difficult, often resulting in non-functional proteins. The tags according to the invention however, especially the preferred ones have targeting activity for the protein of interest at both termini, although not always to the same extent. Anyhow, the tag can also be located on the N-terminal side of the polypeptide of interest. Thus, in another embodiment, the proteinaceous substance is configured such that the lysosomal targeting tag is positioned at the N-terminus of the polypeptide of interest. To minimise steric interference with folding and catalytic function of the protein of interest, the tag is preferably separated from the protein of interest by a linker. Suitablelinkers include glycine-rich spacers such as G4S repeats (for example, (GlyGlyGlyGlySer)n) that confer flexibility and aqueous solubility, or G4P repeats (for example, (GlyGlyGlyGlyPro)n) that introduce defined spacing with modest rigidity. A GAP linker may also be employed to balance flexibility with structural separation. The length and composition of the linker are selected to preserve enzymatic activity, promote efficient secretion, and maintain receptor binding affinity of the tag as much as possible. Where desired, protease-cleavable motifs may be incorporated into or adjacent to the linker to enable conditional release of the tag in endosomal or lysosomal compartments; conversely, non-cleavable designs are preferred when sustained receptor engagement or cotransport is advantageous.

[0099] The choice between N-terminal and C-terminal positioning is guided by structural, functional (including protein / enzyme activity) and trafficking considerations. For example, the following are all considerations for the choice. For secreted lysosomal enzymes synthesised as precursors that undergo proteolytic maturation, C-terminal tags can be advantageous to avoid interfering with pro-peptide processing and / or to situate the tag away from regions participating in catalytic activation. For enzymes or payloads where receptor engagement is enhanced by early N-terminal display one may not want to block this with a tag. However, combining an N-terminal tag with a secretion signal that enhances uptake via specific pathways may be beneficial in other situations. In either case, secretion signals may be native to the polypeptide of interest or may be from a different polypeptide, engineered, or modified; when the tag is N-terminal, the signal peptide precedes the tag to ensure entry into the secretory pathway. Linker selection is tailored empirically to preserve activity and to optimise trafficking, with G4S providing high flexibility and hydrophilicity, G4P offering spacing with modest rigidity, and GAP linkers can be used as a reliable, shorter but soluble linker.

[0100] In further embodiments, dual-tag configurations are contemplated in which an N-terminal tag, for example IGF2-derived to engage CI-M6PR, is combined with a C-terminal tag, for example a progranulin-derived sequence to engage sortilin and / or prosaposin with subsequent CI-M6PR- and LRP1 -mediated pathways. Such redundant or complementary targeting increases the probability of uptake across diverse tissues expressing different receptor repertoires and enhances delivery to lysosomes of recipient cells in vivo. In these dual constructs, independent linkers are used at eachjunction to maintain proper folding and activity, and cleavage or stabilising mutations may be introduced selectively within each tag to tune residence time on receptors versus release in acidic compartments. Dual-tags may also comprise sequential N-terminal tags or sequential C-terminal tags - or “sandwiched” tags where a second tag is positioned in between two parts of another tag. In a further embodiment, the proteinaceous substances of the present invention further comprise a signal peptide for secretion from a cell in which it is expressed. The signal peptide can be native to the polypeptide of interest or can be modified, engineered or a signal peptide from a different protein.

[0101] As is clear from the above, the proteinaceous substances of the present invention are suitable for enzyme replacement and can be administered to a subject in need thereof via routes of administration and dosage regimens that are known to the person skilled in the art. The term “subject” refers to a mammal, preferably a primate, more preferably a human. In enzyme replacement therapy, the proteinaceous substances, upon proper formulation as known to the skilled person, can be administered repeatedly.

[0102] Since most of the LSDs are single protein defects (of monogenic origin), these diseases have long been an area of interest for gene therapy. The present invention can be used both in the context of enzyme replacement and gene therapy to target a protein of interest to a lysosome.

[0103] For gene therapy purposes, nucleic acid constructs encoding the proteinaceous substances of the present invention are provided. Thus, in one embodiment, the invention provides a nucleic acid construct comprising a nucleic acid sequence encoding a polypeptide of interest and a nucleic acid sequence encoding a lysosomal targeting sequence. Preferably, the nucleic acid construct comprises a nucleic acid sequence encoding a proteinaceous substance of the present invention. Preferably, the nucleic acid construct further comprises a promoter. As used herein, a “promoter” is a nucleic acid regulatory sequence operably linked to a coding region that directs transcription initiation by recruiting and positioning RNA polymerase and associated transcription factors. A promoter may be constitutive, inducible or tissue- or celltypespecific, may be native, modified or synthetic, and typically comprises core elements such as a transcription start site and, optionally, motifs including TATA-box, initiator and upstream regulatory sequences. In a preferred embodiment, the promoter is a constitutive promoter suitable for clinical gene therapy, for example EFS, PGK, EF1a, MND or a synthetic promoter. As such, the nucleic acid constructs comprising nucleicacid sequences encoding the proteinaceous substances of the present invention may be arranged to express either N-terminally tagged or C-terminally tagged variants under the control of constitutive promoters suitable for clinical gene therapy, for example EFS, PGK, EF1a, MND or synthetic promoters.

[0104] For gene therapy purposes, codon optimisation of the polypeptide of interest may be used to enhance expression without altering function. As used herein, “codon optimisation” is the process of altering a nucleotide coding sequence without changing the encoded amino acid sequence, to improve expression in a chosen host by adapting codon usage, GC content, mRNA structure, regulatory motifs, and other sequence features to the host’s translational and transcriptional preferences while maintaining the intended protein product. Various algorithms for codon optimisation are well known to the skilled person.

[0105] The skilled person further knows that regulatory elements such as Kozak sequences and (mutated) Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element (WPRE) may be included in the nucleic acid construct as appropriate, as well as a polyA. Typically, when the nucleic acid constructs of the present invention are combined with regulatory elements necessary for correct expression, the skilled person knows that this refers to an expression cassette. As used herein, an “expression cassette” refers to a nucleic acid construct in which a coding sequence is operably linked to the regulatory elements necessary for its transcription and, where applicable, translation in a host cell. The cassette typically comprises a promoter, a transcription start site, the coding region (for example a nucleic acid sequence encoding the proteinaceous substance of the present invention), and a transcription termination / polyadenylation signal, and may further include elements such as a Kozak (or Shine-Dalgarno) sequence, introns, enhancers, posttranscriptional regulatory elements (for example WPRE), and selectable or detectable markers. An expression cassette may be configured for constitutive, inducible, tissue-specific or cell typespecific expression and can be incorporated into a plasmid, viral vector or other gene delivery vehicle.

[0106] Construct selection and optimisation for N-terminal versus C-terminal configurations are determined using in vitro assays of secretion, receptor binding, cellular uptake and lysosomal localisation, as well as functional readouts such as enzyme activity in medium / supernatant or in recipient cells, and substrate depletion. The skilled person will select the orientation and linker architecture based on the structural features of the polypeptide of interest, the receptor specificity of the tag, and empiricalperformance in secretion and cross-correction assays (e.g. as given in the example section), without departing from the scope of the invention. Using either N-tags or C-tags and GFP or GAA as a polypeptide of interest, the inventors have surprisingly shown that fluorescence or enzyme activity could be measured in the supernatant of transfected cells. These data demonstrate that tagged-GAA and tagged-GFP variants are active and secreted upon transfection in cells. Furthermore, cross-corrected recipient cells showed increased intracellular GAA activity levels which were accompanied by decreased glycogen levels, demonstrating successful crosscorrection. The presence of precursor and mature GAA of the N- and C-terminally tagged GAA variants was demonstrated both in cell lysates of transfected cells and in cross-corrected cells. The presence of mature GAA in the lysosome of recipient cells indicated proper processing.

[0107] Gene therapy

[0108] Gene therapy in one aspect relevant for the present invention involves the replacement and / or provision and / or supplementation and / or completion of a (defective and / or absent) gene in the genome of a subject. For example, where the defective and / or absent gene was leading to insufficient presence or function of a required protein, a gene encoding for a functional version of that protein can be provided to a subject in need thereof. In the case of lysosomal storage diseases, this means that the lysosomal enzyme that is functionally missing or insufficiently present / functioning in a subject's cells’ lysosomes must be replaced and / or provided and / or supplemented and / or completed. Either one has to target these cells directly or one has to rely on crosscorrection. This entails that other cells than the cells affected by the disease are provided with a correct copy of the missing or defective gene such that the correct copy encodes for a correct enzyme that is otherwise missing or is otherwise a defective enzyme. These cells, now carrying a correct copy of the gene, then produce the enzyme, secrete it and the secreted protein can be taken up by the cell(s) having the lysosome storing its substrate. In the cell(s), the enzyme needs to be directed to the lysosome as well. If the level of cross-correction needs to be further enhanced, it may be suitable to use hyperactive variants of the defective / missing protein if these are available.Reviews on lysosomal storage diseases and gene therapy methods therefor are presented in Kido et al., 2023 and Kohn et al., 2023. These references are incorporated herein by reference.

[0109] The most common viral vector systems used in gene therapy include retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses. Kohn et al. give a good summary of their characteristics and suitability for various applications of gene therapy. According to the present invention, the preferred gene therapy is lentivirus-based wherein the gene encoding the protein of interest is provided to cells of the subject ex vivo. Typically, the cells to be provided with the lentiviral vector are haematopoietic stem cells.

[0110] The basis for ex vivo gene therapy and haematopoietic stem cell transplantation for LSDs lies in (1) identification of pathways such as the CI-M6P receptor pathway, through which lysosomal enzymes secreted by a cell can bind to the CI-M6P receptor on the cell membrane of surrounding cells with subsequent uptake and transport to the lysosome and (2) transplanted haematopoietic stem cells (HSCs) or their progeny travel through the entire body and can settle in various tissues to contribute to the local populations of haematopoietic progeny, including for example macrophages and microglia, and thus become sources of the required lysosomal enzyme (the protein of interest). The mechanism of cross-correction is summarised in figure 1.

[0111] According to the invention, the targeting of the proteins of interest is improved by providing these proteins with an amino acid sequence that is specifically recognized by e.g. receptors on the cells that upon binding of said amino acid sequence lead to endocytosis of the protein of interest and thus towards the lysosomes that store undesirable substrates for the proteins of interest. Such receptors include CI-M6PR, sortilin and LDL-receptors (e.g. LRP1), with for example the following binding partners containing such targeting sequences: IGF2 and prosaposin (CI-M6PR), progranulin (sortilin), Apolipoproteins and prosaposin (LRP1). In a gene therapy setting, this means that the amino acid sequence of the protein of interest is encoded by a gene that also encodes the targeting sequence (the so-called tag) in a manner such that they are functionally (and / or physically) coupled. How to produce such coupling, such as fusions and chemical linkages, between a tag sequence and a sequence of a polypeptide of interest is well established in the art. In a gene therapy setting, suchfused genes are incorporated in a gene delivery vehicle such as a viral particle, in particular a lentiviral particle. Alternatively, besides fusion of the protein of interest and the tag, the skilled person knows how to create other forms of combinations, such as for example, by covalently linking the two via a chemical linker, by putting the tag on a delivery vehicle of the protein of interest. As used herein, a “combination of a polypeptide of interest and a binding domain” thus refers to any form of functional and / or physical coupling between these two.

[0112] According to the invention, a gene therapy vehicle is provided comprising a nucleic acid construct comprising a nucleic acid sequence encoding the proteinaceous substance of the present invention. The gene therapy vehicle can be any vehicle suitable for delivering a nucleic acid construct to a cell, tissue or organ and can include lipid nanoparticles as well as viral particles. Preferably, the gene therapy vehicle is a viral particle. In a preferred embodiment, lentiviral particles are employed as gene delivery vehicles. Lentiviral vectors offer stable, long term expression of therapeutic transgenes through integration into the host cell genome, thereby supporting durable production of proteins of interest such as enzymes necessary for sustained crosscorrection in lysosomal storage diseases. The skilled person knows how to use or select self-inactivating, third-generation lentiviral designs.

[0113] As stated above, according to the present invention, the preferred gene therapy is lentivirus-based wherein the gene encoding the proteinaceous substance of the present invention is provided to cells of the subject ex vivo. Typically, the cells are haematopoietic stem cells. Unlike non-integrating or episomal systems that may be diluted with cell division, lentiviral integration preserves transgenes across cell division in proliferating haematopoietic stem and progenitor cells and their progeny, including macrophages and microglia that disseminate systemically and contribute to delivery of the proteinaceous substance in peripheral tissues and the central nervous system. Lentiviral vectors efficiently transduce haematopoietic stem cells (HSCs) ex vivo. The transduced HSCs can then be administered to a subject in need thereof for subsequent engraftment of corrected cells.

[0114] Thus, in one embodiment the invention provides for a cell of a subject transduced with a nucleic acid construct of the present invention. Preferably, the cell is transduced with a viral vector, more preferably a lentiviral vector. The transduced cell is preferably of a mammalian subject, preferably a human subject. More preferably the cell is a human stem cell, most preferably a human haematopoietic stem cell.From a regulatory and clinical standpoint, lentiviral ex vivo haematopoietic stem cell gene therapy has an established track record, with multiple approved products demonstrating durable efficacy and manageable risk profiles when combined with careful vector design, defined transduction conditions, and controlled vector copy numbers and monitoring thereof. The ex vivo workflow permits stringent release testing of the cell product, including assessment of identity, purity, potency, and integration metrics, and enables precise dosing by controlling vector copy number per cell. Furthermore, lentiviral vectors accommodate expression cassettes of clinically relevant size, including constitutive promoters, signal peptides, lysosomal targeting tags, polyadenylation signal, and optional regulatory elements such as Kozak motifs and WPRE, without exceeding packaging constraints.

[0115] In the context of lysosomal storage diseases, these features collectively provide sustained systemic supply of the proteinaceous substance from engrafted haematopoietic progeny, support potential immune tolerance through thymic and myeloid antigen presentation, and facilitate cross-correction across diverse tissues. Accordingly, lentiviral particles are preferred to achieve durable, controlled, and clinically translatable expression of proteinaceous substances comprising a polypeptide of interest and lysosomal targeting tags. The sustained systemic supply of proteinaceous substance ensures that it is available for uptake by other cells, including cells that do not produce said substance, allowing for cross-correction. Thus according to the invention, we provide a method for delivering a polypeptide of interest to a lysosomal compartment in a cell of a subject, comprising combining said polypeptide of interest with: 1) a binding domain for a sortilin receptor and / or cationindependent mannose 6 phosphate receptor (CI-M6PR) and / or 2) a binding domain for prosaposin which subsequently can bind CI-M6PR and / or LDL-receptors such as LRP1, and providing said combination to said subject. According to the invention, the protein of interest is targeted to the lysosome of a cell that requires that protein of interest in said lysosome. This is achieved by providing said protein of interest with at least one peptide sequence that directs a protein to said lysosome. Receptors that are transported to lysosomes from other cell organelles or from the cell surface are e.g. LRP1, the Mannose-6-phosphate receptor, and sortilin. Thus, the invention provides proteins of interest provided with a peptide sequence that specifically binds to at least one of these molecules. In a preferred embodiment, the invention provides proteins of interest provided with binding domain for prosaposin and / or sortilin. Preferably, with an N-tagged binding domain for prosaposin, or either an N-tagged or C-tagged bindingdomain for sortilin. As demonstrated in the examples, the present inventors have surprisingly shown that proteinaceous substances comprising N-terminal PSAP-tagged or N-terminal SRT-tagged GAA were among the best performers with good activity of the delivered GAA and vastly improved cross-correction over untagged GAA. N-terminal SRT-tagged GAA also outperformed the N-terminal IGF2-tagged positive control construct, which has shown preclinical success in mice. Interestingly, all variants with a C-terminally positioned SRT-tag also worked well in achieving GAA activity and cross-correction, i.e. improving over untagged GAA. Thus, the SRT-tag works in both N- and C-terminal configurations. Moreover, for a different protein of interest (GFP), the C-SRT tag was most effective, demonstrating the broad and flexible nature of the invention and the possibility for the skilled person to modify the position of tags as needed for different proteins of interest.

[0116] Progranulin is known to bind to sortilin and prosaposin so preferably said targeting peptide sequence is a specific (receptor) binding fragment for either or both of these -proteins. The sequence of progranulin known to bind both these proteins minimally has the sequence of seq id no.1. Preferably it comprises the sequence of seq id no.

[0117] 2, which comprises granulin E, which binds prosaposin. The sequence of progranulin known to bind sortilin is the C-terminus, represented in seq id no. 3. Preferably, it comprises the sortilin binding domain of seq id no. 6.

[0118] In another preferred embodiment, the invention provides proteins of interest provided with a sequence of progranulin that binds prosaposin and / or sortilin. Preferably, an N-tagged binding domain for prosaposin and / or sortilin. As demonstrated in the examples, the present inventors have shown that proteinaceous substances comprising C-terminal tagged GAA were good performers too with sufficient activity of the delivered GAA and with demonstrable cross-correction. Similarly, delivery of fluorescent protein was also shown for C-tagged GFP, albeit outperformed by even better performing C-tagged SRT.

[0119] An IGF2 tag is also considered useful, and can, for example, comprise the sequence of SEQ ID NO. 8. Suitable alternative sequences for IGF2 tags are known in the art. Further useful is a hybrid IGF2 / SRT tag, as it contains an SRT binding site in the IGF central loop, flanked with linkers such as a CSGGGG and SGGGSGC. If used as an N-tag, the hybrid IGF2 / SRT tag is preferably preceded by an IGF2 signal peptide. Preferably, the sequence of the N-terminal IGF2 / SRT hybrid has the sequence of SEQID NO: 11. The IGF2 and SRT tags could also be used in sequence, with optionally additional linkers between the tags.

[0120] In one embodiment, granulin E may be replaced with another granulin that has specific affinity for prosaposin (more than aspecific binding). In an additional embodiment, cleavage sites are removed or replaced from the sequence. Methods for removing or replacing cleavage sites are known in the art. It is a preferred embodiment of the invention that the combination comprises the protein of interest and the peptide of seq ID no1. It is preferred that the combination as invented is a fusion protein, wherein the targeting peptide sequence is fused to the sequence of the protein of interest by a peptide bond. In one embodiment there may be a linker between the targeting peptide sequence and the protein of interest.

[0121] Prosaposin and progranulin are known to bind to each other. A fusion protein can thereby be transported into a cell (organelle) if it has a specific binding sequence for progranulin or prosaposin. It is therefore preferred that said fusion protein comprising said binding domain for targeting to the lysosome further comprises a granulin binding domain, especially a progranulin binding domain, or a prosaposin binding domain (wherein it is preferred that the targeting peptide is not from the same protein as the binding peptide). The combinations according to the invention (herein further referred to as fusion proteins or proteinaceous substances) themselves can be administered as replacement therapy, as they can be taken up by target cells and transported to the lysosome in that manner. It is however preferred to provide the fusion proteins to a subject through gene therapy. In gene therapy, a nucleic acid construct is provided to cells that need to express said construct, normally from an expression cassette that has the necessary elements for correct expression encased in a vector for delivery. The elements for expression (such as promoters, enhancers, etc.) are well known to the skilled person and need no explanation here. Lab manuals such as Maniatis will serve as a source for these elements. When the cells that require the protein of interest can be reached by a gene delivery vehicle, the gene delivery vehicle may be administered parenterally to a subject in need thereof. For such delivery, the usual viral vectors may be used. Alternatively, it is also contemplated to integrate the gene of interest with targeting sequences in a safe harbour locus in a cell, for example through homologous recombination and / or CRISPR Cas9. The nucleic acid constructscan thus be delivered to cells of a subject via ex vivo gene therapy or in vivo gene therapy.

[0122] The present inventors have shown that cross-correction can indeed be achieved using the constructs of the present invention. Briefly, cells were transduced with lentiviral vectors carrying the nucleic acids encoding for the proteinaceous substances of the present invention. Supernatant of these transduced cells was subsequently transferred to recipient cells (either HAP1 GAA (-) cells or fibroblasts from Pompe disease patients) and GAA activity was thereafter significantly present in these recipient cells. Together with increased GAA activity, decreased glycogen levels were observed as compared to untreated recipient cells, demonstrating successful cross-correction. That the cross-correction occurred through uptake of the GAA from the transferred supernatant by the recipient cells, mediated by the envisioned receptors, was demonstrated by adding blocking agents such as small molecules that block SORT1 or LRP1 to the recipient cells. This resulted in reduced GAA uptake as demonstrated in the examples.

[0123] In most lysosomal storage diseases, direct vector delivery to all cells requiring the protein of interest is not easily achieved. It is therefore preferred to provide viral vectors to certain cells ex vivo, that upon reintroduction in the patient express and secrete the protein of interest with the lysosome targeting peptide fused to it. In order to be secreted, the protein of interest may require a signal peptide. This signal peptide may be part of the immature protein of interest itself or a signal peptide sequence may be added to the mature protein of interest. Signal peptides from different proteins may also be used in the constructs of the present invention such that the construct may carry a protein of interest “A” and a signal peptide of protein “B”.

[0124] Such signal peptide sequences are well known to the skilled person. Thus, the invention in a further embodiment provides a method according to the invention, wherein said combination is a fusion protein comprising a peptide signal sequence for secretion from a cell in which it is expressed. According to the invention linking peptide sequences may be present at any of the borders between protein of interest, targeting sequences and signal peptide sequence.

[0125] According to the invention it is important to increase the probability of protein of interest arriving in the lysosomes of the cells of interest. It is therefore a preferredembodiment to provide the nucleic acid encoding the protein of interest with means for secretion from the producing cell, as well as with binding domains recognizing the cells that require the protein of their interest. According to the invention these receptors preferably are the mannose-6-phosphate receptor, the LRP1, and / or sortilin. The probability of targeting can be increased by targeting more than one of these receptors. This may be achieved by providing more targeting sequences or sequences that bind to another protein that binds to one of the receptors just mentioned (so that the protein of interest is dragged along). The nucleic acids, expression cassettes, and gene delivery vehicles that are employed to deliver the protein of interest to the lysosome where it is needed are also part of the invention. Thus, the invention provides a nucleic acid construct comprising a nucleic acid sequence encoding a protein of interest and a nucleic acid sequence encoding a lysosome targeting sequence based on progranulin, apolipoprotein, and / or IGF2. Examples of such nucleic acid constructs are displayed in figure 2, figure 5, and figure 6. Examples of tags derived from IGF2 and apolipoprotein are known in the art (see e.g. Spencer, Brian J., and Inder M. Verma). " Targeted delivery of proteins across the blood–brain barrier." Proceedings of the National Academy of Sciences 104.18 (2007): 7594-7599,. Gleitz, Helene FE, et al. " Brain-targeted stem cell gene therapy corrects mucopolysaccharidosis type II via multiple mechanisms." EMBO molecular medicine 10.7 (2018): e8730. LeBowitz, Jonathan H., et al. " Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice." Proceedings of the National Academy of Sciences 101.9 (2004): 3083-3088. W02003102583A1 )

[0126] The invention further provides a method according to the invention, wherein said nucleic acid construct further encodes a signal sequence for secretion. Also included in the invention are the gene therapy methods (uses) themselves. Preferred is viral (lentiviral) gene therapy, in particular ex vivo gene therapy, wherein Haematopoietic stem cells are contacted with a lentiviral gene delivery vehicle such that the lentiviral particle is internalized in the cell and preferably inserts its nucleic acid cargo in the genome of said haematopoietic stem cell. It is preferred to provide means to the gene delivery vehicle that prevent or at least reduce silencing of the inserted gene therapy sequences. Examples of lentiviral vectors comprising preferred nucleic acid sequences encoding proteins of interest with lysosomal targeting sequence(s) areprovided in figure 2. Such lentiviral vectors can also comprise constructs as provided in figures 5 and 6.

[0127] Upon introduction to the patient, the haematopoietic stem cells will engraft in the patient’s bone marrow where they will self-replicate and populate the patient with their progeny that will express the fusion proteins from their genomes. Because of the presence of the secretion signal peptide, the fusion proteins according to the invention will be secreted into the intracellular space and / or circulation (typically under removal of the signal peptide). The fusion proteins will then circulate until they encounter one of the molecules that is associated with entering the target cell, such as receptors on a cell for which they have a specific binding sequence. The cell encountered will internalize and transport the fusion protein to the lysosome where it is required. This method is making use of the phenomenon of cross-correction. With the fusion proteins of the invention, at least three receptors can be targeted either by a single targeting sequence, or more than one targeting sequence. It is also possible to combine the new targeting sequences with known targeting sequences such as IGF-2, particularly since IGF-2 is typically fused to a protein of interest on the N-terminal side. Sequential targeting sequences, i.e. combining several tags sequentially in a single fusion protein, and / or hybrid targeting sequences, such as an SORT1 binding site in the IGF central loop, are also encompassed by the present invention. It is also possible to have the fusion protein dragged along with another receptor binding protein by providing a binding domain for said receptor binding protein. As an example, a fusion protein may have a signalling sequence, a first targeting sequence (such as IGF-2), a protein of interest and a progranulin sequence comprising a binding domain for prosaposin and a binding domain for sortilin. It is also contemplated to use sequences derived from prosaposin, which comprise binding sequences for progranulin (which in turn comprises a binding sequence for sortilin), CI-M6PR and LRP1, in such at least three receptors can also be targeted either by a single targeting sequence, or more than one targeting sequence (Figure 4). This comprises the linker region between saposins B and C in prosaposin, which comprises the binding domain for progranulin.

[0128] The preferred sequences for the targeting sequences are amino acids 518-593 of progranulin, amino acids 518-573 for the binding domain of progranulin for prosaposin, amino acids 1,8-67 of mature IGF2. Also preferred are the sortilin binding domain of SEQ ID NO: 6 and / or the hybrid IGF2 / SRT tag of SEQ ID NO: 11.Preferred lentiviral vectors are based on self-inactivating 3rdgeneration lentiviral vectors, for example with a pCCL backbone. Promoters are selected preferably from constitutive promoters such as EFS, EF1a, PGK, virally derived promoters such as CAG, CMV or RSV, MND or synthetic promoters.

[0129] Nucleic acid constructs comprise the following elements: promoter, gene of interest with lysosomal targeting sequences on N- and / or C-terminals, signal peptide sequence and a polyadenylation signal. Optionally, nucleic acid constructs may comprise (mutated) WPRE element and Kozak sequence. Further, nucleic acid constructs may comprise marker and selection elements such as sequences for antibiotic resistance genes or fluorescent markers (e.g. GFP, zsGreen or mCherry).

[0130] Expression cassettes preferably comprise the following elements gene of interest with lysosomal targeting sequences on N- and / or C-terminals, signal peptide sequence. Optionally, the expression cassette may comprise a marker in the same expression cassette separated by a 2A peptide to induce ribosomal skipping (e.g. T2A). 2A peptides are a class of 18–22 amino acid-long peptides, which can separate a single open reading frame into several proteins through inducing ribosomal skipping during translation of a protein.

[0131] The gene delivery vehicles can be administered to the target cells in ways well known to the skilled artisan. In vivo gene delivery is not an exact science. The skilled person is however able to calculate and test correct dosages based on animal studies and earlier clinical trials. Dosing is more easily controlled in ex vivo methods, but the required number of vehicles (or indeed number of genome copies) will need to be determined through (pre) clinical trials. Formulation of gene delivery vehicles is within the skill in the art. Typically the vehicles will be delivered to extracellular fluid such as cerebrospinal fluid and / or blood circulation for in vivo gene delivery methods. Excipients apart from water for injection may comprise buffering systems, stability enhancing substances, anti-oxidants, etc. The proteins of interest vary according to the condition to be treated. A non-exhaustive list is provided herein above. Treating or treatment according to the invention includes prophylaxis, partial cures as well as full cures, amelioration of symptoms, etc. In gene therapy, because of the nature of the treatment the effect of the treatment should be relatively long lasting. Typically, it must reduce the burden on the patient dependent on replacement therapy. The effect should be counted in months and years rather than in weeks.Although the focus of this invention is gene therapy, the protein molecules of the invention can also be used for replacement therapy. Formulation of protein drugs is a skill available to the art. Although it is hard to predict which formulation will be suitable, it is usually a given that at least one will be found. Proteins according to the invention will be typically administered parenterally.

[0132] Examples

[0133] Example 1

[0134] Green fluorescent protein (GFP) fusion proteins are made comprising one or more of a prosaposin binding domain (PSPN), a sortilin binding domain (SORT), and a cationindependent mannose 6 phosphate receptor binding domain (M6PR). Such proteins can be ordered commercially if necessary. Fusion proteins with GFP are made, from the N-terminus to the C-terminus, as follows:

[0135] - GFP

[0136] - M6PR— GFP

[0137] - GFP— SORT

[0138] - GFP-PSPN— SORT

[0139] - M6PR— GFP— PSPN— SORT

[0140] The fusion proteins are synthesized and provided to cells in culture. Cells are cultured with fusion proteins for 24-96 hours. Cells are washed with PBS and take up of fusion protein in cells in lysosomes is studied with fluorescence microscopy. Cells used in this experiment express SORT1, and a mannose 6 phosphate receptor. If necessary, the skilled person knows how to make cell lines with forced expression of these receptors. In additional experiments, combined with the fusion protein, prosaposin protein is added to the medium and its effect studied.

[0141] Example 2

[0142] In this experiment, fusion proteins are made as in Example 1, wherein instead of GFP, a protein of interest (POI), i.e. a lysosomal enzyme is used.

[0143] - POI

[0144] - M6PR— POI

[0145] - POI— SORT

[0146] - POI-PSPN— SORT

[0147] - M6PR— POI— PSPN— SORTThe fusion proteins are synthesized and provided to cells in culture and take up in cells in lysosomes is studied as described above. Cells used in this experiment express SORT 1, and a mannose 6 phosphate receptor. If necessary, the skilled person knows how to make cell lines with forced expression of these receptors. In additional experiments, combined with the fusion protein, prosaposin protein is added to the medium and its effect studied. To study uptake and assess enzymatic function of the protein of interest, cells are lysed and enzyme activity is measured in cell lysates using enzyme activity assays (Lysosomal alpha-Glucosidase Activity Assay Kit (Fluorometric), Abeam ab252887; SensoLyte Blue Glucocerebrosidase (GBA) Activity Assay Kit Fluorimetric, AnaSpec AS-72258) following manufacturer’s instructions. Example 3.

[0148] In this experiment, lentiviral vectors encoding fusion proteins, as defined in Example 2 and represented in Figure 2, are made and used to transduce cells in vitro. The fusion proteins expressed comprise an appropriate signal peptide sequence to allow for secretion of the fusion polypeptide. After allowing the cells to express the fusion protein, the supernatant is collected. The supernatant is tested for expressed fusion protein, e.g. enzyme activity is determined. Supernatant is also provided to target cells, which cells express SORT1, prosaposin, and a mannose 6 phosphate receptor. Effective delivery of the fusion proteins to the lysosome of the target cells is studied as described in example 2.

[0149] 3rd generation self-inactivating (SIN) lentiviral (LV) vectors are produced to provide for fusion protein expressing vector(s), i.e. comprising the expression cassettes depicted in Figure 2. 3rdgeneration LV are a class of LV that are characterized by an RCL-deficient (replication competent lentivirus-deficient) backbone and are selfinactivating (SIN). The helper plasmids of 3rd generation lentiviral vectors are gag –pol, rev and env, and are widely commercially available. Gag-pol and rev are packaging plasmids, while env is the envelope plasmid. The vector containing the gene(s) of interest, in this example the fusion protein of for example a lysosomal enzyme and lysosomal targeting sequences, and reporter genes, contains long terminal repeats (LTR) sequences that are self-inactivating on the 5’and 3’ends, i.e. resulting in LTR’s which after reverse transcription and integration, do not have promoter activity. The genes of interest, are separated by regulatory sequences, for instance T2A or P2A, to allow for separate expression of separate proteins from a single transcript. These lentiviral vectors are exemplary gene constructs, comprised inlentiviral vectors, in accordance with the invention. In a therapy setting, reporter genes are preferably omitted.

[0150] Lentivirus production

[0151] Plasmids are designed as shown in Figure 2 and obtained from a commercial manufacturer / VectorBuilder. The nucleic acid construct, i.e. expression cassette, as depicted in Fig 2A is inserted in the lentiviral vector backbone, the transfer plasmid (as provided by Vector Builder, such is commonly (commercially) available, i.a. from Addgene). For further plasmid production Stabl3 E.coli cells are used for transformation and QIAprep Spin Miniprep Kit (#27104), NucleoBond® Xtra Midi EF kit (#740420), EndoFree Plasmid Mega Kit (#12381) for plasmid isolation. Only plasmids isolated with Endotoxin free kits were used for virus production. Helper plasmids pMDLg / pRRE, pRSV-Rev, and pMD2. VSV-G are used for lentiviral production (Available from Addgene, plasmids #12251, #12259, and #12253). HEK293T cells are transiently transfected with the transfer and helper plasmids using X-tremeGENE HP DNA transfection reagent (Sigma-Aldrich #6366236001). Supernatants with lentivirus particles are harvested 40h, and 64 h after transfection, filtered through 0,45 pm pore filters (Corning, Cat#431220). Pooled lentiviral supernatants are concentrated by centrifugation using Vivaspin 20 centrifugal concentrator columns (Sigma–Aldrich, #Z614653-48EA) according to the manufacturer’s instructions. 40-fold concentrated viral supernatant is aliquoted and frozen, to avoid multiple freeze / thaw cycles.

[0152] HEK-293T cells culture

[0153] HEK293T cells (ATCC, #CRL-3216) are cultured in IMDM (Gibco, #12440-053) supplemented with 10% Fetal Calf-Serum (Bodinco, #S00FD10003) and 1% Penicillinstreptomycin (10,000 U / mL) (Gibco, ##15140122).

[0154] Physical titer

[0155] Total RNA is isolated from concentrated viral supernatant using an RNeasy Mini kit (QIAGEN, cat. 74104), with lysate homogenization performed using QIAshredder spincolumns (QIAGEN, #79656) and reverse transcribed into cDNA using a SuperScript III kit (Invitrogen). The physical titer, representing lentiviral vector genomes packaged in particles, is determined by quantitative PCR (Invitrogen QuantStudio 3) targeting theHIVΨ gene located on the transfer plasmid, using TaqMan Fast Advanced Master Mix (Applied Biosystems, #4444557).

[0156] Functional titer on HEK-293T cells

[0157] HEK-293T cells are seeded at 200 000 cells / well using a 6-well plate. 24 hours later, concentrated lentivirus supernatant, supplemented with DMEM / F12 and LentiBOOST (Revvity Health Sciences, #SB-A-LF-901-01) (1 mg / ml) is added to the culture. To aid the transduction efficiency, the cells are spin-oculated for 30 minutes at 300xg and 32°C. Cells are incubated for 5 days at 37°C and 5% CO2 before being harvested and analysed with flow cytometry. The functional titer is calculated as follows; [a*(b / 100)] / c, where a is the number of target cells, b is the percentage of transduced cells and c the volume (mL) of supernatant added, to provide for TU / mL.

[0158] Determination of enzyme activity in supernatant

[0159] For determination of enzyme activity in supernatant to analyse enzyme secretion, HEK 293T cells are transduced with lentiviral vectors described above and cultured as described above. Supernatant medium is sampled regularly, for example every 24 hours and up to one week after transduction. Media samples are used for enzyme activity analysis. At final timepoint, enzyme activity is also measured in transduced cells. To this end, they are washed with PBS and lysed as described below. Enzyme activity is measured in medium samples and cell lysate using enzyme activity assays (Lysosomal alpha-Glucosidase Activity Assay Kit (Fluorometric), Abeam ab252887; SensoLyte Blue Glucocerebrosidase (GBA) Activity Assay Kit Fluorimetric, AnaSpec AS-72258) following manufacturer’s instructions.

[0160] Determination of enzyme activity in cross-correction experiments

[0161] To analyse cross-correction, conditioned media are prepared as described above from medium samples of HEK 293T cells transduced with lentiviral vectors described above. Conditioned media is collected between 24 and 96 hours and can be filtered if necessary (0.22-pm filter, Millipore). Conditioned media are provided to target cells expressing receptors for lysosomal targeting tags (if necessary, the skilled person knows how to create cell lines with forced expression of such receptors), while (potentially) further providing synergistic (e.g. prosaposin) or competitive factors (e.g. IGF2 or PGRN) for mechanistic studies. Target cells are lysed and enzyme activity is measured in cell lysates using enzyme activity assays (Lysosomal alpha-Glucosidase Activity Assay Kit (Fluorometric), Abeam ab252887; SensoLyte BlueGlucocerebrosidase (GBA) Activity Assay Kit Fluorimetric, AnaSpec AS-72258) following manufacturer’s instructions.

[0162] In vitro studies

[0163] DNA constructs

[0164] GAA fusion constructs were generated with either an N- or C-terminal lysosomal targeting peptide Tag. The Myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted (MND) promoter was used to drive expression of human codon-optimised GAA (GAAco). The N-terminally tagged GAA constructs contained the following Tags: amino acids 518-593 of the human Progranulin protein (N-PGRN), amino acids 518-573 of the human Progranulin protein, comprising the prosaposin-binding domain (N-PSAP), or amino acids 570-593 of the human Progranulin protein, comprising the sortilin-binding domain (N-SRT) and a GAP linker in between the GAA and Tag sequence. The C-terminally tagged GAA constructs contained the following Tags: amino acids 518-593 of the human Progranulin protein (C-PGRN), amino acids 518-573 of the human Progranulin protein, comprising the prosaposin-binding domain (C-PSAP), or amino acids 570-593 of the human Progranulin protein, comprising the sortilin-binding domain (C-SRT) and either a 4x G4P or a 4x G4S peptide linker in between the GAA and Tag sequence. Some of the C-terminally tagged GAA variants had a C> S substitution at amino acid 952. Gamillus fusion constructs were generated with either an N- or C-terminal lysosomal targeting peptide Tag. The Cytomegalovirus (CMV) early enhancer fused to modified chicken p-actin promoter (Cbh) was used to drive Gamillus protein expression. The N-terminally tagged Gamillus constructs contained the following Tags: N-PGRN, N-PSAP, or N-SRT and a GAP linker in between the Gamillus and Tag sequence. The C-terminally tagged Gamillus constructs contained the following Tags: C-PGRN, C-PSAP, C-SRT and a 4x G4S peptide linker in between the GAA and Tag sequence.

[0165] Human cells culturing

[0166] Human fibroblasts derived from infantile patients with Pompe disease were obtained from the NIGMS Human Genetic Cell Repository at Coriell Institute for Medical Research. Three infantile onset Pompe disease fibroblasts lines were obtained: GM20089, GM0244 and GM20124 and an unaffected fibroblast line: GM07525. The unaffected cell line GM07525 and Pompe cell line GM00244 were cultured in Eagle's Minimum Essential Medium (EMEM) with Earle's salts and non-essential amino acidswith 2mM L-glutamine containing 15% (v / v) fetal bovine serum (FBS) not heat inactivated in a humidified atmosphere containing 5% CO2 at 37°C. Pompe fibroblasts lines GM20089 and GM20124 were cultured in Dulbecco Modified Eagles Medium (high glucose) with 2mM L-glutamine containing 15% (v / v) FBS not heat inactivated in a humidified atmosphere containing 10% CO2 at 37°C.

[0167] Human HAP1 parental cell line and HAP1 cell line (a near-haploid cell line derived from chronic myelogenous leukaemia (CML) cell line KBM-7, edited by CRISPR / Cas to contain a frameshift mutation in a coding exon of GAA) were obtained from Horizon (Revvity Discovery Limited). HAP1 cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% (v / v) FBS in a humidified atmosphere containing 5% CO2at 37 °C.

[0168] Human embryonic kidney cells modified with the SV40 large T antigen (HEK293T) were obtained from ATCC (American Type Culture Collection) and maintained in IMDM containing 10% of FBS in a humidified atmosphere containing 5% CO2 at 37°C.

[0169] Human microglial cells HMC3 were obtained from ATCC (American Type Culture Collection) and maintained in EMEM containing 10% of FBS in a humidified atmosphere containing 5% CO2 at 37°C.

[0170] Transfection experiments

[0171] HEK293T were maintained in IMDM containing 10% of FBS in a humidified atmosphere containing 5% CO2 at 37°C. For GAA fusion constructs screening, cells were seeded in 24-wells plates at a density of 1*105cells per well in IMDM without phenol. Co-transfections of GAA pDNA (57 or 113fmol / well) and a promoter-driven control secreted NanoLuc luciferase construct (3.9fmol / well) were performed 24-hours post plating using Lipofectamine 2000 reagent according to the manufacturer’s instructions. Supernatants and cell monolayers were collected 48-hours posttransfection. Supernatants were immediately assayed for secreted NanoLuc using Nano-Gio® Luciferase Assay System (Promega) according to manufacturer’s protocol and for GAA activity or stored at -80°C until further analysis. Cell monolayers were harvested with ice cold GAA activity assay buffer (from Abeam kit ab252887) supplemented with 5% glycerol and 0.01% of Triton X-100, cell debris were pelleted by 5 minutes centrifugation at 10000 x g (or maximum speed according to collection format) at 4°C. Cellular lysate was collected and immediately assayed for GAA activity or stored at -80°C until further analysis.

[0172] To investigate GAA expression in supernatants and cell lysates over time, HEK293T cells were transfected as above with selected GAA pDNA (113fmol / well) and asecreted NanoLuc luciferase control plasmid (3.9fmol / well). Supernatants and cell monolayers were collected 24-, 48-, and 72-hours post-transfection. Supernatants and cell monolayers were collected and assayed for GAA activity or stored at -80°C until further use.

[0173] Tagged Gamillus GFP conditioned medium was produced to visualize the tag-mediated uptake and localisation in HMC3 cells. To produce the conditioned medium, 8*106HEK293T cells were seeded into 15 cm petri dishes that were pre-coated with 1:20 Poly-D-Lysine in PBS, in IMDM containing 10% FBS and 100 U / mL Penicillin / streptomycin incubated at 37°C and 5% CO2. The medium was changed to IMDM containing 5% FBS without phenol red after which the cells were transfected with 30 pg of the tagged Gamillus pDNA constructs per dish using Lipofectamine 2000 according to the manufacturer's instructions. Twenty-four hours post-transfection, the medium was refreshed using IMDM containing 5% FBS. Conditioned medium was produced for 48 hours, after which the medium was harvested from the plates and spun down at 2.000 x g for 5 minutes. The supernatant was then filtered using a sterile 0,2 pm filter and concentrated 10x using 10 kDa filtered spin columns at 4.000 x g for 5-10 minutes.

[0174] Lenti virus production

[0175] To produce lentiviral vectors, HEK293T cells were seeded in 15 cm dishes that were pre-coated with Poly-D-Lysine for 1 hour (5 mL with 1:20 dilution in PBS) and washed 2x with sterile water. Per dish, 8*106HEK293T cells were seeded in IMDM with 10% FBS and 1% Penicillin-Streptomycin (pen / strep) and incubated in a humidified atmosphere containing 5% CO2 at 37°C. Pre-warmed 3685pL Opti-MEM are mixed with 90.63pL of Lipofectamine 2000 in a 50mL tube and incubated at room temperature for 5 min. In another tube the DNA and Opti-MEM mix was prepared by adding: 3776pL of Opti-MEM, 14.35pL of pTransfer (1 pg / pL), 3.78pL of pRSV-REV (1 pg / pL), 9.1pL of pMDIg-pRRE (1 pg / pL) and 4.98pL of pMD2-VSVg (1 pg / pL). The Lipofectamine 2000 and Opti-MEM mix were added to the DNA and Opti-MEM mix, gently mixed, and incubated for 20 minutes at room temperature. Next, 8mL of medium was removed from each 15 cm dish and 7.55mL of transfection mix was added to each dish. The dishes were then incubated in a humidified atmosphere containing 5% CO2 at 37°C. After 24-hrs the cells were washed with 10 mL PBS, after which the medium was changed to 11mL Serum-free Opti-MEM™+GlutaMAX™ with 1% pen / strep and transfected dishes were incubated for 24-hours. 24-hours post medium change a firstharvesting of the supernatant containing the virus was performed by collecting the supernatant in a 50mL tube and adding 11 mL of Serum-free Opti-MEM™+GlutaMAX™ with 1% pen / strep. 48-hours post transfection a second and final harvesting of the supernatant was performed. A supernatant total volume of 22mL was filtered through a 0,45 pm filter into a Vivaspin20 concentration column (previously cleaned and sterilized) and approximately a 40x concentration was performed according to manufacturer’s instructions. Concentrated lentiviruses were then aliquoted and stored at -80°C.

[0176] In vitro transduction experiments

[0177] To determine the functional titer of LV productions, 0.3*105HEK293T cells were seeded in a P48 well plate in a volume of 500pL IMDM containing 10% of FBS and 1% pen / strep and incubated in a humidified atmosphere containing 5% CO2 at 37°C. 24 hours post-seeding 250pL of medium were removed from each well, resulting in an end volume of 250pL. Cells were transduced by adding 1, 2.5, 5, 10 or 20pL of lentivirus / well in duplicate and the plate was centrifuged for 1 hour at 32°C and 800 x g. Following centrifugation, the plate was incubated in a humidified atmosphere containing 5% CO2 at 37°C for two days. After two days, the supernatant was aspirated from the wells and monolayers were harvested in 180pL of DNA lysis buffer ATL (supplemented with 20pL of Proteinase K) for DNA isolation using QIAamp DNA Micro kit (Qiagen). DNA concentrations were measured on a N50 nanophotometer (Implen).

[0178] SDS-PAGE and Western Blot

[0179] Samples were prepared in 1x Laemmli sample buffer (Bio-Rad) and heated to 95°C for 5 minutes. Proteins were separated on a Novex wedgewell 4-20% Tris-Glycine Mini Gel (Thermo Fisher Scientific) and transferred to a nitrocellulose membrane using iBIot™ 3 Transfer Stacks mini and iBIot™ 3 Western Blot Transfer System (Thermo Fisher Scientific). The nitrocellulose membrane was blocked with SuperBlock T20 (PBS) Blocking Buffer (Bio-Rad) and stained with the primary antibody in blocking buffer. After washing with 0.05% Tween-20 in PBS, the membrane was incubated with the secondary antibody in SuperBlock T20 (PBS) Blocking Buffer. Following washing with 0.05% Tween-20 in PBS, the proteins were visualized using Thermo Scientific™ SuperSignal™ West Femto Maximum Sensitivity Substrate on the Invitrogen iBright™ Imaging System.The following primary and secondary antibodies were used against the respective protein: GAA (Thermo Fisher Scientific MA5-37870), GAPDH (Thermo Fisher Scientific MA5-15738), Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (Thermo Fisher Scientific, G-21234). Known concentrations of recombinant human GAA (rhGAA) were used as reference. Data analysis was performed with the iBright™ Analysis Software (Thermo Fisher Scientific).

[0180]

[0181] GAA

[0182] GAA enzymatic activity was determined in a fluorometric assay using 4- methylumbelliferyl-a-D-glucoside as a substrate using a GAA assay kit according to manufacturer’s instructions (ab252887, Abeam). Enzyme activity was calculated using a 4-MU standard curve provided by the kit and normalized for the protein sample concentration. GAA activity was measured in cell extracts and supernatants. For samples preparation a lysis buffer was prepared using the GAA activity assay buffer supplemented with 0.1% Triton X-100, 5% glycerol and one tablet of complete™ ULTRA Tablets, Mini, EASYpack Protease Inhibitor Cocktail protease (Merck). Cell debris was removed by centrifuging for 5 minutes at 14000g at 5°C and clarified cell lysates were stored at -80°C. Protein content in the samples was determined using the Bicinchoninic acid (BCA) assay according to manufacturer’s instructions (Pierce™ BCA Protein Assay Kits, Thermo Fisher Scientific).

[0183] For tissue samples, proteins are isolated from frozen tissue with a tissue homogenizer and / or incubated 1 hour in lysis buffer containing protease inhibitor on ice. 5% glycerol is added to the sample to preserve and stabilize the protein prior to storage at -80°C. GAA activity is measured using the fluorometric Lysosomal alpha-Glucosidase Activity Assay Kit according to manufacturer’s instructions (ab252887, Abeam) or a fluorometric method is developed according to Kros et al., 2007 and van der Wai et al., 2017.

[0184] Glycogen content assays

[0185] Glycogen content was quantified by the amount of glucose released from glycogen after hydrolysis using a Glycogen Assay Kit (ab65620, Abeam) and following manufacturer’s instructions. Briefly, in vitro cell samples were washed with PBS and resuspended in 100µL milliQ water on ice. Homogenization was performed by pipetting up and down a few times or with a homogenizer. The homogenate was boiled for 10minutes at 100°C to inactivate enzymes in the sample. Samples were then centrifuged at 4°C at 18000 x g for 10 minutes. If sample was not assayed immediately, storage up to 1 month was applied. For tissue samples the glycogen content can be determined by using or developing one of the following two methods. A first method using the Glycogen Assay Kit (ab65620, Abeam) is applicable to tissue samples between 10-100 mg following the process described above for in vitro cell samples, or for tissue samples larger than 100mg. Tissue samples larger than 100 mg is washed in cold PBS. Sample is then resuspended in a 30% potassium hydroxide solution (KOH) with a final ratio sample to alkaline reagent of 1:4 to 1:2 (weigh / volume), following a boiling step at 100°C for 2 hours. Sample is cooled and 95% ethanol is added twice the initial volume of the sample to precipitate the crude glycogen. Sample is centrifuged for 10 minutes at 4°C at 18,000 x g in a cold microcentrifuge to remove any insoluble material and supernatant is discarded. The pellet is dissolved or resuspended in a minimal amount of ddH2O (enough to dissolve pellet) and acidify sample to pH 3 with hydrochloric acid solution (HCI) with 5 mol / L concentration, drop by drop. Reprecipitation of the sample is done with 1 volume of 95% Ethanol. The steps from the boiling until ethanol re-precipitation are repeated two more times. Finally, wash precipitate in 95% Ethanol and dry. The dried material can be weighed and dissolved in Assay Buffer Vlll / Glycogen Hydrolysis Buffer for analysis. Glycogen content as ng / pg of protein was calculated using a glycogen standard curve provided by the kit and normalized for the protein sample concentration. In a second method to measure the glycogen content in the tissues, amyloglucosidases and a-amylase are added in vitro after boiling the tissues to terminate enzymatic reactions. The amount of glucose will be measured using the amplex red reagent. The amplex reagent (10-acetyl-3,7-dihydroxyphenoxazine) is a colourless, stable, and extremely versatile peroxidase substrate. In the assay, glucose oxidase reacts with D-glucose to form D-gluconolactone and H2O2. In the presence of horseradish peroxidase (HRP), the H2O2 then reacts with the amplex red reagent in a 1:1 stoichiometry to generate the red-fluorescent oxidation product resorufin (fluorescence readout at excitation 530 nm and emission 590 nm). The amount of glucose (pM) is determined using a glucose standard curve.

[0186] Secretion and uptake of Tagged GAA in vitro

[0187] Conditioned medium containing secreted GAA was produced in transduced HEK293T cells. Briefly, 0.6*106HEK293T cells were seeded in a P6 well plate in a volume of2mL IMDM containing 10% of FBS and 1% pen / strep and incubated in a humidified atmosphere containing 5% CO2 at 37°C. 24 hours post-seeding HEK293T cells were transduced with a target vector copy number (VCN) of 2 and incubated overnight in a humidified atmosphere containing 5% CO2 at 37°C. 24 hours post-transduction the medium was changed to IMDM containing 5% of FBS and no phenol and cells were incubated 72 hours in a humidified atmosphere containing 5% CO2 at 37°C. Supernatants were collected and concentrated with Amicon 4mL 10 kDa spin columns, aliquoted and stored at -80°C. GAA activity was measured in conditioned medium samples and cell lysate using 4 MU analysis as described above. Uptake experiments were performed on HAP1 GAA deficient cells and Pompe fibroblasts (GM20089, GM20124 and GM00244) seeded respectively to 40% and 90-100% confluency in P24 well plate format. Enzyme activity in the conditioned media was determined, and a GAA activity input of 1200 nmol / h*mL (150pL in a total well volume of 500pL corresponded to 360 nmol / h*mL) was incubated on HAP1 GAA (-) cells and Pompe fibroblasts. Media and cells were harvested after 24 hours of incubation in conditioned medium, and GAA enzyme assays were performed.

[0188] To study receptor mediated uptake of GAA, uptake experiments in the presence of receptor blocking agents was performed on HAP1 GAA deficient cells in a P48 well plate format. One day post-seeding, cells were treated with various receptor blocking agents. To block CI-M6PR, Prosaposin, and Sortilin 1 (SORT1) receptor: 25mM D-Mannose 6-phosphate (Merck), 125nM RAGE Antagonist Peptide RAP (Merck), 30nM AF38469 (Merck), 10nM of Neurotensin (Merck), or 7pM Human Recombinant IGF-II (StemCell Technologies) was added to the medium of cells, respectively. 24 hours post-treatment, medium was refreshed with replenishment of blocking agents mentioned above. Together with the receptor blocking agents, cells were also incubated with a GAA input activity of 1200 nmol / h*mL (120pL in a total well volume of 240pL, corresponding to 500 nmol / h*mL). Supernatant and cells were harvested 24 hours after GAA protein incubation. GAA enzyme assays and BCA were performed as described above.

[0189] Glycogen depletion in cross-corrected Pompe fibroblasts

[0190] Glycogen depletion experiments in Pompe fibroblasts treated with GAA conditioned medium (GM20089, GM20124 and GM00244) were performed as described below. Fibroblasts were seeded to reach 100% confluency in P24 well plate format after 24 hours in glucose-free DMEM supplemented with 10% non-heat inactivated FBS. 24hours post-seeding, medium was refreshed to 380pL of glucose-free DMEM supplemented with 10% non-heat inactivated FBS and 20pL of conditioned medium was added per well. GAA activity of the conditioned medium was 14316 nmol / h*mL, from which 20 pL was added to a total volume of 400pL on Pompe fibroblasts. 72 hours post-treatment supernatant was removed, and cell monolayer was washed with PBS. Homogenates were harvested in 100pL milliQ water supplemented with 0.01% of Triton X-100 and boiled for 10 min. at 100°C to inactivate enzymes in the samples. Boiled samples were centrifuged for 10 min. at 4°C at 14000 x g to remove any insoluble material. Samples were assayed immediately or stored at -20°C until analysis.

[0191] Uptake of tagged Gamillus GFP in HMC3 in vitro

[0192] Conditioned medium containing the tagged Gamillus GFP constructs was used to determine the tag-mediated uptake of secreted Gamillus GFP into the lysosomes. To assess this, 4,0*104HMC3 cells were seeded into 8-chamber slide dishes in EMDM medium supplemented with 10% FBS. The cells were incubated overnight in a humid environment with 5% CO2 at 37°C, after which the medium was removed to add conditioned medium diluted in EMEM with 10% FBS. The volume of conditioned medium was adjusted individually for each construct to achieve the same fluorescence intensity per mL. The fluorescence intensity was measured by excitation of 25 pL medium at 490-508 nm and detection of fluorescence emission at 523-533 nm using a microplate reader.

[0193] Following 24 hrs of incubation in conditioned medium at 37°C and 5% CO2, the lysosomes of the HMC3 cells were visualized by staining with Lysotracker Red (Invitrogen cat#L7528) according to the manufacturer's instructions. After staining, the cells were imaged using an EVOS M7000 microscope using a 40x objective. A Texas Red cube was used to detect the Lysotracker Red staining, and a GFP cube was used to detect the Gamillus GFP. GFP fluorescence intensity within the lysosome was analysed using Celleste 6 software with Theoretical Point Spread Function 2D deconvolution (Invitrogen).

[0194] De-glycosylation assays

[0195] The cDNA for GAA encodes a protein of 952 amino acids with a predicted molecular mass of 105 kDa. The newly synthesized GAA-precursor has an amino-terminal signal peptide for co-translational transport into the lumen of the endoplasmic reticulum, where it is N-glycosylated at seven glycosylation sites, resulting in a glycosylatedprecursor with an apparent molecular mass of 110 kDa (Moreland et al., 2005; Selvan et al., 2021). To investigate the glycosylation status of the Tagged GAA variants, PNGase F Kit (16347381; Fisher Scientific or P0708; New England Biolabs) and Endoglycosidase H (Endo H) kit (P0702S; New England Biolabs) were used, following the manufacturer’s instructions. Glycoproteins from the supernatants and cell uptake lysates were denatured before releasing the N-glycans by PNGase F, and the chitobiose core of high mannose and some hybrid oligosaccharides from N-linked glycoproteins by Endo H.

[0196] Flow cytometry

[0197] Peripheral blood, bone marrow, and spleen cells (after red blood cell lysis) are stained with fluorescent-labelled monoclonal antibodies against cell surface markers CD117, Sca-1, CD45, CD19, CD3, CD11b and are analysed with an Invitrogen Attune NXT flow cytometer using Fixable Near-IR dead cell staining for dead cell exclusion. Data analysis is done using OMIQ Flow Cytometry software.

[0198] Vector copy number determination and mRNA quantification

[0199] QuantStudio Real-Time PCR Systems was used to determine the expression level of CI-M6PR, prosaposin, sortilin and GAA transcripts in various human cell lines. RNA isolation was performed on cell lysates using PureLink RNA Mini Kit (Thermo Fisher Scientific). Isolated RNA was transcribed into complementary DNA (cDNA) with Maxima Reverse Transcriptase (Thermo Fisher Scientific). qPCR assay mix contained TaqMan assays (Thermo Fisher Scientific) of target genes, as well as housekeeping gene p-actin (ACTB).

[0200] To determine sample VCN, QuantStudio™ Absolute Q™ Digital PCR System (Thermo Fisher Scientific) was used for LV-transduced cells using human p-actin assay (HEX probe) and HIV assay (FAM probe). Obtained VCNs for each lentivirus batch were plotted using linear regression. To determine VCN in mice tissues, dPCR is performed using mouse p-actin (Actb) assay and HIV assay according to manufacturer’s instruction.

[0201] QuantStudio Real-Time PCR Systems is used to determine the expression level of GAAco transcripts in mouse tissues. RNA isolation is performed on homogenized tissues using PureLink RNA Mini Kit (Thermo Fisher Scientific). Isolated RNA is transcribed into complementary DNA (cDNA) with Maxima Reverse Transcriptase (Thermo Fisher Scientific). qPCR assay mix contains mouse TaqMan assays (Thermo Fisher Scientific) of target genes, as well as mouse housekeeping genes e.g.: Actb,glyceraldehyde-3-phosphate dehydrogenase (Gapdh), ATP synthase F1 subunit beta (Atp5f1b), ubiquitin C (Ubc), eukaryotic translation elongation factor 2 (eEF-2), proteasome (prosome, macropain) 26S subunit, non-ATPase, 4 (Psmd4), eukaryotic translation initiation factor 3, subunit A (Eif3a), ribosomal protein L38 (Rpl38), hypoxanthine-guanine phosphoribosyltransferase (Hprt), TATA-Box Binding Protein (Tbp). For copy number quantification, a titration of plasmid with target genes is also performed on the qPCR system, copy number is calculated using formular Copy Number = (Mass (ng) x 6,022*1023) / (Length (bp) x 660 g / mol).

[0202] In vivo

[0203] Pompe mouse studies

[0204] To determine the preconditioning treatment, GAA deficient Pompe 6Neo mice (B6;129-Gaatm1Rabn / J) are used in a pilot study. In this study, 2 Busulfan doses are tested: 2 x 25 mg / kg (at 48hr and 24 hr pre-transplantation) and 4 x 25 mg / kg (96hr, 72hr, 48hr, and 24 hr pre-transplantation). Bone marrow cells are harvested from femurs and tibias of 6-8 weeks old male mice, and hematopoietic stem and progenitor cells enrichment is performed by lineage depletion using Direct Lineage Cell Depletion kit for mouse (Miltenyi Biotech). Lin- cells are seeded in serum free medium (e.g. StemMACS HSPC expansion medium- or StemSpan Serum-Free Expansion Medium) containing pen / strep and supplemented with 50 ng / mL recombinant murine FMS-like tyrosine kinase 3 ligand (Flt-3), 100 ng / ml recombinant murine stem cell factor (SCF) and 10-19 ng / mL recombinant murine thrombopoietin (TPO) and transduced overnight using LV encoding a GFP reporter gene in the presence of LentiBOOST (Revvity Inc). After Busulfan treatment (either 2x 25 mg / kg or 4 x 25 mg / kg), female mice of 3-6 weeks old are transplanted with 5*105transduced Lin- HSPCs via intravenous injections (n=3). Four weeks post-transplantation, animals are sacrificed. Bone marrow, Spleen, and PBMCs are collected to determine GFP expression and VCN. In addition, VCN of the pre-transplanted transduced Lin- HSPCs and bone marrow cells is determined. Optionally, colony forming unit assays are done.

[0205] GAA deficient Pompe 6Neo mice (B6;129-Gaatm1Rabn / J; JAX Laboratory) are used for a proof of mechanism study. Bone marrow cells are harvested from femurs and tibias of 6-8 weeks old male or female Gaa- / - donor mice, and hematopoietic stem and progenitor cells enrichment is performed by lineage depletion using a Direct Lineage Cell Depletion Kit for mouse (Miltenyi Biotech). After enrichment, cells are transduced overnight with lentiviral vectors encoding the tagged GAA in the presence ofLentiBOOST (Revvity Inc) in serum free medium (e.g. StemMACS HSPC expansion medium- or StemSpan Serum-Free Expansion Medium) containing pen / strep and supplemented with 50 ng / mL recombinant murine Flt-3 ligand, 100 ng / ml recombinant murine SCF and 10-19 ng / mL recombinant murine TPO. Lentiviral vector encoding a GFP reporter is used in the control group. Control GAA+ / + mice are used as control group. After preconditioning with Busulfan using 2 x 25 mg / kg (at 48hr and 24 hr pretransplantation) or 4 x 25 mg / kg (96hr, 72hr, 48hr, and 24 hr pre-transplantation), recipient mice of 3-6 weeks of age of the opposing sex receive 5*105transduced Lin-HSPCs via intravenous injections (n= 6-10). Plasma or leukocytes samples are taken at week 6, 12, and at sacrifice to determine GAA activity. Animals are sacrificed at 16 weeks post-treatment. At sacrifice, key organs including heart, brain, diaphragm, gastrocnemius, quadriceps femoris, tibialis anterior, spleen, bone marrow lung and liver are collected for various analyses (biochemical GAA activity, tissue glycogen content, VCN analysis, GAAco mRNA quantification, histological evaluation and pathology scoring in key tissues). LV vector integrations pre- and post-transplantation are determined in the LV-transduced HPSCs and the bone marrow derived cells. Optionally, colony forming unit assays are done.

[0206] For histological evaluation and pathology scoring, mouse tissues are preserved in FFPE blocks. For GAA enzyme activity and glycogen measurements, mouse tissue samples are collected at necropsy and snap frozen in liquid nitrogen. Samples are stored at -80°C until processing.

[0207] In a follow-up study, GAA deficient Pompe 6Neo mice (B6;129-Gaatm1 Rabn / J) are used for a proof-of-concept study. Bone marrow cells are harvested from femurs and tibias of 6-8 weeks old male or female Gaa- / - donor mice, and hematopoietic stem and progenitor cells enrichment is performed by lineage depletion using a Direct Lineage Cell Depletion Kit for mouse (Miltenyi Biotech). After enrichment, cells are transduced overnight with lentiviral vectors encoding the tagged GAA in the presence of LentiBOOST (Revvity Inc) in serum free medium (e.g. StemMACS HSPC expansion medium- or StemSpan Serum-Free Expansion Medium) containing pen / strep and supplemented with 50 ng / mL recombinant murine Flt-3 ligand, 100 ng / ml recombinant murine SCF and 10-19 ng / mL recombinant murine TPO. Lentiviral vector encoding a GFP reporter is used in the control group. Control GAA+ / + mice are used as control group. After preconditioning with Busulfan of 2 x 25 mg / kg (at 48hr and 24 hr pretransplantation) or 4 x 25 mg / kg (96hr, 72hr, 48hr, and 24 hr pre-transplantation),donor mice of 3-6 weeks of age of the opposing sex receive 5*105transduced Lin-HSPCs via intravenous injections (n= 10-16). Plasma or leukocytes samples are taken at week 6, 12, 18 and at sacrifice to determine GAA activity levels. In addition, Neurofilament Light Chain (NFL) levels are determined in the plasma (week 18 and at sacrifice). Phenotypic assays e.g. rotarod, wire hang and beam walk assay are performed to show improvement in motor function and grip strength. Animals are sacrificed at 24-28 weeks post-transplantation. At sacrifice, key organs including heart, brain, spinal cord, diaphragm, gastrocnemius, quadriceps femoris, tibialis anterior, spleen, bone marrow, lung and liver are collected for various analyses (biochemical GAA activity, tissue glycogen content VCN analysis, GAA mRNA quantification, histological evaluation and pathology scoring in key tissues). LV vector integrations pre- and post-transplantation are determined in the LV-transduced HPSCs and the bone marrow derived cells. Optionally, colony forming unit assays are done. For histological evaluation and pathology scoring, mouse tissues are preserved in FFPE blocks.

[0208] Histological staining of mouse tissues

[0209] Mouse tissues are preserved in Formalin-Fixed Paraffin-Embedded (FFPE) blocks. For immunohistochemistry (IHC) staining, FFPE blocks are sectioned into 4µm thick slides with a microtome. Sections go through deparaffinization & rehydration, before being incubated with Periodic Acid Schiff (PAS) and Haematoxylin and Eosin staining to evaluate glycogen accumulation and vacuolation by light microscopy. Vacuolation is determined for scoring of pathology. Sections are stained with primary antibodies targeting markers of macrophages, microglia and lysosomes using anti-lba1 and anti-LAMP1 antibodies. To score for autophagy, sections are stained with anti-LC3-B and anti-p62 primary antibodies. All primary antibodies are incubated overnight at 4°C and washed with PBS and labelled with the appropriate conjugated secondary antibody for 60 minutes at room temperature. All sections are counterstained with Hoechst to stain nuclei. Sections are then rehydrated and mounted with a coverslip.

[0210] Results

[0211] In vitro transfection experiments to assess GAA activity of the Tagged GAA variants HEK293T cells were transfected with different GAA constructs (Fig 5 and 6). All constructs express the GAA transgene either with or without a peptide Tag at either the C-or N-terminal site of the GAA (Fig 5 and 6, respectively). Construct 1 encodes the untagged GAA. Construct 2 encodes the untagged GAA with an amino acidsubstitution of C> S at amino acid (aa) position 952. Construct 3 encodes a GAA with a C-PGRN tag and a 4x G4P linker. Construct 4 encodes a GAA with an amino acid substitution of C> S at amino acid position 952, a C-PGRN tag and a 4x G4P linker. Construct 5 encodes a GAA with a C-PGRN tag and a 4x G4S linker. Construct 6 encodes a GAA with an amino acid substitution of C> S at amino acid position 952, a C-PGRN tag and a 4x G4S linker. Construct 7 encodes a GAA with the C-PSAP tag and a 4x G4P peptide linker. Construct 8 encodes a GAA with an amino acid substitution of C> S at amino acid position 952, C-PSAP and a 4x G4P peptide linker. Construct 9 encodes a GAA with the C-PSAP tag and a 4x G4S peptide linker. Construct 10 encodes a GAA with an amino acid substitution of C> S at amino acid position 952, C-PSAP, and a 4x G4S peptide linker. Construct 11 encodes a GAA with the C-SRT tag and a 4x G4P peptide linker. Construct 12 encodes a GAA with an amino acid substitution of C> S at amino acid position 952 with the C-SRT tag and a 4x G4P peptide linker. Construct 13 encodes a GAA with the C-SRT tag and a 4x G4S peptide linker. Construct 14 encodes a GAA with an amino acid substitution of C> S at amino acid position 952 with the C-SRT tag and a 4x G4S peptide linker. Construct 15 encodes a GAA tagged C-terminally with amino acids ROLL (last 4 aa of human progranulin) and a 4x G4S peptide linker.

[0212] All N-terminal Tagged GAA constructs encode a GAP linker in between the Tag and GAA. Construct 16 encodes a GAA with the N-PGRN tag. Construct 17 encodes a GAA with the N-PSAP tag. Construct 18 encodes a GAA with the N-SRT tag. Construct 19 encodes a GAA tagged N-terminally with an IGF2 peptide linker (1.8-67). Construct 20 encodes a GAA with an N-terminal IGF2 peptide Tag and the IGF2 signal peptide. Construct 21 encodes a GAA linked N-terminally with an IGF2 peptide and the progranulin signal peptide. Construct 22 encodes a GAA tagged N-terminally with a hybrid Tag encoding IGF2 (1.8-67) and aa (518-573) of the human progranulin with a prosaposin binding domain flanked by a GAP peptide linker N- and C-terminally. Figure 7 shows the GAA activity upon transfection of HEK293T cells with the C-terminally Tagged GAA constructs in the cell lysates (Fig 7A) or supernatant (Fig 7B). Untransfected cells and pBluescript (pBS) transfected cells were used as controls to determine the GAA activity background levels. Most of the C-terminally Tagged GAA variants showed similar (construct 3, 4, 5, 8, 11, and 13) or even enhanced (6, 12, and 14) enzymatic activity in the cell (Fig 7A) in this assay. Generally, all C-terminally tagged GAA variants showed a somewhat lower GAA activity as measured in thesupernatant compared to the untagged construct 1 (Fig 7B) in this assay. Highest GAA activity in the supernatant was observed for construct 6, 8, 12, 13, 14, and 15.

[0213] Furthermore, the constructs with the 952 C> S substitution showed a slightly increased GAA activity in this assay in both the cell lysates and the supernatants compared to their counterpart without substitution, suggesting that the 952 C> S substitution can facilitate maintaining or enhancing enzyme activity with C-terminal Tagging of the GAA protein.

[0214] Figure 8 shows the intracellular (Fig 8A) and extracellular (Fig 8B) GAA activity in HEK293T cells upon transfection with N-terminally Tagged GAA constructs. Several N-tagged GAA variants showed similar (construct 17 and 19) or increased (construct 18, 20, and 22) intracellular GAA activity in this assay. In the supernatant, construct 17, 20, and 22 showed similar GAA activity compared to the untagged GAA construct 1. As internal control, we co-transfected the cells with the GAA constructs as well as with a NanoLuc construct (Fig 9). Expression of NanoLuc should be constant throughout all conditions. However, that was not the case for construct 2, 16, and 21 showing lower NanoLuc secretion, therefore the activity data may have been misleading, and these constructs were retested in another transfection experiment, which demonstrated these constructs can drive GAA activity (Fig 11, described below). The intracellular expressed GAA in the transfected HEK293T cells was characterised by western blot analysis (Fig 10). As control, recombinant human GAA protein was used and showed the expected precursor GAA band only. GAPDH protein was stained as loading control. All GAA variants showed both the GAA precursor of ~110-kDa (upper band) and the lysosomal mature GAA forms of 76 / 70 kDa (lower band) on western blots (Fig 10). These results show that the tagged GAA variants retained similar enzymatic activity compared to the untagged GAA and once expressed via transient transfection using plasmids resulted in GAA that undergo correct lysosomal maturation of GAA. Constructs 6, 8, 12, 14, 17, 18, 20, and 22 were selected for further testing as they display similar or enhanced enzymatic activity compared to the untagged GAA.

[0215] Production and characterisation of conditioned medium from lentiviral vector transduced HEK293T cells

[0216] Advantage of a gene therapy for Pompe Disease is that only a small number of cells need to be corrected as secreted GAA can be taken up in neighbouring cells as a result of cross-correction. To proof that the tagged GAA can mediate cross-correctioninto neighbouring cells, conditioned medium containing GAA needed to be produced from LV-transduced cells.

[0217] To determine the optimal timepoint for harvesting the conditioned medium, a transfection experiment was performed in HEK293T cells with a selection of GAA variants: 6, 8, 12, 14, 17, 18, 20, and 22. Construct 1 was taken along as control and constructs 2, 16, and 21 were taken along for retesting. The GAA activity was measured at 24, 48, and 72 hrs post-transfection. At three days post-transfection, the highest GAA activity in the supernatant was detected and was determined as the optimal time point for harvesting the conditioned medium (Fig 11 A). In addition, GAA activity in the cell was determined (Fig 11 B). Notably, construct 20 showed a very high GAA activity level and construct 21 showed a too low GAA activity level that was out of the quantification range of the reference standard, and therefore may not be accurate. Construct 1, 6, 8, 12, 14, 17, 18, 20, and 22 were cloned into pCCL 3rdgeneration lentiviral (LV) vectors and LV vectors were produced. Conditioned media were generated by transducing HEK293T cells and harvesting the supernatant three days post-transduction, which was subsequently concentrated (~4x).

[0218] To characterise the tagged GAA in the conditioned medium, western blotting was performed (Fig 12). As control, recombinant human GAA was used. The conditioned media of the GAA variants were characterized either in PBS pH 7.4 (Fig 12A) or in GAA activity assay buffer at pH 4.5-4.7 (Fig 12B). The conditioned media of all GAA variants contain the precursor GAA of ~ 110 kDa and as expected, all tagged GAA variants showed an increased size compared to the untagged GAA (construct 1) due to the presence of an N- or C-terminal tag (Fig 12A and Fig 12B). In the acidic condition (Fig 12B), some additional bands below ~55kDa were visible that were also present in the non-acidic condition but in much lesser extent. These results indicate that the tagged GAA are stable in a neutral and acidic environment. In addition, the acidic environment alone is not sufficient for GAA maturation as it requires proteolytic processing in the lysosomes. GAA activity was determined in supernatants pre- and post-concentration in 2 independent batches (Fig 13A and 13B). Measured GAA activity in concentrated samples (4x) showed an increased GAA activity confirming the successful concentration in most of the produced conditioned media.

[0219] After endosomal delivery, the single-chain 110-kDa GAA precursor not only undergoes proteolytic processing to attain the mature GAA forms of 76 / 70 kDa, but also glycan trimming. Both have been shown to be essential for attaining maturation-associatedincreases in GAA glycogen hydrolytic activity. Besides, GAA function, glycosylation could also impact proper folding, stability, and lysosomal targeting. Therefore, proper glycosylation of the expressed GAA is crucial. To characterise the glycosylation patterns of the expressed GAA in the conditioned medium, peptide N-glycosidase F (PNGase F), which cleaves complex glycans was used. The different glycosylation types of the proteins were visualised as glycan trimmed proteins at a lower molecular weight compared to untreated GAA (Fig 20A and B), suggesting that the GAA proteins contain high mannose structures. In addition, the GAA variants were treated with Endo H that cleaves high mannose glycan structures. Upon treatment, all GAA variants showed a lower molecular weight indicating the presence of high mannose glycans (Fig 21 A and 21 B). Altogether, these data showed that the expressed tagged GAA contain both high mannose and complex glycan structures.

[0220] Tagged GAA mediated uptake into GAA deficient cells

[0221] To assess the suitability of our cell lines for verifying mechanisms of action of crosscorrection, expression of key receptors targeted by the tags needed to be verified. Therefore, prior to conducting GAA uptake studies, prosaposin, sortilin (SORT1), and mannose 6 phosphate receptor (M6PR) mRNA expression on various Pompe (GM00244, GM20124, and GM20089) and healthy (GM07525) fibroblasts and cell lines (HAP1 GAA deficient, HAP1, HEK293T, and HMC3) were determined (Fig 14). All cells showed M6PR (Fig 14A), prosaposin (Fig 14B), and SORT1 (Fig 14C) mRNA expression, although fibroblasts showed lower SORT1 and M6PR mRNA expression compared to the other cell lines. Interestingly, SORT1 expression in the patients’ fibroblasts was lower compared to M6PR expression, while HAP1 cells showed a higher SORT1 expression vs M6PR. Thus, these results confirmed that the cells express the targeted receptors and, therefore, uptake of tagged GAA can be assessed in these models to verify efficacy of our tags for uptake or cross-correction in either ERT or gene therapy context, respectively.

[0222] To assess the ability of the (un)tagged GAA to be taken up by recipient cells, normalised conditioned media (equal input of 1200 nmol / ml*h resulting in 360 nmol / ml*h in P24 well) were transferred to HAP1 GAA (-) cells. The parental HAP1 GAA expressing cell line was used as a positive control and the HAP1 GAA (-) cell line in the absence of conditioned medium was used as negative control. Conditioned media were incubated for 24 hrs on HAP1 GAA (-) cells and subsequently removed. Cells were washed with PBS and lysed to assess cellular uptake of GAA. Comparedto the untagged GAA construct 1, several Tagged GAA variants showed an increase in GAA activity in the cell e.g. LV-6, LV-12, LV-14, LV-17, LV-18, and LV-20 (Fig 15). The N-terminal Tagged GAA with aa 570-593 of human progranulin Tag (LV-18) and the IGF2 Tag (LV-20) showed a statistically significant increase compared to the untagged GAA (LV-1), respectively p≤0.0001 and p≤0.001 (one-way Anova with Dunnett’s post-test). Thus, several tags showed increased potential in cross-correction compared to untagged GAA and could be promising for use as a gene therapy approach.

[0223] In addition, the Tagged GAA variants were evaluated by comparing their uptake into infantile Pompe patients derived fibroblasts: GM20089 (Fig 16A), GM20124 (Fig 16B), and GM00244 (Fig 16C). LV-12, LV-14, LV-17, LV-18, and LV-20 showed an increase in GAA uptake, with LV-17, LV-18, and LV20 showing the highest uptake into Pompe fibroblasts. The increase in GAA activity in GM20089 for LV-17 is significant versus the untagged GAA, LV-1 (p<0.001, one-way Anova with Dunnett’s post-test). Although, LV-18 showed the highest GAA activity uptake in all Pompe fibroblasts tested, the increase was not significant due to the high variability within experiments. Moreover, the GAA activity in the recipient cells for these GAA variants surpassed the GAA activity observed in healthy fibroblasts (GAA (+)).

[0224] Tagged GAA taken up by recipient cells undergo normal intracellular processing To determine whether the precursor tagged GAA in the conditioned medium retains intracellular trafficking and proteolytic processing events to form mature lysosomal forms of GAA, the GAA taken up by the recipient cells (GM20089 and GM20124) was characterized by western blot analyses (Fig 17A and 18A). As positive control Myozyme was taken along. Upon uptake, the precursor GAA of -110-kDa could be observed for all GAA variants (Fig 17A). The precursor GAA is proteolytically processed at the amino terminus, resulting in a 95-kDa intermediate form. Further processing in the lysosomes results in the mature active forms of 76 and 70 kDa of the GAA enzyme (Wisselaar et al.,1993). As can be observed in Figure 17A and 18A, all GAA are taken up as the precursor form (in conditioned medium: Fig 12) and processed into the lysosomal matured -76 / 70 kDa forms. In Figure 17B and 18B, GAPDH loading control protein is visualized. In Figure 17C and 18C relative quantification of ~ 110 kDa and -76 / 70 kDa band intensity was performed, and GAPDH normalized results are shown as relative units to untreated condition (set to 1). rhGAA and Myozyme positive controls were not included in the quantification analysis asconditions and products differ from GAA conditioned medium treatment (ng of purified protein vs GAA activity in nmol / ml*h). The relative quantification of the mature forms (lowest band) showed a higher relative amount for the constructs with a peptide Tag vs the untagged GAA (construct 1 ) for GM20089 (Fig 17C). For GM20124 the intensity of the bands of the mature forms was also more intense for the constructs 12, 18, and 20 vs the untagged GAA (Fig 18A), which was confirmed by the relative quantification of the bands (Fig 18C). For construct 18 and 20, the intensity of the mature GAA forms was significantly more intense, which resulted in a reduction in intensity of the precursor GAA band. These data demonstrated that the precursor GAA in the conditioned medium is taken up by the cell and routed to the lysosomes undertaking the relevant processing into the lysosomal mature forms and tagged GAA variants were taken up into the lysosome more effectively.

[0225] Tagged GAA taken up by recipient cells mediate lysosomal glycogen reduction Next, glycogen levels were determined in the Pompe fibroblasts (Fig 19). First, the cells were subjected to glucose starvation to deplete the cytosolic glycogen levels at seeding (t=0 hours). Subsequently, GAA conditioned medium was added to the cells (t=24 hours) to examine the effect of GAA uptake on the lysosomal glycogen levels (at t=96 hours) after 3 days of culture in GAA conditioned medium under continued glucose starvation (Fig 19). As positive control, Myozyme was used. There is a trend of glycogen lowering observed when Myozyme was added. However, the effect was not very pronounced, which is most likely due to the continued glycogen accumulation in the lysosomes of infantile Pompe fibroblasts over time when kept in culture, even in glucose-starved conditions (Fig 19C), and previously reported by Umapathysivam and collaborators (2005 Clinica Chimica Acta). Two independent experiments were performed for each Pompe fibroblast line GM20089 and GM20124; the average glycogen depletion data is shown as percentage of glycogen relative to the Untreated condition that was set to 100%. In the Pompe fibroblast line: GM20089 an increase in glycogen reduction could be observed for constructs 17, 18, and 20 compared to the untagged LV-1 GAA (Fig 19A). Untreated group for GM20089 showed respectively in the two experiments a glycogen value of 0.77 (±0.02) and 1.44 ng / pg of protein. Figure 19B showed the glycogen depletion experiment using another Pompe fibroblast line: GM20124. This infantile line showed a higher lysosomal glycogen accumulation compared to the previously tested GM20089 Pompe fibroblasts. A trend of glycogen lowering could be observed in the Tagged GAA variants compared to the untaggedGAA (LV-1). Two independent experiments were performed and Untreated group for GM20124 showed respectively in the two experiments a glycogen value of 7.47 (±0.33) and 3.65 (±0.38) ng / pg of protein.

[0226] Myozyme did not show a significant glycogen decrease due to the high observed variation (Fig 19B). However, LV-17, 18, and 20 showed an enhanced glycogen depletion compared to LV-1 (untagged GAA). Overall, these results suggested that the tagged constructs resulted in an enhanced lysosomal glycogen degradation by facilitating cellular uptake of GAA and lysosomal trafficking.

[0227] Internalization of tagged GAA is M6PR-independent and dependent on the tag targeted receptors

[0228] Next, uptake of the GAA variants in HAP1 -GAA (-) cells was assessed in the presence of various receptor-blocking compounds to study the mechanisms of uptake, and more specifically that the receptors that are targeted with the tags indeed play a role in targeting of the GAA to the lysosome (Fig 22). The following compounds were used: a small molecule that blocks sortilin: AF38469, Receptor-associated protein (RAP) that is a competitive inhibitor of ligand binding to LRP1 and sortilin (Tauris J et al., 1998), neurotensin, a peptide that acts as neuromodulator in the CNS and binds to sortilin, Mannose-6-phosphate (derived from the dissolved sodium salt hydrate) (M6P) that acts as a competitive inhibitor by saturating the M6PRs and insulin-like growth factor 2 (IGF2) that binds CI-M6PR. LV-6 showed a trend in reduction in uptake in HAP1 GAA deficient cells upon blocking the sortilin receptor by addition of AF38469 and Neurotensin but seemed not to be affected by M6P (Fig 22A). The C-terminally tagged GAA variants LV-8 (harbouring a prosaposin binding domain), LV-12 and LV-14 (both harbouring a sortilin binding domain) showed a decrease in GAA uptake in HAP1 GAA deficient cells upon addition of RAP, AF38469, and Neurotensin. Statistical significance was reached for LV-12 and LV-14 with AF38469 and Neurotensin (p<0.001) compared to the condition without blocking agents. Interestingly, M6P addition did not affect GAA uptake for the tagged GAA variants (LV-6, LV-8, LV-12, and LV-14), whereas a drastic reduction in GAA uptake was observed for the untagged GAA LV-1. Thus, these results indicated that sortilin plays a role in internalisation of LV-12 and LV-14 and that the uptake is M6PR independent as M6P addition did not block GAA uptake.

[0229] Uptake of LV-17 that has the N-PSAP tag containing the prosaposin binding domain was diminished in the presence of RAP that could bind LRP1 and sortilin receptor (Fig22B). The sortilin blocking agents did not show a clear reduction in GAA internalisation due to the high variability. Thus, these results suggest that uptake of LV-17 is facilitated by prosaposin (expressed by the cells, see Fig. 14B) and the LRP1 receptor. Internalization of LV-18 that has N-SRT tag with the sortilin binding domain was inhibited by the presence of RAP, AF38469 (p<0.001), and Neurotensin (p<0.001), which are all known to block the sortilin receptor. In conclusion, these data indicate that LV-18 internalisation is mediated by the sortilin receptor. For LV-20 that encodes the IGF2-GAA, uptake was drastically inhibited by the presence of IGF2. These Cl-M6P / IGF2 results are consistent with previous findings from others (Liang et al., 2022, Dogan et al., 2022).

[0230] Testing of the Tags fused to a secretable fluorescent protein

[0231] To show that the Tags can also be used to target other proteins to the lysosome, tagged secretable green fluorescent protein “Gamillus” constructs were designed harbouring either N-terminal or C-terminal Tags derived from progranulin. The “Gamillus” fluorescent protein was selected as it is reported to be an acid tolerant fusion protein that would keep excellent brightness even at low pH and thus should facilitate lysosomal visualisation (Shinoda et al., 2018). Conditioned medium was generated by transfecting the Gamillus constructs into HEK293T cells. The supernatants were harvested and concentrated and Relative Fluorescence Units (RFU) (Fig 23) were determined. Equal RFU input was subsequently transferred to HMC3, microglia cells. Figure 24A shows the images taken from the recipient cells stained for the lysosomes by using a LysoTracker dye, the images of the Gamillus protein expression and the overlay of both images. Gamillus protein uptake could be observed for all Gamillus variants. Overall, the images seem to show an enhanced Gamillus protein uptake when fused to a progranulin derived tag. On the zoomed-in images of certain areas this is more apparent (Fig 24B). The images were analysed and the Pearson’s Mean colocalization correlation was determined (Fig 24C). All Tag variants showed an increase in colocalization with Lysotracker stained areas, except for the N-PGRN variant. In addition, the ratio of Gamillus Fluorescence and Lysotracker Red positive area relative to the total Lysotracker Red positive area was determined (Fig 24D). The colocalized area is increased in all tagged Gamillus variants, except for C-PSAP and N-PGRN. It could be that for this assay differences are more difficult to be picked up compared to the GAA context as the enzymatic assays are more sensitive.These data suggest that the peptide Tag derived from progranulin can also facilitate cellular uptake and targeting to the lysosome in context of an entirely unrelated protein, thus showing generalisability of the tags in their ability to target proteins to the lysosome.SEQ ID 1: (amino acids 519-593 of human progranulin; “PRGN”) VECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTK CLRREAPRWDAPLRDPALRQLL

[0232] SEQ ID 2: (amino acids 519-573 of human progranulin; " PSPN”):

[0233] VECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTK CL SEQ ID 3: C-terminal 20 amino acids of human progranulin (" SORT"):

[0234] RREAPRWDAPLRDPALRQLL SEQ ID 4: (amino acids 518-593 of human progranulin; comprises a prosaposin binding domain and a SORT1 binding domain; 76 AAs; “PGRN” in Figure 5) DVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGT KCLRREAPRWDAPLRDPALRQLL SEQ ID. 5: (amino acids 518-573 of human progranulin; prosaposin binding domain; 56 AAs; “PSAP” in Figure 5):

[0235] DVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGT KCL SEQ ID 6: sortilin binding domain (amino acids 570-593 of human progranulin; comprises a SORT1 binding domain; 24 AAs; “SRT” in Figure 5) TKCLRREAPRWDAPLRDPALRQLL SEQ ID 7: last 4 amino acids on the C-terminal of human progranulin

[0236] RQLL SEQ ID 8: IGF2 tag (amino acids 1,8-67 of mature IGF2) ALCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATP AKSE SEQ ID 9: codon optimized human GAA (hGAAco) (truncated GAA (57-952 aa) without signal peptide CAGCAAGGCGCTTCTAGACCCGGACCTAGAGATGCCCAAGCTCACCCTGGTAGA CCTAGAGCCGTGCCTACACAGTGTGACGTGCCACCTAACAGCAGATTCGACTGC GCCCCTGACAAGGCCATCACACAAGAGCAGTGTGAAGCCAGAGGCTGCTGCTAC ATCCCTGCCAAACAAGGACTGCAGGGCGCCCAGATGGGACAGCCTTGGTGTTTC TTCCCACCATCTTACCCCAGCTACAAGCTGGAAAACCTGAGCAGCAGCGAGATG GGCTACACCGCCACACTGACCAGAACCACACCTACATTCTTCCCGAAGGACATCC TGACACTGCGGCTGGACGTGATGATGGAAACCGAGAACCGGCTGCACTTCACCA TCAAGGACCCCGCCAATCGGAGATACGAGGTGCCACTGGAAACCCCTCACGTGC ACTCTAGAGCCCCATCTCCACTGTACAGCGTGGAATTCAGCGAGGAACCCTTCG GCGTGATCGTGCGGAGACAGCTGGATGGAAGAGTGCTGCTGAACACCACAGTGG CCCCTCTGTTCTTCGCCGACCAGTTTCTGCAGCTGAGCACCAGCCTGCCTAGCC AGTATATCACAGGCCTGGCCGAGCACCTGTCTCCACTGATGCTGAGCACATCCT GGACCAGAATCACCCTGTGGAACAGAGATCTGGCCCCTACACCTGGCGCCAACC TGTATGGCTCTCACCCCTTTTATCTGGCCCTGGAAGATGGCGGATCTGCCCACG GTGTCTTTCTGCTGAACTCCAACGCCATGGACGTGGTGCTGCAGCCATCTCCTGC TCTGTCTTGGAGAAGCACAGGCGGCATCCTGGATGTGTACATCTTTCTGGGCCC CGAGCCTAAGAGCGTGGTGCAGCAGTATCTGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACCTGTGCAGATGGGGATACAGCAGCAC CGCCATCACCAGACAGGTGGTGGAAAACATGACCCGGGCTCACTTCCCACTGGA TGTGCAGTGGAACGACCTGGACTACATGGACAGCAGACGGGACTTCACCTTCAA CAAGGACGGCTTCAGAGACTTCCCCGCCATGGTGCAAGAACTGCACCAAGGCGG CAGACGGTACATGATGATCGTGGACCCTGCCATCAGCTCTAGCGGACCTGCCGG CTCTTACAGACCCTACGATGAGGGACTGAGAAGAGGCGTGTTCATCACCAACGA GACAGGCCAGCCTCTGATCGGCAAAGTGTGGCCTGGCAGCACAGCCTTTCCAGA CTTCACAAACCCCACCGCTCTGGCTTGGTGGGAAGATATGGTGGCCGAGTTTCA CGATCAGGTGCCCTTCGACGGCATGTGGATCGACATGAACGAGCCCAGCAACTT CATCCGGGGCAGCGAGGATGGCTGCCCCAACAACGAACTGGAAAATCCTCCTTA CGTGCCCGGCGTTGTCGGCGGAACACTTCAGGCCGCTACAATCTGTGCCAGCAG CCATCAGTTTCTGAGCACCCACTACAACCTGCACAACCTGTACGGCCTGACCGAG GCCATTGCCTCTCATAGAGCCCTGGTTAAGGCCAGAGGCACCCGGCCTTTTGTG ATCAGCAGAAGCACATTCGCCGGCCACGGCAGATATGCCGGACATTGGACAGGC GACGTGTGGTCTAGTTGGGAGCAGCTGGCTAGCAGCGTGCCAGAGATCCTGCAG TTCAATCTGCTGGGCGTGCCACTCGTGGGAGCCGATGTTTGTGGCTTCCTGGGC AACACCTCCGAGGAACTGTGTGTGCGTTGGACACAGCTGGGCGCCTTCTATCCC TTCATGAGAAACCACAACAGCCTGCTGAGCCTGCCTCAAGAGCCCTACAGCTTTA GCGAGCCTGCACAGCAGGCCATGAGAAAGGCCCTGACTCTGAGATACGCCCTGC TGCCTCACCTGTACACCCTGTTTCATCAGGCCCACGTGGCAGGCGAGACAGTGG CTAGACCTCTGTTCCTGGAATTCCCCAAGGACAGCTCCACCTGGACCGTGGATCA TCAGCTGCTGTGGGGAGAAGCCCTGCTGATTACACCAGTGCTGCAGGCCGGAAA GGCCGAAGTGACAGGCTATTTCCCTCTCGGCACTTGGTACGACCTGCAGACCGT GCCTGTTGAGGCTCTGGGATCTCTTCCTCCACCTCCTGCCGCTCCTAGAGAGCC TGCCATTCACTCTGAAGGCCAGTGGGTTACCCTGCCTGCTCCTCTGGACACCATC AACGTGCACCTGAGAGCTGGCTACATCATCCCTCTGCAAGGCCCTGGCCTGACC ACAACCGAATCTAGACAGCAGCCCATGGCTCTGGCCGTGGCTTTGACAAAAGGC GGAGAGGCTAGAGGCGAGCTGTTCTGGGATGATGGCGAGAGCCTGGAAGTGCT GGAACGGGGCGCTTATACCCAAGTGATCTTCCTGGCCAGAAACAACACCATCGT GAACGAACTCGTGCGCGTGACCAGTGAAGGTGCTGGACTGCAACTGCAGAAAGT GACCGTGCTCGGAGTGGCCACAGCTCCTCAGCAGGTTCTGTCTAATGGCGTGCC CGTGTCCAACTTCACATACAGCCCCGACACCAAGGTCCTGGACATCTGTGTGTCA CTGCTGATGGGCGAGCAGTTCCTGGTGTCCTGGTGT SEQ ID 10: codon optimized human GAA (hGAAco) with 952 C-> S (truncated GAA (57-952 aa) without signal peptide CAGCAAGGCGCTTCTAGACCCGGACCTAGAGATGCCCAAGCTCACCCTGGTAGA CCTAGAGCCGTGCCTACACAGTGTGACGTGCCACCTAACAGCAGATTCGACTGC GCCCCTGACAAGGCCATCACACAAGAGCAGTGTGAAGCCAGAGGCTGCTGCTAC ATCCCTGCCAAACAAGGACTGCAGGGCGCCCAGATGGGACAGCCTTGGTGTTTC TTCCCACCATCTTACCCCAGCTACAAGCTGGAAAACCTGAGCAGCAGCGAGATG GGCTACACCGCCACACTGACCAGAACCACACCTACATTCTTCCCGAAGGACATCC TGACACTGCGGCTGGACGTGATGATGGAAACCGAGAACCGGCTGCACTTCACCA TCAAGGACCCCGCCAATCGGAGATACGAGGTGCCACTGGAAACCCCTCACGTGC ACTCTAGAGCCCCATCTCCACTGTACAGCGTGGAATTCAGCGAGGAACCCTTCG GCGTGATCGTGCGGAGACAGCTGGATGGAAGAGTGCTGCTGAACACCACAGTGG CCCCTCTGTTCTTCGCCGACCAGTTTCTGCAGCTGAGCACCAGCCTGCCTAGCC AGTATATCACAGGCCTGGCCGAGCACCTGTCTCCACTGATGCTGAGCACATCCT GGACCAGAATCACCCTGTGGAACAGAGATCTGGCCCCTACACCTGGCGCCAACC TGTATGGCTCTCACCCCTTTTATCTGGCCCTGGAAGATGGCGGATCTGCCCACG GTGTCTTTCTGCTGAACTCCAACGCCATGGACGTGGTGCTGCAGCCATCTCCTGC TCTGTCTTGGAGAAGCACAGGCGGCATCCTGGATGTGTACATCTTTCTGGGCCC CGAGCCTAAGAGCGTGGTGCAGCAGTATCTGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACCTGTGCAGATGGGGATACAGCAGCAC CGCCATCACCAGACAGGTGGTGGAAAACATGACCCGGGCTCACTTCCCACTGGA TGTGCAGTGGAACGACCTGGACTACATGGACAGCAGACGGGACTTCACCTTCAA CAAGGACGGCTTCAGAGACTTCCCCGCCATGGTGCAAGAACTGCACCAAGGCGG CAGACGGTACATGATGATCGTGGACCCTGCCATCAGCTCTAGCGGACCTGCCGG CTCTTACAGACCCTACGATGAGGGACTGAGAAGAGGCGTGTTCATCACCAACGA GACAGGCCAGCCTCTGATCGGCAAAGTGTGGCCTGGCAGCACAGCCTTTCCAGA CTTCACAAACCCCACCGCTCTGGCTTGGTGGGAAGATATGGTGGCCGAGTTTCA CGATCAGGTGCCCTTCGACGGCATGTGGATCGACATGAACGAGCCCAGCAACTT CATCCGGGGCAGCGAGGATGGCTGCCCCAACAACGAACTGGAAAATCCTCCTTA CGTGCCCGGCGTTGTCGGCGGAACACTTCAGGCCGCTACAATCTGTGCCAGCAG CCATCAGTTTCTGAGCACCCACTACAACCTGCACAACCTGTACGGCCTGACCGAG GCCATTGCCTCTCATAGAGCCCTGGTTAAGGCCAGAGGCACCCGGCCTTTTGTG ATCAGCAGAAGCACATTCGCCGGCCACGGCAGATATGCCGGACATTGGACAGGC GACGTGTGGTCTAGTTGGGAGCAGCTGGCTAGCAGCGTGCCAGAGATCCTGCAG TTCAATCTGCTGGGCGTGCCACTCGTGGGAGCCGATGTTTGTGGCTTCCTGGGC AACACCTCCGAGGAACTGTGTGTGCGTTGGACACAGCTGGGCGCCTTCTATCCC TTCATGAGAAACCACAACAGCCTGCTGAGCCTGCCTCAAGAGCCCTACAGCTTTA GCGAGCCTGCACAGCAGGCCATGAGAAAGGCCCTGACTCTGAGATACGCCCTGC TGCCTCACCTGTACACCCTGTTTCATCAGGCCCACGTGGCAGGCGAGACAGTGG CTAGACCTCTGTTCCTGGAATTCCCCAAGGACAGCTCCACCTGGACCGTGGATCA TCAGCTGCTGTGGGGAGAAGCCCTGCTGATTACACCAGTGCTGCAGGCCGGAAA GGCCGAAGTGACAGGCTATTTCCCTCTCGGCACTTGGTACGACCTGCAGACCGT GCCTGTTGAGGCTCTGGGATCTCTTCCTCCACCTCCTGCCGCTCCTAGAGAGCC TGCCATTCACTCTGAAGGCCAGTGGGTTACCCTGCCTGCTCCTCTGGACACCATC AACGTGCACCTGAGAGCTGGCTACATCATCCCTCTGCAAGGCCCTGGCCTGACC ACAACCGAATCTAGACAGCAGCCCATGGCTCTGGCCGTGGCTTTGACAAAAGGC GGAGAGGCTAGAGGCGAGCTGTTCTGGGATGATGGCGAGAGCCTGGAAGTGCT GGAACGGGGCGCTTATACCCAAGTGATCTTCCTGGCCAGAAACAACACCATCGT GAACGAACTCGTGCGCGTGACCAGTGAAGGTGCTGGACTGCAACTGCAGAAAGT GACCGTGCTCGGAGTGGCCACAGCTCCTCAGCAGGTTCTGTCTAATGGCGTGCC CGTGTCCAACTTCACATACAGCCCCGACACCAAGGTCCTGGACATCTGTGTGTCA CTGCTGATGGGCGAGCAGTTCCTGGTGTCCTGGTCT

[0237] SEQ ID 11: Mature IGF2 / SRT hybrid design (IGF2 / SRT hybrid tag sequence) ALCGGELVDTLQFVCGDRGFYFCSGGGGTKCLRREAPRWDAPLRDPALRQLLSGG GGSGCIVEECCFRSCDLALLETYCATPAKSE SEQ ID 12: GAA signal peptide (aa -27 to 1) MGVRHPPCSHRLLAVCALVSLATAALL SEQ ID 13: PGRN signal peptide (aa -17 to 1)

[0238] MWTLVSWVALTAGLVAG SEQ ID 14: IFN alpha 2 signal peptide (aa -23 to 1) MALTFALLVALLVLSCKSSCSVG SEQ ID 15: 4x G4P linker:

[0239] GGGGPGGGGPGGGGPGGGGP SEQ ID 16: 4x G4S linker:

[0240] GGGGSGGGGSGGGGSGGGGSSEQ ID 17: GAP linker

[0241] GAP SEQ ID 18: DNA sequence of GAAco with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTCAGCAAGGCGCTTCTAGACCCGGACCT AGAGATGCCCAAGCTCACCCTGGTAGACCTAGAGCCGTGCCTACACAGTGTGAC GTGCCACCTAACAGCAGATTCGACTGCGCCCCTGACAAGGCCATCACACAAGAG CAGTGTGAAGCCAGAGGCTGCTGCTACATCCCTGCCAAACAAGGACTGCAGGGC GCCCAGATGGGACAGCCTTGGTGTTTCTTCCCACCATCTTACCCCAGCTACAAGC TGGAAAACCTGAGCAGCAGCGAGATGGGCTACACCGCCACACTGACCAGAACCA CACCTACATTCTTCCCGAAGGACATCCTGACACTGCGGCTGGACGTGATGATGG AAACCGAGAACCGGCTGCACTTCACCATCAAGGACCCCGCCAATCGGAGATACG AGGTGCCACTGGAAACCCCTCACGTGCACTCTAGAGCCCCATCTCCACTGTACA GCGTGGAATTCAGCGAGGAACCCTTCGGCGTGATCGTGCGGAGACAGCTGGATG GAAGAGTGCTGCTGAACACCACAGTGGCCCCTCTGTTCTTCGCCGACCAGTTTCT GCAGCTGAGCACCAGCCTGCCTAGCCAGTATATCACAGGCCTGGCCGAGCACCT GTCTCCACTGATGCTGAGCACATCCTGGACCAGAATCACCCTGTGGAACAGAGAT CTGGCCCCTACACCTGGCGCCAACCTGTATGGCTCTCACCCCTTTTATCTGGCCC TGGAAGATGGCGGATCTGCCCACGGTGTCTTTCTGCTGAACTCCAACGCCATGG ACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTTGGAGAAGCACAGGCGGCATCC TGGATGTGTACATCTTTCTGGGCCCCGAGCCTAAGAGCGTGGTGCAGCAGTATC TGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACC TGTGCAGATGGGGATACAGCAGCACCGCCATCACCAGACAGGTGGTGGAAAACA TGACCCGGGCTCACTTCCCACTGGATGTGCAGTGGAACGACCTGGACTACATGG ACAGCAGACGGGACTTCACCTTCAACAAGGACGGCTTCAGAGACTTCCCCGCCA TGGTGCAAGAACTGCACCAAGGCGGCAGACGGTACATGATGATCGTGGACCCTG CCATCAGCTCTAGCGGACCTGCCGGCTCTTACAGACCCTACGATGAGGGACTGA GAAGAGGCGTGTTCATCACCAACGAGACAGGCCAGCCTCTGATCGGCAAAGTGT GGCCTGGCAGCACAGCCTTTCCAGACTTCACAAACCCCACCGCTCTGGCTTGGT GGGAAGATATGGTGGCCGAGTTTCACGATCAGGTGCCCTTCGACGGCATGTGGA TCGACATGAACGAGCCCAGCAACTTCATCCGGGGCAGCGAGGATGGCTGCCCCA ACAACGAACTGGAAAATCCTCCTTACGTGCCCGGCGTTGTCGGCGGAACACTTC AGGCCGCTACAATCTGTGCCAGCAGCCATCAGTTTCTGAGCACCCACTACAACCT GCACAACCTGTACGGCCTGACCGAGGCCATTGCCTCTCATAGAGCCCTGGTTAA GGCCAGAGGCACCCGGCCTTTTGTGATCAGCAGAAGCACATTCGCCGGCCACGG CAGATATGCCGGACATTGGACAGGCGACGTGTGGTCTAGTTGGGAGCAGCTGGC TAGCAGCGTGCCAGAGATCCTGCAGTTCAATCTGCTGGGCGTGCCACTCGTGGG AGCCGATGTTTGTGGCTTCCTGGGCAACACCTCCGAGGAACTGTGTGTGCGTTG GACACAGCTGGGCGCCTTCTATCCCTTCATGAGAAACCACAACAGCCTGCTGAG CCTGCCTCAAGAGCCCTACAGCTTTAGCGAGCCTGCACAGCAGGCCATGAGAAA GGCCCTGACTCTGAGATACGCCCTGCTGCCTCACCTGTACACCCTGTTTCATCAG GCCCACGTGGCAGGCGAGACAGTGGCTAGACCTCTGTTCCTGGAATTCCCCAAG GACAGCTCCACCTGGACCGTGGATCATCAGCTGCTGTGGGGAGAAGCCCTGCTG ATTACACCAGTGCTGCAGGCCGGAAAGGCCGAAGTGACAGGCTATTTCCCTCTC GGCACTTGGTACGACCTGCAGACCGTGCCTGTTGAGGCTCTGGGATCTCTTCCT CCACCTCCTGCCGCTCCTAGAGAGCCTGCCATTCACTCTGAAGGCCAGTGGGTT ACCCTGCCTGCTCCTCTGGACACCATCAACGTGCACCTGAGAGCTGGCTACATC ATCCCTCTGCAAGGCCCTGGCCTGACCACAACCGAATCTAGACAGCAGCCCATG GCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAGGCTAGAGGCGAGCTGTTCTGG GATGATGGCGAGAGCCTGGAAGTGCTGGAACGGGGCGCTTATACCCAAGTGATC TTCCTGGCCAGAAACAACACCATCGTGAACGAACTCGTGCGCGTGACCAGTGAAGGTGCTGGACTGCAACTGCAGAAAGTGACCGTGCTCGGAGTGGCCACAGCTCCT CAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCCAACTTCACATACAGCCCCGACA CCAAGGTCCTGGACATCTGTGTGTCACTGCTGATGGGCGAGCAGTTCCTGGTGT CCTGGTGTTAGTGA SEQ ID 19: DNA sequence of GAAco 952 C-> S with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTCAGCAAGGCGCTTCTAGACCCGGACCT AGAGATGCCCAAGCTCACCCTGGTAGACCTAGAGCCGTGCCTACACAGTGTGAC GTGCCACCTAACAGCAGATTCGACTGCGCCCCTGACAAGGCCATCACACAAGAG CAGTGTGAAGCCAGAGGCTGCTGCTACATCCCTGCCAAACAAGGACTGCAGGGC GCCCAGATGGGACAGCCTTGGTGTTTCTTCCCACCATCTTACCCCAGCTACAAGC TGGAAAACCTGAGCAGCAGCGAGATGGGCTACACCGCCACACTGACCAGAACCA CACCTACATTCTTCCCGAAGGACATCCTGACACTGCGGCTGGACGTGATGATGG AAACCGAGAACCGGCTGCACTTCACCATCAAGGACCCCGCCAATCGGAGATACG AGGTGCCACTGGAAACCCCTCACGTGCACTCTAGAGCCCCATCTCCACTGTACA GCGTGGAATTCAGCGAGGAACCCTTCGGCGTGATCGTGCGGAGACAGCTGGATG GAAGAGTGCTGCTGAACACCACAGTGGCCCCTCTGTTCTTCGCCGACCAGTTTCT GCAGCTGAGCACCAGCCTGCCTAGCCAGTATATCACAGGCCTGGCCGAGCACCT GTCTCCACTGATGCTGAGCACATCCTGGACCAGAATCACCCTGTGGAACAGAGAT CTGGCCCCTACACCTGGCGCCAACCTGTATGGCTCTCACCCCTTTTATCTGGCCC TGGAAGATGGCGGATCTGCCCACGGTGTCTTTCTGCTGAACTCCAACGCCATGG ACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTTGGAGAAGCACAGGCGGCATCC TGGATGTGTACATCTTTCTGGGCCCCGAGCCTAAGAGCGTGGTGCAGCAGTATC TGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACC TGTGCAGATGGGGATACAGCAGCACCGCCATCACCAGACAGGTGGTGGAAAACA TGACCCGGGCTCACTTCCCACTGGATGTGCAGTGGAACGACCTGGACTACATGG ACAGCAGACGGGACTTCACCTTCAACAAGGACGGCTTCAGAGACTTCCCCGCCA TGGTGCAAGAACTGCACCAAGGCGGCAGACGGTACATGATGATCGTGGACCCTG CCATCAGCTCTAGCGGACCTGCCGGCTCTTACAGACCCTACGATGAGGGACTGA GAAGAGGCGTGTTCATCACCAACGAGACAGGCCAGCCTCTGATCGGCAAAGTGT GGCCTGGCAGCACAGCCTTTCCAGACTTCACAAACCCCACCGCTCTGGCTTGGT GGGAAGATATGGTGGCCGAGTTTCACGATCAGGTGCCCTTCGACGGCATGTGGA TCGACATGAACGAGCCCAGCAACTTCATCCGGGGCAGCGAGGATGGCTGCCCCA ACAACGAACTGGAAAATCCTCCTTACGTGCCCGGCGTTGTCGGCGGAACACTTC AGGCCGCTACAATCTGTGCCAGCAGCCATCAGTTTCTGAGCACCCACTACAACCT GCACAACCTGTACGGCCTGACCGAGGCCATTGCCTCTCATAGAGCCCTGGTTAA GGCCAGAGGCACCCGGCCTTTTGTGATCAGCAGAAGCACATTCGCCGGCCACGG CAGATATGCCGGACATTGGACAGGCGACGTGTGGTCTAGTTGGGAGCAGCTGGC TAGCAGCGTGCCAGAGATCCTGCAGTTCAATCTGCTGGGCGTGCCACTCGTGGG AGCCGATGTTTGTGGCTTCCTGGGCAACACCTCCGAGGAACTGTGTGTGCGTTG GACACAGCTGGGCGCCTTCTATCCCTTCATGAGAAACCACAACAGCCTGCTGAG CCTGCCTCAAGAGCCCTACAGCTTTAGCGAGCCTGCACAGCAGGCCATGAGAAA GGCCCTGACTCTGAGATACGCCCTGCTGCCTCACCTGTACACCCTGTTTCATCAG GCCCACGTGGCAGGCGAGACAGTGGCTAGACCTCTGTTCCTGGAATTCCCCAAG GACAGCTCCACCTGGACCGTGGATCATCAGCTGCTGTGGGGAGAAGCCCTGCTG ATTACACCAGTGCTGCAGGCCGGAAAGGCCGAAGTGACAGGCTATTTCCCTCTC GGCACTTGGTACGACCTGCAGACCGTGCCTGTTGAGGCTCTGGGATCTCTTCCT CCACCTCCTGCCGCTCCTAGAGAGCCTGCCATTCACTCTGAAGGCCAGTGGGTT ACCCTGCCTGCTCCTCTGGACACCATCAACGTGCACCTGAGAGCTGGCTACATC ATCCCTCTGCAAGGCCCTGGCCTGACCACAACCGAATCTAGACAGCAGCCCATG GCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAGGCTAGAGGCGAGCTGTTCTGG GATGATGGCGAGAGCCTGGAAGTGCTGGAACGGGGCGCTTATACCCAAGTGATCTTCCTGGCCAGAAACAACACCATCGTGAACGAACTCGTGCGCGTGACCAGTGAA GGTGCTGGACTGCAACTGCAGAAAGTGACCGTGCTCGGAGTGGCCACAGCTCCT CAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCCAACTTCACATACAGCCCCGACA CCAAGGTCCTGGACATCTGTGTGTCACTGCTGATGGGCGAGCAGTTCCTGGTGT CCTGGTCTTAGTGA SEQ ID 20: DNA sequence of GAAco, 4x G4P linker and C-PGRN tag with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTCAGCAAGGCGCTTCTAGACCCGGACCT AGAGATGCCCAAGCTCACCCTGGTAGACCTAGAGCCGTGCCTACACAGTGTGAC GTGCCACCTAACAGCAGATTCGACTGCGCCCCTGACAAGGCCATCACACAAGAG CAGTGTGAAGCCAGAGGCTGCTGCTACATCCCTGCCAAACAAGGACTGCAGGGC GCCCAGATGGGACAGCCTTGGTGTTTCTTCCCACCATCTTACCCCAGCTACAAGC TGGAAAACCTGAGCAGCAGCGAGATGGGCTACACCGCCACACTGACCAGAACCA CACCTACATTCTTCCCGAAGGACATCCTGACACTGCGGCTGGACGTGATGATGG AAACCGAGAACCGGCTGCACTTCACCATCAAGGACCCCGCCAATCGGAGATACG AGGTGCCACTGGAAACCCCTCACGTGCACTCTAGAGCCCCATCTCCACTGTACA GCGTGGAATTCAGCGAGGAACCCTTCGGCGTGATCGTGCGGAGACAGCTGGATG GAAGAGTGCTGCTGAACACCACAGTGGCCCCTCTGTTCTTCGCCGACCAGTTTCT GCAGCTGAGCACCAGCCTGCCTAGCCAGTATATCACAGGCCTGGCCGAGCACCT GTCTCCACTGATGCTGAGCACATCCTGGACCAGAATCACCCTGTGGAACAGAGAT CTGGCCCCTACACCTGGCGCCAACCTGTATGGCTCTCACCCCTTTTATCTGGCCC TGGAAGATGGCGGATCTGCCCACGGTGTCTTTCTGCTGAACTCCAACGCCATGG ACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTTGGAGAAGCACAGGCGGCATCC TGGATGTGTACATCTTTCTGGGCCCCGAGCCTAAGAGCGTGGTGCAGCAGTATC TGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACC TGTGCAGATGGGGATACAGCAGCACCGCCATCACCAGACAGGTGGTGGAAAACA TGACCCGGGCTCACTTCCCACTGGATGTGCAGTGGAACGACCTGGACTACATGG ACAGCAGACGGGACTTCACCTTCAACAAGGACGGCTTCAGAGACTTCCCCGCCA TGGTGCAAGAACTGCACCAAGGCGGCAGACGGTACATGATGATCGTGGACCCTG CCATCAGCTCTAGCGGACCTGCCGGCTCTTACAGACCCTACGATGAGGGACTGA GAAGAGGCGTGTTCATCACCAACGAGACAGGCCAGCCTCTGATCGGCAAAGTGT GGCCTGGCAGCACAGCCTTTCCAGACTTCACAAACCCCACCGCTCTGGCTTGGT GGGAAGATATGGTGGCCGAGTTTCACGATCAGGTGCCCTTCGACGGCATGTGGA TCGACATGAACGAGCCCAGCAACTTCATCCGGGGCAGCGAGGATGGCTGCCCCA ACAACGAACTGGAAAATCCTCCTTACGTGCCCGGCGTTGTCGGCGGAACACTTC AGGCCGCTACAATCTGTGCCAGCAGCCATCAGTTTCTGAGCACCCACTACAACCT GCACAACCTGTACGGCCTGACCGAGGCCATTGCCTCTCATAGAGCCCTGGTTAA GGCCAGAGGCACCCGGCCTTTTGTGATCAGCAGAAGCACATTCGCCGGCCACGG CAGATATGCCGGACATTGGACAGGCGACGTGTGGTCTAGTTGGGAGCAGCTGGC TAGCAGCGTGCCAGAGATCCTGCAGTTCAATCTGCTGGGCGTGCCACTCGTGGG AGCCGATGTTTGTGGCTTCCTGGGCAACACCTCCGAGGAACTGTGTGTGCGTTG GACACAGCTGGGCGCCTTCTATCCCTTCATGAGAAACCACAACAGCCTGCTGAG CCTGCCTCAAGAGCCCTACAGCTTTAGCGAGCCTGCACAGCAGGCCATGAGAAA GGCCCTGACTCTGAGATACGCCCTGCTGCCTCACCTGTACACCCTGTTTCATCAG GCCCACGTGGCAGGCGAGACAGTGGCTAGACCTCTGTTCCTGGAATTCCCCAAG GACAGCTCCACCTGGACCGTGGATCATCAGCTGCTGTGGGGAGAAGCCCTGCTG ATTACACCAGTGCTGCAGGCCGGAAAGGCCGAAGTGACAGGCTATTTCCCTCTC GGCACTTGGTACGACCTGCAGACCGTGCCTGTTGAGGCTCTGGGATCTCTTCCT CCACCTCCTGCCGCTCCTAGAGAGCCTGCCATTCACTCTGAAGGCCAGTGGGTT ACCCTGCCTGCTCCTCTGGACACCATCAACGTGCACCTGAGAGCTGGCTACATC ATCCCTCTGCAAGGCCCTGGCCTGACCACAACCGAATCTAGACAGCAGCCCATGGCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAGGCTAGAGGCGAGCTGTTCTGG GATGATGGCGAGAGCCTGGAAGTGCTGGAACGGGGCGCTTATACCCAAGTGATC TTCCTGGCCAGAAACAACACCATCGTGAACGAACTCGTGCGCGTGACCAGTGAA GGTGCTGGACTGCAACTGCAGAAAGTGACCGTGCTCGGAGTGGCCACAGCTCCT CAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCCAACTTCACATACAGCCCCGACA CCAAGGTCCTGGACATCTGTGTGTCACTGCTGATGGGCGAGCAGTTCCTGGTGT CCTGGTGTGGTGGCGGAGGACCTGGCGGAGGTGGACCAGGCGGTGGCGGTCC TGGCGGCGGAGGTCCAGATGTTGAGTGTGGCGAGGGCCACTTCTGCCACGACA ATCAGACCTGCTGCCGGGACAACAGACAAGGCTGGGCCTGTTGTCCATACAGAC AGGGCGTGTGCTGCGCCGACAGAAGGCACTGTTGTCCTGCCGGATTCAGATGTG CCGCCAGGGGCACAAAGTGCCTGCGAAGAGAAGCCCCTAGATGGGATGCCCCT CTGAGAGATCCAGCTCTGAGACAGCTCCTGTGATAG SEQ ID 21: DNA sequence of GAAco 952 C> S, 4x G4P linker and C-PGRN tag with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTCAGCAAGGCGCTTCTAGACCCGGACCT AGAGATGCCCAAGCTCACCCTGGTAGACCTAGAGCCGTGCCTACACAGTGTGAC GTGCCACCTAACAGCAGATTCGACTGCGCCCCTGACAAGGCCATCACACAAGAG CAGTGTGAAGCCAGAGGCTGCTGCTACATCCCTGCCAAACAAGGACTGCAGGGC GCCCAGATGGGACAGCCTTGGTGTTTCTTCCCACCATCTTACCCCAGCTACAAGC TGGAAAACCTGAGCAGCAGCGAGATGGGCTACACCGCCACACTGACCAGAACCA CACCTACATTCTTCCCGAAGGACATCCTGACACTGCGGCTGGACGTGATGATGG AAACCGAGAACCGGCTGCACTTCACCATCAAGGACCCCGCCAATCGGAGATACG AGGTGCCACTGGAAACCCCTCACGTGCACTCTAGAGCCCCATCTCCACTGTACA GCGTGGAATTCAGCGAGGAACCCTTCGGCGTGATCGTGCGGAGACAGCTGGATG GAAGAGTGCTGCTGAACACCACAGTGGCCCCTCTGTTCTTCGCCGACCAGTTTCT GCAGCTGAGCACCAGCCTGCCTAGCCAGTATATCACAGGCCTGGCCGAGCACCT GTCTCCACTGATGCTGAGCACATCCTGGACCAGAATCACCCTGTGGAACAGAGAT CTGGCCCCTACACCTGGCGCCAACCTGTATGGCTCTCACCCCTTTTATCTGGCCC TGGAAGATGGCGGATCTGCCCACGGTGTCTTTCTGCTGAACTCCAACGCCATGG ACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTTGGAGAAGCACAGGCGGCATCC TGGATGTGTACATCTTTCTGGGCCCCGAGCCTAAGAGCGTGGTGCAGCAGTATC TGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACC TGTGCAGATGGGGATACAGCAGCACCGCCATCACCAGACAGGTGGTGGAAAACA TGACCCGGGCTCACTTCCCACTGGATGTGCAGTGGAACGACCTGGACTACATGG ACAGCAGACGGGACTTCACCTTCAACAAGGACGGCTTCAGAGACTTCCCCGCCA TGGTGCAAGAACTGCACCAAGGCGGCAGACGGTACATGATGATCGTGGACCCTG CCATCAGCTCTAGCGGACCTGCCGGCTCTTACAGACCCTACGATGAGGGACTGA GAAGAGGCGTGTTCATCACCAACGAGACAGGCCAGCCTCTGATCGGCAAAGTGT GGCCTGGCAGCACAGCCTTTCCAGACTTCACAAACCCCACCGCTCTGGCTTGGT GGGAAGATATGGTGGCCGAGTTTCACGATCAGGTGCCCTTCGACGGCATGTGGA TCGACATGAACGAGCCCAGCAACTTCATCCGGGGCAGCGAGGATGGCTGCCCCA ACAACGAACTGGAAAATCCTCCTTACGTGCCCGGCGTTGTCGGCGGAACACTTC AGGCCGCTACAATCTGTGCCAGCAGCCATCAGTTTCTGAGCACCCACTACAACCT GCACAACCTGTACGGCCTGACCGAGGCCATTGCCTCTCATAGAGCCCTGGTTAA GGCCAGAGGCACCCGGCCTTTTGTGATCAGCAGAAGCACATTCGCCGGCCACGG CAGATATGCCGGACATTGGACAGGCGACGTGTGGTCTAGTTGGGAGCAGCTGGC TAGCAGCGTGCCAGAGATCCTGCAGTTCAATCTGCTGGGCGTGCCACTCGTGGG AGCCGATGTTTGTGGCTTCCTGGGCAACACCTCCGAGGAACTGTGTGTGCGTTG GACACAGCTGGGCGCCTTCTATCCCTTCATGAGAAACCACAACAGCCTGCTGAG CCTGCCTCAAGAGCCCTACAGCTTTAGCGAGCCTGCACAGCAGGCCATGAGAAA GGCCCTGACTCTGAGATACGCCCTGCTGCCTCACCTGTACACCCTGTTTCATCAGGCCCACGTGGCAGGCGAGACAGTGGCTAGACCTCTGTTCCTGGAATTCCCCAAG GACAGCTCCACCTGGACCGTGGATCATCAGCTGCTGTGGGGAGAAGCCCTGCTG ATTACACCAGTGCTGCAGGCCGGAAAGGCCGAAGTGACAGGCTATTTCCCTCTC GGCACTTGGTACGACCTGCAGACCGTGCCTGTTGAGGCTCTGGGATCTCTTCCT CCACCTCCTGCCGCTCCTAGAGAGCCTGCCATTCACTCTGAAGGCCAGTGGGTT ACCCTGCCTGCTCCTCTGGACACCATCAACGTGCACCTGAGAGCTGGCTACATC ATCCCTCTGCAAGGCCCTGGCCTGACCACAACCGAATCTAGACAGCAGCCCATG GCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAGGCTAGAGGCGAGCTGTTCTGG GATGATGGCGAGAGCCTGGAAGTGCTGGAACGGGGCGCTTATACCCAAGTGATC TTCCTGGCCAGAAACAACACCATCGTGAACGAACTCGTGCGCGTGACCAGTGAA GGTGCTGGACTGCAACTGCAGAAAGTGACCGTGCTCGGAGTGGCCACAGCTCCT CAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCCAACTTCACATACAGCCCCGACA CCAAGGTCCTGGACATCTGTGTGTCACTGCTGATGGGCGAGCAGTTCCTGGTGT CCTGGTCTGGTGGCGGAGGACCTGGCGGAGGTGGACCAGGCGGTGGCGGTCCT GGCGGCGGAGGTCCAGATGTTGAGTGTGGCGAGGGCCACTTCTGCCACGACAA TCAGACCTGCTGCCGGGACAACAGACAAGGCTGGGCCTGTTGTCCATACAGACA GGGCGTGTGCTGCGCCGACAGAAGGCACTGTTGTCCTGCCGGATTCAGATGTGC CGCCAGGGGCACAAAGTGCCTGCGAAGAGAAGCCCCTAGATGGGATGCCCCTCT GAGAGATCCAGCTCTGAGACAGCTCCTGTGATAG SEQ ID 22: DNA sequence of GAAco, 4x G4S linker and C-PGRN tag with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTCAGCAAGGCGCTTCTAGACCCGGACCT AGAGATGCCCAAGCTCACCCTGGTAGACCTAGAGCCGTGCCTACACAGTGTGAC GTGCCACCTAACAGCAGATTCGACTGCGCCCCTGACAAGGCCATCACACAAGAG CAGTGTGAAGCCAGAGGCTGCTGCTACATCCCTGCCAAACAAGGACTGCAGGGC GCCCAGATGGGACAGCCTTGGTGTTTCTTCCCACCATCTTACCCCAGCTACAAGC TGGAAAACCTGAGCAGCAGCGAGATGGGCTACACCGCCACACTGACCAGAACCA CACCTACATTCTTCCCGAAGGACATCCTGACACTGCGGCTGGACGTGATGATGG AAACCGAGAACCGGCTGCACTTCACCATCAAGGACCCCGCCAATCGGAGATACG AGGTGCCACTGGAAACCCCTCACGTGCACTCTAGAGCCCCATCTCCACTGTACA GCGTGGAATTCAGCGAGGAACCCTTCGGCGTGATCGTGCGGAGACAGCTGGATG GAAGAGTGCTGCTGAACACCACAGTGGCCCCTCTGTTCTTCGCCGACCAGTTTCT GCAGCTGAGCACCAGCCTGCCTAGCCAGTATATCACAGGCCTGGCCGAGCACCT GTCTCCACTGATGCTGAGCACATCCTGGACCAGAATCACCCTGTGGAACAGAGAT CTGGCCCCTACACCTGGCGCCAACCTGTATGGCTCTCACCCCTTTTATCTGGCCC TGGAAGATGGCGGATCTGCCCACGGTGTCTTTCTGCTGAACTCCAACGCCATGG ACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTTGGAGAAGCACAGGCGGCATCC TGGATGTGTACATCTTTCTGGGCCCCGAGCCTAAGAGCGTGGTGCAGCAGTATC TGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACC TGTGCAGATGGGGATACAGCAGCACCGCCATCACCAGACAGGTGGTGGAAAACA TGACCCGGGCTCACTTCCCACTGGATGTGCAGTGGAACGACCTGGACTACATGG ACAGCAGACGGGACTTCACCTTCAACAAGGACGGCTTCAGAGACTTCCCCGCCA TGGTGCAAGAACTGCACCAAGGCGGCAGACGGTACATGATGATCGTGGACCCTG CCATCAGCTCTAGCGGACCTGCCGGCTCTTACAGACCCTACGATGAGGGACTGA GAAGAGGCGTGTTCATCACCAACGAGACAGGCCAGCCTCTGATCGGCAAAGTGT GGCCTGGCAGCACAGCCTTTCCAGACTTCACAAACCCCACCGCTCTGGCTTGGT GGGAAGATATGGTGGCCGAGTTTCACGATCAGGTGCCCTTCGACGGCATGTGGA TCGACATGAACGAGCCCAGCAACTTCATCCGGGGCAGCGAGGATGGCTGCCCCA ACAACGAACTGGAAAATCCTCCTTACGTGCCCGGCGTTGTCGGCGGAACACTTC AGGCCGCTACAATCTGTGCCAGCAGCCATCAGTTTCTGAGCACCCACTACAACCT GCACAACCTGTACGGCCTGACCGAGGCCATTGCCTCTCATAGAGCCCTGGTTAAGGCCAGAGGCACCCGGCCTTTTGTGATCAGCAGAAGCACATTCGCCGGCCACGG CAGATATGCCGGACATTGGACAGGCGACGTGTGGTCTAGTTGGGAGCAGCTGGC TAGCAGCGTGCCAGAGATCCTGCAGTTCAATCTGCTGGGCGTGCCACTCGTGGG AGCCGATGTTTGTGGCTTCCTGGGCAACACCTCCGAGGAACTGTGTGTGCGTTG GACACAGCTGGGCGCCTTCTATCCCTTCATGAGAAACCACAACAGCCTGCTGAG CCTGCCTCAAGAGCCCTACAGCTTTAGCGAGCCTGCACAGCAGGCCATGAGAAA GGCCCTGACTCTGAGATACGCCCTGCTGCCTCACCTGTACACCCTGTTTCATCAG GCCCACGTGGCAGGCGAGACAGTGGCTAGACCTCTGTTCCTGGAATTCCCCAAG GACAGCTCCACCTGGACCGTGGATCATCAGCTGCTGTGGGGAGAAGCCCTGCTG ATTACACCAGTGCTGCAGGCCGGAAAGGCCGAAGTGACAGGCTATTTCCCTCTC GGCACTTGGTACGACCTGCAGACCGTGCCTGTTGAGGCTCTGGGATCTCTTCCT CCACCTCCTGCCGCTCCTAGAGAGCCTGCCATTCACTCTGAAGGCCAGTGGGTT ACCCTGCCTGCTCCTCTGGACACCATCAACGTGCACCTGAGAGCTGGCTACATC ATCCCTCTGCAAGGCCCTGGCCTGACCACAACCGAATCTAGACAGCAGCCCATG GCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAGGCTAGAGGCGAGCTGTTCTGG GATGATGGCGAGAGCCTGGAAGTGCTGGAACGGGGCGCTTATACCCAAGTGATC TTCCTGGCCAGAAACAACACCATCGTGAACGAACTCGTGCGCGTGACCAGTGAA GGTGCTGGACTGCAACTGCAGAAAGTGACCGTGCTCGGAGTGGCCACAGCTCCT CAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCCAACTTCACATACAGCCCCGACA CCAAGGTCCTGGACATCTGTGTGTCACTGCTGATGGGCGAGCAGTTCCTGGTGT CCTGGTGTGGTGGCGGAGGATCTGGCGGCGGAGGAAGCGGAGGCGGCGGTTC TGGCGGTGGTGGATCTGATGTTGAGTGTGGCGAGGGCCACTTCTGCCACGACAA TCAGACCTGCTGCCGGGACAACAGACAAGGCTGGGCCTGTTGTCCATACAGACA GGGCGTGTGCTGCGCCGACAGAAGGCACTGTTGTCCTGCCGGATTCAGATGTGC CGCCAGGGGCACAAAGTGCCTGCGAAGAGAAGCCCCTAGATGGGATGCCCCTCT GAGAGATCCAGCTCTGAGACAGCTCCTGTGATAG SEQ ID 23: DNA sequence of GAAco 952 C> S, 4x G4P linker and C-PGRN tag with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTCAGCAAGGCGCTTCTAGACCCGGACCT AGAGATGCCCAAGCTCACCCTGGTAGACCTAGAGCCGTGCCTACACAGTGTGAC GTGCCACCTAACAGCAGATTCGACTGCGCCCCTGACAAGGCCATCACACAAGAG CAGTGTGAAGCCAGAGGCTGCTGCTACATCCCTGCCAAACAAGGACTGCAGGGC GCCCAGATGGGACAGCCTTGGTGTTTCTTCCCACCATCTTACCCCAGCTACAAGC TGGAAAACCTGAGCAGCAGCGAGATGGGCTACACCGCCACACTGACCAGAACCA CACCTACATTCTTCCCGAAGGACATCCTGACACTGCGGCTGGACGTGATGATGG AAACCGAGAACCGGCTGCACTTCACCATCAAGGACCCCGCCAATCGGAGATACG AGGTGCCACTGGAAACCCCTCACGTGCACTCTAGAGCCCCATCTCCACTGTACA GCGTGGAATTCAGCGAGGAACCCTTCGGCGTGATCGTGCGGAGACAGCTGGATG GAAGAGTGCTGCTGAACACCACAGTGGCCCCTCTGTTCTTCGCCGACCAGTTTCT GCAGCTGAGCACCAGCCTGCCTAGCCAGTATATCACAGGCCTGGCCGAGCACCT GTCTCCACTGATGCTGAGCACATCCTGGACCAGAATCACCCTGTGGAACAGAGAT CTGGCCCCTACACCTGGCGCCAACCTGTATGGCTCTCACCCCTTTTATCTGGCCC TGGAAGATGGCGGATCTGCCCACGGTGTCTTTCTGCTGAACTCCAACGCCATGG ACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTTGGAGAAGCACAGGCGGCATCC TGGATGTGTACATCTTTCTGGGCCCCGAGCCTAAGAGCGTGGTGCAGCAGTATC TGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACC TGTGCAGATGGGGATACAGCAGCACCGCCATCACCAGACAGGTGGTGGAAAACA TGACCCGGGCTCACTTCCCACTGGATGTGCAGTGGAACGACCTGGACTACATGG ACAGCAGACGGGACTTCACCTTCAACAAGGACGGCTTCAGAGACTTCCCCGCCA TGGTGCAAGAACTGCACCAAGGCGGCAGACGGTACATGATGATCGTGGACCCTG CCATCAGCTCTAGCGGACCTGCCGGCTCTTACAGACCCTACGATGAGGGACTGAGAAGAGGCGTGTTCATCACCAACGAGACAGGCCAGCCTCTGATCGGCAAAGTGT GGCCTGGCAGCACAGCCTTTCCAGACTTCACAAACCCCACCGCTCTGGCTTGGT GGGAAGATATGGTGGCCGAGTTTCACGATCAGGTGCCCTTCGACGGCATGTGGA TCGACATGAACGAGCCCAGCAACTTCATCCGGGGCAGCGAGGATGGCTGCCCCA ACAACGAACTGGAAAATCCTCCTTACGTGCCCGGCGTTGTCGGCGGAACACTTC AGGCCGCTACAATCTGTGCCAGCAGCCATCAGTTTCTGAGCACCCACTACAACCT GCACAACCTGTACGGCCTGACCGAGGCCATTGCCTCTCATAGAGCCCTGGTTAA GGCCAGAGGCACCCGGCCTTTTGTGATCAGCAGAAGCACATTCGCCGGCCACGG CAGATATGCCGGACATTGGACAGGCGACGTGTGGTCTAGTTGGGAGCAGCTGGC TAGCAGCGTGCCAGAGATCCTGCAGTTCAATCTGCTGGGCGTGCCACTCGTGGG AGCCGATGTTTGTGGCTTCCTGGGCAACACCTCCGAGGAACTGTGTGTGCGTTG GACACAGCTGGGCGCCTTCTATCCCTTCATGAGAAACCACAACAGCCTGCTGAG CCTGCCTCAAGAGCCCTACAGCTTTAGCGAGCCTGCACAGCAGGCCATGAGAAA GGCCCTGACTCTGAGATACGCCCTGCTGCCTCACCTGTACACCCTGTTTCATCAG GCCCACGTGGCAGGCGAGACAGTGGCTAGACCTCTGTTCCTGGAATTCCCCAAG GACAGCTCCACCTGGACCGTGGATCATCAGCTGCTGTGGGGAGAAGCCCTGCTG ATTACACCAGTGCTGCAGGCCGGAAAGGCCGAAGTGACAGGCTATTTCCCTCTC GGCACTTGGTACGACCTGCAGACCGTGCCTGTTGAGGCTCTGGGATCTCTTCCT CCACCTCCTGCCGCTCCTAGAGAGCCTGCCATTCACTCTGAAGGCCAGTGGGTT ACCCTGCCTGCTCCTCTGGACACCATCAACGTGCACCTGAGAGCTGGCTACATC ATCCCTCTGCAAGGCCCTGGCCTGACCACAACCGAATCTAGACAGCAGCCCATG GCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAGGCTAGAGGCGAGCTGTTCTGG GATGATGGCGAGAGCCTGGAAGTGCTGGAACGGGGCGCTTATACCCAAGTGATC TTCCTGGCCAGAAACAACACCATCGTGAACGAACTCGTGCGCGTGACCAGTGAA GGTGCTGGACTGCAACTGCAGAAAGTGACCGTGCTCGGAGTGGCCACAGCTCCT CAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCCAACTTCACATACAGCCCCGACA CCAAGGTCCTGGACATCTGTGTGTCACTGCTGATGGGCGAGCAGTTCCTGGTGT CCTGGTCTGGTGGCGGAGGATCTGGCGGCGGAGGAAGCGGAGGCGGCGGTTCT GGCGGTGGTGGATCTGATGTTGAGTGTGGCGAGGGCCACTTCTGCCACGACAAT CAGACCTGCTGCCGGGACAACAGACAAGGCTGGGCCTGTTGTCCATACAGACAG GGCGTGTGCTGCGCCGACAGAAGGCACTGTTGTCCTGCCGGATTCAGATGTGCC GCCAGGGGCACAAAGTGCCTGCGAAGAGAAGCCCCTAGATGGGATGCCCCTCT GAGAGATCCAGCTCTGAGACAGCTCCTGTGATAG SEQ ID 24: DNA sequence of GAAco, 4x G4P linker and C-PSAP tag with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTCAGCAAGGCGCTTCTAGACCCGGACCT AGAGATGCCCAAGCTCACCCTGGTAGACCTAGAGCCGTGCCTACACAGTGTGAC GTGCCACCTAACAGCAGATTCGACTGCGCCCCTGACAAGGCCATCACACAAGAG CAGTGTGAAGCCAGAGGCTGCTGCTACATCCCTGCCAAACAAGGACTGCAGGGC GCCCAGATGGGACAGCCTTGGTGTTTCTTCCCACCATCTTACCCCAGCTACAAGC TGGAAAACCTGAGCAGCAGCGAGATGGGCTACACCGCCACACTGACCAGAACCA CACCTACATTCTTCCCGAAGGACATCCTGACACTGCGGCTGGACGTGATGATGG AAACCGAGAACCGGCTGCACTTCACCATCAAGGACCCCGCCAATCGGAGATACG AGGTGCCACTGGAAACCCCTCACGTGCACTCTAGAGCCCCATCTCCACTGTACA GCGTGGAATTCAGCGAGGAACCCTTCGGCGTGATCGTGCGGAGACAGCTGGATG GAAGAGTGCTGCTGAACACCACAGTGGCCCCTCTGTTCTTCGCCGACCAGTTTCT GCAGCTGAGCACCAGCCTGCCTAGCCAGTATATCACAGGCCTGGCCGAGCACCT GTCTCCACTGATGCTGAGCACATCCTGGACCAGAATCACCCTGTGGAACAGAGAT CTGGCCCCTACACCTGGCGCCAACCTGTATGGCTCTCACCCCTTTTATCTGGCCC TGGAAGATGGCGGATCTGCCCACGGTGTCTTTCTGCTGAACTCCAACGCCATGG ACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTTGGAGAAGCACAGGCGGCATCCTGGATGTGTACATCTTTCTGGGCCCCGAGCCTAAGAGCGTGGTGCAGCAGTATC TGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACC TGTGCAGATGGGGATACAGCAGCACCGCCATCACCAGACAGGTGGTGGAAAACA TGACCCGGGCTCACTTCCCACTGGATGTGCAGTGGAACGACCTGGACTACATGG ACAGCAGACGGGACTTCACCTTCAACAAGGACGGCTTCAGAGACTTCCCCGCCA TGGTGCAAGAACTGCACCAAGGCGGCAGACGGTACATGATGATCGTGGACCCTG CCATCAGCTCTAGCGGACCTGCCGGCTCTTACAGACCCTACGATGAGGGACTGA GAAGAGGCGTGTTCATCACCAACGAGACAGGCCAGCCTCTGATCGGCAAAGTGT GGCCTGGCAGCACAGCCTTTCCAGACTTCACAAACCCCACCGCTCTGGCTTGGT GGGAAGATATGGTGGCCGAGTTTCACGATCAGGTGCCCTTCGACGGCATGTGGA TCGACATGAACGAGCCCAGCAACTTCATCCGGGGCAGCGAGGATGGCTGCCCCA ACAACGAACTGGAAAATCCTCCTTACGTGCCCGGCGTTGTCGGCGGAACACTTC AGGCCGCTACAATCTGTGCCAGCAGCCATCAGTTTCTGAGCACCCACTACAACCT GCACAACCTGTACGGCCTGACCGAGGCCATTGCCTCTCATAGAGCCCTGGTTAA GGCCAGAGGCACCCGGCCTTTTGTGATCAGCAGAAGCACATTCGCCGGCCACGG CAGATATGCCGGACATTGGACAGGCGACGTGTGGTCTAGTTGGGAGCAGCTGGC TAGCAGCGTGCCAGAGATCCTGCAGTTCAATCTGCTGGGCGTGCCACTCGTGGG AGCCGATGTTTGTGGCTTCCTGGGCAACACCTCCGAGGAACTGTGTGTGCGTTG GACACAGCTGGGCGCCTTCTATCCCTTCATGAGAAACCACAACAGCCTGCTGAG CCTGCCTCAAGAGCCCTACAGCTTTAGCGAGCCTGCACAGCAGGCCATGAGAAA GGCCCTGACTCTGAGATACGCCCTGCTGCCTCACCTGTACACCCTGTTTCATCAG GCCCACGTGGCAGGCGAGACAGTGGCTAGACCTCTGTTCCTGGAATTCCCCAAG GACAGCTCCACCTGGACCGTGGATCATCAGCTGCTGTGGGGAGAAGCCCTGCTG ATTACACCAGTGCTGCAGGCCGGAAAGGCCGAAGTGACAGGCTATTTCCCTCTC GGCACTTGGTACGACCTGCAGACCGTGCCTGTTGAGGCTCTGGGATCTCTTCCT CCACCTCCTGCCGCTCCTAGAGAGCCTGCCATTCACTCTGAAGGCCAGTGGGTT ACCCTGCCTGCTCCTCTGGACACCATCAACGTGCACCTGAGAGCTGGCTACATC ATCCCTCTGCAAGGCCCTGGCCTGACCACAACCGAATCTAGACAGCAGCCCATG GCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAGGCTAGAGGCGAGCTGTTCTGG GATGATGGCGAGAGCCTGGAAGTGCTGGAACGGGGCGCTTATACCCAAGTGATC TTCCTGGCCAGAAACAACACCATCGTGAACGAACTCGTGCGCGTGACCAGTGAA GGTGCTGGACTGCAACTGCAGAAAGTGACCGTGCTCGGAGTGGCCACAGCTCCT CAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCCAACTTCACATACAGCCCCGACA CCAAGGTCCTGGACATCTGTGTGTCACTGCTGATGGGCGAGCAGTTCCTGGTGT CCTGGTGTGGTGGCGGAGGACCTGGCGGAGGTGGACCAGGCGGTGGCGGTCC TGGCGGCGGAGGTCCAGATGTTGAGTGTGGCGAGGGCCACTTCTGCCACGACA ATCAGACCTGCTGCCGGGACAACAGACAAGGCTGGGCCTGTTGTCCATACAGAC AGGGCGTGTGCTGCGCCGACAGAAGGCACTGTTGTCCTGCCGGATTCAGATGTG CCGCCAGGGGCACAAAGTGCCTGTGATAG SEQ ID 25: DNA sequence of GAAco 952 C> S, 4x G4P linker and C-PSAP tag with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTCAGCAAGGCGCTTCTAGACCCGGACCT AGAGATGCCCAAGCTCACCCTGGTAGACCTAGAGCCGTGCCTACACAGTGTGAC GTGCCACCTAACAGCAGATTCGACTGCGCCCCTGACAAGGCCATCACACAAGAG CAGTGTGAAGCCAGAGGCTGCTGCTACATCCCTGCCAAACAAGGACTGCAGGGC GCCCAGATGGGACAGCCTTGGTGTTTCTTCCCACCATCTTACCCCAGCTACAAGC TGGAAAACCTGAGCAGCAGCGAGATGGGCTACACCGCCACACTGACCAGAACCA CACCTACATTCTTCCCGAAGGACATCCTGACACTGCGGCTGGACGTGATGATGG AAACCGAGAACCGGCTGCACTTCACCATCAAGGACCCCGCCAATCGGAGATACG AGGTGCCACTGGAAACCCCTCACGTGCACTCTAGAGCCCCATCTCCACTGTACA GCGTGGAATTCAGCGAGGAACCCTTCGGCGTGATCGTGCGGAGACAGCTGGATGGAAGAGTGCTGCTGAACACCACAGTGGCCCCTCTGTTCTTCGCCGACCAGTTTCT GCAGCTGAGCACCAGCCTGCCTAGCCAGTATATCACAGGCCTGGCCGAGCACCT GTCTCCACTGATGCTGAGCACATCCTGGACCAGAATCACCCTGTGGAACAGAGAT CTGGCCCCTACACCTGGCGCCAACCTGTATGGCTCTCACCCCTTTTATCTGGCCC TGGAAGATGGCGGATCTGCCCACGGTGTCTTTCTGCTGAACTCCAACGCCATGG ACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTTGGAGAAGCACAGGCGGCATCC TGGATGTGTACATCTTTCTGGGCCCCGAGCCTAAGAGCGTGGTGCAGCAGTATC TGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACC TGTGCAGATGGGGATACAGCAGCACCGCCATCACCAGACAGGTGGTGGAAAACA TGACCCGGGCTCACTTCCCACTGGATGTGCAGTGGAACGACCTGGACTACATGG ACAGCAGACGGGACTTCACCTTCAACAAGGACGGCTTCAGAGACTTCCCCGCCA TGGTGCAAGAACTGCACCAAGGCGGCAGACGGTACATGATGATCGTGGACCCTG CCATCAGCTCTAGCGGACCTGCCGGCTCTTACAGACCCTACGATGAGGGACTGA GAAGAGGCGTGTTCATCACCAACGAGACAGGCCAGCCTCTGATCGGCAAAGTGT GGCCTGGCAGCACAGCCTTTCCAGACTTCACAAACCCCACCGCTCTGGCTTGGT GGGAAGATATGGTGGCCGAGTTTCACGATCAGGTGCCCTTCGACGGCATGTGGA TCGACATGAACGAGCCCAGCAACTTCATCCGGGGCAGCGAGGATGGCTGCCCCA ACAACGAACTGGAAAATCCTCCTTACGTGCCCGGCGTTGTCGGCGGAACACTTC AGGCCGCTACAATCTGTGCCAGCAGCCATCAGTTTCTGAGCACCCACTACAACCT GCACAACCTGTACGGCCTGACCGAGGCCATTGCCTCTCATAGAGCCCTGGTTAA GGCCAGAGGCACCCGGCCTTTTGTGATCAGCAGAAGCACATTCGCCGGCCACGG CAGATATGCCGGACATTGGACAGGCGACGTGTGGTCTAGTTGGGAGCAGCTGGC TAGCAGCGTGCCAGAGATCCTGCAGTTCAATCTGCTGGGCGTGCCACTCGTGGG AGCCGATGTTTGTGGCTTCCTGGGCAACACCTCCGAGGAACTGTGTGTGCGTTG GACACAGCTGGGCGCCTTCTATCCCTTCATGAGAAACCACAACAGCCTGCTGAG CCTGCCTCAAGAGCCCTACAGCTTTAGCGAGCCTGCACAGCAGGCCATGAGAAA GGCCCTGACTCTGAGATACGCCCTGCTGCCTCACCTGTACACCCTGTTTCATCAG GCCCACGTGGCAGGCGAGACAGTGGCTAGACCTCTGTTCCTGGAATTCCCCAAG GACAGCTCCACCTGGACCGTGGATCATCAGCTGCTGTGGGGAGAAGCCCTGCTG ATTACACCAGTGCTGCAGGCCGGAAAGGCCGAAGTGACAGGCTATTTCCCTCTC GGCACTTGGTACGACCTGCAGACCGTGCCTGTTGAGGCTCTGGGATCTCTTCCT CCACCTCCTGCCGCTCCTAGAGAGCCTGCCATTCACTCTGAAGGCCAGTGGGTT ACCCTGCCTGCTCCTCTGGACACCATCAACGTGCACCTGAGAGCTGGCTACATC ATCCCTCTGCAAGGCCCTGGCCTGACCACAACCGAATCTAGACAGCAGCCCATG GCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAGGCTAGAGGCGAGCTGTTCTGG GATGATGGCGAGAGCCTGGAAGTGCTGGAACGGGGCGCTTATACCCAAGTGATC TTCCTGGCCAGAAACAACACCATCGTGAACGAACTCGTGCGCGTGACCAGTGAA GGTGCTGGACTGCAACTGCAGAAAGTGACCGTGCTCGGAGTGGCCACAGCTCCT CAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCCAACTTCACATACAGCCCCGACA CCAAGGTCCTGGACATCTGTGTGTCACTGCTGATGGGCGAGCAGTTCCTGGTGT CCTGGTCTGGTGGCGGAGGACCTGGCGGAGGTGGACCAGGCGGTGGCGGTCCT GGCGGCGGAGGTCCAGATGTTGAGTGTGGCGAGGGCCACTTCTGCCACGACAA TCAGACCTGCTGCCGGGACAACAGACAAGGCTGGGCCTGTTGTCCATACAGACA GGGCGTGTGCTGCGCCGACAGAAGGCACTGTTGTCCTGCCGGATTCAGATGTGC CGCCAGGGGCACAAAGTGCCTGTGATAG SEQ ID 26: DNA sequence of GAAco, 4x G4S linker and C-PSAP tag with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTCAGCAAGGCGCTTCTAGACCCGGACCT AGAGATGCCCAAGCTCACCCTGGTAGACCTAGAGCCGTGCCTACACAGTGTGAC GTGCCACCTAACAGCAGATTCGACTGCGCCCCTGACAAGGCCATCACACAAGAG CAGTGTGAAGCCAGAGGCTGCTGCTACATCCCTGCCAAACAAGGACTGCAGGGCGCCCAGATGGGACAGCCTTGGTGTTTCTTCCCACCATCTTACCCCAGCTACAAGC TGGAAAACCTGAGCAGCAGCGAGATGGGCTACACCGCCACACTGACCAGAACCA CACCTACATTCTTCCCGAAGGACATCCTGACACTGCGGCTGGACGTGATGATGG AAACCGAGAACCGGCTGCACTTCACCATCAAGGACCCCGCCAATCGGAGATACG AGGTGCCACTGGAAACCCCTCACGTGCACTCTAGAGCCCCATCTCCACTGTACA GCGTGGAATTCAGCGAGGAACCCTTCGGCGTGATCGTGCGGAGACAGCTGGATG GAAGAGTGCTGCTGAACACCACAGTGGCCCCTCTGTTCTTCGCCGACCAGTTTCT GCAGCTGAGCACCAGCCTGCCTAGCCAGTATATCACAGGCCTGGCCGAGCACCT GTCTCCACTGATGCTGAGCACATCCTGGACCAGAATCACCCTGTGGAACAGAGAT CTGGCCCCTACACCTGGCGCCAACCTGTATGGCTCTCACCCCTTTTATCTGGCCC TGGAAGATGGCGGATCTGCCCACGGTGTCTTTCTGCTGAACTCCAACGCCATGG ACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTTGGAGAAGCACAGGCGGCATCC TGGATGTGTACATCTTTCTGGGCCCCGAGCCTAAGAGCGTGGTGCAGCAGTATC TGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACC TGTGCAGATGGGGATACAGCAGCACCGCCATCACCAGACAGGTGGTGGAAAACA TGACCCGGGCTCACTTCCCACTGGATGTGCAGTGGAACGACCTGGACTACATGG ACAGCAGACGGGACTTCACCTTCAACAAGGACGGCTTCAGAGACTTCCCCGCCA TGGTGCAAGAACTGCACCAAGGCGGCAGACGGTACATGATGATCGTGGACCCTG CCATCAGCTCTAGCGGACCTGCCGGCTCTTACAGACCCTACGATGAGGGACTGA GAAGAGGCGTGTTCATCACCAACGAGACAGGCCAGCCTCTGATCGGCAAAGTGT GGCCTGGCAGCACAGCCTTTCCAGACTTCACAAACCCCACCGCTCTGGCTTGGT GGGAAGATATGGTGGCCGAGTTTCACGATCAGGTGCCCTTCGACGGCATGTGGA TCGACATGAACGAGCCCAGCAACTTCATCCGGGGCAGCGAGGATGGCTGCCCCA ACAACGAACTGGAAAATCCTCCTTACGTGCCCGGCGTTGTCGGCGGAACACTTC AGGCCGCTACAATCTGTGCCAGCAGCCATCAGTTTCTGAGCACCCACTACAACCT GCACAACCTGTACGGCCTGACCGAGGCCATTGCCTCTCATAGAGCCCTGGTTAA GGCCAGAGGCACCCGGCCTTTTGTGATCAGCAGAAGCACATTCGCCGGCCACGG CAGATATGCCGGACATTGGACAGGCGACGTGTGGTCTAGTTGGGAGCAGCTGGC TAGCAGCGTGCCAGAGATCCTGCAGTTCAATCTGCTGGGCGTGCCACTCGTGGG AGCCGATGTTTGTGGCTTCCTGGGCAACACCTCCGAGGAACTGTGTGTGCGTTG GACACAGCTGGGCGCCTTCTATCCCTTCATGAGAAACCACAACAGCCTGCTGAG CCTGCCTCAAGAGCCCTACAGCTTTAGCGAGCCTGCACAGCAGGCCATGAGAAA GGCCCTGACTCTGAGATACGCCCTGCTGCCTCACCTGTACACCCTGTTTCATCAG GCCCACGTGGCAGGCGAGACAGTGGCTAGACCTCTGTTCCTGGAATTCCCCAAG GACAGCTCCACCTGGACCGTGGATCATCAGCTGCTGTGGGGAGAAGCCCTGCTG ATTACACCAGTGCTGCAGGCCGGAAAGGCCGAAGTGACAGGCTATTTCCCTCTC GGCACTTGGTACGACCTGCAGACCGTGCCTGTTGAGGCTCTGGGATCTCTTCCT CCACCTCCTGCCGCTCCTAGAGAGCCTGCCATTCACTCTGAAGGCCAGTGGGTT ACCCTGCCTGCTCCTCTGGACACCATCAACGTGCACCTGAGAGCTGGCTACATC ATCCCTCTGCAAGGCCCTGGCCTGACCACAACCGAATCTAGACAGCAGCCCATG GCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAGGCTAGAGGCGAGCTGTTCTGG GATGATGGCGAGAGCCTGGAAGTGCTGGAACGGGGCGCTTATACCCAAGTGATC TTCCTGGCCAGAAACAACACCATCGTGAACGAACTCGTGCGCGTGACCAGTGAA GGTGCTGGACTGCAACTGCAGAAAGTGACCGTGCTCGGAGTGGCCACAGCTCCT CAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCCAACTTCACATACAGCCCCGACA CCAAGGTCCTGGACATCTGTGTGTCACTGCTGATGGGCGAGCAGTTCCTGGTGT CCTGGTGTGGTGGCGGAGGATCTGGCGGCGGAGGAAGCGGAGGCGGCGGTTC TGGCGGTGGTGGATCTGATGTTGAGTGTGGCGAGGGCCACTTCTGCCACGACAA TCAGACCTGCTGCCGGGACAACAGACAAGGCTGGGCCTGTTGTCCATACAGACA GGGCGTGTGCTGCGCCGACAGAAGGCACTGTTGTCCTGCCGGATTCAGATGTGC CGCCAGGGGCACAAAGTGCCTGTGATAGSEQ ID 27: DNA sequence of GAAco 952 C> S, 4x G4S linker and C-PSAP tag with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTCAGCAAGGCGCTTCTAGACCCGGACCT AGAGATGCCCAAGCTCACCCTGGTAGACCTAGAGCCGTGCCTACACAGTGTGAC GTGCCACCTAACAGCAGATTCGACTGCGCCCCTGACAAGGCCATCACACAAGAG CAGTGTGAAGCCAGAGGCTGCTGCTACATCCCTGCCAAACAAGGACTGCAGGGC GCCCAGATGGGACAGCCTTGGTGTTTCTTCCCACCATCTTACCCCAGCTACAAGC TGGAAAACCTGAGCAGCAGCGAGATGGGCTACACCGCCACACTGACCAGAACCA CACCTACATTCTTCCCGAAGGACATCCTGACACTGCGGCTGGACGTGATGATGG AAACCGAGAACCGGCTGCACTTCACCATCAAGGACCCCGCCAATCGGAGATACG AGGTGCCACTGGAAACCCCTCACGTGCACTCTAGAGCCCCATCTCCACTGTACA GCGTGGAATTCAGCGAGGAACCCTTCGGCGTGATCGTGCGGAGACAGCTGGATG GAAGAGTGCTGCTGAACACCACAGTGGCCCCTCTGTTCTTCGCCGACCAGTTTCT GCAGCTGAGCACCAGCCTGCCTAGCCAGTATATCACAGGCCTGGCCGAGCACCT GTCTCCACTGATGCTGAGCACATCCTGGACCAGAATCACCCTGTGGAACAGAGAT CTGGCCCCTACACCTGGCGCCAACCTGTATGGCTCTCACCCCTTTTATCTGGCCC TGGAAGATGGCGGATCTGCCCACGGTGTCTTTCTGCTGAACTCCAACGCCATGG ACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTTGGAGAAGCACAGGCGGCATCC TGGATGTGTACATCTTTCTGGGCCCCGAGCCTAAGAGCGTGGTGCAGCAGTATC TGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACC TGTGCAGATGGGGATACAGCAGCACCGCCATCACCAGACAGGTGGTGGAAAACA TGACCCGGGCTCACTTCCCACTGGATGTGCAGTGGAACGACCTGGACTACATGG ACAGCAGACGGGACTTCACCTTCAACAAGGACGGCTTCAGAGACTTCCCCGCCA TGGTGCAAGAACTGCACCAAGGCGGCAGACGGTACATGATGATCGTGGACCCTG CCATCAGCTCTAGCGGACCTGCCGGCTCTTACAGACCCTACGATGAGGGACTGA GAAGAGGCGTGTTCATCACCAACGAGACAGGCCAGCCTCTGATCGGCAAAGTGT GGCCTGGCAGCACAGCCTTTCCAGACTTCACAAACCCCACCGCTCTGGCTTGGT GGGAAGATATGGTGGCCGAGTTTCACGATCAGGTGCCCTTCGACGGCATGTGGA TCGACATGAACGAGCCCAGCAACTTCATCCGGGGCAGCGAGGATGGCTGCCCCA ACAACGAACTGGAAAATCCTCCTTACGTGCCCGGCGTTGTCGGCGGAACACTTC AGGCCGCTACAATCTGTGCCAGCAGCCATCAGTTTCTGAGCACCCACTACAACCT GCACAACCTGTACGGCCTGACCGAGGCCATTGCCTCTCATAGAGCCCTGGTTAA GGCCAGAGGCACCCGGCCTTTTGTGATCAGCAGAAGCACATTCGCCGGCCACGG CAGATATGCCGGACATTGGACAGGCGACGTGTGGTCTAGTTGGGAGCAGCTGGC TAGCAGCGTGCCAGAGATCCTGCAGTTCAATCTGCTGGGCGTGCCACTCGTGGG AGCCGATGTTTGTGGCTTCCTGGGCAACACCTCCGAGGAACTGTGTGTGCGTTG GACACAGCTGGGCGCCTTCTATCCCTTCATGAGAAACCACAACAGCCTGCTGAG CCTGCCTCAAGAGCCCTACAGCTTTAGCGAGCCTGCACAGCAGGCCATGAGAAA GGCCCTGACTCTGAGATACGCCCTGCTGCCTCACCTGTACACCCTGTTTCATCAG GCCCACGTGGCAGGCGAGACAGTGGCTAGACCTCTGTTCCTGGAATTCCCCAAG GACAGCTCCACCTGGACCGTGGATCATCAGCTGCTGTGGGGAGAAGCCCTGCTG ATTACACCAGTGCTGCAGGCCGGAAAGGCCGAAGTGACAGGCTATTTCCCTCTC GGCACTTGGTACGACCTGCAGACCGTGCCTGTTGAGGCTCTGGGATCTCTTCCT CCACCTCCTGCCGCTCCTAGAGAGCCTGCCATTCACTCTGAAGGCCAGTGGGTT ACCCTGCCTGCTCCTCTGGACACCATCAACGTGCACCTGAGAGCTGGCTACATC ATCCCTCTGCAAGGCCCTGGCCTGACCACAACCGAATCTAGACAGCAGCCCATG GCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAGGCTAGAGGCGAGCTGTTCTGG GATGATGGCGAGAGCCTGGAAGTGCTGGAACGGGGCGCTTATACCCAAGTGATC TTCCTGGCCAGAAACAACACCATCGTGAACGAACTCGTGCGCGTGACCAGTGAA GGTGCTGGACTGCAACTGCAGAAAGTGACCGTGCTCGGAGTGGCCACAGCTCCT CAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCCAACTTCACATACAGCCCCGACA CCAAGGTCCTGGACATCTGTGTGTCACTGCTGATGGGCGAGCAGTTCCTGGTGTCCTGGTCTGGTGGCGGAGGATCTGGCGGCGGAGGAAGCGGAGGCGGCGGTTCT GGCGGTGGTGGATCTGATGTTGAGTGTGGCGAGGGCCACTTCTGCCACGACAAT CAGACCTGCTGCCGGGACAACAGACAAGGCTGGGCCTGTTGTCCATACAGACAG GGCGTGTGCTGCGCCGACAGAAGGCACTGTTGTCCTGCCGGATTCAGATGTGCC GCCAGGGGCACAAAGTGCCTGTGATAG SEQ ID 28: DNA sequence of GAAco, 4x G4P linker and C-SRT tag with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTCAGCAAGGCGCTTCTAGACCCGGACCT AGAGATGCCCAAGCTCACCCTGGTAGACCTAGAGCCGTGCCTACACAGTGTGAC GTGCCACCTAACAGCAGATTCGACTGCGCCCCTGACAAGGCCATCACACAAGAG CAGTGTGAAGCCAGAGGCTGCTGCTACATCCCTGCCAAACAAGGACTGCAGGGC GCCCAGATGGGACAGCCTTGGTGTTTCTTCCCACCATCTTACCCCAGCTACAAGC TGGAAAACCTGAGCAGCAGCGAGATGGGCTACACCGCCACACTGACCAGAACCA CACCTACATTCTTCCCGAAGGACATCCTGACACTGCGGCTGGACGTGATGATGG AAACCGAGAACCGGCTGCACTTCACCATCAAGGACCCCGCCAATCGGAGATACG AGGTGCCACTGGAAACCCCTCACGTGCACTCTAGAGCCCCATCTCCACTGTACA GCGTGGAATTCAGCGAGGAACCCTTCGGCGTGATCGTGCGGAGACAGCTGGATG GAAGAGTGCTGCTGAACACCACAGTGGCCCCTCTGTTCTTCGCCGACCAGTTTCT GCAGCTGAGCACCAGCCTGCCTAGCCAGTATATCACAGGCCTGGCCGAGCACCT GTCTCCACTGATGCTGAGCACATCCTGGACCAGAATCACCCTGTGGAACAGAGAT CTGGCCCCTACACCTGGCGCCAACCTGTATGGCTCTCACCCCTTTTATCTGGCCC TGGAAGATGGCGGATCTGCCCACGGTGTCTTTCTGCTGAACTCCAACGCCATGG ACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTTGGAGAAGCACAGGCGGCATCC TGGATGTGTACATCTTTCTGGGCCCCGAGCCTAAGAGCGTGGTGCAGCAGTATC TGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACC TGTGCAGATGGGGATACAGCAGCACCGCCATCACCAGACAGGTGGTGGAAAACA TGACCCGGGCTCACTTCCCACTGGATGTGCAGTGGAACGACCTGGACTACATGG ACAGCAGACGGGACTTCACCTTCAACAAGGACGGCTTCAGAGACTTCCCCGCCA TGGTGCAAGAACTGCACCAAGGCGGCAGACGGTACATGATGATCGTGGACCCTG CCATCAGCTCTAGCGGACCTGCCGGCTCTTACAGACCCTACGATGAGGGACTGA GAAGAGGCGTGTTCATCACCAACGAGACAGGCCAGCCTCTGATCGGCAAAGTGT GGCCTGGCAGCACAGCCTTTCCAGACTTCACAAACCCCACCGCTCTGGCTTGGT GGGAAGATATGGTGGCCGAGTTTCACGATCAGGTGCCCTTCGACGGCATGTGGA TCGACATGAACGAGCCCAGCAACTTCATCCGGGGCAGCGAGGATGGCTGCCCCA ACAACGAACTGGAAAATCCTCCTTACGTGCCCGGCGTTGTCGGCGGAACACTTC AGGCCGCTACAATCTGTGCCAGCAGCCATCAGTTTCTGAGCACCCACTACAACCT GCACAACCTGTACGGCCTGACCGAGGCCATTGCCTCTCATAGAGCCCTGGTTAA GGCCAGAGGCACCCGGCCTTTTGTGATCAGCAGAAGCACATTCGCCGGCCACGG CAGATATGCCGGACATTGGACAGGCGACGTGTGGTCTAGTTGGGAGCAGCTGGC TAGCAGCGTGCCAGAGATCCTGCAGTTCAATCTGCTGGGCGTGCCACTCGTGGG AGCCGATGTTTGTGGCTTCCTGGGCAACACCTCCGAGGAACTGTGTGTGCGTTG GACACAGCTGGGCGCCTTCTATCCCTTCATGAGAAACCACAACAGCCTGCTGAG CCTGCCTCAAGAGCCCTACAGCTTTAGCGAGCCTGCACAGCAGGCCATGAGAAA GGCCCTGACTCTGAGATACGCCCTGCTGCCTCACCTGTACACCCTGTTTCATCAG GCCCACGTGGCAGGCGAGACAGTGGCTAGACCTCTGTTCCTGGAATTCCCCAAG GACAGCTCCACCTGGACCGTGGATCATCAGCTGCTGTGGGGAGAAGCCCTGCTG ATTACACCAGTGCTGCAGGCCGGAAAGGCCGAAGTGACAGGCTATTTCCCTCTC GGCACTTGGTACGACCTGCAGACCGTGCCTGTTGAGGCTCTGGGATCTCTTCCT CCACCTCCTGCCGCTCCTAGAGAGCCTGCCATTCACTCTGAAGGCCAGTGGGTT ACCCTGCCTGCTCCTCTGGACACCATCAACGTGCACCTGAGAGCTGGCTACATC ATCCCTCTGCAAGGCCCTGGCCTGACCACAACCGAATCTAGACAGCAGCCCATGGCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAGGCTAGAGGCGAGCTGTTCTGG GATGATGGCGAGAGCCTGGAAGTGCTGGAACGGGGCGCTTATACCCAAGTGATC TTCCTGGCCAGAAACAACACCATCGTGAACGAACTCGTGCGCGTGACCAGTGAA GGTGCTGGACTGCAACTGCAGAAAGTGACCGTGCTCGGAGTGGCCACAGCTCCT CAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCCAACTTCACATACAGCCCCGACA CCAAGGTCCTGGACATCTGTGTGTCACTGCTGATGGGCGAGCAGTTCCTGGTGT CCTGGTGTGGTGGCGGAGGACCTGGCGGAGGTGGACCAGGCGGTGGCGGTCC TGGCGGCGGAGGTCCAACAAAGTGCCTGCGAAGAGAAGCCCCTAGATGGGATG CCCCTCTGAGAGATCCAGCTCTGAGACAGCTCCTGTGATAG SEQ ID 29: DNA sequence of GAAco 952 C> S, 4x G4P linker and C-SRT tag with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTCAGCAAGGCGCTTCTAGACCCGGACCT AGAGATGCCCAAGCTCACCCTGGTAGACCTAGAGCCGTGCCTACACAGTGTGAC GTGCCACCTAACAGCAGATTCGACTGCGCCCCTGACAAGGCCATCACACAAGAG CAGTGTGAAGCCAGAGGCTGCTGCTACATCCCTGCCAAACAAGGACTGCAGGGC GCCCAGATGGGACAGCCTTGGTGTTTCTTCCCACCATCTTACCCCAGCTACAAGC TGGAAAACCTGAGCAGCAGCGAGATGGGCTACACCGCCACACTGACCAGAACCA CACCTACATTCTTCCCGAAGGACATCCTGACACTGCGGCTGGACGTGATGATGG AAACCGAGAACCGGCTGCACTTCACCATCAAGGACCCCGCCAATCGGAGATACG AGGTGCCACTGGAAACCCCTCACGTGCACTCTAGAGCCCCATCTCCACTGTACA GCGTGGAATTCAGCGAGGAACCCTTCGGCGTGATCGTGCGGAGACAGCTGGATG GAAGAGTGCTGCTGAACACCACAGTGGCCCCTCTGTTCTTCGCCGACCAGTTTCT GCAGCTGAGCACCAGCCTGCCTAGCCAGTATATCACAGGCCTGGCCGAGCACCT GTCTCCACTGATGCTGAGCACATCCTGGACCAGAATCACCCTGTGGAACAGAGAT CTGGCCCCTACACCTGGCGCCAACCTGTATGGCTCTCACCCCTTTTATCTGGCCC TGGAAGATGGCGGATCTGCCCACGGTGTCTTTCTGCTGAACTCCAACGCCATGG ACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTTGGAGAAGCACAGGCGGCATCC TGGATGTGTACATCTTTCTGGGCCCCGAGCCTAAGAGCGTGGTGCAGCAGTATC TGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACC TGTGCAGATGGGGATACAGCAGCACCGCCATCACCAGACAGGTGGTGGAAAACA TGACCCGGGCTCACTTCCCACTGGATGTGCAGTGGAACGACCTGGACTACATGG ACAGCAGACGGGACTTCACCTTCAACAAGGACGGCTTCAGAGACTTCCCCGCCA TGGTGCAAGAACTGCACCAAGGCGGCAGACGGTACATGATGATCGTGGACCCTG CCATCAGCTCTAGCGGACCTGCCGGCTCTTACAGACCCTACGATGAGGGACTGA GAAGAGGCGTGTTCATCACCAACGAGACAGGCCAGCCTCTGATCGGCAAAGTGT GGCCTGGCAGCACAGCCTTTCCAGACTTCACAAACCCCACCGCTCTGGCTTGGT GGGAAGATATGGTGGCCGAGTTTCACGATCAGGTGCCCTTCGACGGCATGTGGA TCGACATGAACGAGCCCAGCAACTTCATCCGGGGCAGCGAGGATGGCTGCCCCA ACAACGAACTGGAAAATCCTCCTTACGTGCCCGGCGTTGTCGGCGGAACACTTC AGGCCGCTACAATCTGTGCCAGCAGCCATCAGTTTCTGAGCACCCACTACAACCT GCACAACCTGTACGGCCTGACCGAGGCCATTGCCTCTCATAGAGCCCTGGTTAA GGCCAGAGGCACCCGGCCTTTTGTGATCAGCAGAAGCACATTCGCCGGCCACGG CAGATATGCCGGACATTGGACAGGCGACGTGTGGTCTAGTTGGGAGCAGCTGGC TAGCAGCGTGCCAGAGATCCTGCAGTTCAATCTGCTGGGCGTGCCACTCGTGGG AGCCGATGTTTGTGGCTTCCTGGGCAACACCTCCGAGGAACTGTGTGTGCGTTG GACACAGCTGGGCGCCTTCTATCCCTTCATGAGAAACCACAACAGCCTGCTGAG CCTGCCTCAAGAGCCCTACAGCTTTAGCGAGCCTGCACAGCAGGCCATGAGAAA GGCCCTGACTCTGAGATACGCCCTGCTGCCTCACCTGTACACCCTGTTTCATCAG GCCCACGTGGCAGGCGAGACAGTGGCTAGACCTCTGTTCCTGGAATTCCCCAAG GACAGCTCCACCTGGACCGTGGATCATCAGCTGCTGTGGGGAGAAGCCCTGCTG ATTACACCAGTGCTGCAGGCCGGAAAGGCCGAAGTGACAGGCTATTTCCCTCTCGGCACTTGGTACGACCTGCAGACCGTGCCTGTTGAGGCTCTGGGATCTCTTCCT CCACCTCCTGCCGCTCCTAGAGAGCCTGCCATTCACTCTGAAGGCCAGTGGGTT ACCCTGCCTGCTCCTCTGGACACCATCAACGTGCACCTGAGAGCTGGCTACATC ATCCCTCTGCAAGGCCCTGGCCTGACCACAACCGAATCTAGACAGCAGCCCATG GCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAGGCTAGAGGCGAGCTGTTCTGG GATGATGGCGAGAGCCTGGAAGTGCTGGAACGGGGCGCTTATACCCAAGTGATC TTCCTGGCCAGAAACAACACCATCGTGAACGAACTCGTGCGCGTGACCAGTGAA GGTGCTGGACTGCAACTGCAGAAAGTGACCGTGCTCGGAGTGGCCACAGCTCCT CAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCCAACTTCACATACAGCCCCGACA CCAAGGTCCTGGACATCTGTGTGTCACTGCTGATGGGCGAGCAGTTCCTGGTGT CCTGGTCTGGTGGCGGAGGACCTGGCGGAGGTGGACCAGGCGGTGGCGGTCCT GGCGGCGGAGGTCCAACAAAGTGCCTGCGAAGAGAAGCCCCTAGATGGGATGC CCCTCTGAGAGATCCAGCTCTGAGACAGCTCCTGTGATAG SEQ ID 30: DNA sequence of GAAco, 4x G4S linker and C-SRT tag with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTCAGCAAGGCGCTTCTAGACCCGGACCT AGAGATGCCCAAGCTCACCCTGGTAGACCTAGAGCCGTGCCTACACAGTGTGAC GTGCCACCTAACAGCAGATTCGACTGCGCCCCTGACAAGGCCATCACACAAGAG CAGTGTGAAGCCAGAGGCTGCTGCTACATCCCTGCCAAACAAGGACTGCAGGGC GCCCAGATGGGACAGCCTTGGTGTTTCTTCCCACCATCTTACCCCAGCTACAAGC TGGAAAACCTGAGCAGCAGCGAGATGGGCTACACCGCCACACTGACCAGAACCA CACCTACATTCTTCCCGAAGGACATCCTGACACTGCGGCTGGACGTGATGATGG AAACCGAGAACCGGCTGCACTTCACCATCAAGGACCCCGCCAATCGGAGATACG AGGTGCCACTGGAAACCCCTCACGTGCACTCTAGAGCCCCATCTCCACTGTACA GCGTGGAATTCAGCGAGGAACCCTTCGGCGTGATCGTGCGGAGACAGCTGGATG GAAGAGTGCTGCTGAACACCACAGTGGCCCCTCTGTTCTTCGCCGACCAGTTTCT GCAGCTGAGCACCAGCCTGCCTAGCCAGTATATCACAGGCCTGGCCGAGCACCT GTCTCCACTGATGCTGAGCACATCCTGGACCAGAATCACCCTGTGGAACAGAGAT CTGGCCCCTACACCTGGCGCCAACCTGTATGGCTCTCACCCCTTTTATCTGGCCC TGGAAGATGGCGGATCTGCCCACGGTGTCTTTCTGCTGAACTCCAACGCCATGG ACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTTGGAGAAGCACAGGCGGCATCC TGGATGTGTACATCTTTCTGGGCCCCGAGCCTAAGAGCGTGGTGCAGCAGTATC TGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACC TGTGCAGATGGGGATACAGCAGCACCGCCATCACCAGACAGGTGGTGGAAAACA TGACCCGGGCTCACTTCCCACTGGATGTGCAGTGGAACGACCTGGACTACATGG ACAGCAGACGGGACTTCACCTTCAACAAGGACGGCTTCAGAGACTTCCCCGCCA TGGTGCAAGAACTGCACCAAGGCGGCAGACGGTACATGATGATCGTGGACCCTG CCATCAGCTCTAGCGGACCTGCCGGCTCTTACAGACCCTACGATGAGGGACTGA GAAGAGGCGTGTTCATCACCAACGAGACAGGCCAGCCTCTGATCGGCAAAGTGT GGCCTGGCAGCACAGCCTTTCCAGACTTCACAAACCCCACCGCTCTGGCTTGGT GGGAAGATATGGTGGCCGAGTTTCACGATCAGGTGCCCTTCGACGGCATGTGGA TCGACATGAACGAGCCCAGCAACTTCATCCGGGGCAGCGAGGATGGCTGCCCCA ACAACGAACTGGAAAATCCTCCTTACGTGCCCGGCGTTGTCGGCGGAACACTTC AGGCCGCTACAATCTGTGCCAGCAGCCATCAGTTTCTGAGCACCCACTACAACCT GCACAACCTGTACGGCCTGACCGAGGCCATTGCCTCTCATAGAGCCCTGGTTAA GGCCAGAGGCACCCGGCCTTTTGTGATCAGCAGAAGCACATTCGCCGGCCACGG CAGATATGCCGGACATTGGACAGGCGACGTGTGGTCTAGTTGGGAGCAGCTGGC TAGCAGCGTGCCAGAGATCCTGCAGTTCAATCTGCTGGGCGTGCCACTCGTGGG AGCCGATGTTTGTGGCTTCCTGGGCAACACCTCCGAGGAACTGTGTGTGCGTTG GACACAGCTGGGCGCCTTCTATCCCTTCATGAGAAACCACAACAGCCTGCTGAG CCTGCCTCAAGAGCCCTACAGCTTTAGCGAGCCTGCACAGCAGGCCATGAGAAAGGCCCTGACTCTGAGATACGCCCTGCTGCCTCACCTGTACACCCTGTTTCATCAG GCCCACGTGGCAGGCGAGACAGTGGCTAGACCTCTGTTCCTGGAATTCCCCAAG GACAGCTCCACCTGGACCGTGGATCATCAGCTGCTGTGGGGAGAAGCCCTGCTG ATTACACCAGTGCTGCAGGCCGGAAAGGCCGAAGTGACAGGCTATTTCCCTCTC GGCACTTGGTACGACCTGCAGACCGTGCCTGTTGAGGCTCTGGGATCTCTTCCT CCACCTCCTGCCGCTCCTAGAGAGCCTGCCATTCACTCTGAAGGCCAGTGGGTT ACCCTGCCTGCTCCTCTGGACACCATCAACGTGCACCTGAGAGCTGGCTACATC ATCCCTCTGCAAGGCCCTGGCCTGACCACAACCGAATCTAGACAGCAGCCCATG GCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAGGCTAGAGGCGAGCTGTTCTGG GATGATGGCGAGAGCCTGGAAGTGCTGGAACGGGGCGCTTATACCCAAGTGATC TTCCTGGCCAGAAACAACACCATCGTGAACGAACTCGTGCGCGTGACCAGTGAA GGTGCTGGACTGCAACTGCAGAAAGTGACCGTGCTCGGAGTGGCCACAGCTCCT CAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCCAACTTCACATACAGCCCCGACA CCAAGGTCCTGGACATCTGTGTGTCACTGCTGATGGGCGAGCAGTTCCTGGTGT CCTGGTGTGGTGGCGGAGGATCTGGCGGCGGAGGAAGCGGAGGCGGCGGTTC TGGCGGTGGTGGATCTACAAAGTGCCTGCGAAGAGAAGCCCCTAGATGGGATGC CCCTCTGAGAGATCCAGCTCTGAGACAGCTCCTGTGATAG SEQ ID 31: DNA sequence of GAAco 952 C> S, 4x G4S linker and C-SRT tag with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTCAGCAAGGCGCTTCTAGACCCGGACCT AGAGATGCCCAAGCTCACCCTGGTAGACCTAGAGCCGTGCCTACACAGTGTGAC GTGCCACCTAACAGCAGATTCGACTGCGCCCCTGACAAGGCCATCACACAAGAG CAGTGTGAAGCCAGAGGCTGCTGCTACATCCCTGCCAAACAAGGACTGCAGGGC GCCCAGATGGGACAGCCTTGGTGTTTCTTCCCACCATCTTACCCCAGCTACAAGC TGGAAAACCTGAGCAGCAGCGAGATGGGCTACACCGCCACACTGACCAGAACCA CACCTACATTCTTCCCGAAGGACATCCTGACACTGCGGCTGGACGTGATGATGG AAACCGAGAACCGGCTGCACTTCACCATCAAGGACCCCGCCAATCGGAGATACG AGGTGCCACTGGAAACCCCTCACGTGCACTCTAGAGCCCCATCTCCACTGTACA GCGTGGAATTCAGCGAGGAACCCTTCGGCGTGATCGTGCGGAGACAGCTGGATG GAAGAGTGCTGCTGAACACCACAGTGGCCCCTCTGTTCTTCGCCGACCAGTTTCT GCAGCTGAGCACCAGCCTGCCTAGCCAGTATATCACAGGCCTGGCCGAGCACCT GTCTCCACTGATGCTGAGCACATCCTGGACCAGAATCACCCTGTGGAACAGAGAT CTGGCCCCTACACCTGGCGCCAACCTGTATGGCTCTCACCCCTTTTATCTGGCCC TGGAAGATGGCGGATCTGCCCACGGTGTCTTTCTGCTGAACTCCAACGCCATGG ACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTTGGAGAAGCACAGGCGGCATCC TGGATGTGTACATCTTTCTGGGCCCCGAGCCTAAGAGCGTGGTGCAGCAGTATC TGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACC TGTGCAGATGGGGATACAGCAGCACCGCCATCACCAGACAGGTGGTGGAAAACA TGACCCGGGCTCACTTCCCACTGGATGTGCAGTGGAACGACCTGGACTACATGG ACAGCAGACGGGACTTCACCTTCAACAAGGACGGCTTCAGAGACTTCCCCGCCA TGGTGCAAGAACTGCACCAAGGCGGCAGACGGTACATGATGATCGTGGACCCTG CCATCAGCTCTAGCGGACCTGCCGGCTCTTACAGACCCTACGATGAGGGACTGA GAAGAGGCGTGTTCATCACCAACGAGACAGGCCAGCCTCTGATCGGCAAAGTGT GGCCTGGCAGCACAGCCTTTCCAGACTTCACAAACCCCACCGCTCTGGCTTGGT GGGAAGATATGGTGGCCGAGTTTCACGATCAGGTGCCCTTCGACGGCATGTGGA TCGACATGAACGAGCCCAGCAACTTCATCCGGGGCAGCGAGGATGGCTGCCCCA ACAACGAACTGGAAAATCCTCCTTACGTGCCCGGCGTTGTCGGCGGAACACTTC AGGCCGCTACAATCTGTGCCAGCAGCCATCAGTTTCTGAGCACCCACTACAACCT GCACAACCTGTACGGCCTGACCGAGGCCATTGCCTCTCATAGAGCCCTGGTTAA GGCCAGAGGCACCCGGCCTTTTGTGATCAGCAGAAGCACATTCGCCGGCCACGG CAGATATGCCGGACATTGGACAGGCGACGTGTGGTCTAGTTGGGAGCAGCTGGCTAGCAGCGTGCCAGAGATCCTGCAGTTCAATCTGCTGGGCGTGCCACTCGTGGG AGCCGATGTTTGTGGCTTCCTGGGCAACACCTCCGAGGAACTGTGTGTGCGTTG GACACAGCTGGGCGCCTTCTATCCCTTCATGAGAAACCACAACAGCCTGCTGAG CCTGCCTCAAGAGCCCTACAGCTTTAGCGAGCCTGCACAGCAGGCCATGAGAAA GGCCCTGACTCTGAGATACGCCCTGCTGCCTCACCTGTACACCCTGTTTCATCAG GCCCACGTGGCAGGCGAGACAGTGGCTAGACCTCTGTTCCTGGAATTCCCCAAG GACAGCTCCACCTGGACCGTGGATCATCAGCTGCTGTGGGGAGAAGCCCTGCTG ATTACACCAGTGCTGCAGGCCGGAAAGGCCGAAGTGACAGGCTATTTCCCTCTC GGCACTTGGTACGACCTGCAGACCGTGCCTGTTGAGGCTCTGGGATCTCTTCCT CCACCTCCTGCCGCTCCTAGAGAGCCTGCCATTCACTCTGAAGGCCAGTGGGTT ACCCTGCCTGCTCCTCTGGACACCATCAACGTGCACCTGAGAGCTGGCTACATC ATCCCTCTGCAAGGCCCTGGCCTGACCACAACCGAATCTAGACAGCAGCCCATG GCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAGGCTAGAGGCGAGCTGTTCTGG GATGATGGCGAGAGCCTGGAAGTGCTGGAACGGGGCGCTTATACCCAAGTGATC TTCCTGGCCAGAAACAACACCATCGTGAACGAACTCGTGCGCGTGACCAGTGAA GGTGCTGGACTGCAACTGCAGAAAGTGACCGTGCTCGGAGTGGCCACAGCTCCT CAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCCAACTTCACATACAGCCCCGACA CCAAGGTCCTGGACATCTGTGTGTCACTGCTGATGGGCGAGCAGTTCCTGGTGT CCTGGTCTGGTGGCGGAGGATCTGGCGGCGGAGGAAGCGGAGGCGGCGGTTCT GGCGGTGGTGGATCTACAAAGTGCCTGCGAAGAGAAGCCCCTAGATGGGATGCC CCTCTGAGAGATCCAGCTCTGAGACAGCTCCTGTGATAG SEQ ID 32: DNA sequence of GAAco, 4x G4S linker, and C-terminal RQLL tag with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTCAGCAAGGCGCTTCTAGACCCGGACCT AGAGATGCCCAAGCTCACCCTGGTAGACCTAGAGCCGTGCCTACACAGTGTGAC GTGCCACCTAACAGCAGATTCGACTGCGCCCCTGACAAGGCCATCACACAAGAG CAGTGTGAAGCCAGAGGCTGCTGCTACATCCCTGCCAAACAAGGACTGCAGGGC GCCCAGATGGGACAGCCTTGGTGTTTCTTCCCACCATCTTACCCCAGCTACAAGC TGGAAAACCTGAGCAGCAGCGAGATGGGCTACACCGCCACACTGACCAGAACCA CACCTACATTCTTCCCGAAGGACATCCTGACACTGCGGCTGGACGTGATGATGG AAACCGAGAACCGGCTGCACTTCACCATCAAGGACCCCGCCAATCGGAGATACG AGGTGCCACTGGAAACCCCTCACGTGCACTCTAGAGCCCCATCTCCACTGTACA GCGTGGAATTCAGCGAGGAACCCTTCGGCGTGATCGTGCGGAGACAGCTGGATG GAAGAGTGCTGCTGAACACCACAGTGGCCCCTCTGTTCTTCGCCGACCAGTTTCT GCAGCTGAGCACCAGCCTGCCTAGCCAGTATATCACAGGCCTGGCCGAGCACCT GTCTCCACTGATGCTGAGCACATCCTGGACCAGAATCACCCTGTGGAACAGAGAT CTGGCCCCTACACCTGGCGCCAACCTGTATGGCTCTCACCCCTTTTATCTGGCCC TGGAAGATGGCGGATCTGCCCACGGTGTCTTTCTGCTGAACTCCAACGCCATGG ACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTTGGAGAAGCACAGGCGGCATCC TGGATGTGTACATCTTTCTGGGCCCCGAGCCTAAGAGCGTGGTGCAGCAGTATC TGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACC TGTGCAGATGGGGATACAGCAGCACCGCCATCACCAGACAGGTGGTGGAAAACA TGACCCGGGCTCACTTCCCACTGGATGTGCAGTGGAACGACCTGGACTACATGG ACAGCAGACGGGACTTCACCTTCAACAAGGACGGCTTCAGAGACTTCCCCGCCA TGGTGCAAGAACTGCACCAAGGCGGCAGACGGTACATGATGATCGTGGACCCTG CCATCAGCTCTAGCGGACCTGCCGGCTCTTACAGACCCTACGATGAGGGACTGA GAAGAGGCGTGTTCATCACCAACGAGACAGGCCAGCCTCTGATCGGCAAAGTGT GGCCTGGCAGCACAGCCTTTCCAGACTTCACAAACCCCACCGCTCTGGCTTGGT GGGAAGATATGGTGGCCGAGTTTCACGATCAGGTGCCCTTCGACGGCATGTGGA TCGACATGAACGAGCCCAGCAACTTCATCCGGGGCAGCGAGGATGGCTGCCCCA ACAACGAACTGGAAAATCCTCCTTACGTGCCCGGCGTTGTCGGCGGAACACTTCAGGCCGCTACAATCTGTGCCAGCAGCCATCAGTTTCTGAGCACCCACTACAACCT GCACAACCTGTACGGCCTGACCGAGGCCATTGCCTCTCATAGAGCCCTGGTTAA GGCCAGAGGCACCCGGCCTTTTGTGATCAGCAGAAGCACATTCGCCGGCCACGG CAGATATGCCGGACATTGGACAGGCGACGTGTGGTCTAGTTGGGAGCAGCTGGC TAGCAGCGTGCCAGAGATCCTGCAGTTCAATCTGCTGGGCGTGCCACTCGTGGG AGCCGATGTTTGTGGCTTCCTGGGCAACACCTCCGAGGAACTGTGTGTGCGTTG GACACAGCTGGGCGCCTTCTATCCCTTCATGAGAAACCACAACAGCCTGCTGAG CCTGCCTCAAGAGCCCTACAGCTTTAGCGAGCCTGCACAGCAGGCCATGAGAAA GGCCCTGACTCTGAGATACGCCCTGCTGCCTCACCTGTACACCCTGTTTCATCAG GCCCACGTGGCAGGCGAGACAGTGGCTAGACCTCTGTTCCTGGAATTCCCCAAG GACAGCTCCACCTGGACCGTGGATCATCAGCTGCTGTGGGGAGAAGCCCTGCTG ATTACACCAGTGCTGCAGGCCGGAAAGGCCGAAGTGACAGGCTATTTCCCTCTC GGCACTTGGTACGACCTGCAGACCGTGCCTGTTGAGGCTCTGGGATCTCTTCCT CCACCTCCTGCCGCTCCTAGAGAGCCTGCCATTCACTCTGAAGGCCAGTGGGTT ACCCTGCCTGCTCCTCTGGACACCATCAACGTGCACCTGAGAGCTGGCTACATC ATCCCTCTGCAAGGCCCTGGCCTGACCACAACCGAATCTAGACAGCAGCCCATG GCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAGGCTAGAGGCGAGCTGTTCTGG GATGATGGCGAGAGCCTGGAAGTGCTGGAACGGGGCGCTTATACCCAAGTGATC TTCCTGGCCAGAAACAACACCATCGTGAACGAACTCGTGCGCGTGACCAGTGAA GGTGCTGGACTGCAACTGCAGAAAGTGACCGTGCTCGGAGTGGCCACAGCTCCT CAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCCAACTTCACATACAGCCCCGACA CCAAGGTCCTGGACATCTGTGTGTCACTGCTGATGGGCGAGCAGTTCCTGGTGT CCTGGTGTGGTGGCGGAGGATCTGGCGGCGGAGGAAGCGGAGGCGGCGGTTC TGGCGGTGGTGGATCTAGACAGCTCCTGTGATAG SEQ ID 33: DNA sequence of GAAco, N-PGRN tag and GAP linker with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTGATGTGGAATGTGGCGAGGGCCACTTC TGCCACGACAATCAGACCTGCTGCCGGGACAACAGACAAGGCTGGGCCTGCTGT CCTTACAGACAGGGCGTGTGCTGTGCCGACAGAAGGCACTGTTGTCCTGCCGGC TTTAGATGTGCCGCCAGGGGCACAAAGTGCCTGAGAAGAGAAGCCCCTAGATGG GACGCCCCTCTGAGAGATCCTGCTCTGAGACAGCTGCTTGGCGCTCCTCAGCAA GGCGCTTCTAGACCCGGACCTAGAGATGCCCAAGCTCACCCTGGTAGACCTAGA GCCGTGCCTACACAGTGTGACGTGCCACCTAACAGCAGATTCGACTGCGCCCCT GACAAGGCCATCACACAAGAGCAGTGTGAAGCCAGAGGCTGCTGCTACATCCCT GCCAAACAAGGACTGCAGGGCGCCCAGATGGGACAGCCTTGGTGTTTCTTCCCA CCATCTTACCCCAGCTACAAGCTGGAAAACCTGAGCAGCAGCGAGATGGGCTAC ACCGCCACACTGACCAGAACCACACCTACATTCTTCCCGAAGGACATCCTGACAC TGCGGCTGGACGTGATGATGGAAACCGAGAACCGGCTGCACTTCACCATCAAGG ACCCCGCCAATCGGAGATACGAGGTGCCACTGGAAACCCCTCACGTGCACTCTA GAGCCCCATCTCCACTGTACAGCGTGGAATTCAGCGAGGAACCCTTCGGCGTGA TCGTGCGGAGACAGCTGGATGGAAGAGTGCTGCTGAACACCACAGTGGCCCCTC TGTTCTTCGCCGACCAGTTTCTGCAGCTGAGCACCAGCCTGCCTAGCCAGTATAT CACAGGCCTGGCCGAGCACCTGTCTCCACTGATGCTGAGCACATCCTGGACCAG AATCACCCTGTGGAACAGAGATCTGGCCCCTACACCTGGCGCCAACCTGTATGG CTCTCACCCCTTTTATCTGGCCCTGGAAGATGGCGGATCTGCCCACGGTGTCTTT CTGCTGAACTCCAACGCCATGGACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTT GGAGAAGCACAGGCGGCATCCTGGATGTGTACATCTTTCTGGGCCCCGAGCCTA AGAGCGTGGTGCAGCAGTATCTGGACGTCGTGGGCTACCCCTTCATGCCTCCTT ATTGGGGCCTGGGCTTCCACCTGTGCAGATGGGGATACAGCAGCACCGCCATCA CCAGACAGGTGGTGGAAAACATGACCCGGGCTCACTTCCCACTGGATGTGCAGT GGAACGACCTGGACTACATGGACAGCAGACGGGACTTCACCTTCAACAAGGACGGCTTCAGAGACTTCCCCGCCATGGTGCAAGAACTGCACCAAGGCGGCAGACGGT ACATGATGATCGTGGACCCTGCCATCAGCTCTAGCGGACCTGCCGGCTCTTACA GACCCTACGATGAGGGACTGAGAAGAGGCGTGTTCATCACCAACGAGACAGGCC AGCCTCTGATCGGCAAAGTGTGGCCTGGCAGCACAGCCTTTCCAGACTTCACAA ACCCCACCGCTCTGGCTTGGTGGGAAGATATGGTGGCCGAGTTTCACGATCAGG TGCCCTTCGACGGCATGTGGATCGACATGAACGAGCCCAGCAACTTCATCCGGG GCAGCGAGGATGGCTGCCCCAACAACGAACTGGAAAATCCTCCTTACGTGCCCG GCGTTGTCGGCGGAACACTTCAGGCCGCTACAATCTGTGCCAGCAGCCATCAGT TTCTGAGCACCCACTACAACCTGCACAACCTGTACGGCCTGACCGAGGCCATTG CCTCTCATAGAGCCCTGGTTAAGGCCAGAGGCACCCGGCCTTTTGTGATCAGCA GAAGCACATTCGCCGGCCACGGCAGATATGCCGGACATTGGACAGGCGACGTGT GGTCTAGTTGGGAGCAGCTGGCTAGCAGCGTGCCAGAGATCCTGCAGTTCAATC TGCTGGGCGTGCCACTCGTGGGAGCCGATGTTTGTGGCTTCCTGGGCAACACCT CCGAGGAACTGTGTGTGCGTTGGACACAGCTGGGCGCCTTCTATCCCTTCATGA GAAACCACAACAGCCTGCTGAGCCTGCCTCAAGAGCCCTACAGCTTTAGCGAGC CTGCACAGCAGGCCATGAGAAAGGCCCTGACTCTGAGATACGCCCTGCTGCCTC ACCTGTACACCCTGTTTCATCAGGCCCACGTGGCAGGCGAGACAGTGGCTAGAC CTCTGTTCCTGGAATTCCCCAAGGACAGCTCCACCTGGACCGTGGATCATCAGCT GCTGTGGGGAGAAGCCCTGCTGATTACACCAGTGCTGCAGGCCGGAAAGGCCG AAGTGACAGGCTATTTCCCTCTCGGCACTTGGTACGACCTGCAGACCGTGCCTGT TGAGGCTCTGGGATCTCTTCCTCCACCTCCTGCCGCTCCTAGAGAGCCTGCCATT CACTCTGAAGGCCAGTGGGTTACCCTGCCTGCTCCTCTGGACACCATCAACGTG CACCTGAGAGCTGGCTACATCATCCCTCTGCAAGGCCCTGGCCTGACCACAACC GAATCTAGACAGCAGCCCATGGCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAG GCTAGAGGCGAGCTGTTCTGGGATGATGGCGAGAGCCTGGAAGTGCTGGAACG GGGCGCTTATACCCAAGTGATCTTCCTGGCCAGAAACAACACCATCGTGAACGAA CTCGTGCGCGTGACCAGTGAAGGTGCTGGACTGCAACTGCAGAAAGTGACCGTG CTCGGAGTGGCCACAGCTCCTCAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCC AACTTCACATACAGCCCCGACACCAAGGTCCTGGACATCTGTGTGTCACTGCTGA TGGGCGAGCAGTTCCTGGTGTCCTGGTGTTAGTGA SEQ ID 34: DNA sequence of GAAco, N-PSAP tag and GAP linker with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTGACGTGGAGTGTGGGGAAGGACACTTC TGCCATGATAACCAGACCTGCTGCCGAGACAACCGACAGGGCTGGGCCTGCTGT CCCTACCGCCAGGGCGTCTGTTGTGCTGATCGGCGCCACTGCTGTCCTGCTGGC TTCCGCTGCGCAGCCAGGGGTACCAAGTGTTTGGGCGCTCCTCAGCAAGGCGCT TCTAGACCCGGACCTAGAGATGCCCAAGCTCACCCTGGTAGACCTAGAGCCGTG CCTACACAGTGTGACGTGCCACCTAACAGCAGATTCGACTGCGCCCCTGACAAG GCCATCACACAAGAGCAGTGTGAAGCCAGAGGCTGCTGCTACATCCCTGCCAAA CAAGGACTGCAGGGCGCCCAGATGGGACAGCCTTGGTGTTTCTTCCCACCATCT TACCCCAGCTACAAGCTGGAAAACCTGAGCAGCAGCGAGATGGGCTACACCGCC ACACTGACCAGAACCACACCTACATTCTTCCCGAAGGACATCCTGACACTGCGGC TGGACGTGATGATGGAAACCGAGAACCGGCTGCACTTCACCATCAAGGACCCCG CCAATCGGAGATACGAGGTGCCACTGGAAACCCCTCACGTGCACTCTAGAGCCC CATCTCCACTGTACAGCGTGGAATTCAGCGAGGAACCCTTCGGCGTGATCGTGC GGAGACAGCTGGATGGAAGAGTGCTGCTGAACACCACAGTGGCCCCTCTGTTCT TCGCCGACCAGTTTCTGCAGCTGAGCACCAGCCTGCCTAGCCAGTATATCACAG GCCTGGCCGAGCACCTGTCTCCACTGATGCTGAGCACATCCTGGACCAGAATCA CCCTGTGGAACAGAGATCTGGCCCCTACACCTGGCGCCAACCTGTATGGCTCTC ACCCCTTTTATCTGGCCCTGGAAGATGGCGGATCTGCCCACGGTGTCTTTCTGCT GAACTCCAACGCCATGGACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTTGGAGAAGCACAGGCGGCATCCTGGATGTGTACATCTTTCTGGGCCCCGAGCCTAAGAG CGTGGTGCAGCAGTATCTGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTG GGGCCTGGGCTTCCACCTGTGCAGATGGGGATACAGCAGCACCGCCATCACCAG ACAGGTGGTGGAAAACATGACCCGGGCTCACTTCCCACTGGATGTGCAGTGGAA CGACCTGGACTACATGGACAGCAGACGGGACTTCACCTTCAACAAGGACGGCTT CAGAGACTTCCCCGCCATGGTGCAAGAACTGCACCAAGGCGGCAGACGGTACAT GATGATCGTGGACCCTGCCATCAGCTCTAGCGGACCTGCCGGCTCTTACAGACC CTACGATGAGGGACTGAGAAGAGGCGTGTTCATCACCAACGAGACAGGCCAGCC TCTGATCGGCAAAGTGTGGCCTGGCAGCACAGCCTTTCCAGACTTCACAAACCC CACCGCTCTGGCTTGGTGGGAAGATATGGTGGCCGAGTTTCACGATCAGGTGCC CTTCGACGGCATGTGGATCGACATGAACGAGCCCAGCAACTTCATCCGGGGCAG CGAGGATGGCTGCCCCAACAACGAACTGGAAAATCCTCCTTACGTGCCCGGCGT TGTCGGCGGAACACTTCAGGCCGCTACAATCTGTGCCAGCAGCCATCAGTTTCT GAGCACCCACTACAACCTGCACAACCTGTACGGCCTGACCGAGGCCATTGCCTC TCATAGAGCCCTGGTTAAGGCCAGAGGCACCCGGCCTTTTGTGATCAGCAGAAG CACATTCGCCGGCCACGGCAGATATGCCGGACATTGGACAGGCGACGTGTGGTC TAGTTGGGAGCAGCTGGCTAGCAGCGTGCCAGAGATCCTGCAGTTCAATCTGCT GGGCGTGCCACTCGTGGGAGCCGATGTTTGTGGCTTCCTGGGCAACACCTCCGA GGAACTGTGTGTGCGTTGGACACAGCTGGGCGCCTTCTATCCCTTCATGAGAAA CCACAACAGCCTGCTGAGCCTGCCTCAAGAGCCCTACAGCTTTAGCGAGCCTGC ACAGCAGGCCATGAGAAAGGCCCTGACTCTGAGATACGCCCTGCTGCCTCACCT GTACACCCTGTTTCATCAGGCCCACGTGGCAGGCGAGACAGTGGCTAGACCTCT GTTCCTGGAATTCCCCAAGGACAGCTCCACCTGGACCGTGGATCATCAGCTGCT GTGGGGAGAAGCCCTGCTGATTACACCAGTGCTGCAGGCCGGAAAGGCCGAAG TGACAGGCTATTTCCCTCTCGGCACTTGGTACGACCTGCAGACCGTGCCTGTTGA GGCTCTGGGATCTCTTCCTCCACCTCCTGCCGCTCCTAGAGAGCCTGCCATTCAC TCTGAAGGCCAGTGGGTTACCCTGCCTGCTCCTCTGGACACCATCAACGTGCAC CTGAGAGCTGGCTACATCATCCCTCTGCAAGGCCCTGGCCTGACCACAACCGAA TCTAGACAGCAGCCCATGGCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAGGCT AGAGGCGAGCTGTTCTGGGATGATGGCGAGAGCCTGGAAGTGCTGGAACGGGG CGCTTATACCCAAGTGATCTTCCTGGCCAGAAACAACACCATCGTGAACGAACTC GTGCGCGTGACCAGTGAAGGTGCTGGACTGCAACTGCAGAAAGTGACCGTGCTC GGAGTGGCCACAGCTCCTCAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCCAAC TTCACATACAGCCCCGACACCAAGGTCCTGGACATCTGTGTGTCACTGCTGATGG GCGAGCAGTTCCTGGTGTCCTGGTGTTAGTGA SEQ ID 35: DNA sequence of GAAco, N-SRT tag and GAP linker with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTACCAAGTGTTTGCGCAGGGAGGCCCCG CGCTGGGACGCCCCTTTGAGGGACCCAGCCTTGAGACAGCTGCTGGGCGCTCC TCAGCAAGGCGCTTCTAGACCCGGACCTAGAGATGCCCAAGCTCACCCTGGTAG ACCTAGAGCCGTGCCTACACAGTGTGACGTGCCACCTAACAGCAGATTCGACTG CGCCCCTGACAAGGCCATCACACAAGAGCAGTGTGAAGCCAGAGGCTGCTGCTA CATCCCTGCCAAACAAGGACTGCAGGGCGCCCAGATGGGACAGCCTTGGTGTTT CTTCCCACCATCTTACCCCAGCTACAAGCTGGAAAACCTGAGCAGCAGCGAGAT GGGCTACACCGCCACACTGACCAGAACCACACCTACATTCTTCCCGAAGGACAT CCTGACACTGCGGCTGGACGTGATGATGGAAACCGAGAACCGGCTGCACTTCAC CATCAAGGACCCCGCCAATCGGAGATACGAGGTGCCACTGGAAACCCCTCACGT GCACTCTAGAGCCCCATCTCCACTGTACAGCGTGGAATTCAGCGAGGAACCCTT CGGCGTGATCGTGCGGAGACAGCTGGATGGAAGAGTGCTGCTGAACACCACAGT GGCCCCTCTGTTCTTCGCCGACCAGTTTCTGCAGCTGAGCACCAGCCTGCCTAG CCAGTATATCACAGGCCTGGCCGAGCACCTGTCTCCACTGATGCTGAGCACATCCTGGACCAGAATCACCCTGTGGAACAGAGATCTGGCCCCTACACCTGGCGCCAA CCTGTATGGCTCTCACCCCTTTTATCTGGCCCTGGAAGATGGCGGATCTGCCCAC GGTGTCTTTCTGCTGAACTCCAACGCCATGGACGTGGTGCTGCAGCCATCTCCT GCTCTGTCTTGGAGAAGCACAGGCGGCATCCTGGATGTGTACATCTTTCTGGGC CCCGAGCCTAAGAGCGTGGTGCAGCAGTATCTGGACGTCGTGGGCTACCCCTTC ATGCCTCCTTATTGGGGCCTGGGCTTCCACCTGTGCAGATGGGGATACAGCAGC ACCGCCATCACCAGACAGGTGGTGGAAAACATGACCCGGGCTCACTTCCCACTG GATGTGCAGTGGAACGACCTGGACTACATGGACAGCAGACGGGACTTCACCTTC AACAAGGACGGCTTCAGAGACTTCCCCGCCATGGTGCAAGAACTGCACCAAGGC GGCAGACGGTACATGATGATCGTGGACCCTGCCATCAGCTCTAGCGGACCTGCC GGCTCTTACAGACCCTACGATGAGGGACTGAGAAGAGGCGTGTTCATCACCAAC GAGACAGGCCAGCCTCTGATCGGCAAAGTGTGGCCTGGCAGCACAGCCTTTCCA GACTTCACAAACCCCACCGCTCTGGCTTGGTGGGAAGATATGGTGGCCGAGTTT CACGATCAGGTGCCCTTCGACGGCATGTGGATCGACATGAACGAGCCCAGCAAC TTCATCCGGGGCAGCGAGGATGGCTGCCCCAACAACGAACTGGAAAATCCTCCT TACGTGCCCGGCGTTGTCGGCGGAACACTTCAGGCCGCTACAATCTGTGCCAGC AGCCATCAGTTTCTGAGCACCCACTACAACCTGCACAACCTGTACGGCCTGACCG AGGCCATTGCCTCTCATAGAGCCCTGGTTAAGGCCAGAGGCACCCGGCCTTTTG TGATCAGCAGAAGCACATTCGCCGGCCACGGCAGATATGCCGGACATTGGACAG GCGACGTGTGGTCTAGTTGGGAGCAGCTGGCTAGCAGCGTGCCAGAGATCCTGC AGTTCAATCTGCTGGGCGTGCCACTCGTGGGAGCCGATGTTTGTGGCTTCCTGG GCAACACCTCCGAGGAACTGTGTGTGCGTTGGACACAGCTGGGCGCCTTCTATC CCTTCATGAGAAACCACAACAGCCTGCTGAGCCTGCCTCAAGAGCCCTACAGCTT TAGCGAGCCTGCACAGCAGGCCATGAGAAAGGCCCTGACTCTGAGATACGCCCT GCTGCCTCACCTGTACACCCTGTTTCATCAGGCCCACGTGGCAGGCGAGACAGT GGCTAGACCTCTGTTCCTGGAATTCCCCAAGGACAGCTCCACCTGGACCGTGGA TCATCAGCTGCTGTGGGGAGAAGCCCTGCTGATTACACCAGTGCTGCAGGCCGG AAAGGCCGAAGTGACAGGCTATTTCCCTCTCGGCACTTGGTACGACCTGCAGAC CGTGCCTGTTGAGGCTCTGGGATCTCTTCCTCCACCTCCTGCCGCTCCTAGAGA GCCTGCCATTCACTCTGAAGGCCAGTGGGTTACCCTGCCTGCTCCTCTGGACAC CATCAACGTGCACCTGAGAGCTGGCTACATCATCCCTCTGCAAGGCCCTGGCCT GACCACAACCGAATCTAGACAGCAGCCCATGGCTCTGGCCGTGGCTTTGACAAA AGGCGGAGAGGCTAGAGGCGAGCTGTTCTGGGATGATGGCGAGAGCCTGGAAG TGCTGGAACGGGGCGCTTATACCCAAGTGATCTTCCTGGCCAGAAACAACACCAT CGTGAACGAACTCGTGCGCGTGACCAGTGAAGGTGCTGGACTGCAACTGCAGAA AGTGACCGTGCTCGGAGTGGCCACAGCTCCTCAGCAGGTTCTGTCTAATGGCGT GCCCGTGTCCAACTTCACATACAGCCCCGACACCAAGGTCCTGGACATCTGTGT GTCACTGCTGATGGGCGAGCAGTTCCTGGTGTCCTGGTGTTAGTGA SEQ ID 36: DNA sequence of GAAco, N-IGF2 tag and GAP linker with GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTGCTCTGTGTGGCGGAGAGCTGGTGGAT ACCCTGCAGTTCGTGTGTGGCGACCGGGGCTTCTACTTTAGCAGACCTGCCAGC CGGGTTTCCAGACGGTCTAGAGGAATCGTGGAAGAGTGCTGCTTCAGAAGCTGC GATCTGGCCCTGCTGGAAACCTACTGTGCCACACCAGCCAAGTCTGAAGGCGCT CCTCAGCAAGGCGCTTCTAGACCCGGACCTAGAGATGCCCAAGCTCACCCTGGT AGACCTAGAGCCGTGCCTACACAGTGTGACGTGCCACCTAACAGCAGATTCGAC TGCGCCCCTGACAAGGCCATCACACAAGAGCAGTGTGAAGCCAGAGGCTGCTGC TACATCCCTGCCAAACAAGGACTGCAGGGCGCCCAGATGGGACAGCCTTGGTGT TTCTTCCCACCATCTTACCCCAGCTACAAGCTGGAAAACCTGAGCAGCAGCGAGA TGGGCTACACCGCCACACTGACCAGAACCACACCTACATTCTTCCCGAAGGACAT CCTGACACTGCGGCTGGACGTGATGATGGAAACCGAGAACCGGCTGCACTTCACCATCAAGGACCCCGCCAATCGGAGATACGAGGTGCCACTGGAAACCCCTCACGT GCACTCTAGAGCCCCATCTCCACTGTACAGCGTGGAATTCAGCGAGGAACCCTT CGGCGTGATCGTGCGGAGACAGCTGGATGGAAGAGTGCTGCTGAACACCACAGT GGCCCCTCTGTTCTTCGCCGACCAGTTTCTGCAGCTGAGCACCAGCCTGCCTAG CCAGTATATCACAGGCCTGGCCGAGCACCTGTCTCCACTGATGCTGAGCACATC CTGGACCAGAATCACCCTGTGGAACAGAGATCTGGCCCCTACACCTGGCGCCAA CCTGTATGGCTCTCACCCCTTTTATCTGGCCCTGGAAGATGGCGGATCTGCCCAC GGTGTCTTTCTGCTGAACTCCAACGCCATGGACGTGGTGCTGCAGCCATCTCCT GCTCTGTCTTGGAGAAGCACAGGCGGCATCCTGGATGTGTACATCTTTCTGGGC CCCGAGCCTAAGAGCGTGGTGCAGCAGTATCTGGACGTCGTGGGCTACCCCTTC ATGCCTCCTTATTGGGGCCTGGGCTTCCACCTGTGCAGATGGGGATACAGCAGC ACCGCCATCACCAGACAGGTGGTGGAAAACATGACCCGGGCTCACTTCCCACTG GATGTGCAGTGGAACGACCTGGACTACATGGACAGCAGACGGGACTTCACCTTC AACAAGGACGGCTTCAGAGACTTCCCCGCCATGGTGCAAGAACTGCACCAAGGC GGCAGACGGTACATGATGATCGTGGACCCTGCCATCAGCTCTAGCGGACCTGCC GGCTCTTACAGACCCTACGATGAGGGACTGAGAAGAGGCGTGTTCATCACCAAC GAGACAGGCCAGCCTCTGATCGGCAAAGTGTGGCCTGGCAGCACAGCCTTTCCA GACTTCACAAACCCCACCGCTCTGGCTTGGTGGGAAGATATGGTGGCCGAGTTT CACGATCAGGTGCCCTTCGACGGCATGTGGATCGACATGAACGAGCCCAGCAAC TTCATCCGGGGCAGCGAGGATGGCTGCCCCAACAACGAACTGGAAAATCCTCCT TACGTGCCCGGCGTTGTCGGCGGAACACTTCAGGCCGCTACAATCTGTGCCAGC AGCCATCAGTTTCTGAGCACCCACTACAACCTGCACAACCTGTACGGCCTGACCG AGGCCATTGCCTCTCATAGAGCCCTGGTTAAGGCCAGAGGCACCCGGCCTTTTG TGATCAGCAGAAGCACATTCGCCGGCCACGGCAGATATGCCGGACATTGGACAG GCGACGTGTGGTCTAGTTGGGAGCAGCTGGCTAGCAGCGTGCCAGAGATCCTGC AGTTCAATCTGCTGGGCGTGCCACTCGTGGGAGCCGATGTTTGTGGCTTCCTGG GCAACACCTCCGAGGAACTGTGTGTGCGTTGGACACAGCTGGGCGCCTTCTATC CCTTCATGAGAAACCACAACAGCCTGCTGAGCCTGCCTCAAGAGCCCTACAGCTT TAGCGAGCCTGCACAGCAGGCCATGAGAAAGGCCCTGACTCTGAGATACGCCCT GCTGCCTCACCTGTACACCCTGTTTCATCAGGCCCACGTGGCAGGCGAGACAGT GGCTAGACCTCTGTTCCTGGAATTCCCCAAGGACAGCTCCACCTGGACCGTGGA TCATCAGCTGCTGTGGGGAGAAGCCCTGCTGATTACACCAGTGCTGCAGGCCGG AAAGGCCGAAGTGACAGGCTATTTCCCTCTCGGCACTTGGTACGACCTGCAGAC CGTGCCTGTTGAGGCTCTGGGATCTCTTCCTCCACCTCCTGCCGCTCCTAGAGA GCCTGCCATTCACTCTGAAGGCCAGTGGGTTACCCTGCCTGCTCCTCTGGACAC CATCAACGTGCACCTGAGAGCTGGCTACATCATCCCTCTGCAAGGCCCTGGCCT GACCACAACCGAATCTAGACAGCAGCCCATGGCTCTGGCCGTGGCTTTGACAAA AGGCGGAGAGGCTAGAGGCGAGCTGTTCTGGGATGATGGCGAGAGCCTGGAAG TGCTGGAACGGGGCGCTTATACCCAAGTGATCTTCCTGGCCAGAAACAACACCAT CGTGAACGAACTCGTGCGCGTGACCAGTGAAGGTGCTGGACTGCAACTGCAGAA AGTGACCGTGCTCGGAGTGGCCACAGCTCCTCAGCAGGTTCTGTCTAATGGCGT GCCCGTGTCCAACTTCACATACAGCCCCGACACCAAGGTCCTGGACATCTGTGT GTCACTGCTGATGGGCGAGCAGTTCCTGGTGTCCTGGTGTTAGTGA SEQ ID 37: DNA sequence of GAAco, N-IGF2 tag and GAP linker with IGF2 signal peptide ATGGGCATCCCTATGGGCAAGAGCATGCTGGTGCTGCTGACCTTCCTGGCCTTC GCCAGCTGTTGTATCGCTGCTCTGTGTGGCGGAGAGCTGGTGGATACCCTGCAG TTCGTGTGTGGCGACCGGGGCTTCTACTTTAGCAGACCTGCCAGCCGGGTTTCC AGACGGTCTAGAGGAATCGTGGAAGAGTGCTGCTTCAGAAGCTGCGATCTGGCC CTGCTGGAAACCTACTGTGCCACACCAGCCAAGTCTGAAGGCGCTCCTCAGCAA GGCGCTTCTAGACCCGGACCTAGAGATGCCCAAGCTCACCCTGGTAGACCTAGA GCCGTGCCTACACAGTGTGACGTGCCACCTAACAGCAGATTCGACTGCGCCCCTGACAAGGCCATCACACAAGAGCAGTGTGAAGCCAGAGGCTGCTGCTACATCCCT GCCAAACAAGGACTGCAGGGCGCCCAGATGGGACAGCCTTGGTGTTTCTTCCCA CCATCTTACCCCAGCTACAAGCTGGAAAACCTGAGCAGCAGCGAGATGGGCTAC ACCGCCACACTGACCAGAACCACACCTACATTCTTCCCGAAGGACATCCTGACAC TGCGGCTGGACGTGATGATGGAAACCGAGAACCGGCTGCACTTCACCATCAAGG ACCCCGCCAATCGGAGATACGAGGTGCCACTGGAAACCCCTCACGTGCACTCTA GAGCCCCATCTCCACTGTACAGCGTGGAATTCAGCGAGGAACCCTTCGGCGTGA TCGTGCGGAGACAGCTGGATGGAAGAGTGCTGCTGAACACCACAGTGGCCCCTC TGTTCTTCGCCGACCAGTTTCTGCAGCTGAGCACCAGCCTGCCTAGCCAGTATAT CACAGGCCTGGCCGAGCACCTGTCTCCACTGATGCTGAGCACATCCTGGACCAG AATCACCCTGTGGAACAGAGATCTGGCCCCTACACCTGGCGCCAACCTGTATGG CTCTCACCCCTTTTATCTGGCCCTGGAAGATGGCGGATCTGCCCACGGTGTCTTT CTGCTGAACTCCAACGCCATGGACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTT GGAGAAGCACAGGCGGCATCCTGGATGTGTACATCTTTCTGGGCCCCGAGCCTA AGAGCGTGGTGCAGCAGTATCTGGACGTCGTGGGCTACCCCTTCATGCCTCCTT ATTGGGGCCTGGGCTTCCACCTGTGCAGATGGGGATACAGCAGCACCGCCATCA CCAGACAGGTGGTGGAAAACATGACCCGGGCTCACTTCCCACTGGATGTGCAGT GGAACGACCTGGACTACATGGACAGCAGACGGGACTTCACCTTCAACAAGGACG GCTTCAGAGACTTCCCCGCCATGGTGCAAGAACTGCACCAAGGCGGCAGACGGT ACATGATGATCGTGGACCCTGCCATCAGCTCTAGCGGACCTGCCGGCTCTTACA GACCCTACGATGAGGGACTGAGAAGAGGCGTGTTCATCACCAACGAGACAGGCC AGCCTCTGATCGGCAAAGTGTGGCCTGGCAGCACAGCCTTTCCAGACTTCACAA ACCCCACCGCTCTGGCTTGGTGGGAAGATATGGTGGCCGAGTTTCACGATCAGG TGCCCTTCGACGGCATGTGGATCGACATGAACGAGCCCAGCAACTTCATCCGGG GCAGCGAGGATGGCTGCCCCAACAACGAACTGGAAAATCCTCCTTACGTGCCCG GCGTTGTCGGCGGAACACTTCAGGCCGCTACAATCTGTGCCAGCAGCCATCAGT TTCTGAGCACCCACTACAACCTGCACAACCTGTACGGCCTGACCGAGGCCATTG CCTCTCATAGAGCCCTGGTTAAGGCCAGAGGCACCCGGCCTTTTGTGATCAGCA GAAGCACATTCGCCGGCCACGGCAGATATGCCGGACATTGGACAGGCGACGTGT GGTCTAGTTGGGAGCAGCTGGCTAGCAGCGTGCCAGAGATCCTGCAGTTCAATC TGCTGGGCGTGCCACTCGTGGGAGCCGATGTTTGTGGCTTCCTGGGCAACACCT CCGAGGAACTGTGTGTGCGTTGGACACAGCTGGGCGCCTTCTATCCCTTCATGA GAAACCACAACAGCCTGCTGAGCCTGCCTCAAGAGCCCTACAGCTTTAGCGAGC CTGCACAGCAGGCCATGAGAAAGGCCCTGACTCTGAGATACGCCCTGCTGCCTC ACCTGTACACCCTGTTTCATCAGGCCCACGTGGCAGGCGAGACAGTGGCTAGAC CTCTGTTCCTGGAATTCCCCAAGGACAGCTCCACCTGGACCGTGGATCATCAGCT GCTGTGGGGAGAAGCCCTGCTGATTACACCAGTGCTGCAGGCCGGAAAGGCCG AAGTGACAGGCTATTTCCCTCTCGGCACTTGGTACGACCTGCAGACCGTGCCTGT TGAGGCTCTGGGATCTCTTCCTCCACCTCCTGCCGCTCCTAGAGAGCCTGCCATT CACTCTGAAGGCCAGTGGGTTACCCTGCCTGCTCCTCTGGACACCATCAACGTG CACCTGAGAGCTGGCTACATCATCCCTCTGCAAGGCCCTGGCCTGACCACAACC GAATCTAGACAGCAGCCCATGGCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAG GCTAGAGGCGAGCTGTTCTGGGATGATGGCGAGAGCCTGGAAGTGCTGGAACG GGGCGCTTATACCCAAGTGATCTTCCTGGCCAGAAACAACACCATCGTGAACGAA CTCGTGCGCGTGACCAGTGAAGGTGCTGGACTGCAACTGCAGAAAGTGACCGTG CTCGGAGTGGCCACAGCTCCTCAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCC AACTTCACATACAGCCCCGACACCAAGGTCCTGGACATCTGTGTGTCACTGCTGA TGGGCGAGCAGTTCCTGGTGTCCTGGTGTTAGTGA SEQ ID 38: DNA sequence of GAAco, N-IGF2 tag and GAP linker with PGRN signal peptide ATGTGGACCCTGGTGTCTTGGGTTGCCCTGACAGCTGGACTTGTTGCCGGTGCT CTGTGTGGCGGAGAGCTGGTGGATACCCTGCAGTTCGTGTGTGGCGACCGGGGCTTCTACTTTAGCAGACCTGCCAGCCGGGTTTCCAGACGGTCTAGAGGAATCGTG GAAGAGTGCTGCTTCAGAAGCTGCGATCTGGCCCTGCTGGAAACCTACTGTGCC ACACCAGCCAAGTCTGAAGGCGCTCCTCAGCAAGGCGCTTCTAGACCCGGACCT AGAGATGCCCAAGCTCACCCTGGTAGACCTAGAGCCGTGCCTACACAGTGTGAC GTGCCACCTAACAGCAGATTCGACTGCGCCCCTGACAAGGCCATCACACAAGAG CAGTGTGAAGCCAGAGGCTGCTGCTACATCCCTGCCAAACAAGGACTGCAGGGC GCCCAGATGGGACAGCCTTGGTGTTTCTTCCCACCATCTTACCCCAGCTACAAGC TGGAAAACCTGAGCAGCAGCGAGATGGGCTACACCGCCACACTGACCAGAACCA CACCTACATTCTTCCCGAAGGACATCCTGACACTGCGGCTGGACGTGATGATGG AAACCGAGAACCGGCTGCACTTCACCATCAAGGACCCCGCCAATCGGAGATACG AGGTGCCACTGGAAACCCCTCACGTGCACTCTAGAGCCCCATCTCCACTGTACA GCGTGGAATTCAGCGAGGAACCCTTCGGCGTGATCGTGCGGAGACAGCTGGATG GAAGAGTGCTGCTGAACACCACAGTGGCCCCTCTGTTCTTCGCCGACCAGTTTCT GCAGCTGAGCACCAGCCTGCCTAGCCAGTATATCACAGGCCTGGCCGAGCACCT GTCTCCACTGATGCTGAGCACATCCTGGACCAGAATCACCCTGTGGAACAGAGAT CTGGCCCCTACACCTGGCGCCAACCTGTATGGCTCTCACCCCTTTTATCTGGCCC TGGAAGATGGCGGATCTGCCCACGGTGTCTTTCTGCTGAACTCCAACGCCATGG ACGTGGTGCTGCAGCCATCTCCTGCTCTGTCTTGGAGAAGCACAGGCGGCATCC TGGATGTGTACATCTTTCTGGGCCCCGAGCCTAAGAGCGTGGTGCAGCAGTATC TGGACGTCGTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACC TGTGCAGATGGGGATACAGCAGCACCGCCATCACCAGACAGGTGGTGGAAAACA TGACCCGGGCTCACTTCCCACTGGATGTGCAGTGGAACGACCTGGACTACATGG ACAGCAGACGGGACTTCACCTTCAACAAGGACGGCTTCAGAGACTTCCCCGCCA TGGTGCAAGAACTGCACCAAGGCGGCAGACGGTACATGATGATCGTGGACCCTG CCATCAGCTCTAGCGGACCTGCCGGCTCTTACAGACCCTACGATGAGGGACTGA GAAGAGGCGTGTTCATCACCAACGAGACAGGCCAGCCTCTGATCGGCAAAGTGT GGCCTGGCAGCACAGCCTTTCCAGACTTCACAAACCCCACCGCTCTGGCTTGGT GGGAAGATATGGTGGCCGAGTTTCACGATCAGGTGCCCTTCGACGGCATGTGGA TCGACATGAACGAGCCCAGCAACTTCATCCGGGGCAGCGAGGATGGCTGCCCCA ACAACGAACTGGAAAATCCTCCTTACGTGCCCGGCGTTGTCGGCGGAACACTTC AGGCCGCTACAATCTGTGCCAGCAGCCATCAGTTTCTGAGCACCCACTACAACCT GCACAACCTGTACGGCCTGACCGAGGCCATTGCCTCTCATAGAGCCCTGGTTAA GGCCAGAGGCACCCGGCCTTTTGTGATCAGCAGAAGCACATTCGCCGGCCACGG CAGATATGCCGGACATTGGACAGGCGACGTGTGGTCTAGTTGGGAGCAGCTGGC TAGCAGCGTGCCAGAGATCCTGCAGTTCAATCTGCTGGGCGTGCCACTCGTGGG AGCCGATGTTTGTGGCTTCCTGGGCAACACCTCCGAGGAACTGTGTGTGCGTTG GACACAGCTGGGCGCCTTCTATCCCTTCATGAGAAACCACAACAGCCTGCTGAG CCTGCCTCAAGAGCCCTACAGCTTTAGCGAGCCTGCACAGCAGGCCATGAGAAA GGCCCTGACTCTGAGATACGCCCTGCTGCCTCACCTGTACACCCTGTTTCATCAG GCCCACGTGGCAGGCGAGACAGTGGCTAGACCTCTGTTCCTGGAATTCCCCAAG GACAGCTCCACCTGGACCGTGGATCATCAGCTGCTGTGGGGAGAAGCCCTGCTG ATTACACCAGTGCTGCAGGCCGGAAAGGCCGAAGTGACAGGCTATTTCCCTCTC GGCACTTGGTACGACCTGCAGACCGTGCCTGTTGAGGCTCTGGGATCTCTTCCT CCACCTCCTGCCGCTCCTAGAGAGCCTGCCATTCACTCTGAAGGCCAGTGGGTT ACCCTGCCTGCTCCTCTGGACACCATCAACGTGCACCTGAGAGCTGGCTACATC ATCCCTCTGCAAGGCCCTGGCCTGACCACAACCGAATCTAGACAGCAGCCCATG GCTCTGGCCGTGGCTTTGACAAAAGGCGGAGAGGCTAGAGGCGAGCTGTTCTGG GATGATGGCGAGAGCCTGGAAGTGCTGGAACGGGGCGCTTATACCCAAGTGATC TTCCTGGCCAGAAACAACACCATCGTGAACGAACTCGTGCGCGTGACCAGTGAA GGTGCTGGACTGCAACTGCAGAAAGTGACCGTGCTCGGAGTGGCCACAGCTCCT CAGCAGGTTCTGTCTAATGGCGTGCCCGTGTCCAACTTCACATACAGCCCCGACA CCAAGGTCCTGGACATCTGTGTGTCACTGCTGATGGGCGAGCAGTTCCTGGTGT CCTGGTGTTAGTGASEQ ID 39: DNA sequence of GAAco, N-IGF2 / PSAP hybrid Tag (1.8-67 of IGF2 and 518-573 of progranulin) flanked by a GAP peptide linker with a GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTTGCTCTGTGTGGCGGAGAGCTGGTGGAT ACCCTGCAGTTCGTGTGTGGCGACCGGGGCTTCTACTTTAGCAGACCTGCCAGC CGGGTTTCCAGACGGTCTAGAGGAATCGTGGAAGAGTGCTGCTTCAGAAGCTGC GATCTGGCCCTGCTGGAAACCTACTGTGCCACACCAGCCAAGTCTGAAGGCGCT CCTGATGTGGAATGTGGCGAGGGCCACTTCTGCCACGACAATCAGACCTGCTGC CGGGACAACAGACAAGGCTGGGCCTGCTGTCCTTACAGACAGGGCGTGTGCTGT GCCGACAGAAGGCACTGTTGTCCTGCCGGCTTTAGATGTGCCGCCAGGGGCACA AAGTGCCTGGGCGCTCCTCAGCAAGGCGCTTCTAGACCCGGACCTAGAGATGCC CAAGCTCACCCTGGTAGACCTAGAGCCGTGCCTACACAGTGTGACGTGCCACCT AACAGCAGATTCGACTGCGCCCCTGACAAGGCCATCACACAAGAGCAGTGTGAA GCCAGAGGCTGCTGCTACATCCCTGCCAAACAAGGACTGCAGGGCGCCCAGATG GGACAGCCTTGGTGTTTCTTCCCACCATCTTACCCCAGCTACAAGCTGGAAAACC TGAGCAGCAGCGAGATGGGCTACACCGCCACACTGACCAGAACCACACCTACAT TCTTCCCGAAGGACATCCTGACACTGCGGCTGGACGTGATGATGGAAACCGAGA ACCGGCTGCACTTCACCATCAAGGACCCCGCCAATCGGAGATACGAGGTGCCAC TGGAAACCCCTCACGTGCACTCTAGAGCCCCATCTCCACTGTACAGCGTGGAATT CAGCGAGGAACCCTTCGGCGTGATCGTGCGGAGACAGCTGGATGGAAGAGTGC TGCTGAACACCACAGTGGCCCCTCTGTTCTTCGCCGACCAGTTTCTGCAGCTGAG CACCAGCCTGCCTAGCCAGTATATCACAGGCCTGGCCGAGCACCTGTCTCCACT GATGCTGAGCACATCCTGGACCAGAATCACCCTGTGGAACAGAGATCTGGCCCC TACACCTGGCGCCAACCTGTATGGCTCTCACCCCTTTTATCTGGCCCTGGAAGAT GGCGGATCTGCCCACGGTGTCTTTCTGCTGAACTCCAACGCCATGGACGTGGTG CTGCAGCCATCTCCTGCTCTGTCTTGGAGAAGCACAGGCGGCATCCTGGATGTG TACATCTTTCTGGGCCCCGAGCCTAAGAGCGTGGTGCAGCAGTATCTGGACGTC GTGGGCTACCCCTTCATGCCTCCTTATTGGGGCCTGGGCTTCCACCTGTGCAGA TGGGGATACAGCAGCACCGCCATCACCAGACAGGTGGTGGAAAACATGACCCGG GCTCACTTCCCACTGGATGTGCAGTGGAACGACCTGGACTACATGGACAGCAGA CGGGACTTCACCTTCAACAAGGACGGCTTCAGAGACTTCCCCGCCATGGTGCAA GAACTGCACCAAGGCGGCAGACGGTACATGATGATCGTGGACCCTGCCATCAGC TCTAGCGGACCTGCCGGCTCTTACAGACCCTACGATGAGGGACTGAGAAGAGGC GTGTTCATCACCAACGAGACAGGCCAGCCTCTGATCGGCAAAGTGTGGCCTGGC AGCACAGCCTTTCCAGACTTCACAAACCCCACCGCTCTGGCTTGGTGGGAAGATA TGGTGGCCGAGTTTCACGATCAGGTGCCCTTCGACGGCATGTGGATCGACATGA ACGAGCCCAGCAACTTCATCCGGGGCAGCGAGGATGGCTGCCCCAACAACGAAC TGGAAAATCCTCCTTACGTGCCCGGCGTTGTCGGCGGAACACTTCAGGCCGCTA CAATCTGTGCCAGCAGCCATCAGTTTCTGAGCACCCACTACAACCTGCACAACCT GTACGGCCTGACCGAGGCCATTGCCTCTCATAGAGCCCTGGTTAAGGCCAGAGG CACCCGGCCTTTTGTGATCAGCAGAAGCACATTCGCCGGCCACGGCAGATATGC CGGACATTGGACAGGCGACGTGTGGTCTAGTTGGGAGCAGCTGGCTAGCAGCGT GCCAGAGATCCTGCAGTTCAATCTGCTGGGCGTGCCACTCGTGGGAGCCGATGT TTGTGGCTTCCTGGGCAACACCTCCGAGGAACTGTGTGTGCGTTGGACACAGCT GGGCGCCTTCTATCCCTTCATGAGAAACCACAACAGCCTGCTGAGCCTGCCTCAA GAGCCCTACAGCTTTAGCGAGCCTGCACAGCAGGCCATGAGAAAGGCCCTGACT CTGAGATACGCCCTGCTGCCTCACCTGTACACCCTGTTTCATCAGGCCCACGTG GCAGGCGAGACAGTGGCTAGACCTCTGTTCCTGGAATTCCCCAAGGACAGCTCC ACCTGGACCGTGGATCATCAGCTGCTGTGGGGAGAAGCCCTGCTGATTACACCA GTGCTGCAGGCCGGAAAGGCCGAAGTGACAGGCTATTTCCCTCTCGGCACTTGG TACGACCTGCAGACCGTGCCTGTTGAGGCTCTGGGATCTCTTCCTCCACCTCCTGCCGCTCCTAGAGAGCCTGCCATTCACTCTGAAGGCCAGTGGGTTACCCTGCCTG CTCCTCTGGACACCATCAACGTGCACCTGAGAGCTGGCTACATCATCCCTCTGCA AGGCCCTGGCCTGACCACAACCGAATCTAGACAGCAGCCCATGGCTCTGGCCGT GGCTTTGACAAAAGGCGGAGAGGCTAGAGGCGAGCTGTTCTGGGATGATGGCGA GAGCCTGGAAGTGCTGGAACGGGGCGCTTATACCCAAGTGATCTTCCTGGCCAG AAACAACACCATCGTGAACGAACTCGTGCGCGTGACCAGTGAAGGTGCTGGACT GCAACTGCAGAAAGTGACCGTGCTCGGAGTGGCCACAGCTCCTCAGCAGGTTCT GTCTAATGGCGTGCCCGTGTCCAACTTCACATACAGCCCCGACACCAAGGTCCT GGACATCTGTGTGTCACTGCTGATGGGCGAGCAGTTCCTGGTGTCCTGGTGTTA GTGA

[0242] SEQ ID 40: DNA sequence of Gamillus without signal peptide ATGGTGAGCAAGGGCGAGGAGGCATCTGGCAGAGCCCTGTTCCAGTACCCCATG ACCAGCAAGATCGAGCTGAACGGCGAGATCAACGGCAAGAAATTCAAGGTGGCC GGCGAGGGCTTCACCCCCAGCAGCGGCAGATTCAACATGCACGCCTACTGCACC ACCGGCGACCTGCCTATGAGCTGGGTCGTGATTGCCAGCCCCCTCCAGTACGGC TTCCACATGTTCGCCCACTACCCCGAGGACATCACACACTTTTTCCAGGAATGCT TCCCCGGCAGCTACACCCTGGACCGGACCCTGAGAATGGAAGGCGACGGCACC CTGACCACCCACCACGAGTACAGCCTGGAGGACGGCTGCGTGACCTCCAAGACC ACCCTGAATGCCAGCGGCTTCGACCCTAAGGGCGCCACCATGACCAAGAGCTTC GTGAAACAACTGCCTAACGAGGTGAAGATCACCCCCCACGGCCCCAACGGCATC AGACTGACCAGCACCGTGCTGTACCTGAAGGAGGATGGCACCATCCAGATCGGC ACCCAGGACTGCATCGTGACCCCTGTGGGCGGAAGGAAAGTGACCCAGCCCAA GGCCCACTTCCTGCACACCCAGATCATCCAGAAGAAGGACCCCAACGACACCCG GGACCACATCGTGCAGACAGAACTGGCCGTGGCCGGCAATCTGTGGCACGGCAT GGACGAGCTGTACAAGTAATAG

[0243] SEQ ID 41: DNA sequence of SecGamillus with IFN alpha 2 signal peptide ATGGCCTTGACCTTTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAAGTCAA GCTGCTCTGTGGGCATGGTGAGCAAGGGCGAGGAGGCATCTGGCAGAGCCCTG TTCCAGTACCCCATGACCAGCAAGATCGAGCTGAACGGCGAGATCAACGGCAAG AAATTCAAGGTGGCCGGCGAGGGCTTCACCCCCAGCAGCGGCAGATTCAACATG CACGCCTACTGCACCACCGGCGACCTGCCTATGAGCTGGGTCGTGATTGCCAGC CCCCTCCAGTACGGCTTCCACATGTTCGCCCACTACCCCGAGGACATCACACACT TTTTCCAGGAATGCTTCCCCGGCAGCTACACCCTGGACCGGACCCTGAGAATGG AAGGCGACGGCACCCTGACCACCCACCACGAGTACAGCCTGGAGGACGGCTGC GTGACCTCCAAGACCACCCTGAATGCCAGCGGCTTCGACCCTAAGGGCGCCACC ATGACCAAGAGCTTCGTGAAACAACTGCCTAACGAGGTGAAGATCACCCCCCAC GGCCCCAACGGCATCAGACTGACCAGCACCGTGCTGTACCTGAAGGAGGATGGC ACCATCCAGATCGGCACCCAGGACTGCATCGTGACCCCTGTGGGCGGAAGGAAA GTGACCCAGCCCAAGGCCCACTTCCTGCACACCCAGATCATCCAGAAGAAGGAC CCCAACGACACCCGGGACCACATCGTGCAGACAGAACTGGCCGTGGCCGGCAA TCTGTGGCACGGCATGGACGAGCTGTACAAGTAATAG

[0244] SEQ ID 42: DNA sequence of SecGamillus, 4x G4S linker and C-PGRN tag with IFN alpha 2 signal peptide ATGGCCTTGACCTTTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAAGTCAA GCTGCTCTGTGGGCATGGTGAGCAAGGGCGAGGAGGCATCTGGCAGAGCCCTG TTCCAGTACCCCATGACCAGCAAGATCGAGCTGAACGGCGAGATCAACGGCAAGAAATTCAAGGTGGCCGGCGAGGGCTTCACCCCCAGCAGCGGCAGATTCAACATG CACGCCTACTGCACCACCGGCGACCTGCCTATGAGCTGGGTCGTGATTGCCAGC CCCCTCCAGTACGGCTTCCACATGTTCGCCCACTACCCCGAGGACATCACACACT TTTTCCAGGAATGCTTCCCCGGCAGCTACACCCTGGACCGGACCCTGAGAATGG AAGGCGACGGCACCCTGACCACCCACCACGAGTACAGCCTGGAGGACGGCTGC GTGACCTCCAAGACCACCCTGAATGCCAGCGGCTTCGACCCTAAGGGCGCCACC ATGACCAAGAGCTTCGTGAAACAACTGCCTAACGAGGTGAAGATCACCCCCCAC GGCCCCAACGGCATCAGACTGACCAGCACCGTGCTGTACCTGAAGGAGGATGGC ACCATCCAGATCGGCACCCAGGACTGCATCGTGACCCCTGTGGGCGGAAGGAAA GTGACCCAGCCCAAGGCCCACTTCCTGCACACCCAGATCATCCAGAAGAAGGAC CCCAACGACACCCGGGACCACATCGTGCAGACAGAACTGGCCGTGGCCGGCAA TCTGTGGCACGGCATGGACGAGCTGTACAAGGGTGGCGGAGGATCTGGCGGCG GAGGAAGCGGAGGCGGCGGTTCTGGCGGTGGTGGATCTGATGTTGAGTGTGGC GAGGGCCACTTCTGCCACGACAATCAGACCTGCTGCCGGGACAACAGACAAGGC TGGGCCTGTTGTCCATACAGACAGGGCGTGTGCTGCGCCGACAGAAGGCACTGT TGTCCTGCCGGATTCAGATGTGCCGCCAGGGGCACAAAGTGCCTGCGAAGAGAA GCCCCTAGATGGGATGCCCCTCTGAGAGATCCAGCTCTGAGACAGCTCCTGTGA TAG SEQ ID 43: DNA sequence of SecGamillus, 4x G4S linker and C-PSAP tag with IFN alpha 2 signal peptide ATGGCCTTGACCTTTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAAGTCAA GCTGCTCTGTGGGCATGGTGAGCAAGGGCGAGGAGGCATCTGGCAGAGCCCTG TTCCAGTACCCCATGACCAGCAAGATCGAGCTGAACGGCGAGATCAACGGCAAG AAATTCAAGGTGGCCGGCGAGGGCTTCACCCCCAGCAGCGGCAGATTCAACATG CACGCCTACTGCACCACCGGCGACCTGCCTATGAGCTGGGTCGTGATTGCCAGC CCCCTCCAGTACGGCTTCCACATGTTCGCCCACTACCCCGAGGACATCACACACT TTTTCCAGGAATGCTTCCCCGGCAGCTACACCCTGGACCGGACCCTGAGAATGG AAGGCGACGGCACCCTGACCACCCACCACGAGTACAGCCTGGAGGACGGCTGC GTGACCTCCAAGACCACCCTGAATGCCAGCGGCTTCGACCCTAAGGGCGCCACC ATGACCAAGAGCTTCGTGAAACAACTGCCTAACGAGGTGAAGATCACCCCCCAC GGCCCCAACGGCATCAGACTGACCAGCACCGTGCTGTACCTGAAGGAGGATGGC ACCATCCAGATCGGCACCCAGGACTGCATCGTGACCCCTGTGGGCGGAAGGAAA GTGACCCAGCCCAAGGCCCACTTCCTGCACACCCAGATCATCCAGAAGAAGGAC CCCAACGACACCCGGGACCACATCGTGCAGACAGAACTGGCCGTGGCCGGCAA TCTGTGGCACGGCATGGACGAGCTGTACAAGGGTGGCGGAGGATCTGGCGGCG GAGGAAGCGGAGGCGGCGGTTCTGGCGGTGGTGGATCTGATGTTGAGTGTGGC GAGGGCCACTTCTGCCACGACAATCAGACCTGCTGCCGGGACAACAGACAAGGC TGGGCCTGTTGTCCATACAGACAGGGCGTGTGCTGCGCCGACAGAAGGCACTGT TGTCCTGCCGGATTCAGATGTGCCGCCAGGGGCACAAAGTGCCTGTGATAG SEQ ID 44: DNA sequence of SecGamillus, 4x G4S linker and C-SRT tag with IFN alpha 2 signal peptide ATGGCCTTGACCTTTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAAGTCAA GCTGCTCTGTGGGCATGGTGAGCAAGGGCGAGGAGGCATCTGGCAGAGCCCTG TTCCAGTACCCCATGACCAGCAAGATCGAGCTGAACGGCGAGATCAACGGCAAG AAATTCAAGGTGGCCGGCGAGGGCTTCACCCCCAGCAGCGGCAGATTCAACATG CACGCCTACTGCACCACCGGCGACCTGCCTATGAGCTGGGTCGTGATTGCCAGC CCCCTCCAGTACGGCTTCCACATGTTCGCCCACTACCCCGAGGACATCACACACT TTTTCCAGGAATGCTTCCCCGGCAGCTACACCCTGGACCGGACCCTGAGAATGG AAGGCGACGGCACCCTGACCACCCACCACGAGTACAGCCTGGAGGACGGCTGC GTGACCTCCAAGACCACCCTGAATGCCAGCGGCTTCGACCCTAAGGGCGCCACC ATGACCAAGAGCTTCGTGAAACAACTGCCTAACGAGGTGAAGATCACCCCCCACGGCCCCAACGGCATCAGACTGACCAGCACCGTGCTGTACCTGAAGGAGGATGGC ACCATCCAGATCGGCACCCAGGACTGCATCGTGACCCCTGTGGGCGGAAGGAAA GTGACCCAGCCCAAGGCCCACTTCCTGCACACCCAGATCATCCAGAAGAAGGAC CCCAACGACACCCGGGACCACATCGTGCAGACAGAACTGGCCGTGGCCGGCAA TCTGTGGCACGGCATGGACGAGCTGTACAAGGGTGGCGGAGGATCTGGCGGCG GAGGAAGCGGAGGCGGCGGTTCTGGCGGTGGTGGATCTACAAAGTGCCTGCGA AGAGAAGCCCCTAGATGGGATGCCCCTCTGAGAGATCCAGCTCTGAGACAGCTC CTGTGATAG SEQ ID 45: DNA sequence of SecGamillus, N-PGRN tag and a GAP linker with IFN alpha 2 signal peptide ATGGCCTTGACCTTTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAAGTCAA GCTGCTCTGTGGGCGATGTGGAATGTGGCGAGGGCCACTTCTGCCACGACAATC AGACCTGCTGCCGGGACAACAGACAAGGCTGGGCCTGCTGTCCTTACAGACAGG GCGTGTGCTGTGCCGACAGAAGGCACTGTTGTCCTGCCGGCTTTAGATGTGCCG CCAGGGGCACAAAGTGCCTGAGAAGAGAAGCCCCTAGATGGGACGCCCCTCTGA GAGATCCTGCTCTGAGACAGCTGCTTGGCGCTCCTATGGTGAGCAAGGGCGAGG AGGCATCTGGCAGAGCCCTGTTCCAGTACCCCATGACCAGCAAGATCGAGCTGA ACGGCGAGATCAACGGCAAGAAATTCAAGGTGGCCGGCGAGGGCTTCACCCCCA GCAGCGGCAGATTCAACATGCACGCCTACTGCACCACCGGCGACCTGCCTATGA GCTGGGTCGTGATTGCCAGCCCCCTCCAGTACGGCTTCCACATGTTCGCCCACT ACCCCGAGGACATCACACACTTTTTCCAGGAATGCTTCCCCGGCAGCTACACCCT GGACCGGACCCTGAGAATGGAAGGCGACGGCACCCTGACCACCCACCACGAGT ACAGCCTGGAGGACGGCTGCGTGACCTCCAAGACCACCCTGAATGCCAGCGGCT TCGACCCTAAGGGCGCCACCATGACCAAGAGCTTCGTGAAACAACTGCCTAACG AGGTGAAGATCACCCCCCACGGCCCCAACGGCATCAGACTGACCAGCACCGTGC TGTACCTGAAGGAGGATGGCACCATCCAGATCGGCACCCAGGACTGCATCGTGA CCCCTGTGGGCGGAAGGAAAGTGACCCAGCCCAAGGCCCACTTCCTGCACACCC AGATCATCCAGAAGAAGGACCCCAACGACACCCGGGACCACATCGTGCAGACAG AACTGGCCGTGGCCGGCAATCTGTGGCACGGCATGGACGAGCTGTACAAGTAAT AG SEQ ID 46: DNA sequence of SecGamillus, N-PSAP tag and a GAP linker with IFN alpha 2 signal peptide ATGGCCTTGACCTTTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAAGTCAA GCTGCTCTGTGGGCGACGTGGAGTGTGGGGAAGGACACTTCTGCCATGATAACC AGACCTGCTGCCGAGACAACCGACAGGGCTGGGCCTGCTGTCCCTACCGCCAG GGCGTCTGTTGTGCTGATCGGCGCCACTGCTGTCCTGCTGGCTTCCGCTGCGCA GCCAGGGGTACCAAGTGTTTGGGCGCTCCTATGGTGAGCAAGGGCGAGGAGGC ATCTGGCAGAGCCCTGTTCCAGTACCCCATGACCAGCAAGATCGAGCTGAACGG CGAGATCAACGGCAAGAAATTCAAGGTGGCCGGCGAGGGCTTCACCCCCAGCAG CGGCAGATTCAACATGCACGCCTACTGCACCACCGGCGACCTGCCTATGAGCTG GGTCGTGATTGCCAGCCCCCTCCAGTACGGCTTCCACATGTTCGCCCACTACCC CGAGGACATCACACACTTTTTCCAGGAATGCTTCCCCGGCAGCTACACCCTGGAC CGGACCCTGAGAATGGAAGGCGACGGCACCCTGACCACCCACCACGAGTACAG CCTGGAGGACGGCTGCGTGACCTCCAAGACCACCCTGAATGCCAGCGGCTTCGA CCCTAAGGGCGCCACCATGACCAAGAGCTTCGTGAAACAACTGCCTAACGAGGT GAAGATCACCCCCCACGGCCCCAACGGCATCAGACTGACCAGCACCGTGCTGTA CCTGAAGGAGGATGGCACCATCCAGATCGGCACCCAGGACTGCATCGTGACCCC TGTGGGCGGAAGGAAAGTGACCCAGCCCAAGGCCCACTTCCTGCACACCCAGAT CATCCAGAAGAAGGACCCCAACGACACCCGGGACCACATCGTGCAGACAGAACT GGCCGTGGCCGGCAATCTGTGGCACGGCATGGACGAGCTGTACAAGTAATAGSEQ ID 47: DNA sequence of SecGamillus, N-SRT tag and a GAP linker with IFN alpha 2 signal peptide ATGGCCTTGACCTTTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAAGTCAA GCTGCTCTGTGGGCACCAAGTGTTTGCGCAGGGAGGCCCCGCGCTGGGACGCC CCTTTGAGGGACCCAGCCTTGAGACAGCTGCTGGGCGCTCCTATGGTGAGCAAG GGCGAGGAGGCATCTGGCAGAGCCCTGTTCCAGTACCCCATGACCAGCAAGATC GAGCTGAACGGCGAGATCAACGGCAAGAAATTCAAGGTGGCCGGCGAGGGCTT CACCCCCAGCAGCGGCAGATTCAACATGCACGCCTACTGCACCACCGGCGACCT GCCTATGAGCTGGGTCGTGATTGCCAGCCCCCTCCAGTACGGCTTCCACATGTT CGCCCACTACCCCGAGGACATCACACACTTTTTCCAGGAATGCTTCCCCGGCAG CTACACCCTGGACCGGACCCTGAGAATGGAAGGCGACGGCACCCTGACCACCCA CCACGAGTACAGCCTGGAGGACGGCTGCGTGACCTCCAAGACCACCCTGAATGC CAGCGGCTTCGACCCTAAGGGCGCCACCATGACCAAGAGCTTCGTGAAACAACT GCCTAACGAGGTGAAGATCACCCCCCACGGCCCCAACGGCATCAGACTGACCAG CACCGTGCTGTACCTGAAGGAGGATGGCACCATCCAGATCGGCACCCAGGACTG CATCGTGACCCCTGTGGGCGGAAGGAAAGTGACCCAGCCCAAGGCCCACTTCCT GCACACCCAGATCATCCAGAAGAAGGACCCCAACGACACCCGGGACCACATCGT GCAGACAGAACTGGCCGTGGCCGGCAATCTGTGGCACGGCATGGACGAGCTGT ACAAGTAATAG SEQ ID 48: SEQ ID: DNA sequence of C-PGRN Tag GATGTTGAGTGTGGCGAGGGCCACTTCTGCCACGACAATCAGACCTGCTGCCGG GACAACAGACAAGGCTGGGCCTGTTGTCCATACAGACAGGGCGTGTGCTGCGCC GACAGAAGGCACTGTTGTCCTGCCGGATTCAGATGTGCCGCCAGGGGCACAAAG TGCCTGCGAAGAGAAGCCCCTAGATGGGATGCCCCTCTGAGAGATCCAGCTCTG AGACAGCTCCTG SEQ ID 49: DNA sequence of N-PGRN Tag GATGTGGAATGTGGCGAGGGCCACTTCTGCCACGACAATCAGACCTGCTGCCGG GACAACAGACAAGGCTGGGCCTGCTGTCCTTACAGACAGGGCGTGTGCTGTGCC GACAGAAGGCACTGTTGTCCTGCCGGCTTTAGATGTGCCGCCAGGGGCACAAAG TGCCTGAGAAGAGAAGCCCCTAGATGGGACGCCCCTCTGAGAGATCCTGCTCTG AGACAGCTGCTT SEQ ID 50: DNA sequence of C-PSAP Tag GATGTTGAGTGTGGCGAGGGCCACTTCTGCCACGACAATCAGACCTGCTGCCGG GACAACAGACAAGGCTGGGCCTGTTGTCCATACAGACAGGGCGTGTGCTGCGCC GACAGAAGGCACTGTTGTCCTGCCGGATTCAGATGTGCCGCCAGGGGCACAAAG TGCCTG SEQ ID 51: DNA sequence of N-PSAP Tag GACGTGGAGTGTGGGGAAGGACACTTCTGCCATGATAACCAGACCTGCTGCCGA GACAACCGACAGGGCTGGGCCTGCTGTCCCTACCGCCAGGGCGTCTGTTGTGCT GATCGGCGCCACTGCTGTCCTGCTGGCTTCCGCTGCGCAGCCAGGGGTACCAA GTGTTTG SEQ ID 52: DNA sequence of C-SRT Tag ACAAAGTGCCTGCGAAGAGAAGCCCCTAGATGGGATGCCCCTCTGAGAGATCCA GCTCTGAGACAGCTCCTG

[0245] SEQ ID 53: DNA sequence of N-SRT TagACCAAGTGTTTGCGCAGGGAGGCCCCGCGCTGGGACGCCCCTTTGAGGGACCC AGCCTTGAGACAGCTGCTG SEQ ID 54 DNA sequence of RQLL

[0246] AGACAGCTCCTG SEQ ID 55: DNA sequence of IGF2 Tag GCTCTGTGTGGCGGAGAGCTGGTGGATACCCTGCAGTTCGTGTGTGGCGACCG GGGCTTCTACTTTAGCAGACCTGCCAGCCGGGTTTCCAGACGGTCTAGAGGAAT CGTGGAAGAGTGCTGCTTCAGAAGCTGCGATCTGGCCCTGCTGGAAACCTACTG TGCCACACCAGCCAAGTCTGAA

[0247] SEQ ID 56: DNA sequence of IGF2 / SRT hybrid Tag GCTCTGTGTGGCGGAGAGCTGGTGGATACCCTGCAGTTCGTGTGTGGCGACCG GGGCTTCTACTTTTGTTCTGGCGGCGGAGGAACCAAGTGTTTGCGCAGGGAGGC CCCGCGCTGGGACGCCCCTTTGAGGGACCCAGCCTTGAGACAGCTGCTGAGCG GAGGCGGCGGTTCTGGCTGTATCGTGGAAGAGTGCTGCTTCAGAAGCTGCGATC TGGCCCTGCTGGAAACCTACTGTGCCACACCAGCCAAGTCTGAA SEQ ID 57: DNA sequence of IGF2 signal peptide ATGGGCATCCCTATGGGCAAGAGCATGCTGGTGCTGCTGACCTTCCTGGCCTTC GCCAGCTGTTGTATCGCT SEQ ID 58: DNA sequence of GAA signal peptide ATGGGCGTTAGACACCCTCCTTGCAGCCATAGACTGCTGGCCGTGTGTGCTCTG GTGTCTCTGGCTACAGCTGCCCTGCTT SEQ ID 59: DNA sequence of progranulin signal peptide ATGTGGACCCTGGTGTCTTGGGTTGCCCTGACAGCTGGACTTGTTGCCGGT SEQ ID 60: DNA sequence of IFN alpha 2 signal peptide ATGGCCTTGACCTTTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAAGTCAA GCTGCTCTGTGGGC SEQ ID 61: DNA sequence of 4 x G4P peptide linker GGTGGCGGAGGACCTGGCGGAGGTGGACCAGGCGGTGGCGGTCCTGGCGGCG GAGGTCCA SEQ ID 62: DNA sequence of 4 x G4S peptide linker GGTGGCGGAGGATCTGGCGGCGGAGGAAGCGGAGGCGGCGGTTCTGGCGGTG GTGGATCT SEQ ID 63: DNA sequence of the GAP peptide linker

[0248] GGCGCTCCT SEQ ID 64: Human IGF2 signal peptide (aa -24 to 1)

[0249] MGIPMGKSMLVLLTFLAFASCCIAReferences

[0250] Dogan Y, Barese CN, Schindler JW, Yoon JK, Unnisa Z, Guda S, Jacobs ME, Oborski C, Maiwald T, Clarke DL, Schambach A, Pfeifer R, Harper C, Mason C, van Til NP. Screening chimeric GAA variants in preclinical study results in hematopoietic stem cell gene therapy candidate vectors for Pompe disease. Mol Ther Meth & Clin Dev 2022, 27: 464-487

[0251] Kido, Jun, Keishin Sugawara, and Kimitoshi Nakamura. " Gene therapy for lysosomal storage diseases: Current clinical trial prospects." Frontiers in genetics 14 (2023): 1064924.

[0252] Kohn, Donald B., Yvonne Y. Chen, and Melissa J. Spencer. " Successes and challenges in clinical gene therapy." Gene Therapy 30.10 (2023): 738-746.

[0253] Massaro, Giulia, et al. " Gene therapy for lysosomal storage disorders: ongoing studies and clinical development." Biomolecules 11.4 (2021 ): 611.

[0254] Kroos MA, Pomponio RJ, Hagemans ML, Keulemans JL, Phipps M, DeRiso M, Palmer RE, Ausems MG, Van der Beek NA, Van Diggelen OP, Halley DJ, Van der Ploeg AT, Reuser AJ. Broad spectrum of Pompe disease in patients with the same c.-32-13T-> G haplotype. Neurology. 2007 Jan 9;68(2):110-5

[0255] Liang Q, Catalano F, Vlaar EC, Pijnenburg JM, Stok M, van Helsdingen Y, Vulto AG, van der Ploeg AT, van Til NP, Pijnappel WWMP. IGF2-tagging of GAA promotes full correction of murine Pompe disease at a clinically relevant dosage of lentiviral gene therapy. Mol Ther Meth & Clin Dev 2022, 27:109-130

[0256] Moreland RJ, Jin X, Zhang XK, Decker RW, Albee KL, Lee KL, Cauthron RD, Brewer K, Edmunds T, Canfield WM. Lysosomal acid alpha-glucosidase consists of four different peptides processed from a single chain precursor. J Biol Chem. 2005 Feb 25;280(8):6780-91.

[0257] Selvan N, Mehta N, Venkateswaran S, Brignol N, Graziano M, Sheikh MO, McAnany Y, Hung F, Madrid M, Krampetz R, Siano N, Mehta A, Brudvig J, Gotschall R, Weimer JM, Do HV. Endolysosomal N-glycan processing is critical to attain the most active form of the enzyme acid alpha-glucosidase. J Biol Chem. 2021 Jan-Jun;296:100769.Shinoda H, Ma Y, Nakashima R, Sakurai K, Matsuda T, Nagai T. Acid-Tolerant Monomeric GFP from Olindias Formosa. Cell Chem Biol 2018; 25, 330-338

[0258] Tauris J, Ellegaard L, Jacobsen C, Nielsen MS, Madsen P, Thøgersen HC, Gliemann J, Petersen CM, and Moestrup SK. The carboxy-terminal domain of the receptor-associated protein binds to the Vps10p domain of sortilin. FEBS Lett. 1998; 429, 27-30

[0259] van der Wal E, Bergsma AJ, Pijnenburg JM, van der Ploeg AT, Pijnappel WWMP. Antisense Oligonucleotides Promote Exon Inclusion and Correct the Common c.-32-13T> G GAA Splicing Variant in Pompe Disease. Mol Ther Nucleic Acids. 2017 Jun 16;7:90-100

[0260] Wisselaar H, Kroos MA, Hermans MMP, van Beeumen J, and Reuser AJJ. Structural and Functional Changes of Lysosomal Acid a-Glycosidase during Intracellular Transport and Maturation*. J Biol. Chem 1993 Jan; 2223-2231

[0261] Umapathysivam K, Hopwood JJ, Meikle PJ. Correlation of acid alpha-glucosidase and glycogen content in skin fibroblasts with age of onset in Pompe disease. Clin Chim Acta. 2005 Nov;361(1-2):191-8.

Claims

1. CLAIMS1. A method for delivering a polypeptide of interest to a lysosomal compartment in a cell of a subject, comprising providing a proteinaceous substance comprising a polypeptide of interest and at least a binding domain for a membrane receptor and / or a binding domain for prosaposin to said subject.

2. A method according to claim 1 wherein the membrane receptor is selected from a mannose 6 phosphate receptor, an LRP1, and a sortilin.

3. A method according to claim 1 or 2, wherein at least one of the binding domains comprises a binding domain for prosaposin and / or a sortilin binding domain.

4. A method according to claims 1-3 wherein said proteinaceous substance is a fusion protein.

5. A method according to claim 3 or 4, wherein said binding domain for prosaposin and / or a sortilin binding domain is tagged to the N-terminus of the polypeptide of interest.

6. A method according to any one of claims 1-5, wherein said at least one binding domain further comprises a granulin binding domain.

7. A method according to any one of claims 1-6, wherein said proteinaceous substance is further comprising a signal peptide sequence for secretion from a cell in which it is expressed.

8. A method according to any one of claims 1 -7, wherein said polypeptide of interest is a lysosomal enzyme.

9. A method according to any one of claims 1 -8, wherein said polypeptide of interest is GAA.

10. A method according to any one of the afore going claims, wherein said proteinaceous substance is expressed from a nucleic acid construct in a cell under conditions allowing for expression.

11. A method according to claim 10, wherein said nucleic acid construct encodes a signal sequence for secretion.

12. A nucleic acid construct comprising a nucleic acid sequence encoding a polypeptide of interest and a nucleic acid sequence encoding a lysosome targeting sequence based on progranulin and / or prosaposin.

13. An expression cassette comprising a construct according to claim 12 and regulatory elements necessary for correct expression.

14. A gene delivery vehicle comprising a construct according to claim 12 or an expression cassette according to claim 13.

15. A gene delivery vehicle according to claim 14 derived from a lentivirus.

16. A gene delivery vehicle according to claim 14 or 15 for use in gene therapy.

17. A gene delivery vehicle for use according to claim 16 wherein said gene therapy is administered to target cells ex vivo.

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