Therapeutic adeno-associated virus for treating pompe disease

Abstract

To provide gene therapy constructs, methods and compositions for treating Pompe disease.SOLUTION: Provided herein is an rAAV vector including a recombinant AAV (rAAV) genome having a heterologous nucleic acid encoding a signal peptide and optionally an IGF-2 sequence fused to an acid α-glucosidase (GAA) polypeptide to enable the GAA polypeptide to be secreted from the liver and targeted to the lysosome. A particular aspect relates to an rAAV vector encoding a GAA polypeptide, where the rAAV vector comprises a liver secretory signal peptide and a targeting IGF2 sequence that binds to human cation-independent mannose-6-phosphate receptor (CI-MPR) or IGF2 receptor to enable proper subcellular localization of the GAA polypeptide to lysosomes.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This invention claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 768,449, filed November 16, 2018, and U.S. Provisional Patent Application No. 62 / 769,702, filed November 20, 2018, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on November 15, 2019, is named 046192-093900WOPT_SL.txt and is 189,408 bytes in size.

[0003] FIELD OF THE INVENTION The present invention relates to adeno-associated virus (AAV) particles, virions, and vectors for target-specific delivery of alpha-glucosidase (GAA) polypeptides, and methods of use for the treatment of Pompe disease. [Background technology]

[0004] background Acid α-glucosidase (GAA) is a lysosomal enzyme that hydrolyzes the α1-4 linkage in maltose and other linear oligosaccharides, such as the outer branches of glycogen, thereby degrading excess glycogen in lysosomes (Hirschhorn et al. (2001) in The Metabolic and Molecular Basis of Inherited Disease (Non-Patent Document 1); Scriver, et al., eds. (2001), McGraw-Hill: New York, pp. 3389-3420 (Non-Patent Document 2)). Like other mammalian lysosomal enzymes, GAA is synthesized in the cytosol and transits the ER, where it is glycosylated with N-linked high-mannose carbohydrates. In the Golgi, high-mannose carbohydrates on lysosomal proteins are modified by the addition of mannose-6-phosphate (M6P), thereby targeting these proteins to lysosomes. M6P-modified proteins are delivered to lysosomes via interaction with one of two M6P receptors. The most favorable type of modification occurs when two M6P moieties are attached to a high-mannose carbohydrate.

[0005] Insufficient GAA activity in lysosomes leads to Pompe disease, also known as acid maltase deficiency (AMD), glycogen storage disease type II (GSDII), glycogen storage disease type II, or GAA deficiency. Reduced enzyme activity is caused by a variety of missense and nonsense mutations in the gene encoding GAA. Consequently, in patients with Pompe disease, glycogen accumulates in the lysosomes of all cells. Specifically, glycogen accumulation is most pronounced in the lysosomes of cardiac and skeletal muscle, liver, and other tissues. Accumulated glycogen ultimately impairs muscle function. In the most severe forms of Pompe disease, death occurs before the age of two due to cardiopulmonary failure.

[0006] There is a need for an effective treatment for Pompe disease. Enzyme replacement therapy for Pompe disease requires that recombinant GAA protein be administered, taken up by the subject's muscle and liver cells, and then transported to the lysosomes of those cells in an M6P-dependent manner. That is, recombinant GAA protein with exposed M6P binds to the M6P receptor in the trans-Golgi, is transported to endosomes, and then to lysosomes. However, two of the main sources of recombinant GAA protein used for enzyme replacement therapy, recombinant GAA produced in engineered CHO cells or in the milk of transgenic rabbits, contain very little M6P, which is required for targeting the protein to lysosomes (Van Hove et al. (1996) Proc Natl Acad Sci USA, 93(1):65-70 (Non-Patent Document 3); and U.S. Patent No. 6,537,785 (Patent Document 1)). Thus, M6P-dependent delivery of recombinant GAA protein to lysosomes is inefficient, requiring both high dosages and frequent injections.

[0007] Thus, although enzyme therapy has shown reasonable efficacy for severe infantile GSD II, the benefit of GAA enzyme therapy is limited due to the need for frequent infusions and the development in subjects of inhibitors or neutralizing antibodies against recombinant hGAA protein (Amalfitano, A., et al. (2001) Genet. In Med. 3:132-138 (Non-Patent Document 4)).

[0008] Viral gene therapy has the potential not only to cure genetic disorders but also to facilitate long-term, non-invasive treatment of acquired degenerative diseases. One gene therapy vector is the adeno-associated virus (AAV). AAV itself is a nonpathogenic, dependent parvovirus that requires a helper virus for efficient replication. Due to its safety and simplicity, AAV has been used as a viral vector for gene therapy. AAV has a broad host and cell type tropism, capable of transducing both dividing and non-dividing cells. To date, 12 AAV serotypes and over 100 variants have been identified. Different AAV serotypes have different abilities to infect cells of various tissues either in vivo or in vitro, and it has been shown that these differences in infectivity are likely related to specific receptors and coreceptors located on the cell surface of each AAV serotype or may be related to the intracellular trafficking pathways themselves.

[0009] Therefore, the feasibility of gene therapy approaches to treat GSD-II as an alternative or adjunct to enzyme therapy is being investigated (Amalfitano, A., et al., (1999) Proc. Natl. Acad. Sci. USA 96:8861-8866 (Non-Patent Document 5); Ding, E., et al. (2002) Mol. Ther. 5:436-446 (Non-Patent Document 6); Fraites, TJ, et al., (2002) Mol. Ther. 5:571-578 (Non-Patent Document 7); Tsujino, S., et al. (1998) Hum. Gene Ther. 9:1609-1616 (Non-Patent Document 8)).

[0010] However, AAV delivery of GAA polypeptide has some problems in terms of achieving sufficient expression in the liver and / or delivery to lysosomes, and patients have been reported to experience glycemia.In vivo research, it has been reported that the use of adenovirus (Ad) vectors encoding hGAA targeted to the mouse liver in GAA-KO mouse models reverses glycogen accumulation in skeletal muscle and cardiac muscle within 12 days through the secretion of hGAA from the liver and uptake in other tissues (Amalfitano, A., et al., (1999) Proc.Natl.Acad.Sci.USA 96:8861-8866 (Non-Patent Document 5)). Introduction of an adeno-associated virus 2 (AAV2) vector encoding GAA normalized GAA activity in injected skeletal muscle and injected cardiac muscle, and when an AAV1-pseudotyped vector with improved muscle transduction was administered, glycogen content in muscle was normalized (Fraites, TJ, et al. (2002) Mol. Ther. 5:571-578 (Non-Patent Document 7)). Gene therapy with muscle-targeted Ad vectors was attempted in a Japanese quail model, but only a limited reversal of glycogen accumulation at the site of vector injection was achieved (Tsujino, S., et al. (1998) Hum. Gene Ther. 9:1609-1616 (Non-Patent Document 8)).

[0011] However, in human subjects, administration of rAAV vectors encoding the GAA polypeptide results in many patients experiencing hypoglycemia or developing hyperglycemia due to nonspecific cellular uptake (see, e.g., Byrne et al., A study on the safety and efficacy of Reveglucosidease alfa in patients with late-onset Pompe disease; Orphanet J. of Rare diseases; 2017; 12:144).

[0012] Therefore, there is a need in the art for an improved method for producing lysosomal polypeptides such as GAA in vitro and in vivo, for example, to treat lysosomal polypeptide deficiency.In addition, there is a need for improved secretion from the liver, and for improving the targeting of GAA to lysosomes, in order to help reduce the side effects caused by overexpression of GAA polypeptide and reduce the risk of hypoglycemia.In addition, there is a need for a method that can deliver GAA and other lysosomal polypeptides to affected tissues and organs in a systemic manner.In particular, there is a need for a more efficient method for administering GAA protein to subjects and targeting GAA protein to the lysosomes of patients, while reducing possible side effects. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] U.S. Patent No. 6,537,785 [Non-patent literature]

[0014] [Non-Patent Document 1] Hirschhorn et al. (2001), The Metabolic and Molecular Basis of Inherited Disease [Non-patent document 2] Scriver, et al., eds. (2001), McGraw-Hill: New York, p. 3389-3420 [Non-patent document 3] Van Hove et al. (1996) Proc Natl Acad Sci USA,93(1):65-70 [Non-patent document 4] Amalfitano, A., et al. (2001) Genet. In Med. 3:132-138 [Non-patent document 5] Amalfitano, A., et al., (1999) Proc. Natl. Acad. Sci. USA 96:8861-8866 [Non-patent document 6] Ding, E., et al. (2002) Mol. Ther. 5:436-446 [Non-Patent Document 7] Fraites, TJ, et al., (2002) Mol.Ther.5:571-578 [Non-patent document 8] Tsujino, S., et al. (1998) Hum. Gene Ther. 9:1609-1616 [Non-Patent Document 9] Byrne et al.,A study on the safety and efficacy of Reveglucosidease alfa in patients with late-onset Pompe disease;Orphanet J.of Rare diseases;2017;12:144 Summary of the Invention

[0015] The technology described herein generally relates to gene therapy constructs, methods, and compositions for the treatment of Pompe disease. More specifically, the technology relates to adeno-associated (AAV) virions configured to deliver GAA polypeptides to subjects. Thus, a rAAV vector for use in treating Pompe disease is described herein, which includes a nucleotide sequence containing an inverted terminal repeat (ITR), a promoter, a heterologous gene, a polyA tail, and optionally other regulatory elements, wherein the heterologous gene encodes an acid alpha-glucosidase (GAA) protein, and the rAAV expressing the GAA protein can be administered to a patient at a therapeutically effective dose to express the heterologous gene encoding the GAA protein in the appropriate tissue and / or organ for the treatment of a subject with Pompe disease.

[0016] Therefore, the technology described herein generally relates to a means for expressing GAA protein in the liver using rAAV vectors, and for effectively targeting the expressed GAA protein to the lysosomes of mammalian cells, such as human cardiac muscle cells and skeletal muscle cells.Described herein are rAAV vectors, rAAV genomes, and isolated nucleic acid compositions that encode lysosomal polypeptides (such as GAA) fused with signal peptides, where the signal peptide enhances the targeting of the GAA polypeptide to the secretory pathway, and the GAA polypeptide is optionally fused with a targeting sequence to aid in uptake into lysosomes.Therefore, the method and composition enable the secretion of GAA polypeptides from cells, such as hepatocytes, and the lysosomal targeting of GAA protein to the lysosomes of muscle.Lysosomal targeting of GAA protein provides many advantages. For example, administration of rAAV vectors encoding a GAA polypeptide results in many patients experiencing hypoglycemia or hyperglycemia due to nonspecific cellular uptake (see, e.g., Byrne et al., A study on the safety and efficacy of Reveglucosidease alfa in patients with late-onset Pompe disease; Orphanet J. of Rare diseases; 2017; 12:144). Optimizing or improving GAA secretion from the liver and improving its targeting to lysosomes would allow GAA to be expressed at lower levels, helping to reduce side effects of overexpression of the GAA polypeptide, such as reducing the risk of hypoglycemia.

[0017] Thus, we describe herein an rAAV vector that contains within its genome a heterologous nucleic acid encoding a chimeric gene that encodes a secretory signal peptide (SS) operably linked to an IGF2 sequence (e.g., a targeting peptide or TP) fused to the N-terminus of a GAA polypeptide at the native signal peptide cleavage site or a suitable downstream site. Expression of such a chimeric gene will direct the production of a recombinant GAA fusion protein that is secreted at high levels and contains a high-affinity ligand for the M6P / IGF2 receptor.

[0018] In some embodiments of the compositions and methods described herein, the rAAV vector disclosed herein comprises within its genome 5' and 3' AAV inverted terminal repeats (ITRs) and a heterologous nucleic acid sequence encoding a fusion polypeptide comprising (i) a secretory signal peptide, (ii) an IGF2 sequence, and (iii) an alpha-glucosidase (GAA) polypeptide located between the 5' and 3' ITRs, wherein the heterologous nucleic acid is operably linked to a promoter, such as, but not limited to, a liver-specific promoter. In some embodiments, the rAAV vector disclosed herein comprises within its genome 5' and 3' AAV inverted terminal repeats (ITRs) and a heterologous nucleic acid sequence encoding a fusion polypeptide comprising (i) a secretory signal peptide and (ii) an alpha-glucosidase (GAA) polypeptide located between the 5' and 3' ITRs, wherein the heterologous nucleic acid is operably linked to a promoter, such as, but not limited to, a liver-specific promoter.

[0019] In some embodiments of the compositions and methods described herein, the secretory signal peptide is selected from the AAT signal peptide, fibronectin signal peptide (FN1), GAA signal peptide, or any of their active fragments having secretory signal activity.

[0020] In some embodiments of the compositions and methods described herein, the alpha-glucosidase (GAA) polypeptide is linked to an IGF2 sequence at the N-terminus of the GAA polypeptide. In some embodiments, the IGF2 sequence is linked N-terminally at amino acid 70 of the human acid alpha-glucosidase (GAA) polypeptide (SEQ ID NO: 10) (i.e., linked to the N-terminus of residues 70-952 of the human acid alpha-glucosidase (GAA) polypeptide). In other embodiments, the IGF2 sequence is linked N-terminally at amino acid 40 of the human acid alpha-glucosidase (GAA) polypeptide (SEQ ID NO: 10) (i.e., linked to the N-terminus of residues 40-952 of the human acid alpha-glucosidase (GAA) polypeptide). In some embodiments of the compositions and methods described herein, the GAA polypeptide may be encoded by a wild-type GAA nucleic acid sequence (e.g., SEQ ID NO:11 or SEQ ID NO:72) or may be a GAA nucleic acid sequence that has been codon-optimized, e.g., for any one of increased in vivo expression, reduced CpG islands, and / or reduced innate immune response in a subject. Exemplary codon-optimized GAA nucleic acid sequences include, but are not limited to, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76.

[0021] In some embodiments of the compositions and methods described herein, the IGF2 sequence is a nucleic acid sequence encoding either residues 1 followed by residues 8-67 of wild-type mature human insulin-like growth factor II (IGF2) of SEQ ID NO:5 (i.e., IGF2-delta 2-7 or IGF2Δ2-7, corresponding to SEQ ID NO:6); residues 8-67 of wild-type mature human insulin-like growth factor II (IGF2) of SEQ ID NO:5 (i.e., IGF2-delta 1-7 or IGF2Δ1-7, corresponding to SEQ ID NO:7), or residues 43-67 of wild-type mature human insulin-like growth factor II (IGF2) of SEQ ID NO:5 (i.e., IGF2 delta 1-42 or IGF2Δ1-42, corresponding to SEQ ID NO:8). In some embodiments of the compositions and methods described herein, the IGF2 sequence is a nucleic acid sequence having a modification of amino acid residue 43, e.g., residue 43 modified to the start codon, e.g., IGF2-V43M (corresponding to SEQ ID NO:9).

[0022] In some embodiments of the compositions and methods described herein, the IGF2 sequence is a nucleic acid sequence comprising any of SEQ ID NO:2 (i.e., IGF2-delta 2-7); SEQ ID NO:3 (i.e., IGF2-delta 1-7), or SEQ ID NO:4 (i.e., IGF2-V43M).

[0023] In some embodiments of the compositions and methods described herein, a fusion protein comprising a GAA polypeptide and an IGF2 sequence comprises amino acid residues 40-952 or residues 70-952 of a human acid alpha glucosidase (GAA) polypeptide (SEQ ID NO:10) attached to an IGF2 sequence comprising residue 1 followed by residues 8-67 of wild-type mature human insulin-like growth factor II (IGF2) (SEQ ID NO:5) (i.e., residues 2-7 of mature human IGF2 (SEQ ID NO:5) are absent), where the IGF2 sequence is linked to amino acid residue 70 of human GAA (SEQ ID NO:10).

[0024] In some embodiments of the compositions and methods described herein, a fusion protein comprising a GAA polypeptide and an IGF2 sequence comprises amino acid residues 40-952 or residues 70-952 of a human acid alpha glucosidase (GAA) polypeptide (SEQ ID NO:10) attached to an IGF2 sequence comprising residues 8-67 of wild-type mature human insulin-like growth factor II (IGF2) (SEQ ID NO:5) (i.e., residues 1-7 of mature human IGF2 (i.e., YRPSET; SEQ ID NO:63) are absent), where the IGF2 sequence is linked to amino acid residue 70 of human GAA (SEQ ID NO:10).

[0025] In some embodiments of the compositions and methods described herein, a fusion protein comprising a GAA polypeptide and an IGF2 sequence comprises amino acid residues 40-952 or residues 70-952 of a human acid alpha glucosidase (GAA) polypeptide (SEQ ID NO:10) attached to a modified IGF2 sequence comprising residues 43-67 of wild-type mature human insulin-like growth factor II (IGF2) (SEQ ID NO:5) (residues 1-42 of mature human IGF2 (SEQ ID NO:5) are absent), where the IGF2 sequence is linked to amino acid residue 70 of human GAA (SEQ ID NO:10).

[0026] In some embodiments of the compositions and methods described herein, the IGF2 sequence (i.e., Delta 1-7, Delta 2-7, or Delta 1-42 disclosed herein) binds to the cation-independent mannose-6-phosphate receptor (CI-MPR). In one embodiment, the IGF2 sequence is fused directly to the N- or C-terminus of the GAA polypeptide. In another embodiment, the IGF2 sequence is fused to the N- or C-terminus of the GAA polypeptide via a spacer. In one specific embodiment, the IGF2 sequence is fused to the GAA polypeptide via a spacer of 10 to 25 amino acids. In another specific embodiment, the IGF2 sequence is fused to the GAA polypeptide via a spacer comprising a glycine residue. In another specific embodiment, the IGF2 sequence is fused to the GAA polypeptide via a spacer comprising a helix structure. In another specific embodiment, the IGF2 sequence is fused to the GAA polypeptide via a spacer at least 50% identical to the sequence GGGTVGDDDDK (SEQ ID NO:35).

[0027] In some embodiments of the compositions and methods described herein, the secretion signal serves the general purpose of supporting the secretion of a fusion polypeptide, such as an IGF2 sequence-GAA fusion polypeptide, from hepatocytes into the blood, and the fusion polypeptide can be transported and targeted to the lysosomes of mammalian cells, such as human cardiac muscle cells and skeletal muscle cells, as described herein. In some embodiments, the secretion signal is selected from the AAT signal peptide, fibronectin signal peptide (FN1), GAA signal peptide, or an active fragment of the AAT, FN1, or GAA signal peptide that has secretion signal activity.

[0028] All aspects of the compositions and methods of the presently disclosed technology are described below.

[0029] In some embodiments, the present technology relates to recombinant adeno-associated virus (AAV) vector compositions and methods of use thereof, wherein the rAAV vector comprises within its genome (a) 5' and 3' AAV inverted terminal repeats (ITRs), and (b) a heterologous nucleic acid sequence encoding a fusion polypeptide comprising a secretory signal peptide and an alpha-glucosidase (GAA) polypeptide, located between the 5' ITR and the 3' ITR, wherein the heterologous nucleic acid is operably linked to a promoter. In some embodiments of the methods and compositions disclosed herein, the rAAV vector comprises a heterologous nucleic acid sequence encoding a fusion polypeptide further comprising an IGF-2 sequence located between the secretory signal peptide and the alpha-glucosidase (GAA) polypeptide. In some embodiments of the methods and compositions disclosed herein, the rAAV composition comprises an AAV genome comprising, in the 5' to 3' direction, (a) a 5' ITR, (b) a promoter sequence, (c) an intron sequence, (d) a nucleic acid encoding a secretory signal peptide, (e) a nucleic acid encoding an IGF-2 sequence, a nucleic acid encoding an alpha glucosidase (GAA) polypeptide, (f) a polyA sequence, and (g) a 3' ITR.

[0030] In some embodiments, the present technology relates to recombinant adeno-associated virus (AAV) vector compositions and methods for their use, wherein the recombinant AAV (rAAV) vector comprises within its genome (a) 5' and 3' AAV inverted terminal repeats (ITRs), and (b) a heterologous nucleic acid sequence encoding a fusion polypeptide comprising an alpha-glucosidase (GAA) polypeptide located between the 5' and 3' ITRs, the heterologous nucleic acid being operably linked to a liver-specific promoter, and the recombinant AAV vector comprises a capsid protein of the AAV3b serotype. In such embodiments, the fusion polypeptide further comprises a secretory signal peptide located at the N-terminus of the GAA polypeptide. In some embodiments of the methods and compositions disclosed herein, such a recombinant AAV vector comprises a heterologous nucleic acid sequence encoding a fusion polypeptide further comprising an IGF-2 sequence located between the secretory signal peptide and the alpha-glucosidase (GAA) polypeptide.

[0031] In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector comprises, in a 5' to 3' direction within its genome, (a) a 5' ITR, (b) a liver-specific promoter sequence, (c) an intron sequence, (d) a nucleic acid encoding a secretory signal peptide, (e) a nucleic acid encoding an IGF-2 sequence, (f) a nucleic acid encoding an alpha-glucosidase (GAA) polypeptide, (g) a polyA sequence, and (h) a 3' ITR.

[0032] In some embodiments of the methods and compositions disclosed herein, the rAAV vector composition comprises a nucleic acid encoding a secretory signal peptide, e.g., selected from the group consisting of the AAT signal peptide (e.g., SEQ ID NO:17), fibronectin signal peptide (FN) (e.g., SEQ ID NOs:18-21), the GAA signal peptide, the hIGF2 signal peptide (e.g., SEQ ID NO:22), or an active fragment thereof having secretory signal activity, e.g., a nucleic acid encoding an amino acid sequence having at least about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99% sequence identity to SEQ ID NOs:17-22. In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector comprises a heterologous nucleic acid sequence encoding an IGF-2 leader sequence that binds to the human cation-independent mannose-6-phosphate receptor (CI-MPR) or IGF-2 receptor, e.g., the heterologous nucleic acid sequence encodes an IGF-2 sequence having the amino acid sequence of SEQ ID NO:5 or comprising at least one amino modification in SEQ ID NO:5 that binds to the IGF-2 receptor. In some embodiments, the recombinant AAV vector comprises a heterologous nucleic acid sequence encoding an IGF-2 leader sequence having at least one amino modification in SEQ ID NO:5 that is a V43M amino acid modification (SEQ ID NO:8 or SEQ ID NO:9), Δ2-7 (SEQ ID NO:6), or Δ1-7 (SEQ ID NO:7), or an IGF2 peptide having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs:5-9.

[0033] In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector comprises a constitutive, cell-specific, or inducible promoter. In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector comprises a liver-specific promoter, for example, but not limited to, the liver-specific promoter is selected from the group consisting of a transthyretin promoter (TTR), an LSP promoter (LSP), and a synthetic liver-specific promoter.

[0034] In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector comprises a heterologous nucleic acid sequence encoding a wild-type GAA polypeptide (wtGAA) or a modified GAA polypeptide. In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector comprises a heterologous nucleic acid sequence encoding a GAA polypeptide, the heterologous nucleic acid sequence being a human GAA gene or a human codon-optimized GAA gene (coGAA) or a modified GAA nucleic acid sequence. In all aspects of the methods and compositions disclosed herein, the nucleic acid sequence encoding the GAA polypeptide is codon-optimized for one or more of enhanced in vivo expression, reduced CpG islands, or reduced innate immune response. In all aspects of the methods and compositions disclosed herein, the nucleic acid sequence encoding the GAA polypeptide is codon-optimized to reduce CpG islands and reduce innate immune response.

[0035] In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector comprises a heterologous nucleic acid sequence, wherein the encoded fusion polypeptide further comprises a spacer comprising a nucleotide sequence for at least one amino acid located amino-terminal to the GAA polypeptide and C-terminal to the IGF-2 sequence. In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector comprises a heterologous nucleic acid sequence, wherein the heterologous nucleic acid sequence comprises a nucleic acid encoding a spacer of at least one amino acid located between the nucleic acid encoding the IGF-2 sequence and the nucleic acid encoding the GAA polypeptide.

[0036] In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector comprises at least one polyA sequence located 3' to the nucleic acid encoding the GAA gene and 5' to the 3' ITR sequence.

[0037] In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector comprises a heterologous nucleic acid sequence further comprising a collagen stability (CS) sequence located 3' to the nucleic acid encoding the GAA polypeptide and 5' to the 3' ITR sequence. In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector further comprises a nucleic acid encoding a collagen stability (CS) sequence located between the nucleic acid encoding the GAA polypeptide and the polyA sequence.

[0038] In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector comprises a heterologous nucleic acid sequence further comprising an intron sequence located 5' to the sequence encoding the secretory signal peptide and 3' to the promoter. In some embodiments, the intron sequence comprises an MVM sequence or an HBB2 sequence, wherein the MVN sequence comprises the nucleic acid sequence of SEQ ID NO:13, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:13, and the HBB2 sequence comprises the nucleic acid sequence of SEQ ID NO:14, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:14.

[0039] In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector comprises an ITR sequence that contains an insertion, deletion, or substitution. In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector comprises at least one ITR sequence in which one or more CpG islands within the ITR have been removed.

[0040] In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector comprises a heterologous nucleic acid sequence encoding a secretory signal sequence that is a fibronectin signal peptide (FN1) or an active fragment thereof having secretory signal activity (e.g., the FN1 signal peptide has any of the sequence of SEQ ID NOs:18-21, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any of SEQ ID NOs:18-21), and the heterologous nucleic acid sequence encodes an IGF-2 sequence selected from any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or an IGF2 peptide having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs:5-9. In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector comprises a heterologous nucleic acid sequence encoding a secretory signal sequence that is an AAT signal peptide or an active fragment thereof having secretory signal activity (e.g., the AAT signal peptide has the sequence of SEQ ID NO:17, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:17), and the heterologous nucleic acid sequence encodes an IGF-2 sequence selected from any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or an IGF2 peptide having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs:5-9.

[0041] In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector comprises a heterologous nucleic acid sequence encoding an IGF2 peptide, wherein the IGF2 peptide sequence is SEQ ID NO:8 or SEQ ID NO:9, or an IGF2 peptide having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:8 or 9.

[0042] In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector is a chimeric AAV vector, a haploid AAV vector, a hybrid AAV vector, or a polyploid AAV vector, for example, but not limited to, the recombinant AAV vector comprises a capsid protein selected from any AAV serotype in the group consisting of those listed in Table 1 and any combination thereof. In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector is of the AAV3b serotype. In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector is of the AAV3b serotype comprising one or more mutations in the capsid protein selected from 265D, 549A, or Q263Y. In some embodiments of the methods and compositions disclosed herein, the recombinant AAV vector is of the AAV3b serotype selected from AAV3b265D, AAV3b265D549A, AAV3b549A, or AAV3bQ263Y, or AAV3bSASTG.

[0043] Another aspect of the technology herein relates to a pharmaceutical composition comprising any of the recombinant AAV vector compositions disclosed herein and a pharmaceutically acceptable carrier.

[0044] Another aspect of the technology herein relates to a composition comprising a nucleic acid sequence comprising a liver-specific promoter operably linked to a nucleic acid sequence comprising, in the following order: (a) a nucleic acid encoding a secretory signal peptide; (b) a nucleic acid encoding an IGF-2 sequence; and (c) a nucleic acid encoding a GAA polypeptide.

[0045] Another aspect of the technology herein relates to compositions comprising a nucleic acid sequence for a recombinant adeno-associated virus (rAAV) vector genome, wherein the nucleic acid sequence comprises (a) 5' and 3' AAV inverted terminal repeat (ITR) nucleic acid sequences, and (b) a heterologous nucleic acid sequence encoding a fusion polypeptide comprising a secretory signal peptide and an alpha-glucosidase (GAA) polypeptide, located between the 5' ITR sequence and the 3' ITR sequence, wherein the heterologous nucleic acid is operably linked to a promoter.

[0046] In some embodiments of the methods and compositions disclosed herein, the nucleic acid sequence comprises a heterologous nucleic acid sequence encoding a fusion polypeptide further comprising an IGF-2 sequence positioned between the secretory signal peptide and the alpha glucosidase (GAA) polypeptide. In some embodiments of the methods and compositions disclosed herein, the nucleic acid sequence comprises a nucleic acid encoding a secretory signal selected from any of SEQ ID NOs: 17, 22-26, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any of SEQ ID NOs: 17 or 22-26.

[0047] In some embodiments of the methods and compositions disclosed herein, the nucleic acid sequence comprises a heterologous nucleic acid sequence encoding an IGF-2 sequence selected from any of SEQ ID NO:2 (IGF2-Δ2-7), SEQ ID NO:3 (IGF2-Δ1-7), or SEQ ID NO:4 (IGF2 V43M), or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any of SEQ ID NO:2, 3, or 4.

[0048] In some embodiments of the methods and compositions disclosed herein, the nucleic acid sequence comprises a heterologous nucleic acid sequence encoding a GAA polypeptide, wherein the nucleic acid sequence is a human GAA gene or a human codon-optimized GAA gene (coGAA) or a modified GAA nucleic acid sequence. In some embodiments of the methods and compositions disclosed herein, the nucleic acid sequence comprises a heterologous nucleic acid sequence that is a codon-optimized (coGAA) GAA gene for one or more of enhanced in vivo expression, reduced CpG islands, or reduced innate immune response. In some embodiments of the methods and compositions disclosed herein, the nucleic acid sequence comprises a heterologous nucleic acid sequence that is a codon-optimized (coGAA) GAA gene for reduced CpG islands and reduced innate immune response.

[0049] In some embodiments of the methods and compositions disclosed herein, the nucleic acid sequence comprises a heterologous nucleic acid sequence encoding a GAA polypeptide selected from any of SEQ ID NO:11 (full-length hGAA), SEQ ID NO:55 (Dwight cDNA), SEQ ID NO:56 (hGAAΔ1-66), or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any of SEQ ID NO:11, 55, or 56.

[0050] In some embodiments of the methods and compositions disclosed herein, the nucleic acid sequence comprises a heterologous nucleic acid sequence encoding a GAA polypeptide, wherein the heterologous nucleic acid sequence encoding the GAA polypeptide is selected from any of SEQ ID NO:74 (codon optimized 1), SEQ ID NO:75 (codon optimized 2), and SEQ ID NO:76 (codon optimized 3), or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any of SEQ ID NO:74, 75, or 76.

[0051] In some embodiments of the methods and compositions disclosed herein, the nucleic acid sequence is selected from the group consisting of SEQ ID NO:57 (AAT-V43M-wtGAA(delta 1-69 aa)); SEQ ID NO:58 (rat FN1-IGF2V43M-wtGAA(delta 1-69 aa)); SEQ ID NO:59 (hFN1-IGF2V43M-wtGAA(delta 1-69 aa)); SEQ ID NO:60 (ATT-IGF2Δ2-7-wtGAA(delta 1-69)); SEQ ID NO:61 (FN1 rat-IGFΔ2-7-wtGAA(delta 1-69)); SEQ ID NO:62 (hFN1-IGFΔ2-7-wtGAA(delta 1-69)), SEQ ID NO: SEQ ID NO:79 (AAT_hIGF2-V43M_wtGAA_del1-69_Stuffer.V02); SEQ ID NO:80 (FIB Rat_hIGF2-V43M_wtGAA_del1-69_Stuffer.V02); SEQ ID NO:81 (FIBhum_hIGF2-V43M_wtGAA_del1-69_Stuffer.V02); SEQ ID NO:82 (AAT_GILT_wtGAA_del1-69_Stuffer.V02); SEQ ID NO:83 (FIB Rat_GILT_wtGAA_del1-69_Stuffer.V02); SEQ ID NO:84 (FIBhum_GILT_wtGAA_del1-69_Stuffer.V02), or SEQ ID The nucleic acid sequence may be selected from any of the nucleic acid sequences having at least 80%, 85%, 90%, 95%, or 98% identity to NO:57, 58, 59, 60, 61, 62, 79, 80, 81, 82, 83, or 84.

[0052] In some embodiments of the methods and compositions disclosed herein, the rAAV vector is selected from the group consisting of SEQ ID NO:57 (AAT-V43M-wtGAA(delta 1-69 aa)); SEQ ID NO:58 (ratFN1-IGF2V43M-wtGAA(delta 1-69 aa)); SEQ ID NO:59 (hFN1-IGF2V43M-wtGAA(delta 1-69 aa)); SEQ ID NO:60 (ATT-IGF2Δ2-7-wtGAA(delta 1-69)); SEQ ID NO:61 (FN1 rat-IGFΔ2-7-wtGAA(delta 1-69)); SEQ ID NO:62 (hFN1-IGFΔ2-7-wtGAA(delta 1-69)), SEQ ID NO: SEQ ID NO:79 (AAT_hIGF2-V43M_wtGAA_del1-69_stuffer.V02); SEQ ID NO:80 (FIB rat_hIGF2-V43M_wtGAA_del1-69_stuffer.V02); SEQ ID NO:81 (FIB hum_hIGF2-V43M_wtGAA_del1-69_stuffer.V02); SEQ ID NO:82 (AAT_GILT_wtGAA_del1-69_stuffer.V02); SEQ ID NO:83 (FIB rat_GILT_wtGAA_del1-69_stuffer.V02); SEQ ID NO:84 (FIB hum_GILT_wtGAA_del1-69_stuffer.V02), or SEQ ID The present invention also includes a nucleic acid sequence selected from any of the nucleic acid sequences having at least 80%, 85%, 90%, 95%, or 98% identity to NOs:57, 58, 59, 60, 61, 62, 79, 80, 81, 82, 83, or 84.

[0053] Another aspect of the technology herein relates to the use of the rAAV compositions and nucleic acid compositions disclosed herein in methods for treating diseases. Specifically, one aspect of the technology herein relates to the use of the rAAV vector compositions and nucleic acid compositions disclosed herein in methods for treating subjects with glycogen storage disease type II (GSD II, Pompe disease, acid maltase deficiency) or subjects with a deficiency of alpha-glucosidase (GAA) polypeptide, the method comprising administering to the subject a recombinant AAV vector or rAAV genome or nucleic acid sequence disclosed herein. In some embodiments of the methods disclosed herein, the expressed GAA polypeptide is secreted from the liver of the subject, and there is uptake of the secreted GAA by skeletal muscle tissue, cardiac muscle tissue, diaphragm muscle tissue, or a combination thereof, wherein the uptake of the secreted GAA results in a decrease in lysosomal glycogen storage in the tissue. In some embodiments of the disclosed methods, the recombinant AAV vector or rAAV genome or nucleic acid sequence is administered to the subject by any suitable administration method, including, but not limited to, intramuscular, subcutaneous, intraspinal, intracisternal, intrathecal, or intravenous administration. In some embodiments, the pharmaceutical compositions disclosed herein can be used in the methods disclosed herein.

[0054] Another aspect of the technology herein relates to cells comprising one or more of the rAAV compositions, rAAV genome compositions, or nucleic acid compositions disclosed herein. In some embodiments, the cells are human cells or non-human mammalian cells or insect cells.

[0055] Another aspect of the technology herein relates to a host animal comprising one or more of the rAAV compositions, rAAV genome compositions, or nucleic acid compositions disclosed herein. In some embodiments, the host animal is a mammal, a non-human mammal, or a human.

[0056] Another aspect of the technology herein relates to a host animal comprising at least one cell comprising one or more of the rAAV compositions, rAAV genome compositions, or nucleic acid compositions disclosed herein. In some embodiments, the host animal comprising such a modified cell is a mammal, a non-human mammal, or a human.

[0057] Aspects of the present invention teach certain advantages in construction and use that result in the following exemplary benefits.

[0058] Disclosed herein, in some embodiments, is a pharmaceutical formulation comprising an rAAV vector disclosed herein, a nucleic acid encoding the rAAV genome, and a pharmaceutically acceptable carrier.

[0059] [The present invention 1001] Within its genome (a) 5' and 3' AAV inverted terminal repeats (ITRs), and (b) a heterologous nucleic acid sequence encoding a fusion polypeptide comprising a secretory signal peptide and an alpha-glucosidase (GAA) polypeptide, the heterologous nucleic acid sequence being located between the 5'ITR and the 3'ITR, the heterologous nucleic acid being operably linked to a promoter; A recombinant adeno-associated virus (AAV) vector comprising: [The present invention 1002] 1001. The recombinant AAV vector of the present invention, wherein said heterologous nucleic acid sequence encoding a fusion polypeptide further comprises an IGF-2 sequence located between said secretory signal peptide and said alpha glucosidase (GAA) polypeptide. [The present invention 1003] The AAV genome, in a 5' to 3' direction, (a) 5'ITR, (b) promoter sequence; (c) intron sequences; (d) a nucleic acid encoding a secretory signal peptide; (e) a nucleic acid encoding an IGF-2 sequence; (f) a nucleic acid encoding an alpha-glucosidase (GAA) polypeptide; (g) a polyA sequence, and (h) 3' ITR 1002. A recombinant AAV vector of the present invention, comprising: [The present invention 1004] The recombinant AAV vector of any of claims 1001 to 1003, wherein the secretory signal peptide is selected from an AAT signal peptide, a fibronectin signal peptide (FN), a GAA signal peptide, or an active fragment thereof having secretory signal activity. [The present invention 1005] The recombinant AAV vector of any of claims 1001 to 1003, wherein the IGF-2 leader sequence binds to the human cation-independent mannose-6-phosphate receptor (CI-MPR) or the IGF-2 receptor. [The present invention 1006] 1005. The recombinant AAV vector of the present invention, wherein said IGF-2 sequence comprises SEQ ID NO:5 or comprises at least one amino modification in SEQ ID NO:5 that binds to said IGF-2 receptor. [The present invention 1007] 1006. The recombinant AAV vector of the present invention, wherein said at least one amino acid modification in SEQ ID NO:5 is a V43M amino acid modification (SEQ ID NO:8 or SEQ ID NO:9) or Δ2-7 (SEQ ID NO:6) or Δ1-7 (SEQ ID NO:7). [The present invention 1008] The recombinant AAV vector of any one of claims 1001 to 1003, wherein the promoter is constitutive, cell-specific, or inducible. [The present invention 1009] The recombinant AAV vector of any of claims 1001 to 1003, wherein the promoter is a liver-specific promoter. [The present invention 1010] 1009. The recombinant AAV vector of the present invention, wherein the liver-specific promoter is selected from the group consisting of a transthyretin promoter (TTR), an LSP promoter (LSP), and a synthetic liver-specific promoter. [The present invention 1011] The recombinant AAV vector of the present invention 1001 or 1002, wherein the nucleic acid sequence encodes a wild-type GAA polypeptide or a modified GAA polypeptide. [The present invention 1012] The recombinant AAV vector of any of claims 1001 to 1003, wherein the nucleic acid sequence encoding the GAA polypeptide is a human GAA gene, a human codon-optimized GAA gene (coGAA), or a modified GAA nucleic acid sequence. [The present invention 1013] The recombinant AAV vector of any of claims 1001 to 1003 or 1012, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized for enhanced expression in vivo. [The present invention 1014] The recombinant AAV vector of any of claims 1001 to 1003 or 1012, wherein the nucleic acid sequence encoding the GAA polypeptide has been codon-optimized to reduce CpG islands. [The present invention 1015] The recombinant AAV vector of any of claims 1001 to 1003 or 1012, wherein the nucleic acid sequence encoding the GAA polypeptide has been codon-optimized to reduce the innate immune response. [The present invention 1016] A recombinant AAV vector of any of claims 1001 to 1003 or 1012, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized to reduce CpG islands and lower the innate immune response. [The present invention 1017] A recombinant AAV vector of any of claims 1001 to 1003, wherein the encoded fusion polypeptide further comprises a spacer comprising a nucleotide sequence for at least one amino acid located on the amino-terminal side of the GAA polypeptide and on the C-terminal side of the IGF-2 sequence. [The present invention 1018] 1003. The recombinant AAV vector of the present invention, further comprising a nucleic acid encoding a spacer of at least one amino acid located between the nucleic acid encoding the IGF-2 sequence and the nucleic acid encoding the GAA polypeptide. [The present invention 1019] The recombinant AAV vector of any of the present inventions 1001 to 1003, further comprising at least one polyA sequence located on the 3' side of the nucleic acid encoding the GAA gene and on the 5' side of the 3' ITR sequence. [The present invention 1020] A recombinant AAV vector of any of claims 1001 to 1003, wherein the heterologous nucleic acid sequence further comprises a collagen stability (CS) sequence located 3' to the nucleic acid encoding the GAA polypeptide and 5' to the 3' ITR sequence. [The present invention 1021] The recombinant AAV vector of the present invention 1003 or 1020, further comprising a nucleic acid encoding a collagen stability (CS) sequence located between the nucleic acid encoding the GAA polypeptide and the polyA sequence. [The present invention 1022] The recombinant AAV vector of any of claims 1001 to 1003, further comprising an intron sequence located on the 5' side of the sequence encoding the secretory signal peptide and on the 3' side of the promoter. [The present invention 1023] 1022. The recombinant AAV vector of the present invention, wherein the intron sequence comprises an MVM sequence or an HBB2 sequence. [The present invention 1024] The recombinant AAV vector of any of claims 1001 to 1003, wherein the ITR sequence comprises an insertion, deletion, or substitution. [The present invention 1025] 1024. The recombinant AAV vector of the present invention, wherein one or more CpG islands within said ITRs have been removed. [The present invention 1026] A recombinant AAV vector of any of the present inventions 1001 to 1003, wherein the secretory signal peptide is a fibronectin signal peptide (FN1) or an active fragment thereof having secretory signal activity, and the IGF-2 sequence is selected from any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. [The present invention 1027] A recombinant AAV vector of any of the present inventions 1001 to 1003, wherein the encoded secretory signal peptide is an AAT signal peptide or an active fragment thereof having secretory signal activity, and the IGF-2 sequence is selected from any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. [The present invention 1028] The recombinant AAV vector of the present invention 1026 or 1027, wherein the IGF-2 sequence is SEQ ID NO:8 or SEQ ID NO:9. [The present invention 1029] The recombinant AAV vector of any one of 1001 to 1003, which is a chimeric AAV vector, a haploid AAV vector, a hybrid AAV vector, or a polyploid AAV vector. [The present invention 1030] The recombinant AAV vector of any of claims 1001 to 1003, comprising a capsid protein selected from any AAV serotype in the group consisting of those listed in Table 1, and any combination thereof. [The present invention 1031] The recombinant AAV vector of the present invention 1030, wherein the serotype is AAV3b. [The present invention 1032] 1031. The recombinant AAV vector of the present invention, wherein the AAV3b serotype comprises one or more mutations in the capsid protein selected from 265D, 549A, and Q263Y. [The present invention 1033] 1031. The recombinant AAV vector of the present invention, wherein the AAV3b serotype is selected from any of AAV3b265D, AAV3b265D549A, AAV3b549A, or AAV3bQ263Y, or AAV3bSASTG. [The present invention 1034] Within its genome (a) 5' and 3' AAV inverted terminal repeats (ITRs), and (b) a heterologous nucleic acid sequence encoding a fusion polypeptide comprising an alpha-glucosidase (GAA) polypeptide, the heterologous nucleic acid sequence being located between the 5'ITR and the 3'ITR, the heterologous nucleic acid being operably linked to a liver-specific promoter; Including, containing the capsid protein of the AAV3b serotype, Recombinant adeno-associated virus (AAV) vectors. [This invention 1035] 1034. The recombinant AAV vector of the present invention, wherein the fusion polypeptide further comprises a secretory signal peptide located N-terminally of the GAA polypeptide. [The present invention 1036] 1034. The recombinant AAV vector of the present invention, wherein the heterologous nucleic acid sequence encoding a fusion polypeptide further comprises an IGF-2 sequence located between the secretory signal peptide and the alpha glucosidase (GAA) polypeptide. [This invention 1037] The AAV genome, in a 5' to 3' direction, (a) 5'ITR, (b) a liver-specific promoter sequence; (c) intron sequences; (d) a nucleic acid encoding a secretory signal peptide; (e) a nucleic acid encoding an IGF-2 sequence; (f) a nucleic acid encoding an alpha-glucosidase (GAA) polypeptide; (g) a polyA sequence, and (h) 3' ITR 1034. The recombinant AAV vector of the present invention, comprising: [The present invention 1038] The recombinant AAV vector of any of the present inventions 1034 to 1037, wherein the secretory signal peptide is selected from the group consisting of AAT signal peptide, fibronectin signal peptide (FN), GAA signal peptide, and active fragments thereof having secretory signal activity. [This invention 1039] The recombinant AAV vector of any of claims 1034 to 1037, wherein the IGF-2 leader sequence binds to the human cation-independent mannose-6-phosphate receptor (CI-MPR) or the IGF-2 receptor. [The present invention 1040] 1039. The recombinant AAV vector of the present invention, wherein the IGF-2 sequence comprises SEQ ID NO:5 or comprises at least one amino modification in SEQ ID NO:5 that affects binding to the IGF-2 receptor. [This invention 1041] 1040. The recombinant AAV vector of the present invention, wherein said at least one amino acid modification in SEQ ID NO:5 is a V43M amino acid modification (SEQ ID NO:8 or SEQ ID NO:9) or Δ2-7 (SEQ ID NO:6) or Δ1-7 (SEQ ID NO:7). [The present invention 1042] The recombinant AAV vector of any one of claims 1034 to 1041, wherein the liver-specific promoter is selected from the group consisting of a transthyretin promoter (TTR), an LSP promoter (LSP), and a synthetic liver-specific promoter. [This invention 1043] The recombinant AAV vector of any of claims 1034 to 1042, wherein the nucleic acid sequence encodes a wild-type GAA polypeptide or a modified GAA polypeptide. [This invention 1044] The recombinant AAV vector of any of claims 1034 to 1043, wherein the nucleic acid sequence encoding the GAA polypeptide is a human GAA gene, a human codon-optimized GAA gene (coGAA), or a modified GAA nucleic acid sequence. [This invention 1045] The recombinant AAV vector of any of claims 1034 to 1044, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized for enhanced in vivo expression. [The present invention 1046] The recombinant AAV vector of any of claims 1034 to 1044, wherein the nucleic acid sequence encoding the GAA polypeptide has been codon-optimized to reduce CpG islands. [This invention 1047] The recombinant AAV vector of any of claims 1034 to 1044, wherein the nucleic acid sequence encoding the GAA polypeptide has been codon-optimized to reduce the innate immune response. [This invention 1048] The recombinant AAV vector of any of claims 1034 to 1044, wherein the nucleic acid sequence encoding the GAA polypeptide has been codon-optimized to reduce CpG islands and lower the innate immune response. [This invention 1049] The recombinant AAV vector of any of claims 1034 to 1049, wherein the intron sequence comprises an MVM sequence or an HBB2 sequence. [The present invention 1050] The recombinant AAV vector of any of claims 1034 to 1049, wherein the ITRs comprise an insertion, deletion, or substitution. [This invention 1051] 1040. The recombinant AAV vector of the present invention, wherein one or more CpG islands within said ITRs have been removed. [This invention 1052] The recombinant AAV vector of any of the present inventions 1034 to 1049, wherein the secretory signal peptide is a fibronectin signal peptide (FN1) or an active fragment thereof having secretory signal activity, and the IGF-2 sequence is selected from any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. [This invention 1053] The recombinant AAV vector of any of the present inventions 1034 to 1049, wherein the encoded secretory signal peptide is an AAT signal peptide or an active fragment thereof having secretory signal activity, and the IGF-2 sequence is selected from any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. [This invention 1054] The recombinant AAV vector of any of claims 1034 to 1049, wherein the IGF-2 sequence is SEQ ID NO:8 or SEQ ID NO:9. [This invention 1055] A pharmaceutical composition comprising any of the recombinant AAV vectors of the present invention and a pharmaceutically acceptable carrier. [This invention 1056] A liver-specific promoter operably linked to a nucleic acid sequence, the nucleic acid sequence comprising, in the following order: a nucleic acid encoding a secretory signal peptide; a nucleic acid encoding an IGF-2 sequence; and a nucleic acid encoding a GAA polypeptide. A nucleic acid sequence comprising: [This invention 1057] (a) 5' and 3' AAV inverted terminal repeat (ITR) nucleic acid sequences, and (b) a heterologous nucleic acid sequence encoding a fusion polypeptide comprising a secretory signal peptide and an alpha-glucosidase (GAA) polypeptide, the heterologous nucleic acid sequence being located between a 5'ITR sequence and a 3'ITR sequence, the heterologous nucleic acid being operably linked to a promoter; A nucleic acid sequence for a recombinant adeno-associated virus (rAAV) vector genome, comprising: [This invention 1058] 1057. The nucleic acid sequence of claim 1057, wherein said heterologous nucleic acid sequence encoding a fusion polypeptide further comprises an IGF-2 sequence located between said secretory signal peptide and said alpha glucosidase (GAA) polypeptide. [This invention 1059] The nucleic acid sequence of the present invention 1056 or 1057, wherein the nucleic acid encoding the secretion signal is selected from any of SEQ ID NOs: 17, 22 to 26, or nucleic acids having at least about 85% sequence identity thereto. [The present invention 1060] 1059. The nucleic acid sequence of the present invention, wherein the nucleic acid encoding the IGF-2 sequence is selected from SEQ ID NO:2 (IGF2-Δ2-7), SEQ ID NO:3 (IGF2-Δ1-7), or SEQ ID NO:4 (IGF2 V43M), or a nucleic acid having at least about 85% sequence identity thereto. [This invention 1061] The nucleic acid sequence of invention 1056 or 1057, wherein the nucleic acid sequence encoding the GAA polypeptide is a human GAA gene or a human codon-optimized GAA gene (coGAA) or a modified GAA nucleic acid sequence. [This invention 1062] The nucleic acid sequence of claim 1056 or 1057, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized for enhanced expression in vivo. [This invention 1063] 1056 or 1057, wherein the nucleic acid sequence encoding the GAA polypeptide has been codon-optimized to reduce CpG islands. [This invention 1064] The nucleic acid sequence of invention 1056 or 1057, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized to reduce the innate immune response. [This invention 1065] The nucleic acid sequence of claim 1056 or 1057, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized to reduce CpG islands and decrease the innate immune response. [The present invention 1066] The nucleic acid sequence of the present invention 1056 or 1057, wherein the nucleic acid encoding the GAA polypeptide is selected from SEQ ID NO:11 (full-length hGAA), SEQ ID NO:55 (Dwight cDNA), and SEQ ID NO:56 (hGAAΔ1-66). [This invention 1067] The nucleic acid sequence of the present invention 1056 or 1057, wherein the nucleic acid encoding the GAA polypeptide is selected from any of SEQ ID NO:74 (codon optimization 1), SEQ ID NO:75 (codon optimization 2), and SEQ ID NO:76 (codon optimization 3). [The present invention 1068] SEQ ID NO:57 (AAT-V43M-wtGAA(delta 1-69aa)); SEQ ID NO:58 (rat FN1-IGF2V43M-wtGAA(delta 1-69aa)); SEQ ID NO:59 (hFN1-IGF2V43M-wtGAA(delta 1-69aa)); SEQ ID NO:60 (ATT-IGF2Δ2-7-wtGAA(delta 1-69)); SEQ ID NO:61 (FN1 rat-IGFΔ2-7-wtGAA(delta 1-69)); SEQ ID NO:62 (hFN1-IGFΔ2-7-wtGAA(delta 1-69)), SEQ ID NO:79 (AAT_hIGF2-V43M_wtGAA_del1-69_stuffer.V02); SEQ ID The nucleic acid sequence of the present invention 1056 or 1057 is selected from any of SEQ ID NO:80 (FIBrat_hIGF2-V43M_wtGAA_del1-69_stuffer.V02); SEQ ID NO:81 (FIBhum_hIGF2-V43M_wtGAA_del1-69_stuffer.V02); SEQ ID NO:82 (AAT_GILT_wtGAA_del1-69_stuffer.V02); SEQ ID NO:83 (FIBrat_GILT_wtGAA_del1-69_stuffer.V02); SEQ ID NO:84 (FIBhum_GILT_wtGAA_del1-69_stuffer.V02), or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity thereto. [The present invention 1069] A method for treating a subject having glycogen storage disease type II (GSD II, Pompe disease, acid maltase deficiency) or a deficiency of alpha-glucosidase (GAA) polypeptide, comprising administering any of the recombinant AAV vectors or rAAV genomes or nucleic acid sequences of the present invention to the subject. [The present invention 1070] The method of claim 1069, wherein a GAA polypeptide is secreted from the liver of the subject and there is uptake of the secreted GAA by skeletal muscle tissue, cardiac muscle tissue, diaphragm muscle tissue, or a combination thereof, wherein the uptake of the secreted GAA results in a reduction in lysosomal glycogen storage in the tissue. [This invention 1071] 1069. The method of claim 1069, wherein the administration to the subject is selected from intramuscular administration, subcutaneous administration, intraspinal administration, intracisternal administration, intrathecal administration, and intravenous administration. Other features and advantages of aspects of the present invention will become apparent from the following more detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of aspects of the present invention. [Brief explanation of the drawings]

[0060] The present application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The accompanying drawings illustrate aspects of the invention. In such drawings: [Figure 1] 1 is a graph illustrating the number of vector genomes per diploid genome on the y-axis and the x-axis of different AAV serotypes, AAV3b, AAV3ST, AAV8, and AAV9, as measured in whole blood, according to at least one embodiment. [Figure 2]1 is a graph illustrating the number of vector genomes per diploid genome on the y-axis and the x-axis of different AAV serotypes, AAV3b, AAV3ST, AAV8, and AAV9, measured in the left, middle, and right lobes of the liver, according to at least one embodiment. [Figure 3] 1 is an illustration of a plasmid map of an adeno-associated virus vector plasmid, according to at least one embodiment. [Figure 4] 1 is an illustration of a plasmid map of the pAAV-LSPhGAA plasmid, according to at least one embodiment. [Figure 5-1]

[0033] Figure 5A illustrates an exemplary nucleic acid construct for a rAAV genome disclosed herein. Figure 5A shows a nucleic acid construct for a rAAV genome including a 5' ITR, a promoter, a nucleic acid encoding a secretory signal peptide (SS), a targeting peptide, and a human GAA polypeptide operably linked thereto, and a 3' ITR. Figure 5A shows a nucleic acid construct for a rAAV genome including a 5' ITR, a promoter, a nucleic acid encoding a secretory signal peptide (SS), a targeting peptide (TP), and a human GAA (hGAA) polypeptide operably linked thereto, and a 3' ITR. Figure 5B shows an exemplary nucleic acid construct for a rAAV genome disclosed herein that includes the same elements as Figure 5A, but additionally includes at least one polyA signal 3' to the hGAA polypeptide and 5' to the -ITR. Figure 5C shows an exemplary nucleic acid construct for a rAAV genome disclosed herein that includes the same elements as Figure 5B, except that it includes an intron sequence 3' to the promoter. Figure 5D shows an exemplary nucleic acid construct for the rAAV genome disclosed herein, which includes the same elements as Figure 5C, except that it includes a collagen stability (CS) sequence located 3' to the hGAA polypeptide nucleic acid sequence and before the polyA sequence. [Figure 5-2]5A-5D illustrate exemplary nucleic acid constructs for the rAAV genome disclosed herein. Figure 5E shows an exemplary nucleic acid construct for the rAAV genome disclosed herein, which includes the same elements as Figure 5D, except that it also includes a nucleic acid encoding a spacer of at least one amino acid located between the nucleic acid encoding the hGAA polypeptide and the nucleic acid encoding a targeting peptide (TP), e.g., an IGF2 sequence. Figure 5F shows an exemplary nucleic acid construct for the rAAV genome disclosed herein, which includes the same elements as Figure 5E, in which the promoter is a liver promoter, the intron sequence is selected from an MVM or HBB2 intron sequence, the secretory signal peptide is selected from an FN1 signal peptide (e.g., hFN1, rat FN1), an AAT signal peptide, or an hGAA signal peptide; the targeting peptide is the IGF2 sequence disclosed herein, and at least the polyA sequence is selected from an hGHpA or synPA polyA sequence. Figure 5G shows an exemplary nucleic acid construct for a rAAV genome disclosed herein, including the same elements as Figure 5F, except that the IGF2 sequence is a nucleic acid sequence selected from SEQ ID NO:2 (IGF2Δ2-7), SEQ ID NO:3 (IGF2Δ1-7), or SEQ ID NO:4 (IGF2 V43M). [Figure 6] An exemplary nucleic acid construct for an rAAV genome is shown, comprising a 5' ITR, a liver-specific promoter, operably linked thereto, an intron sequence (e.g., an MVM or HBB2 intron sequence), a nucleic acid encoding a signal secretory peptide selected from any of the FN1, ATT, or GAA signal peptides, a nucleic acid encoding a human GAA polypeptide, a collagen stability (CS) sequence, at least one polyA sequence (e.g., an hGHpA and / or synPA polyA sequence), and a 3' ITR. [Figure 7]An example of the Gibson cloning method for generating the rAAV genome disclosed herein is shown. Specifically, a triple ligation is performed to ligate together three blocks of nucleic acid sequence, which can then be cloned into a vector together with a promoter, e.g., a liver-specific promoter, and 5' and 3' ITRs to generate the rAAV genome. Gibson cloning methodology was used to generate the following rAAV genomes: SEQ ID NO: 57 (AAT-V43M-wtGAA(delta 1-69 aa)); SEQ ID NO: 58 (rat FN1-IGF2V43M-wtGAA(delta 1-69 aa)); SEQ ID NO: 59 (hFN1-IGF2V43M-wtGAA(delta 1-69 aa)); SEQ ID NO: 60 (ATT-IGF2Δ2-7-wtGAA(delta 1-69)); SEQ ID NO: 61 (FN1 rat-IGFΔ2-7-wtGAA(delta 1-69)); SEQ ID NO: 62 (hFN1-IGFΔ2-7-wtGAA(delta 1-69)). [Figure 8] 8 shows the generation of an exemplary rAAV genome of SEQ ID NO:57 containing AAT-V43M-wtGAA(Delta 1-69aa) using Gibson cloning of nucleic acid sequence blocks (1, 2, and 3). Also shown are the locations of the three amino acid (3aa) spacer nucleic acid sequence (shown as the exemplary 3aa sequence "GAP" as SEQ ID NO:31) located 3' to the nucleic acid sequence encoding the IGF(V42M) targeting peptide and 5' to the nucleic acid encoding the wtGAA(Delta 1-69) enzyme, and the stuffer nucleic acid sequence (referred to as the "spacer" sequence in FIG. 8) located 3' to the polyA sequence and 5' to the 3' ITR sequence in the AAT-V43M-wtGAA(Delta 1-69aa) vector. [Figure 9]9 shows the generation of an exemplary rAAV genome of SEQ ID NO:58 containing rat FN1-IGF2V43M-wtGAA(Delta 1-69aa) using Gibson cloning of nucleic acid sequence blocks (4, 2, and 3). Also shown in the rat FN1-IGF2V43M-wtGAA(Delta 1-69aa) vector are the positions of a three amino acid (3aa) spacer nucleic acid sequence (shown as the exemplary 3aa sequence "GAP" as SEQ ID NO:31) located 3' to the nucleic acid sequence encoding the IGF(V42M) targeting peptide and 5' to the nucleic acid encoding the wtGAA(Delta 1-69) enzyme, and a stuffer nucleic acid sequence (referred to as the "spacer" sequence in FIG. 9) located 3' to the polyA sequence and 5' to the 3' ITR sequence. [Figure 10] 10 shows the generation of an exemplary rAAV genome of SEQ ID NO:59 containing hFN1-IGF2V43M-wtGAA(Delta 1-69aa) using Gibson cloning of nucleic acid sequence blocks (5, 2, and 3). Also shown are the locations of the three amino acid (3aa) spacer nucleic acid sequence (shown as the exemplary 3aa sequence "GAP" as SEQ ID NO:31) located 3' to the nucleic acid sequence encoding the IGF(V42M) targeting peptide and 5' to the nucleic acid encoding the wtGAA(Delta 1-69) enzyme, and the stuffer nucleic acid sequence (referred to as the "spacer" sequence in FIG. 10) located 3' to the polyA sequence and 5' to the 3' ITR sequence in the hFN1-IGF2V43M-wtGAA(Delta 1-69aa) vector. [Figure 11]11 shows the generation of an exemplary rAAV genome of SEQ ID NO:60 containing ATT-IGF2Δ2-7-wtGAA(Delta 1-69) using Gibson cloning of nucleic acid sequence blocks (6, 2, and 3). Also shown in the ATT-IGF2Δ2-7-wtGAA(Delta 1-69) vector are the positions of a three amino acid (3aa) spacer nucleic acid sequence (shown as the exemplary 3aa sequence "GAP" as SEQ ID NO:31) located 3' to the nucleic acid sequence encoding the IGF2Δ2-7 targeting peptide and 5' to the nucleic acid encoding the wtGAA(Δ1-69) enzyme, and a stuffer nucleic acid sequence (referred to as the "spacer" sequence in FIG. 11) located 3' to the polyA sequence and 5' to the 3' ITR sequence. [Figure 12] 12 shows the generation of the rAAV genome of SEQ ID NO:61 containing FN1rat-IGFΔ2-7-wtGAA(Delta 1-69) using Gibson cloning of nucleic acid sequence blocks (7, 2, and 3). Also shown in the FN1rat-IGFΔ2-7-wtGAA(Delta 1-69) vector are the positions of the three amino acid (3aa) spacer nucleic acid sequence (shown as an exemplary 3aa sequence "GAP" as SEQ ID NO:31) located 3' to the nucleic acid sequence encoding the IGFΔ2-7 targeting peptide and 5' to the nucleic acid encoding the wtGAA(Δ1-69) enzyme, and the stuffer nucleic acid sequence (referred to as the "spacer" sequence in FIG. 12) located 3' to the polyA sequence and 5' to the 3' ITR sequence. [Figure 13]13 shows the generation of the rAAV genome of SEQ ID NO:62 containing hFN1-IGFΔ2-7-wtGAA(Delta 1-69) using Gibson cloning of nucleic acid sequence blocks (8, 2, and 3). Also shown in the hFN1-IGFΔ2-7-wtGAA(Delta 1-69) vector are the positions of the three amino acid (3aa) spacer nucleic acid sequence (shown as an exemplary 3aa sequence "GAP" as SEQ ID NO:31) located 3' to the nucleic acid sequence encoding the IGFΔ2-7 targeting peptide and 5' to the nucleic acid encoding the wtGAA(Δ1-69) enzyme, and the stuffer nucleic acid sequence (referred to as the "spacer" sequence in FIG. 13) located 3' to the polyA sequence and 5' to the 3' ITR sequence. [Figure 14A] Figure 14A shows a schematic diagram of an exemplary rAAV genome construct expressing wild-type GAA. Figure 14A shows a schematic diagram of an exemplary rAAV genome construct for candidate 1_AAT_hIGF2-V43M_wtGAA_del1-69_stuffer.V02 (SEQ ID NO:79). [Figure 14B] Figure 14B shows a schematic diagram of an exemplary rAAV genome construct expressing wild-type GAA. Figure 14B shows a schematic diagram of an exemplary rAAV genome construct of candidate 2_FIB rat_hIGF2-V43M_wtGAA_del1-69_stuffer.V02 (SEQ ID NO:80). [Figure 14C] Figure 14C shows a schematic diagram of an exemplary rAAV genome construct expressing wild-type GAA. Figure 14C shows a schematic diagram of an exemplary rAAV genome construct for candidate 3_FIBhum_hIGF2-V43M_wtGAA_del-69_stuffer.V02 (SEQ ID NO:81). [Figure 14D] Figure 14D shows a schematic diagram of an exemplary rAAV genome construct expressing wild-type GAA. Figure 14D shows a schematic diagram of an exemplary rAAV genome construct of candidate 4_AAT_GILT_wtGAA_del1-69_stuffer.V02 (SEQ ID NO:82). [Figure 14E]Figure 14E shows a schematic diagram of an exemplary rAAV genome construct expressing wild-type GAA. Figure 14F shows a schematic diagram of an exemplary rAAV genome construct of candidate 5_FIBrat_GILT_wtGAA_del1-69_stuffer.V02 (SEQ ID NO:83). [Figure 14F] Figure 14F shows a schematic diagram of an exemplary rAAV genome construct expressing wild-type GAA. Figure 14F shows a schematic diagram of an exemplary rAAV genome construct of candidate 6_FIBhum_GILT_wtGAA_del1-69_stuffer.V02 (SEQ ID NO:84). DETAILED DESCRIPTION OF THE INVENTION

[0061] The foregoing drawings illustrate aspects of the present invention in at least one of the exemplary embodiments defined in more detail in the following description. Features, elements, and aspects of the present invention referenced by the same numerals in different drawings represent the same, equivalent, or similar features, elements, or aspects according to one or more embodiments.

[0062] Detailed Description The disclosure described herein generally relates to recombinant AAV (rAAV) vectors and constructs for gene therapy, and the rAAV genome for delivering GAA polypeptide to a subject.Specifically, the technology described herein generally relates to the rAAV vector or rAAV genome for producing GAA polypeptide that is expressed in the liver and effectively targeted to the lysosomes of mammalian cells, such as human cardiac muscle cells and skeletal muscle cells.For example, the technology relates to the rAAV vector for transducing hepatocytes, wherein the transduced hepatocytes secrete GAA polypeptide, and the secreted GAA polypeptide is targeted to the lysosomes in skeletal muscle tissue, cardiac muscle tissue, diaphragm muscle tissue, or a combination thereof.

[0063] Thus, one aspect of the technology described herein provides rAAV vectors containing an rAAV genome that can be used to produce GAA that is more efficiently secreted from cells, e.g., hepatocytes, and then targeted to the lysosomes of mammalian cells, e.g., human cardiac and skeletal muscle cells.

[0064] Specifically, in some embodiments, GAA polypeptide is expressed as fusion protein, comprising at least the signal peptide that promotes the secretion of GAA polypeptide from liver.In some embodiments, GAA polypeptide is expressed as fusion protein, comprising at least the signal peptide that promotes the secretion of GAA polypeptide from liver and the targeting sequence that allows effective targeting to the lysosome of mammalian cell, for example, muscle cell, for example, human cardiac muscle cell and skeletal muscle cell.In some embodiments, targeting peptide is IGF2 sequence as described herein.

[0065] One aspect of the technology described herein relates to an rAAV vector comprising a nucleotide sequence containing an inverted terminal repeat (ITR), a promoter, a heterologous gene, a polyA tail, and optionally other regulatory elements, for use in treating diseases such as Pompe disease, and further for the treatment of Pompe disease, wherein the heterologous gene is GAA, and the rAAV GAA can be administered to a patient at a therapeutically effective dose for delivery to the appropriate tissues and / or organs for expression of the heterologous gene and treatment of the disease.

[0066] One aspect of the technology described herein relates to an rAAV vector, wherein the rAAV vector comprises, in a 5' to 3' direction, 5' and 3' AAV inverted terminal repeat (ITR) sequences within its genome, and a heterologous nucleic acid sequence located between the 5' ITR and the 3' ITR, encoding a fusion polypeptide comprising (i) a secretory signal peptide (SS), (ii) an IGF2 sequence, and (iii) an alpha-glucosidase (GAA) polypeptide, wherein the heterologous nucleic acid is operably linked to a promoter. In some embodiments of the methods and compositions disclosed herein, the secretory signal peptide is selected from the group consisting of an AAT signal peptide, a fibronectin signal peptide (FN1), a GAA signal peptide, or an active fragment thereof having secretory signal activity.

[0067] In some embodiments, the rAAV vectors described herein are derived from any serotype, ie, an AAV3b serotype, including but not limited to, an AAV3b265D virion, an AAV3b265D549A virion, an AAV3b549A virion, an AAV3bQ263Y virion, or an AAV3bSASTG virion (i.e., a virion comprising an AAV3b capsid containing the Q263A / T265 mutation).

[0068] An aspect of the present technology relates to the use of the rAAV vector described herein in a method for treating a deficiency of GAA polypeptide in a subject, comprising administering to the subject a therapeutically effective amount of the rAAV vector disclosed herein contained in a pharmaceutically acceptable carrier. In some embodiments, the rAAV vector is for use in the treatment or prevention of Pompe disease (also known as glycogen storage disease type 2 or GSD II). In some embodiments, the subject is a mammal, and the mammal is a human, a primate, a dog, a horse, a cow, or a cat.

[0069] In some embodiments, the rAAV vector comprises a nucleic acid encoding a GAA polypeptide, which comprises at least an N-terminal secretory signal peptide, wherein the hepatocytes transduced by the rAAV vector express the GAA polypeptide and the N-terminal secretory peptide and secrete the GAA polypeptide.In addition, the secreted GAA polypeptide can optionally comprise a targeting sequence, such as an IGF2 sequence, attached to the N-terminus or C-terminus of the GAA polypeptide, to enhance the uptake and targeting of the GAA polypeptide to lysosomes in skeletal muscle tissue, cardiac muscle tissue, diaphragm muscle tissue, nerve cells innervating muscle tissue, or a combination thereof.In addition, in one embodiment, the uptake of the secreted GAA polypeptide into muscle cells leads to a reduction in lysosomal glycogen storage in tissue, and a reduction or elimination of symptoms associated with Pompe disease.

[0070] In one embodiment, the rAAV vector comprises a capsid, and within the capsid is a nucleotide sequence referred to herein as the "rAAV vector genome." The rAAV vector genome comprises two inverted terminal repeats (ITRs, e.g., 5'-ITR and 3'-ITR), and multiple elements located between the ITRs, including, but not limited to, additional elements including a promoter, a heterologous gene, and a polyA tail. In a further embodiment, additional elements, such as a seed region sequence for binding of an miRNA or shRNA sequence, may be present between the ITRs.

[0071] I. Definition The following terminology is used in the description and appended claims.

[0072] The terms "a," "an," "the," and similar references used in describing the present invention (particularly with respect to the claims that follow) should be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. Furthermore, ordinal terms such as "first," "second," "third," etc., used with respect to identified elements are used to distinguish elements and do not denote or imply a required or limited number of such elements, nor do they denote a specific location or order of such elements unless otherwise specifically stated. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to facilitate understanding of the invention and does not limit the scope of the invention as set forth in the claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0073] Furthermore, the term "about," as used herein, when referring to a measurable value such as the length of a polynucleotide or polypeptide sequence, amount of dosage, time, temperature, etc., is intended to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0074] Also, as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination ("or") when interpreted in the alternative.

[0075] As used herein, the transitional phrase "consisting essentially of" means that the scope of a claim should be construed to include the specified materials or steps recited in the claim and "which do not materially affect the basic and novel characteristics" of the claimed invention. See In re Herz, 537 F.2d 549,551-52,190 USPQ 461,463 (CCPA 1976) (emphasis in original); see also MPEP § 2111.03. Thus, the term "consisting essentially of," when used in the claims of the invention, should not be construed as equivalent to "comprising." In particular, it is intended that the various features of the invention described herein may be used in any combination unless the context indicates otherwise.

[0076] Furthermore, the present invention also contemplates that in some aspects of the invention, any feature or combination of features set forth herein may be excluded or omitted.

[0077] To further illustrate, for example, if the specification indicates that a particular amino acid may be selected from A, G, I, L, and / or V, this language also indicates that an amino acid may be selected from any subset of these amino acids, e.g., A, G, I, or L; A, G, I, or V; A or G; L only; etc., as if each such subcombination were specifically set forth herein. Furthermore, such language also indicates that one or more of the specified amino acids may be discarded (e.g., by negative qualification). For example, in specific embodiments, amino acids are discarded as if each possible discard, such as not A, G, or I; not A; not G or V; etc., were specifically set forth herein.

[0078] The term "parvovirus," as used herein, encompasses the Parvoviridae family, which includes autonomously replicating parvoviruses and dependoviruses. Autonomous parvoviruses include members of the Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Contravirus genera. Exemplary autonomous parvoviruses include, but are not limited to, minute virus of mice, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, H1 parvovirus, Muscovy duck parvovirus, B19 virus, and other autonomous parvoviruses now known or later discovered. Other autonomous parvoviruses will be known to those skilled in the art. See, for example, Bernard N. Fields et al., Virology, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers).

[0079] As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV types 1, 2, 3 (including 3A and 3B), 4, 5, 6, 7, 8, 9, 10, 11, avian, bovine, canine, equine, ovine, and other AAVs now known or later discovered. See, e.g., Bernard N. Fields et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). A number of relatively new AAV serotypes and clades have been identified (see, e.g., Gao et al., (2004) J. Virology 78:6381-6388; Morris et al., (2004) Virology 33:375-383; and Table 1).

[0080] The genome sequences of various serotypes of AAV and autonomous parvoviruses, as well as the sequences of native inverted terminal repeats (ITRs), Rep protein and capsid subunits, are known in the art.Such sequences can be found in literature or public databases such as GenBank.For example, GenBank accession numbers NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U89790, AF043303, AF028705, AF028704, J0227 5, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC_001358, NC_001540, AF513851, AF513852, AY530579 (the disclosures of which are incorporated by reference herein as they teach the nucleic acid and amino acid sequences of parvoviruses and AAV). For example, Srivistava et al.,(1983)J Virology 45:555;Chiarini et al.,(1998)J.Virology 71:6823;Chiarini et al.,(1999)J.Virology 73:1309;Bantel-Schaal et al.,(1999)J.Virology 73:939;Xiao et al. al.,(1999)J.Virology 73:3994;Muramatsu et al.,(1996)Virology 221:208;Shade et al.,(1986)J.Viral.58:921;Gao et al.,(2002)Proc.Nat.Acad.Sci.USA 99:11854;Morris et al. al.,(2004) Virology 33-:375-383; International Patent Publications WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and U.S. Patent No. 6,156,303 (the disclosures of which are incorporated herein by reference as they teach the nucleic acid and amino acid sequences of parvoviruses and AAV).See also Tables 1 and 5 disclosed herein.

[0081] The capsid structure of autonomous parvoviruses and AAV is described in more detail in Bernard N. Fields et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). See also the descriptions of the crystal structures of AAV2 (Xie et al., (2002) Proc. Nat. Acad. Sci. 99:10405-10), AAV4 (Padron et al., (2005) J. Viral. 79:5047-58), AAV5 (Walters et al., (2004) J. Viral. 78:3361-71), and CPV (Xie et al., (1996) J. Mal. Biol. 6:497-520 and Tsao et al., (1991) Science 251:1456-64).

[0082] The term "tropism," as used herein, refers to the preferential invasion of a virus into a particular cell or tissue, optionally followed by expression (e.g., transcription and, optionally, translation) of sequences carried by the viral genome in the cell, e.g., in the case of a recombinant virus, expression of a heterologous nucleic acid of interest.

[0083] As used herein, "systemic tropism" and "systemic transduction" (and equivalent terms) indicate that the viral capsids or viral vectors of the invention exhibit tropism for and / or transduce tissues throughout the body (e.g., brain, lung, skeletal muscle, heart, liver, kidney, and / or pancreas). In embodiments of the invention, systemic transduction of the central nervous system (e.g., brain, neuronal cells, etc.) is observed. In other embodiments, systemic transduction of cardiac muscle tissue is achieved.

[0084] As used herein, "selective tropism" or "specific tropism" refers to the delivery of a viral vector to, and / or the specific transduction of, specific target cells and / or specific tissues.

[0085] In some embodiments of the present invention, AAV particles comprising capsids of the present invention may exhibit multiple phenotypes: efficient transduction of 30 specific tissues / cells, and very low levels of transduction (e.g., reduced transduction) of specific tissues / cells where transduction is not desirable.

[0086] As used herein, unless otherwise indicated, the term "polypeptide" includes both peptides and proteins.

[0087] A "polynucleotide" is a sequence of nucleotide bases and may be an RNA, DNA, or DNA-RNA hybrid sequence (containing both naturally occurring and non-naturally occurring nucleotides), but in typical embodiments is either a single-stranded or double-stranded DNA sequence.

[0088] A "chimeric nucleic acid" comprises two or more nucleic acid sequences covalently linked together to encode a fusion polypeptide. The nucleic acids can be DNA, RNA, or a hybrid thereof.

[0089] The term "fusion polypeptide" includes two or more polypeptides covalently linked together, typically by peptide bonds.

[0090] As used herein, an "isolated" polynucleotide (e.g., "isolated DNA" or "isolated RNA") means a polynucleotide that is at least partially separated from other components of the naturally occurring organism or virus, e.g., at least a portion of a cellular or viral structural component or other polypeptide or nucleic acid that is typically found in association with the polynucleotide. In representative embodiments, the "isolated" nucleotide is enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more, compared to the starting material.

[0091] Similarly, an "isolated" polypeptide refers to a polypeptide that is at least partially separated from other components of the naturally occurring organism or virus, e.g., at least a portion of cellular or viral structural components or other polypeptides or nucleic acids that are typically found in association with the polypeptide. In representative embodiments, an "isolated" polypeptide is enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more, compared to the starting material.

[0092] An "isolated cell" refers to a cell that has been separated from other components with which it is normally associated in its natural state. For example, an isolated cell can be a cell in culture medium and / or a cell in a pharmaceutically acceptable carrier of the present invention. Thus, the isolated cell can be delivered and / or introduced into a subject. In some embodiments, the isolated cell can be a cell that has been removed from a subject, manipulated ex vivo as described herein, and then returned to the subject.

[0093] As used herein, "isolating" or "purifying" (or grammatical equivalents) a viral vector or viral particle or population of viral particles means that the viral vector or viral particle or population of viral particles is at least partially separated from at least some of the other components in the starting material. In representative embodiments, the "isolated" or "purified" viral vector or viral particle or population of viral particles is at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more enriched compared to the starting material.

[0094] Unless otherwise indicated, "efficient transduction" or "efficient tropism" or similar terms may be determined relative to an appropriate control (e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 500%, or more of the transduction or tropism of the control, respectively). In specific embodiments, the viral vector efficiently transduces or has efficient tropism for neuronal cells and cardiomyocytes. An appropriate control will depend on a variety of factors, including the desired tropism and / or transduction profile.

[0095] A "therapeutic polypeptide" is a polypeptide that alleviates, reduces, prevents, delays, and / or stabilizes symptoms resulting from a lack or defect in a protein in a cell or subject, and / or confers some other benefit to the subject, e.g., enzyme replacement to reduce or eliminate disease symptoms, or improved graft viability, or induction of an immune response.

[0096] The terms "treat," "treating," or "treatment of" (and grammatical variations thereof) mean that the severity of a subject's condition is reduced, at least partially improved, or stabilized, and / or some alleviation, reduction, decrease, or stabilization of at least one clinical symptom is achieved, and / or a delay in the progression of a disease or disorder occurs.

[0097] The terms "prevent," "preventing," and "prevention" (and grammatical variations thereof) refer to preventing and / or delaying the onset of a disease, disorder, and / or clinical symptom in a subject and / or reducing the severity of the onset of a disease, disorder, and / or clinical symptom compared to that which would occur in the absence of the methods of the present invention. Prevention can be complete, e.g., the total absence of a disease, disorder, and / or clinical symptom. Prevention can also be partial, such that the occurrence of a disease, disorder, and / or clinical symptom in a subject is substantially less than that which would occur in the absence of the present invention.

[0098] As used herein, " effective amount for treatment " is an amount sufficient to provide some improvement or benefit to the subject.In other words, " effective amount for treatment " is an amount that provides some relief, alleviation, reduction or stabilization of at least one clinical symptom in the subject.Those skilled in the art will recognize that the therapeutic effect may not be complete or curative, as long as some benefit is provided to the subject.

[0099] A "prophylactically effective" amount, as used herein, is an amount sufficient to prevent and / or delay the onset of a disease, disorder, and / or clinical symptom in a subject and / or to reduce the severity and / or delay the onset of a disease, disorder, and / or clinical symptom in a subject compared to what would occur in the absence of the method of the present invention. One of skill in the art will recognize that the level of prevention need not be complete, as long as some prophylactic benefit is provided to the subject.

[0100] The terms "heterologous nucleotide sequence" and "heterologous nucleic acid molecule" are used interchangeably herein to refer to a nucleic acid sequence that does not naturally occur in a virus. Generally, a heterologous nucleic acid molecule or heterologous nucleotide sequence comprises an open reading frame encoding a polypeptide and / or untranslated RNA of interest (e.g., for delivery to a cell and / or subject), such as a GAA polypeptide.

[0101] As used herein, the terms "viral vector," "vector," or "gene delivery vector" refer to a viral (e.g., AAV) particle that functions as a nucleic acid delivery vehicle and contains a vector genome (e.g., viral DNA [vDNA]) packaged within the virion. Alternatively, in some circumstances, the term "vector" may be used to refer to the vector genome / vDNA alone.

[0102] A "rAAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA) containing one or more heterologous nucleic acid sequences. rAAV vectors generally require only inverted terminal repeats (TRs) in cis to generate virus. All other viral sequences are non-essential and can be supplied in trans (Muzyczka, (1992) Curr. Topics Microbial. Immunol. 158:97). Typically, rAAV vector genomes will retain only one or more TR sequences to maximize the size of the transgene that can be efficiently packaged by the vector. Sequences encoding structural and nonstructural proteins can be supplied in trans (e.g., from a vector such as a plasmid or by stable integration of the sequences into the packaging cell). In embodiments of the present invention, the rAAV vector genome comprises at least one ITR sequence (e.g., an AAV TR sequence), and optionally two ITRs (e.g., two AAV TRs), which are typically present at the 5' and 3' ends of the vector genome and are adjacent to, but not necessarily contiguous with, the heterologous nucleic acid. The TRs may be identical to one another or different.

[0103] The term "terminal repeat" or "TR" includes any viral or synthetic terminal repeat sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., an ITR that mediates a desired function, such as replication, viral packaging, integration, and / or proviral rescue). TRs can be AAV or non-AAV TRs. For example, non-AAV TR sequences, such as those of other parvoviruses (e.g., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19), or other suitable viral sequences (e.g., the SV40 hairpin that serves as the SV40 origin of replication), can be used as TRs, which may be further modified by truncation, substitution, deletion, insertion, and / or addition. Furthermore, TRs may be partially or completely synthetic, such as the "double D sequence" described in U.S. Patent No. 5,478,745 to Samulski et al.

[0104] "AAV terminal repeats" or "AAV TRs," e.g., "AAV inverted terminal sequences" or "AAV ITRs," can be derived from any AAV, including, but not limited to, serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or any other AAV now known or later discovered (see, e.g., Table 3). The AAV terminal repeats do not have the native terminal repeat sequence (e.g., the native AAV TR or AAV ITR sequence may be altered by insertion, deletion, truncation, and / or missense mutation), so long as they mediate the desired function, such as replication, viral packaging, integration, and / or proviral rescue.

[0105] AAV proteins VP1, VP2, and VP3 are capsid proteins that interact together to form the icosahedral symmetric AAV capsid. VP1.5 is an AAV capsid protein described in U.S. Patent Application Publication No. 2014 / 0037585.

[0106] The viral vectors of the present invention may further be "target-specific" viral vectors (e.g., with a directed tropism) and / or "hybrid" parvoviruses (i.e., in which the viral TR and viral capsid are derived from different parvoviruses) as described in International Patent Publication WO 00 / 28004 and Chao et al., (2000) Molecular Therapy 2:619.

[0107] The viral vectors of the present invention may further be double-stranded parvovirus particles as described in International Patent Publication WO 01 / 92551, the disclosure of which is incorporated herein by reference in its entirety. Thus, in some embodiments, a double-stranded (duplex) genome may be packaged into the viral capsids of the present invention.

[0108] Additionally, the viral capsid or genomic elements may contain other modifications, including insertions, deletions, and / or substitutions.

[0109] As used herein, a "chimeric" capsid protein refers to an AAV capsid protein that has been modified by the substitution of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues within the amino acid sequence of the capsid protein relative to the wild-type, as well as an AAV capsid protein that has been modified by the insertion and / or deletion of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues within the amino acid sequence relative to the wild-type. In some embodiments, complete or partial domains, functional regions, epitopes, etc. from one AAV serotype can be exchanged for corresponding wild-type domains, functional regions, epitopes, etc. from a different AAV serotype in any combination to create a chimeric capsid protein of the present invention. The production of chimeric capsid proteins can be carried out according to protocols well known in the art, and a number of chimeric capsid proteins that can be included in the capsids of the invention have been described in the literature and herein.

[0110] As used herein, the term "haploid AAV" is intended to mean an AAV as described in PCT / US18 / 22725, which is incorporated herein.

[0111] The term "hybrid" AAV vector or parvovirus refers to an rAAV vector in which the viral TRs or ITRs and viral capsid are derived from different parvoviruses. Hybrid vectors are described in International Patent Publication WO 00 / 28004 and Chao et al., (2000) Molecular Therapy 2:619. For example, hybrid AAV vectors typically contain sufficient adenovirus 5' and 3' cis-ITR sequences (i.e., adenovirus terminal repeat and PAC sequences) for adenovirus replication and packaging.

[0112] The term "polyploid AAV" refers to an AAV vector that is composed of capsids derived from, for example, two or more AAV serotypes and can utilize individual serotypes for higher transduction potential, while in certain embodiments eliminating parental tropism.

[0113] The term "GAA" or "GAA polypeptide," as used herein, encompasses mature GAA (approximately 76 or approximately 67 kDa) and precursor GAA (e.g., approximately 110 kDa), modified (e.g., truncated or mutated by insertion, deletion, and / or substitution) GAA proteins or fragments thereof that retain biological function (i.e., have at least one biological activity of the native GAA protein as defined above, e.g., are capable of hydrolyzing glycogen), as well as GAA variants (e.g., GAA II as described by Kunita et al., (1997) Biochemica et Biophysica Acta 1362:269; GAA polymorphisms and SNPs are described in Hirschhorn, R. and Reuser, AJ. (2001) in The Metabolic and Molecular Basis for Inherited Disease (Scriver, CR, Beaudet, AL, Sly, WS & Also encompassed are those described by GAA in "Genetical Sequences of GAA Genes," by G. Valle, D. Eds., pp. 3389-3419, McGraw-Hill, New York (see pages 3403-3405). Any GAA coding sequence known in the art can be used, see, for example, the coding sequence of Figures 8 and 9; GenBank Accession No. NM_00152 and Hoefsloot et al., (1988) EMBO J. 7:1697 and Van Hove et al., (1996) Proc. Natl. Acad. Sci. USA 93:65 (human), GenBank Accession No. NM_008064 (mouse), and Kunita et al., (1997) Biochemica et Biophysics Acta 1362:269 (quail), the disclosures of which are incorporated herein by reference for their teaching of the coding and non-coding sequences of GAA.

[0114] The term "targeting peptide," also referred to as a "targeting sequence," as used herein, refers to a peptide that targets a specific intracellular compartment, e.g., mammalian lysosomes. Targeting peptides encompassed for use in the present invention are mannose-6-phosphate-independent lysosomal targeting peptides.

[0115] The terms "IGF2 sequence" or "IGF-2 sequence," used in conjunction with "IGF2 leader sequence" and "IGF-2 leader sequence," are used interchangeably herein and refer to a sequence of an IGF2 polypeptide that binds to CI-MBR on the surface of a cell. Specifically, an IGF2 sequence is a peptide that includes a portion of the IGF-2 uptake sequence of SEQ ID NO:5 or includes modifications of the amino acids of SEQ ID NO:5. An IGF2 sequence refers to a peptide sequence that binds to a receptor domain consisting essentially of repeats 11-12, repeat 11, or amino acids 1508-1566 of the human cation-independent mannose-6-phosphate receptor (CI-MPR or CA-M6P receptor).

[0116] The terms "secretory signal sequence" or "signal sequence," and variations thereof, are used interchangeably herein to refer to an amino acid sequence that functions to enhance (as defined above) the secretion of an operably linked polypeptide, e.g., a GAA or GAA fusion protein, from a cell compared to the level of secretion seen with the native polypeptide. As defined above, "enhanced" secretion means that the relative proportion of lysosomal polypeptides synthesized by the cell that are secreted from the cell is increased; the absolute amount of protein secreted need not be increased either. In specific embodiments of the invention, essentially all (i.e., at least 95%, 97%, 98%, 99%, or more) of the GAA polypeptide is secreted. However, essentially all, or even most, of the GAA polypeptide need not be secreted, so long as the level of secretion is enhanced compared to the native GAA polypeptide.

[0117] As used herein, the term "amino acid" includes any naturally occurring amino acid, modified forms thereof, and synthetic amino acids.

[0118] Additional patents incorporated herein by reference that relate to, disclose, or describe AAV, or aspects of AAV, such as DNA vectors containing a gene of interest to be expressed, are U.S. Patent Nos. 6,491,907; 7,229,823; 7,790,154; 7,201898; 7,071,172; 7,892,809; 7,867,484; 8,889,641; 9,169,494; 9,169,492; 9,441,206; 9,409,953; and 9,447,433; 9,592,247; and 9,737,618.

[0119] II. rAAV Genomic Elements As disclosed herein, one aspect of the present technology relates to an rAAV vector comprising a capsid and a nucleotide sequence within the capsid referred to as the "rAAV vector genome." The rAAV vector genome (also referred to as the "rAAV genome") comprises two inverted terminal repeats (ITRs, e.g., 5'-ITR and 3'-ITR), as well as multiple elements located between the ITRs, including, but not limited to, a promoter, a heterologous gene, and a polyA tail.

[0120] In some embodiments, the rAAV genome disclosed herein comprises 5' ITR and 3' ITR sequences, and a promoter, e.g., a liver-specific promoter sequence, operably linked to a heterologous nucleic acid encoding a nucleic acid encoding an alpha glucosidase (GAA) polypeptide, located between the 5' ITR and 3' ITR, wherein the heterologous nucleic acid sequence may further comprise one or more of the following elements: an intron sequence, a nucleic acid encoding a secretory signal peptide, a nucleic acid encoding an IGF2 sequence, and a polyA sequence.

[0121] In some embodiments, the rAAV genome disclosed herein comprises 5' ITR and 3' ITR sequences, and a promoter operably linked to a heterologous nucleic acid encoding a secretory peptide and a nucleic acid encoding an alpha glucosidase (GAA) polypeptide (i.e., the heterologous nucleic acid encodes a GAA fusion polypeptide comprising a signal peptide-GAA polypeptide) located between the 5' ITR and 3' ITR, wherein the rAAV genome optionally further comprises one or more of a nucleic acid encoding an intron sequence, a collagen stability (CS) sequence, a polyA tail, and a spacer of at least one amino acid. In some embodiments, the rAAV genome disclosed herein comprises 5'ITR and 3'ITR sequences, and a liver promoter operably linked to a heterologous nucleic acid encoding a secretory peptide (e.g., an FIV, ATT, or GAA signal peptide) and a nucleic acid encoding an alpha glucosidase (GAA) polypeptide located between the 5'ITR and 3'ITR, wherein the rAAV genome optionally further comprises one or more of an intron sequence (e.g., an MVM or HBB2 intron sequence), a collagen stability (CS) sequence, a polyA tail, and a nucleic acid encoding a spacer of at least one amino acid.

[0122] In some embodiments, the rAAV genome disclosed herein comprises 5'ITR and 3'ITR sequences, and a promoter operably linked to a heterologous nucleic acid located between the 5'ITR and 3'ITR, encoding a secretory peptide, a targeting peptide, and a GAA polypeptide (i.e., the heterologous nucleic acid encodes a GAA fusion polypeptide comprising a signal peptide-targeting sequence-GAA polypeptide), wherein the targeting peptide is an IGF2 sequence described herein, and the rAAV genome optionally further comprises one or more of an intron sequence, a collagen stability (CS) sequence, a polyA tail, and a nucleic acid encoding a spacer of at least one amino acid.

[0123] Each of the elements within the rAAV genome is described herein.

[0124] A. Alpha-glucosidase (GAA) polypeptide The alpha-glucosidase (GAA) polypeptide is a member of the glycosidase family 31. Human GAA is synthesized as a 110 kDa precursor (Wisselaar et al. (1993) J. Biol. Chem. 268(3):2223-31). The mature form of the enzyme is a mixture of 70 and 76 kDa monomers (Wisselaar et al. (1993) J. Biol. Chem. 268(3):2223-31). The precursor enzyme has seven potential glycosylation sites, four of which are retained in the mature enzyme (Wisselaar et al. (1993) J. Biol. Chem. 268(3):2223-31). The proteolytic cleavage events that produce the mature enzyme occur in late endosomes or lysosomes (Wisselaar et al. (1993) J. Biol. Chem. 268(3):2223-31).

[0125] The rAAV vector genome can encode a GAA polypeptide that can include, for example, amino acid residues 40-952 or 70-952 of human GAA, or a smaller portion such as amino acid residues 40-790 or 70-790.

[0126] In one embodiment, the IGF2 sequence is fused to amino acid 40, or amino acid 70, or an amino acid at one or two positions of amino acid 40 or 70. In some embodiments, the IGF2 sequence is a ligand for an extracellular receptor, e.g., the IGF2 sequence binds to the human cation-independent mannose-6-phosphate receptor (CI-MPR) or the IGF2 receptor.

[0127] The first 27 amino acids of the human GAA polypeptide are typical of signal peptides of lysosomal and secretory proteins. GAA can be targeted to lysosomes via phosphomannosyl receptors and / or by sequences associated with delayed cleavage of the signal peptide (Hirschhorn, R. and Reuser, AJ (2001), in *The Metabolic and Molecular Basis for Inherited Disease*, (eds., Scriver, CR et al.) pages 3389-3419 (McGraw-Hill, New York)). A membrane-bound (i.e., anchored by an uncleaved signal peptide) precursor form of the enzyme has been identified in the lumen of the endoplasmic reticulum (see, e.g., Wisselaar et al., (1993) J. Biol. Chem. 268:2223-31).

[0128] The C-terminal 160 amino acids are absent from the 70- and 76-kDa mature GAA polypeptide species. However, certain Pompe alleles, e.g., Val949Asp, that result in a complete loss of GAA activity have been mapped to this region (Becker et al. (1998) J. Hum. Genet. 62:991). The phenotype of this mutant indicates that the C-terminal portion of the protein, although not included in the 70- or 76-kDa species, plays an important role in protein function. It has also been reported that the C-terminal portion of the protein is cleaved from the rest of the protein during processing but remains associated with the major species (Moreland et al. (Nov. 1, 2004) J. Biol. Chem., Manuscript 404008200). Thus, the C-terminal residues may play a direct role in protein catalysis and / or may be involved in promoting proper folding of the N-terminal portion of the protein.

[0129] The native GAA gene encodes a precursor polypeptide possessing a signal sequence and adjacent putative transmembrane domain, a trefoil domain (PFAM PF00088) (Thim (1989) FEBS Lett. 250:85), a cysteine-rich domain of approximately 45 amino acids containing three disulfide bonds, a domain defined by the mature 70 / 76 kDa polypeptide, and a C-terminal domain. It has been reported that both the trefoil domain and the C-terminal domain are required for the production of functional GAA, and that the C-terminal domain may interact with the trefoil domain during protein folding, possibly facilitating proper disulfide bond formation in the trefoil domain.

[0130] GAA polypeptides are described in U.S. Patents 5,962,313 and 6,537,785, which are incorporated herein by reference in their entireties. Those skilled in the art will recognize specific positions in GAA to which a secretory signal peptide (SS) or a targeting peptide (e.g., an IGF2 sequence) can be fused. Thus, in one aspect, the present invention relates to GAA fusion proteins in which an SP or IGF2 sequence is fused to amino acids 40, 68, 69, 70, 71, 72, 779, 787, 789, 790, 791, 792, 793, or 796 of human GAA or a portion thereof.

[0131] In some embodiments of the methods and compositions disclosed herein, the human GAA protein expressed by AAV comprises the amino acids of SEQ ID NO:10, or a fragment thereof, such as a human GAA protein starting at residue 40, 68, 69, 70, 71, 72, 779, 787, 789, 790, 791, 792, 793, or 796 of SEQ ID NO:10. In some embodiments of the methods and compositions disclosed herein, the human GAA protein expressed by AAV comprises the amino acids of SEQ ID NO:10, or a protein that is at least 60%, or 70%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99% identical to SEQ ID NO:10. In some embodiments of the methods and compositions disclosed herein, the human GAA protein expressed by AAV comprises amino acids of a human GAA protein starting at residue 40, 68, 69, 70, 71, 72, 779, 787, 789, 790, 791, 792, 793, or 796 of SEQ ID NO:10, or a protein at least 60%, or 70%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99% identical thereto.

[0132] In some embodiments, one skilled in the art will recognize specific positions in GAA to which a secretory signal peptide (SS) or a targeting peptide (e.g., an IGF2 sequence) can be fused. For example, International Patent Application WO2018046774A1, the entire contents of which are incorporated herein, discloses truncated GAA polypeptides to which a secretory signal peptide (SS) or a targeting peptide (e.g., an IGF2 sequence) can be attached, and discloses the following truncated GAA polypeptide variants: Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ14, Δ15, Δ16, Δ17, Δ18, Δ19, Δ20, Δ21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42, Δ43, Δ44, Δ45, Δ46, Δ47, Δ48, Δ49, Δ50, Δ51, Δ52, Δ53, Δ54, Δ55, Δ56, Δ57, Δ58, Δ59, Δ60, Δ61, Δ62, Δ63, Δ64, Δ65, Δ66, Δ67, Δ68, Δ69, Δ70, Δ71, Δ72, Δ73, Δ74, Δ75, Δ76, Δ77, Δ7 21, Δ22, Δ23, Δ24, Δ25, Δ26, Δ27, Δ28, Δ29, Δ30, Δ31, Δ32, Δ33, Δ34, Δ35, Δ36, Δ37, Δ38, Δ39, Δ40, Δ41, Δ42, Δ43, Δ44, Δ45, Δ46, Δ47, Δ48 , Δ49, Δ50, Δ51, Δ52, Δ53, Δ54, Δ55, Δ56, Δ57, Δ58, Δ59, Δ60, Δ61, Δ62, Δ63, Δ64, Δ65, Δ66, Δ67, Δ68, Δ69, Δ70, Δ71, Δ72, Δ73, Δ74, or Δ75 GAA shortened version.

[0133] In some embodiments, the GAA fusion polypeptides encoded by the rAAV genomes described herein can include a smaller portion, such as, for example, amino acid residues 40-952 or residues 70-952 of human GAA, or amino acid residues 40-790 or 70-790. In one embodiment, a secretory signal peptide (SS) or targeting peptide, e.g., an IGF2 sequence, is fused to amino acid 40, or amino acid 70, or amino acids at one or two positions of amino acid 40 or 70.

[0134] In some embodiments, a fusion protein comprising a secretory signal peptide (SS), a GAA polypeptide, and optionally an IGF2 sequence (i.e., an SS-GAA fusion polypeptide or an SS-IGF2-GAA fusion protein) comprises amino acid residues 40-952 or residues 70-952 of human acid alpha-glucosidase (GAA) (SEQ ID NO:10). In some embodiments, the N-terminus of the GAA polypeptide is attached to the C-terminus of the SS, and in some embodiments, the N-terminus of the GAA polypeptide is attached to the C-terminus of the IGF2 sequence, and the N-terminus of the IGF2 sequence is attached to the C-terminus of the secretory signal peptide.

[0135] In one embodiment, the rAAV genome comprises a heterologous nucleic acid sequence encoding a secretory signal peptide or an IGF2 sequence fused in frame to the 3' end of a GAA nucleic acid sequence encoding the entire GAA polypeptide (e.g., the N-terminal / catalytic domain and the C-terminal domain). For example, a heterologous nucleic acid sequence encoding a secretory signal peptide or an IGF2 sequence is fused in frame to the 3' end of a GAA nucleic acid sequence encoding the 70 kDa and 76 kDa GAA polypeptides, and both such polypeptides are expressed from the rAAV genome when the rAAV vector transduces a mammalian cell. In some embodiments, expression of the GAA nucleic acid can be driven by two promoters within the rAAV genome, or by one promoter driving expression of a bicistronic construct.

[0136] In some embodiments of the methods and compositions disclosed herein, the rAAV vector comprises a nucleic acid sequence encoding a GAA protein that is a wild-type GAA nucleic acid sequence, e.g., SEQ ID NO: 11 or SEQ ID NO: 72. In some embodiments of the methods and compositions disclosed herein, the rAAV vector comprises a nucleic acid sequence encoding a GAA protein that is a GAA nucleic acid sequence that has been codon-optimized for enhanced expression in vivo, and / or to reduce CpG islands, and / or to reduce the innate immune response. Exemplary codon-optimized GAA nucleic acid sequences encompassed for use in the methods and rAAV compositions disclosed herein can be selected from any of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76, or a nucleic acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76.

[0137] The C-terminal domain of GAA functions in trans with the 70 / 76 kDa species to generate active GAA. The boundary between the catalytic and C-terminal domains is absent in most members of family 31 hydrolases and appears to be at approximately amino acid residue 791 in GAA, based on its presence in a short region of less than 18 amino acids containing four consecutive proline residues. The C-terminal domain associated with the mature species has been reported to begin at amino acid residue 792 (Moreland et al. (Nov. 1, 2004) J. Biol. Chem., Manuscript 404008200). Thus, in some embodiments, the GAA nucleic acid sequence encodes the entire GAA polypeptide, excluding the C-terminal domain. Thus, in such embodiments, rAAV vectors can be used to transduce mammalian cells that express the C-terminal domain of GAA as a separate polypeptide.

[0138] Furthermore, patients with Pompe disease or GSD may benefit from the administration of optimized forms of GAA. For example, administration of GAA has been shown to reduce glycogen in primary myoblasts derived from patients with glycogen storage disease type III (GSD III) (Sun et al. (2013) Mol Genet Metab 108(2):145; WO2010 / 005565).

[0139] B. Secretory signal peptide The native GAA signal peptide is not cleaved in the ER, and therefore the native GAA polypeptide is membrane-bound in the ER (Tsuji et al. (1987) Biochem. Int. 15(5):945-952). In some cell types, the GAA polypeptide can be found bound to the plasma membrane, presumably because the signal peptide cannot be cleaved, preserving the membrane topology of the ER (Hirschhorn et al., in *The Metabolic and Molecular Basis of Inherited Disease*, Valle, ed., 2001, McGraw-Hill: New York, pp. 3389-3420).

[0140] Disruption of membrane association of GAA can be achieved by exchanging the endogenous GAA signal peptide (and, optionally, adjacent sequences) for an alternative signal peptide for GAA.

[0141] Thus, the rAAV genomes disclosed herein comprise a heterologous nucleic acid sequence encoding a secretory signal peptide. In representative embodiments, the rAAV vectors and rAAV genomes disclosed herein further comprise a heterologous nucleic acid encoding a GAA polypeptide to be transferred to a target cell. The heterologous nucleic acid is operably associated with a segment encoding a secretory signal peptide such that transcription and translation produce a fusion polypeptide containing a secretory signal sequence operably associated with (e.g., directing the secretion of) the GAA polypeptide.

[0142] In some embodiments, the secretory signal peptide is heterologous (i.e., foreign or exogenous) to the polypeptide of interest. For example, if the secretory signal peptide is a fibronectin secretory signal peptide, the polypeptide of interest is not fibronectin. In some embodiments, the secretory signal peptide is selected from AAT signal peptide, fibronectin signal peptide (FN1), or the active fragment of AAT, FN1, or GAA signal peptide that has secretory signal activity. In another embodiment, the secretory signal peptide is not heterologous to GAA, i.e., the signal peptide is GAA signal peptide (i.e., residues 1-27 of native GAA polypeptide).

[0143] Generally, the secretory signal peptide is present at the amino-terminus (N-terminus) of the fusion polypeptide (i.e., in the rAAV vectors or rAAV genomes disclosed herein, the nucleic acid segment encoding the secretory signal peptide is present 5' to the heterologous nucleic acid encoding the GAA peptide or GAA fusion peptide). Alternatively, the secretory signal can be present at the carboxy-terminus or embedded within the GAA polypeptide or GAA fusion polypeptide (e.g., an IGF2-GAA fusion polypeptide), so long as the secretory signal is functionally associated with the GAA polypeptide or GAA fusion polypeptide of interest and directs its secretion from the cell (with or without cleavage of the signal peptide from the GAA polypeptide).

[0144] The secretory signal is functionally associated with the polypeptide of interest so that the GAA polypeptide or GAA fusion polypeptide is targeted to the secretory pathway. In other words, the secretory signal is functionally associated with the GAA polypeptide so that the GAA polypeptide or GAA fusion polypeptide is secreted from cells at a higher level (i.e., in a larger amount) than in the absence of the secretory signal peptide. The extent to which the secretory signal peptide directs the secretion of the GAA polypeptide or GAA fusion polypeptide is not important, as long as it provides the desired level of secretion and / or control of expression of the GAA polypeptide. Those skilled in the art will recognize that when a secretory protein is overexpressed, it often saturates the cellular secretory machinery and is retained within the cell. Generally, typically, at least about 20%, 30%, 40%, 50%, 70%, 80%, 85%, 90%, 95% or more of the GAA polypeptide or IGF2-GAA fusion polypeptide (alone and / or fused with a signal peptide) is secreted from cells. In other embodiments, essentially all of the detectable polypeptide (alone and / or in the form of a fusion polypeptide) is secreted from the cell.

[0145] By the phrase "secreted from the cell," the polypeptide may be secreted into any compartment (e.g., body fluid or space) outside the cell, including, but not limited to, the interstitial space, blood, lymph, cerebrospinal fluid, renal tubules, the respiratory tract (e.g., alveoli, bronchioles, bronchi, nasal cavity, etc.), the gastrointestinal tract (e.g., esophagus, stomach, small intestine, colon, etc.), the vitreous humor of the eye, and the endolymphatic fluid of the cochlea.

[0146] In one embodiment, the rAAV genome comprises a heterologous nucleic acid encoding a secretory signal peptide (SP) fused to a GAA polypeptide. In another embodiment, the rAAV genome comprises a heterologous nucleic acid encoding a secretory signal peptide (SP) fused to a GAA fusion polypeptide, wherein the GAA fusion polypeptide comprises a targeting peptide (e.g., an IGF2 sequence) fused to the GAA polypeptide. Thus, the signal peptides disclosed herein increase the efficiency of secretion of a GAA polypeptide or an IGF2-GAA fusion polypeptide from cells transduced by an rAAV vector or containing an rAAV genome, as described herein.

[0147] Thus, in some embodiments, the rAAV genome disclosed herein comprises 5' ITR and 3' ITR sequences, and a promoter operably linked to a heterologous nucleic acid encoding a secretory peptide and a nucleic acid encoding an alpha glucosidase (GAA) polypeptide (i.e., the heterologous nucleic acid encodes a GAA fusion polypeptide comprising a signal peptide-GAA polypeptide) located between the 5' ITR and 3' ITR.

[0148] In another embodiment, the rAAV genome disclosed herein comprises 5' ITR and 3' ITR sequences, and a promoter operably linked to a heterologous nucleic acid encoding a secretory peptide and a nucleic acid encoding an alpha glucosidase (GAA) fusion polypeptide located between the 5' ITR and 3' ITR, wherein the fusion protein comprises an IGF2 sequence and a GAA polypeptide (i.e., the heterologous nucleic acid encodes a GAA fusion polypeptide comprising a signal peptide-IGF2-GAA polypeptide).

[0149] In some embodiments, the secretory signal peptide (also referred to as a signal peptide) results in the secretion of at least about 50%, 60%, 75%, 85%, 90%, 95%, 98%, or more of the GAA polypeptide or GAA fusion polypeptide from the cell. The relative proportion of a GAA polypeptide expressed from an rAAV genome (e.g., a fusion polypeptide comprising signal peptide-GAA (SP-GAA), or a fusion protein comprising signal peptide-targeting peptide-GAA, e.g., an SP-IGF2-GAA fusion polypeptide) that is secreted from the cell can be determined conventionally by methods known in the art, such as by measuring GAA activity in the supernatant, as described in the Examples. Secreted protein can be detected in cell culture medium, serum, milk, etc., by directly measuring the protein itself (e.g., by Western blot) or by protein activity assays (e.g., enzyme assays).

[0150] Generally, secretory signal peptides are cleaved in the endoplasmic reticulum, and in some embodiments, the secretory signal peptide is cleaved from the GAA polypeptide before secretion. However, as long as the secretion of the GAA polypeptide or IGF2-GAA fusion polypeptide from cells is enhanced and the GAA polypeptide is functional, it is not necessary for the secretory signal peptide to be cleaved. Thus, in some embodiments, the secretory signal peptide is partially or completely retained.

[0151] In some embodiments, the rAAV genome or isolated nucleic acid disclosed herein comprises a nucleic acid encoding a chimeric polypeptide comprising a GAA polypeptide operably linked to a secretory signal peptide, wherein the chimeric polypeptide is expressed and produced from a cell transduced with the rAAV vector, and the GAA polypeptide is secreted from the cell. The GAA polypeptide or GAA fusion polypeptide (e.g., an IGF2-GAA fusion polypeptide) may be secreted after cleavage of all or part of the secretory signal peptide. Alternatively, the GAA polypeptide or GAA fusion polypeptide (e.g., an IGF2-GAA fusion polypeptide) may retain the secretory signal peptide (i.e., the secretory signal is not cleaved). Thus, in this context, a "GAA polypeptide or GAA fusion polypeptide" may be a chimeric polypeptide comprising a secretory peptide.

[0152] Those skilled in the art will further understand that chimeric polypeptides can contain additional amino acids, for example, as a result of manipulation of the nucleic acid construct, such as the addition of restriction sites, so long as these additional amino acids do not render the secretory signal sequence or the GAA polypeptide or GAA fusion polypeptide (e.g., an IGF2-GAA fusion polypeptide) non-functional. The additional amino acids may be cleaved or retained in the mature GAA polypeptide, so long as such retention does not result in a non-functional GAA polypeptide.

[0153] In representative embodiments, the secretory signal peptide replaces most, essentially all, or all of the sequence found in the native GAA polypeptide. In specific embodiments, most or all of the native sequence of GAA is retained, so long as secretion of the GAA polypeptide or GAA fusion polypeptide (e.g., an IGF2-GAA fusion polypeptide) is enhanced and the mature GAA polypeptide is functional.

[0154] Without wishing to be limited by theory, it is generally believed that the secretory signal sequence directs the insertion of the nascent polypeptide into the endoplasmic reticulum, from where the nascent polypeptide is transported to the Golgi, which then fuses with the cell membrane to secrete the polypeptide from the cell. Typically, the secretory signal is cleaved from the polypeptide during processing, which is believed to occur in the endoplasmic reticulum. In the case of the fusion polypeptide of the present invention, it is not necessary for the secretory signal peptide to be completely or completely cleaved from the chimeric GAA polypeptide or chimeric IGF2-GAA fusion polypeptide. In some embodiments, the secretory signal peptide may be essentially completely cleaved; alternatively, in some cells, incomplete cleavage may occur, or essentially no cleavage may occur. Without wishing to be limited by a particular theory, in some embodiments, retention of part or all of the secretory signal peptide (i.e., non-cleavage) appears to stabilize the resulting chimeric GAA polypeptide or chimeric IGF2-GAA fusion polypeptide.

[0155] In some embodiments, the secretory signal peptide is only partially removed from the polypeptide, i.e., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or even 15 or more amino acid residues are retained in the secreted polypeptide. For illustrative purposes only, using the fibronectin signal peptide as an exemplary signal peptide, amino acids 22(Val)-32(Arg), 23(Arg)-32(Arg), 24(Cys)-32(Arg), 25(Thr)-32(Arg), or 26(Glu)-32(Arg) of SEQ ID NO:18 may be retained in the secreted polypeptide.

[0156] Secretory signal peptides included for use in the rAAV genomes disclosed herein may be derived in whole or in part from the secretory signal of a secreted polypeptide (i.e., precursor) and / or may be synthetic in whole or in part. As those skilled in the art will recognize, secretory signal sequences generally function cross-species. Thus, secretory signal peptides can be derived from any species of origin, including animals (e.g., birds, and mammals such as humans, monkeys and other non-human primates, cattle, sheep, goats, horses, pigs, dogs, cats, rats, mice, and rabbits), plants, yeast, bacteria, protozoa, or fungi. The length of the secretory signal sequence is not critical; generally, known secretory signal sequences are from about 10-15 amino acids to 50-60 amino acids in length. Furthermore, known secretory signals from secreted polypeptides may be altered or modified (e.g., by amino acid substitution, deletion, truncation, or insertion) so long as the resulting secretory signal sequence functions to enhance secretion of an operably linked GAA polypeptide or GAA fusion polypeptide (e.g., an IGF2-GAA fusion polypeptide).

[0157] The secretory signal sequence of the present invention is not limited to a particular length, so long as it directs the polypeptide of interest into the secretory pathway. In representative embodiments, the signal peptide is at least about 6, 8, 10, 12, 15, 20, 25, 30, or 35 amino acids in length, up to about 40, 50, 60, 75, or 100 amino acids in length or more.

[0158] The secretory signal peptide encoded in the rAAV vector by the rAAV genome disclosed herein can comprise, consist essentially of, or consist of a naturally occurring secretory signal sequence or a modification thereof. Numerous secreted proteins and sequences that direct secretion from cells are known in the art. Exemplary secreted proteins (and their secretory signals) include erythropoietin, coagulation factor IX, cystatin, lactotransferrin, plasma protease C1 inhibitor, apolipoproteins (e.g., APO A, C, E), MCP-1, α-2-HS-glycoprotein, α-1-microglobulin, complement (e.g., C1Q, C3), vitronectin, lymphotoxin α, azurocidin, VIP, metalloproteinase inhibitor 2, glypican 1, pancreatic hormones, clusterin, hepatocyte growth factor, insulin, α1-antichymotrypsin, growth hormone, type IV collagenase, guanylin, properdin, proenkephalin A, inhibin β (e.g., A chain), prealbumin, angiocenin, lutropin (e.g., β chain), insulin-like growth factor binding protein 1 and 2, proactivator polypeptide, fibrinogen (e.g., β chain), gastric triacylglycerol lipase, midkine, neutrophil defensins 1, 2, and 3, α- l-antitrypsin, matrix gla-protein, alpha tryptase, bile-salt-activated lipase, chymotrypsinogen B, elastin, interferon lambda chain V region, platelet factor 4 variant, chromogranin A, WNT-1 proto-oncogene protein, oncostatin M, beta neoendorphin-dynorphin, von Willebrand factor, plasma serine protease inhibitor, serum amyloid A protein, nidogen, fibronectin, rennin, osteonectin, histatin 3, phospholipase A2, cartilage matrix protein, GM-CSF, matrilysin, neuroendocrine protein 7B2, placental protein 11, gelsolin, M-CSF, transcobalamin I, lactase-phlorizin hydrolase, elastase 2B, pepsinogen A, MIP1β, prolactin, trypsinogen II, gastrin-releasing peptide II, atrial natriuretic factor, secretory alkaline phosphatase, pancreatic α-amylase, secretogranin I, β-casein, serotransferrin, tissue factor pathway inhibitor, follitropin β-chain, coagulation factor XII, growth hormone-releasing factor, prostate seminal plasma protein, interleukins (e.g., 2, 3, 4, 5, 9, 11), inhibins (e.g., α-chain), angiotensinogen, thyroglobulin, IG heavy or light chain, plasminogen activator inhibitor 1, lysozyme C, plasminogen activator, antileukoproteinase 1, statherin, fibrin 1, isoform B, Romodulin, thyroxine-binding globulin, axonin-1, endometrial alpha-2 globulin, interferons (e.g., alpha, beta, gamma), beta-2-microglobulin, procholecystokinin, progastricsin, prostatic acid phosphatase, bone sialoprotein II, colipase, Alzheimer's amyloid A4 protein, PDGF (e.g., A or B chain), coagulation factor V, triacylglycerol lipase, haptoglobin-2, corticosteroid-binding globulin, triacylglycerol lipase, prorelaxin H2, follistatin 1 and 2, platelet glycoprotein IX, GCSF, VEGF, heparin cofactor II, antithrombin-III, leukemia inhibitory factor, interstitial collagenase, pleiotrophin, small inducibleInducible cytokine A1, melanin-concentrating hormone, angiotensin-converting enzyme, pancreatic trypsin inhibitor, coagulation factor VIII, alpha-fetoprotein, alpha-lactalbumin, senogelin II, kappa-casein, glucagon, thyroid-stimulating hormone beta chain, transcobalamin II, thrombospondin 1, parathyroid hormone, vasopressin copeptin, tissue factor, motilin, MPIF-1, kininogen, neuroendocrine convertase 2, stem cell factor procollagen alpha 1 chain, plasma kallikrein, keratinocyte growth factor, and any other secreted hormone, growth factor, cytokine, enzyme, clotting factor, milk protein, immunoglobulin chain, and the like.

[0159] In some embodiments, other secretory signal peptides encoded in the rAAV vector by the rAAV genome disclosed herein can be selected from, but are not limited to, preprocathepsin L (e.g., GenBank Accession Nos. KHRTL, NP_037288; NP_034114, AAB81616, AAA39984, P07154, CAA68691; the disclosures of which are incorporated herein by reference in their entireties) and prepro-alpha2 collagen (e.g., GenBank Accession Nos. CAA98969, CAA26320, CGHU2S, NP_000080, BAA25383, P08123; the disclosures of which are incorporated herein by reference in their entireties), and allelic variations, modifications, and functional fragments thereof (as described above with respect to the fibronectin secretory signal sequence). Exemplary secretory signal sequences include that of preprocathepsin L (Rattus norvegicus), TIFF2025032149000001.tif4128; accession number CAA68691), and that of prepro-α2 collagen (Homo sapiens, TIFF2025032149000002.tif4128; Accession No. CAA98969). Longer amino acid sequences containing full-length secretory signal sequences derived from preprocathepsin L and prepro-α2 collagen, or functional fragments thereof (as described for the fibronectin secretory signal sequence above), are also encompassed.

[0160] In some embodiments, the secretory signal peptide is derived in whole or in part from a secretory polypeptide produced by hepatocytes. In some embodiments, the secretory signal peptide may further be synthetic or artificial in whole or in part. Synthetic or artificial secretory signal peptides are known in the art; see, for example, Barash et al., "Human secretory signal peptide description by hidden Markov model and generation of a strong artificial signal peptide for secretory protein expression," Biochem.Biophys.Res.Comm.294:835-42(2002) (the disclosure of which is incorporated herein in its entirety). In a specific embodiment, the secretory signal peptide is an artificial secretory signal: TIFF2025032149000003.tif4128 or a variation thereof having 1, 2, 3, 4, or 5 amino acid substitutions (optionally conservative amino acid substitutions; conservative amino acid substitutions are known in the art).

[0161] Fibronectin secretory signal peptide: In some embodiments, the secretory signal peptide is a fibronectin secretory signal peptide, which term includes modifications of the naturally occurring sequence (as described in more detail below).

[0162] In some embodiments, the secretory signal peptide is a fibronectin signal peptide, such as the signal sequence of human fibronectin or a signal sequence derived from rat fibronectin. The fibronectin (FN1) signal sequence and modified FN1 signal peptides included for use in the rAAV genomes and rAAV vectors described herein are disclosed in U.S. Patent No. 7,071,172, which is incorporated herein by reference in its entirety.

[0163] Thus, the fibronectin secretory signal sequence of the present invention can be derived from any species, including, but not limited to, birds (e.g., chicken, duck, turkey, quail, etc.), mammals (e.g., humans, monkeys, mice, rats, cows, sheep, goats, horses, pigs, rabbits, cats, dogs, etc.), and other animals, including Caenorhabditis elegans, Xenopus laevis, and zebrafish (Danio rerio). Exemplary fibronectin secretory signal sequences include, but are not limited to, those listed in Table 1 of U.S. Patent No. 7,071,172, the entire contents of which are incorporated herein by reference.

[0164] Table 3. Exemplary fibronectin (FN1) secretory signal peptides TIFF2025032149000004.tif151170

[0165] An exemplary nucleotide sequence encoding the Rattus norvegicus fibronectin secretory signal sequence is found in GenBank Accession No. X15906 (the disclosure of which is incorporated herein by reference). Yet another exemplary sequence is the nucleotide sequence encoding the secretory signal peptide of human fibronectin 1 transcript variant 1 (Accession No. NM_002026, nucleotides 268-345; the disclosure of Accession No. NM_002026 is incorporated herein by reference in its entirety). Another exemplary secretory signal sequence is encoded by the nucleotide sequence encoding the secretory signal peptide of Xenopus laevis fibronectin protein (Accession No. M77820, nucleotides 98-190; the disclosure of Accession No. M77820 is incorporated herein by reference in its entirety).

[0166] In another embodiment, the fibronectin signal sequence (FN1, nucleotides 208-303, TIFF2025032149000005.tif17158) is derived from the rat fibronectin mRNA sequence (Genbank accession #X15906) and encodes the following peptide signal sequence: Met Leu Arg Gly Pro Gly Pro Gly Arg Leu Leu Leu Leu Ala Val Leu Cys Leu Gly Thr Ser Val Arg Cys Thr Glu Thr Gly Lys Ser Lys Arg (SEQ ID NO:18).

[0167] In some embodiments, the nucleic acid sequence encoding the rat fibronectin signal peptide does not include the nucleotide sequence 3' to the cleavage site (i.e., encoding the amino acids C-terminal to the cleavage site). As those skilled in the art will recognize, the fibronectin secretory signal peptide is typically cleaved from the fibronectin precursor by the cleavage action of intracellular peptidases.

[0168] Those of skill in the art will recognize that a secretory signal sequence can encode one, two, three, four, five, or all six or more of the amino acids C-terminal to the peptidase cleavage site (identified by ↑) (see, e.g., SEQ ID NOs:19 and 24 in Table 3). Those of skill in the art will recognize that additional amino acids (e.g., one, two, three, four, five, six, or more amino acids) carboxy-terminal to the cleavage site can also be included in the secretory signal sequence.

[0169] In some embodiments, the rAAV genome may encode a fibronectin secretory signal peptide derived from a species other than those specifically disclosed herein, or may encode allelic variations and modifications thereof that retain secretory signal activity (e.g., confer a level (i.e., amount) of secretion of the associated polypeptide greater than that observed in the absence of the secretory signal peptide; in other words, have at least 50%, 70%, 80%, or 90% or more of the secretory signal activity of the secretory signal peptides specifically disclosed herein, or even have a greater level of secretory signal activity). For illustrative purposes only, the fibronectin secretory signal peptide encoded in the rAAV genome disclosed herein may also include a functional portion or fragment of a full-length secretory signal peptide (e.g., a functional fragment of the amino acid sequence (fibronectin signal sequence) shown in Table 3). The length of the fragment is not critical, as long as it has secretory signal activity (e.g., confer a level (i.e., amount) of secretion of the associated polypeptide greater than that observed in the absence of the secretory signal peptide). Exemplary fragments include at least 10, 12, 15, 18, 20, 25, or 27 consecutive amino acids of the full-length secretory signal peptide (e.g., fragments of the amino acid sequences shown in Table 3, i.e., the FN1 signal peptides of SEQ ID NOs: 18, 19, 20, 22, encoded by the nucleic acids of SEQ ID NOs: 23, 24, 25, and 26, respectively).

[0170] In embodiments of the present invention, functional fragments have at least about 50%, 70%, 80%, 90% or more secretory signal activity, or even greater levels of secretory signal activity, compared to the sequences specifically disclosed herein.

[0171] Similarly, those skilled in the art will recognize that longer amino acid sequences (and the nucleotide sequences encoding them) including the full-length fibronectin secretory signal (or fragments thereof having secretory signal activity) are encompassed by the term "fibronectin signal sequence" according to the present invention. Additional amino acids (e.g., 1, 2, 4, 6, 8, 10, 15, or even more amino acids) can be added to a fibronectin secretory signal sequence without unduly affecting secretory signal activity (e.g., conferring a level (i.e., amount) of secretion of the associated polypeptide greater than that observed in the absence of the secretory signal peptide; in other words, having at least about 50%, 70%, 80%, 90%, or more secretory signal activity, or even greater levels of secretory signal activity, compared to the sequences specifically disclosed herein). For example, those skilled in the art will recognize that peptide cleavage sites (as described above) or restriction enzyme sites can be added, typically at either end of the secretory signal sequence. Additional sequences with other functions (e.g., sequences encoding FLAG sequences or poly-His tails that facilitate polypeptide purification, or spacer sequences) can also be fused to the fibronectin secretion signal sequence. Furthermore, sequences encoding polypeptides that enhance the stability of the polypeptide of interest, such as sequences encoding maltose-binding protein (MBP) or glutathione-S-transferase, can also be added.

[0172] The secretory signal sequence may also be derived from any species, as described above for the fibronectin secretory signal sequence. Comparison of the fibronectin secretory signal sequence with the secretory signal sequences derived from cathepsin L and the alpha2 collagen precursor led to the identification of a core or canonical amino acid sequence: LLLLAVLCLGT (SEQ ID NO:64). Thus, in some embodiments, the rAAV genome comprises a chimeric nucleic acid sequence comprising the canonical amino acid sequence of LLLLAVLCLGT (SEQ ID NO:64).

[0173] Similarly, one of skill in the art will recognize that the secretory signal sequences specifically disclosed herein typically tolerate amino acid sequence substitutions that retain secretory signal activity (e.g., at least 50%, 70%, 80%, 90%, 95%, or more of the secretory signal activity of the secretory signal peptides specifically disclosed herein). Amino acid substitutions for identifying secretory signal peptides of the invention other than those specifically disclosed herein can be based on any characteristic known in the art, including the relative similarities or differences of amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc.

[0174] Peptidase cleavage site In some embodiments, one or more exogenous peptidase cleavage sites can be inserted into the secretory signal peptide-GAA fusion polypeptide, for example, between the secretory signal peptide and the GAA polypeptide. In specific embodiments, an autoprotease (e.g., foot-and-mouth disease virus 2A autoprotease) is inserted between the secretory signal peptide and the GAA polypeptide or the IGF2-GAA fusion polypeptide. In other embodiments, protease recognition sites that can be regulated by the addition of exogenous proteases (e.g., Lys-Arg recognition sites for trypsin, Lys-Arg recognition sites for Aspergillus KEX2-like proteases, recognition sites for metalloproteases, recognition sites for serine proteases, etc.) are used.

[0175] In some embodiments, the signal peptide is flanked by peptidase cleavage sites so that the signal peptide can be removed. Thus, in some embodiments, the rAAV genome comprises a nucleic acid encoding a signal peptide with an N-terminal or C-terminal cleavage site, or an N-terminal and a C-terminal cleavage site. In some embodiments, the N-terminal cleavage site is cleaved by the same enzyme as the C-terminal cleavage site, and in some embodiments, the N-terminal cleavage site and the C-terminal cleavage site are cleaved by different enzymes.

[0176] In specific embodiments of the invention, although not necessarily, the heterologous nucleic acid encoding a GAA polypeptide of the rAAV genome encodes a mature form of the GAA polypeptide (e.g., excluding precursor sequences that are normally removed during processing of the polypeptide). Similarly, the GAA polypeptide sequence may be modified to delete or inactivate native targeting or processing signals (e.g., if they interfere with the desired level of secretion of the polypeptide in accordance with the present invention).

[0177] C. IGF2 sequence In one embodiment, the rAAV genome contains a heterologous nucleic acid encoding a targeting peptide fused to a GAA polypeptide. In some embodiments, the targeting peptide is a ligand for an extracellular receptor. In some embodiments, the targeting peptide is a targeting domain that binds to the extracellular domain of a receptor on the surface of a target cell and enables the polypeptide to be localized to human lysosomes by internalization of the receptor. In one embodiment, the targeting peptide includes a urokinase-type plasminogen receptor portion that can bind to the cation-independent mannose-6-phosphate receptor. In some embodiments, the targeting peptide incorporates one or more amino acid sequences of an IGF2 sequence.

[0178] IGF2 is also known by the following aliases: chromosome 11 open reading frame 43, insulin-like growth factor 2, IGF-II, FLJ44734; IGF2, somatomedin A, and preptin. The mRNA for the wild-type human IGF2 sequence is Corresponds to TIFF2025032149000006.tif24160. The full length IGF2 protein (including the IGF2 leader sequence) is encoded by the nucleic acid sequence of NM_000612.6, which encodes the full length IGF2 protein NP_000603.1.

[0179] The coding sequence for human IGF2 is disclosed in U.S. Pat. No. 8,492,388 (see, e.g., Figure 2), which is incorporated herein by reference in its entirety. The IGF2 protein is synthesized as a preproprotein containing an amino-terminal 24-amino acid signal peptide and an 89-amino acid carboxy-terminal region, both of which are removed post-translationally (reviewed in O'Dell et al. (1998) Int. J. Biochem Cell Biol. 30(7):767-71). The mature protein is 67 amino acids. A Leishmania codon-optimized version of mature IGF2 is disclosed in U.S. Pat. No. 8,492,388 (see, e.g., Figure 3 of U.S. Pat. No. 8,492,388) (see Langford et al. (1992) Exp. Parasitol. 74(3):360-1). Additional cassettes containing a deletion of amino acids 1-7 of the mature polypeptide (Δ1-7), a change of residue 27 from tyrosine to leucine (Y27L), or both mutations (Δ1-7, Y27L) were constructed to generate IGF-II cassettes with specificity for only the desired receptor, as described below. Wild-type, IGF2 variants Y27L, Δ1-7, and Y27LΔ1-7 are encompassed for use in the present invention.

[0180] The mature human IGF2 sequence is: Shown in TIFF2025032149000007.tif11159.

[0181] In some embodiments, rAAV genomes are generated that contain a nucleic acid encoding a mature IGF2 polypeptide (SEQ ID NO:5) or an IGF2 sequence variant (e.g., SEQ ID NO:6 (IGF2-Δ2-7); SEQ ID NO:7 (IGF2-Δ1-7); SEQ ID NO:8 (IGF2-Δ1-42), SEQ ID NO:9 (IGF2-V43M)), or a sequence having at least 85%, 90%, or 95% sequence identity to SEQ ID NOs:5-9, that is taken up by a variety of cell types and transported to lysosomes, fused to the 5' end of a nucleic acid encoding a GAA protein, a fusion protein (e.g., an IGF2-GAA fusion polypeptide). Alternatively, a nucleic acid encoding a precursor IGF2 polypeptide can be fused to the 3' end of the GAA gene; the precursor includes a carboxy-terminal portion that is cleaved in mammalian cells to yield the mature IGF2 polypeptide, but the IGF2 signal peptide is preferably omitted (or moved to the 5' end of the GAA gene). This method has numerous advantages over methods involving glycosylation, including simplicity and cost-effectiveness, since further modification after protein isolation is not required.

[0182] The rAAV genome can encode a targeting peptide derived from IGF2 to target the CI-MPR. Alternatively, in some embodiments, a targeting peptide that preferentially binds to a receptor on the surface of myotubes can be used. Such peptides have been described (Samoylova et al. (1999) Muscle and Nerve 22:460; U.S. Patent No. 6,329,501). Other cell surface receptors, such as Fc receptors, LDL receptors, or transferrin receptors, are also suitable targets and can promote the targeting of GAA.

[0183] In some embodiments, IGF2 sequences encompassed for use in the present invention are described in US Pat. Nos. 7,785,856 and 9,873,868, each of which is incorporated by reference in its entirety.

[0184] IGF2 deletion mutants: In some embodiments, the IGF sequence comprises the minimal region of IGF2 capable of binding with high affinity to the M6P / IGF2 receptor. Residues associated with IGF2 binding to the M6P / IGF2 receptor are primarily clustered on one face of IGF2 (Terasawa et al. (1994) EMBO J. 13(23):5590-7). Although the tertiary structure of IGF2 is typically maintained by three intramolecular disulfide bonds, peptides incorporating the amino acid sequence of the M6P / IGF2 receptor-binding face of IGF2 can be designed to fold properly and possess binding activity. Such minimal binding peptides are highly preferred targeting moieties. Peptides designed based on the region surrounding amino acids 48-55 can be tested for binding to the M6P / IGF2 receptor. Alternatively, random peptide libraries can be screened for their ability to bind to the M6P / IGF2 receptor via either a yeast two-hybrid assay or a phage display-type assay.

[0185] In some embodiments, the IGF2 sequence is the minimal region of IGF2 capable of binding with high affinity to the M6P / IGF2 receptor. Residues associated with IGF2 binding to the M6P / IGF2 receptor are primarily clustered on one face of IGF2 (Terasawa et al. (1994) EMBO J. 13(23):5590-7). The tertiary structure of IGF2 is typically maintained by three intramolecular disulfide bonds, but peptides incorporating the amino acid sequence of the M6P / IGF2 receptor-binding surface of IGF2 can be designed to fold properly and have binding activity. Such minimal binding peptides are highly preferred IGF2 sequences herein. Peptides designed based on the region around amino acids 43-55 or 48-55 can be tested for binding to the M6P / IGF2 receptor.

[0186] In a specific embodiment, the IGF2 sequence includes a modification at valine 43, where valine is modified to met (V43M) such that translation initiation begins at amino acid 43. IGF2 sequences with the V43M modification encompassed for use in the present invention as targeting peptides or IGF2 sequences bind to the cation-independent mannose-6-phosphate receptor. In another embodiment, the IGF2 sequence is IGF2 delta 1-42 with V43 changed to Met (i.e., IGF2-Δ1-42 (SEQ ID NO:8) or IGF2-V43M (SEQ ID NO:9)).

[0187] The binding surfaces for IGF-I and the cation-independent M6P receptor reside on separate faces of IGF2, allowing the construction of a functional cation-independent M6P-binding domain that is substantially smaller than that of human IGF2. For example, the amino-terminal amino acids 2-7 or 1-7 and / or the carboxy-terminal residues 62-67 of the human IGF2 protein can be deleted or replaced. Additionally, optionally, amino acids 29-40 can be eliminated or replaced without altering the folding of the remainder of the polypeptide or its binding to the cation-independent M6P receptor. Thus, in some embodiments, the IGF2 sequence for fusion with a GAA polypeptide can include amino acids 8-28 and 41-61 of IGF2. In some embodiments, these stretches of amino acids can be directly joined or separated by a linker. Alternatively, amino acids 8-28 and 41-61 can be provided on separate polypeptide chains. In some embodiments, amino acids 8-28 of IGF2 or a conservatively substituted variant thereof may be fused to a GAA polypeptide to express an IGF2-GAA fusion protein from an rAAV vector, and another rAAV vector may express IGF2 amino acids 41-61 or a conservatively substituted variant thereof.

[0188] To facilitate proper presentation and folding of the IGF2 sequence, a longer portion of the IGF2 protein can be used, for example, an IGF2 tag comprising amino acid residues 1-67, 1-87, or the entire precursor form.

[0189] In some embodiments, the IGF2 sequence is a nucleic acid sequence encoding any of the following IGF2 targeting peptides: residue 1 followed by residues 8-67 of wild-type mature human insulin-like growth factor II (IGF2) of SEQ ID NO:5 (i.e., SEQ ID NO:6; i.e., IGF2-delta 2-7); residues 8-67 of wild-type mature human insulin-like growth factor II (IGF2) of SEQ ID NO:5 (i.e., SEQ ID NO:7; IGF2-delta 1-7), or residues 43-67 of wild-type mature human insulin-like growth factor II (IGF2) of SEQ ID NO:5 (i.e., IGF2-V43M (SEQ ID NO:9) or IGF-delta 1-42 (SEQ ID NO:8)).

[0190] In some embodiments of the methods and compositions disclosed herein, the IGF2 sequence is a nucleic acid sequence selected from any nucleic acid sequence including SEQ ID NO:2 (i.e., IGF2-delta 2-7); SEQ ID NO:3 (i.e., IGF2-delta 1-7), or SEQ ID NO:4 (i.e., IGF2-V43M), or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0191] SEQ ID NO:2 (i.e., IGF2-delta 2-7) has the following structure: As shown in TIFF2025032149000008.tif24145.

[0192] SEQ ID NO:3 (i.e., IGF2-delta 1-7) has the following structure: As shown in TIFF2025032149000009.tif24145.

[0193] SEQ ID NO:4 (i.e., IGF2-V43M) has the following structure: As shown in TIFF2025032149000010.tif24145.

[0194] In some embodiments, to facilitate proper presentation and folding of the IGF2 sequence, a longer portion of the IGF2 protein can be used, for example, an IGF2 sequence including amino acid residues 1-67, 1-87, or the entire precursor form can be used.

[0195] Modified IGF2 sequences and IGF2 homologues In some embodiments, the nucleic acid encoding IGF2 can be modified to reduce its affinity for IGFBP and / or to reduce its binding affinity to the IGF-I receptor, thereby improving targeting of the fused GAA-polypeptide to lysosomes and increasing its bioavailability.

[0196] The IGF2 sequence is preferably specifically targeted to the M6P receptor. Particularly useful are IGF2 sequences that have mutations in the IGF2 polypeptide that result in the protein binding with high affinity to the CI-MPR / M6P receptor but not with appreciable affinity to the other two receptors.

[0197] The IGF2 sequence can be modified to avoid sequestration of the IGF2 construct and minimize binding to serum IGF-binding proteins (IGFBPs) (Baxter (2000) Am. J. Physiol Endocrinol Metab. 278(6):967-76) and IGF-I receptors. Numerous studies have identified the residues of IGF-I and IGF-2 required for binding to IGF-binding proteins. Constructs containing mutations at these residues can be screened for retention of high-affinity binding to the M6P / IGF2 receptor and reduced affinity for IGF-binding proteins. For example, it has been reported that exchanging Phe 26 of IGF2 to Ser reduces the affinity of IGF2 for IGFBP-I and IGFBP-6, but has no effect on binding to the M6P / IGF2 receptor (Bach et al. (1993) J. Biol. Chem. 268(13):9246-54). Other substitutions, such as Ser for Phe 19 and Lys for Glu 9, may also be advantageous. Similar mutations in regions of IGF-I that are highly conserved with IGF2, either separately or in combination, result in large decreases in IGF-BP binding (Magee et al. (1999) Biochemistry 38(48):15863-70).

[0198] IGF2 binds with relatively high affinity to the IGF2 / M6P receptor and the IGF-I receptor, and with lower affinity to the insulin receptor. Substitution of IGF2 residues 48-50 (Phe Arg Ser) with the corresponding residues from insulin (Thr Ser Ile) or residues 54-55 (Ala Leu) with the corresponding residues from IGF-I (Arg Arg) reduces binding to the IGF2 / M6P receptor, but retains binding to the IGF-I receptor and the insulin receptor (Sakano et al. (1991) J. Biol. Chem. 266(31):20626-35).

[0199] IGF2 binds to repeat 11 of the cation-independent M6P receptor. In fact, a minireceptor in which only repeat 11 is fused to the transmembrane and cytoplasmic domains of the cation-independent M6P receptor binds to IGF2 (with approximately one-tenth the affinity of the full-length receptor) and can mediate IGF2 internalization and delivery to lysosomes (Grimme et al. (2000) J. Biol. Chem. 275(43):33697-33703). The structure of domain 11 of the M6P receptor is known (Protein Data Base entries 1GP0 and 1GP3; Brown et al. (2002) EMBO J. 21(5):1054-1062). The putative IGF2-binding site is a hydrophobic pocket that is thought to interact with hydrophobic amino acids of IGF2; candidate amino acids of IGF2 include leucine 8, phenylalanine 48, alanine 54, and leucine 55. Repeat 11 is sufficient for IGF2 binding, but constructs containing larger portions of the cation-independent M6P receptor (e.g., repeats 10-13 or 1-15) generally bind IGF2 with greater affinity and increased pH dependence (see, e.g., Linnell et al. (2001) J. Biol. Chem. 276(26):23986-23991).

[0200] Substitution of IGF2 residues Tyr 27 with Leu or Ser 26 with Phe reduces the affinity of IGF2 for the IGF-I receptor by 94-fold, 56-fold, and 4-fold, respectively (Torres et al. (1995) J. Mol. Biol. 248(2):385-401). Deletion of residues 1-7 of human IGF2 resulted in a 30-fold decrease in affinity for the human IGF-I receptor, while simultaneously resulting in a 12-fold increase in affinity for the rat IGF2 receptor (Hashimoto et al. (1995) J. Biol. Chem. 270(30):18013-8). Truncating the C-terminus of IGF2 (residues 62-67) also appears to reduce the affinity of IGF2 for the IGF-I receptor by 5-fold (Roth et al. (1991) Biochem. Biophys. Res. Commun. 181(2):907-14).

[0201] Substitution of IGF2 residue phenylalanine 26 with serine reduces binding to IGFBPs 1-5 by 5-75 fold (Bach et al. (1993) J. Biol. Chem. 268(13):9246-54). Exchange of IGF2 residues 48-50 with threonine-serine-isoleucine reduces binding to most IGFBPs by more than 100 fold (Bach et al. (1993) J. Biol. Chem. 268(13):9246-54); however, these residues are also important for binding to the cation-independent mannose-6-phosphate receptor. The Y27L substitution, which disrupts binding to the IGF-I receptor, interferes with the formation of a ternary complex with IGFBP3 and the acid-labile subunit (Hashimoto et al. (1997) J. Biol. Chem. 272(44):27936-42); this ternary complex accounts for the majority of circulating IGF2 (Yu et al. (1999) J. Clin. Lab Anal. 13(4):166-72). Deletion of the first six residues of IGF2 also interferes with IGFBP binding (Luthi et al. (1992) Eur. J. Biochem. 205(2):483-90).

[0202] Studies on the interaction of IGF-I with IGFBPs further revealed that substitution of phenylalanine 16 with serine reduced IGFBP binding by 40-300-fold, without affecting secondary structure (Magee et al. (1999) Biochemistry 38(48):15863-70). Changing glutamic acid 9 to lysine also resulted in a significant reduction in IGFBP binding. Furthermore, the double mutant lysine 9 / serine 16 showed the lowest affinity for IGFBPs. The sequence conservation of this region between IGF-I and IGF2 suggests that similar effects would be observed when analogous mutations are made in IGF2 (glutamic acid 12 lysine / phenylalanine 19 serine).

[0203] In some embodiments, the IGF2 sequence includes at least amino acids 48-55; at least amino acids 8-28 and 41-61; or at least amino acids 8-87, or a sequence variant thereof (e.g., R68A) or truncated form thereof (e.g., truncated C-terminally from position 62) that binds to the cation-independent mannose-6-phosphate receptor.

[0204] IGF2 sequence reduces binding to IGF-I receptor: Substitution of IGF2 residues Tyr 27 with Leu or Ser 26 with Phe reduces the affinity of IGF2 for the IGF-I receptor by 94-fold, 56-fold, and 4-fold, respectively (Torres et al. (1995) J. Mol. Biol. 248(2):385-401). Deletion of residues 1-7 of human IGF2 resulted in a 30-fold decrease in affinity for the human IGF-I receptor, while simultaneously increasing affinity for the rat IGF2 receptor by 12-fold (Hashimoto et al. (1995) J. Biol. Chem. 270(30):18013-8). The NMR structure of IGF2 shows that Thr 7 is located near residues 48 Phe and 50 Ser, and also near the 9 Cys-47 Cys disulfide bridge. The interaction of Thr 7 with these residues is thought to stabilize the flexible N-terminal hexapeptide required for IGF-I receptor binding (Terasawa et al. (1994) EMBO J. 13(23)5590-7). At the same time, this interaction can modulate binding to the IGF2 receptor. Truncation of the C-terminus of IGF2 (residues 62-67) also appears to reduce the affinity of IGF2 for the IGF-I receptor by 5-fold (Roth et al. (1991) Biochem. Biophys. Res. Commun. 181(2):907-14).

[0205] In some embodiments, targeting peptides (e.g., IGF2 sequences) encompassed for use in the present invention bind to CI-MPR with a dissociation constant of submicromolar. Generally, lower dissociation constants (e.g., less than 10 M, less than 10 M, or less than 10 M) are preferred. Determination of the dissociation constant can be determined by one skilled in the art, for example, by surface plasmon resonance as described in Linnell et al. (2001) J. Biol. Chem. 276(26):23986-23991. In some embodiments, assessment of the ability of a targeting peptide (e.g., IGF2 sequence) to bind to CI-MPR can be determined using an assay involving a soluble form of the extracellular domain of CI-MPR (e.g., repeats 1-15 of the cation-independent M6P receptor) immobilized to a chip via avidin-biotin interactions. A targeting peptide (eg, an IGF2 sequence) is passed over the chip and kinetic and equilibrium constants are detected and calculated by measuring the change in mass associated with the chip surface.

[0206] In another embodiment of the present invention, the rAAV genome encoding a targeting peptide (e.g., an IGF2 sequence) is inserted into the native GAA coding sequence at the junction between the mature 70 / 76 kDa polypeptide and the C-terminal domain, e.g., at position 791. This creates a single chimeric polypeptide. Because the targeting peptide (e.g., an IGF2 sequence) cannot bind to its cognate receptor in this configuration, a protease cleavage site can be inserted immediately downstream of the targeting peptide (e.g., an IGF2 sequence). After the protein is produced in the correctly folded form, the C-terminal domain can be cleaved by protease treatment.

[0207] It may be desirable to use a protease cleavage site that is acted upon by a protease normally found in human serum. In this way, a targeting peptide (e.g., an IGF2 sequence) tagged GAA can be introduced into the bloodstream in a prodrug form and activated for uptake by a protease normally present in serum. This can improve the distribution of the enzyme. As before, the peptide tag can be an IGF2 sequence tag or a muscle-specific tag.

[0208] In another embodiment of the invention, a targeting peptide (e.g., an IGF2 sequence) is fused to the N-terminus of GAA so that enzymatic activity is maintained. In the case of N-terminal fusion, the secretion level of the enzyme can be influenced by replacing the native GAA signal peptide with a heterologous signal peptide.

[0209] In some embodiments, the rAAV genome encoding a targeting peptide (e.g., an IGF2 sequence) is inserted into the native GAA coding sequence at the junction of the mature 70 / 76 kDa polypeptide and the C-terminal domain, e.g., at position 791. This creates a single fusion (or chimeric) polypeptide. Because the targeting peptide (e.g., an IGF2 sequence) cannot bind to its cognate receptor in this configuration, a protease cleavage site can be inserted immediately downstream of the targeting peptide (e.g., an IGF2 sequence). After the GAA polypeptide is produced in the correctly folded form, the C-terminal domain can be cleaved by protease treatment.

[0210] Therefore, in some embodiments, it may be desirable to use a protease cleavage site that is acted upon by a protease normally found in human serum. In this way, a targeting peptide (e.g., an IGF2 sequence) fused to a GAA polypeptide can be introduced into the bloodstream in a prodrug form and activated for uptake by a protease normally present in serum. This can improve the distribution of the GAA polypeptide. As before, the targeting peptide is an IGF2 sequence or a muscle-specific sequence disclosed herein.

[0211] In another embodiment of the invention, a targeting peptide (e.g., an IGF2 sequence) is fused at the N-terminus of GAA so that enzymatic activity is retained (see, e.g., the Examples describing an assay for measuring GAA activity). In the case of an N-terminal fusion, the level of secretion of GAA can be increased by replacing the native GAA signal peptide with a heterologous signal peptide described herein.

[0212] In one embodiment, a targeting peptide as defined herein, e.g., an IGF2 sequence, is directly fused to the N-terminus or C-terminus of a GAA polypeptide. In another embodiment, the IGF2 sequence is fused to the N-terminus or C-terminus of a GAA polypeptide via a spacer. In one specific embodiment, the IGF2 sequence is fused to the GAA polypeptide via a spacer of 10 to 25 amino acids. In another embodiment, the IGF2 sequence is fused to the GAA polypeptide via a spacer containing a glycine residue.

[0213] In some embodiments, the IGF2 sequence is fused to the GAA polypeptide by a spacer of at least 1, 2, or 3 amino acids. In some embodiments, the spacer comprises the amino acids GAP or Gly-Ala-Pro (SEQ ID NO: 31), or an amino acid sequence at least 50% identical thereto. In some embodiments, the spacer is GGG or GA or AP or GP, or a variant thereof. In some embodiments, the spacer is encoded by the nucleic acid ggc gcg ccg (SEQ ID NO: 30).

[0214] In some embodiments, the IGF2 sequence is fused to the GAA polypeptide by a spacer comprising a helical structure. In another specific embodiment, the IGF2 sequence is TIFF2025032149000011.tif4128. In some embodiments of the methods and compositions disclosed herein, the spacer is SEQ ID NO:31 (encoded by the nucleic acid of SEQ ID NO:30). In some embodiments of the methods and compositions disclosed herein, the spacer is selected from any of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0215] Cation-independent M6P receptor In some embodiments, the targeting peptide is a lysosomal targeting peptide or protein or other moiety that binds to the cation-independent M6P / IGF2 receptor (CI-MPR) in a mannose-6-phosphate-independent manner. Advantageously, this embodiment mimics the normal biological mechanism for uptake of LSD proteins, but does so in a mannose-6-phosphate-independent manner.

[0216] The cation-independent M6P receptor is a 275 kDa single-chain transmembrane glycoprotein that is widely expressed in mammalian tissues. It is one of two mammalian receptors that bind M6P; the second is called the cation-dependent M6P receptor. The cation-dependent M6P receptor requires divalent cations for M6P binding; the cation-independent M6P receptor does not. These receptors play an important role in the trafficking of lysosomal enzymes through recognition of the M6P moiety on high-mannose carbohydrates on lysosomal enzymes. The extracellular domain of the cation-independent M6P receptor contains 15 homologous domains ("repeats") at discrete locations on the receptor that bind to a diverse group of ligands.

[0217] The cation-independent M6P receptor (CI-MPR) contains two binding sites for M6P, one located in repeats 1-3 and the other in repeats 7-9. The receptor binds monovalent M6P ligands with dissociation constants in the μM range, but bivalent M6P ligands with dissociation constants in the nM range, presumably due to receptor oligomerization. IGF2 uptake by the CI-MPR is enhanced by simultaneous binding of multivalent M6P ligands, such as lysosomal enzymes, to the receptor.

[0218] The CI-MPR contains binding sites for at least three different ligands that can be used as targeting peptides. As disclosed herein, IGF2 ligands bind to the CI-MPR at or about pH 7.4 with a dissociation constant of about 14 nM, primarily through interactions with repeat 11. Consistent with its function in targeting IGF2 to lysosomes, the dissociation constant increases approximately 100-fold at or about pH 5.5, promoting dissociation of IGF2 in acidic late endosomes. The CI-MPR can bind high molecular weight O-glycosylated forms of IGF2. Thus, in some embodiments, the IGF2 sequence contains O-glycosylation.

[0219] In another embodiment, the targeting peptide that binds to CI-MPR is retinoic acid. Retinoic acid binds to the receptor with a dissociation constant of 2.5 nM. Affinity photolabeling of the cation-independent M6P receptor with retinoic acid does not interfere with the binding of IGF2 or M6P to the receptor, indicating that retinoic acid binds to a different site on the receptor. Binding of retinoic acid to the receptor alters the intracellular distribution of the receptor, resulting in greater accumulation of the receptor in cytoplasmic vesicles, and also enhances the uptake of M6P-modified β-glucuronidase. Retinoic acid has a photoactivatable moiety that can be used to link it to a therapeutic agent without interfering with its ability to bind to the cation-independent M6P receptor.

[0220] Urokinase-type plasminogen receptor (uPAR) also binds to CI-MPR with a dissociation constant of 9 μM. uPAR is a GPI-anchored receptor on the surface of most cell types, where it functions as an adhesion molecule and in the proteolytic activation of plasminogen and TGFβ. Binding of uPAR to the CI-M6P receptor targets it to lysosomes, thereby modulating its activity. Fusing the extracellular domain of uPAR, or a portion thereof capable of binding to the cation-independent M6P receptor, with a therapeutic agent allows for targeting of the agent to lysosomes.

[0221] In some embodiments, the IGF2 sequence is modified to be furin-resistant, i.e., resistant to degradation by the furin protease, which recognizes the Arg-XX-Arg cleavage site. Such IGF2 sequences are disclosed in U.S. Patent Application Publication No. 22012 / 0213762, which is incorporated by reference in its entirety. In some embodiments, the furin-resistant IGF2 sequences for use in the rAAV genome described herein contain mutations, which may be substitutions or deletions for other amino acids, in the region corresponding to amino acids 30-40 (e.g., 31-40, 32-40, 33-40, 34-40, 30-39, 31-39, 32-39, 34-37, 32-39, 33-39, 34-39, 35-39, 36-39, 37-40, 34-40) of SEQ ID NO:5 (the wt IGF2 sequence). For example, a substitution at position 34 can affect furin recognition of the first cleavage site. Insertion of one or more additional amino acids at each recognition site can eliminate one or both furin cleavage sites. Deletion of one or more residues at degenerate positions can also eliminate both furin cleavage sites.

[0222] In some embodiments, the furin-resistant IGF2 sequence contains an amino acid substitution at a position corresponding to Arg37 (R37) or Arg40 (R40) of SEQ ID NO: 5. In some embodiments, the furin-resistant IGF2 sequence contains a Lys (K) or Ala (A) substitution at position Arg37 or Arg40 of SEQ ID NO: 5. Other substitutions are also possible, including combinations of Lys and / or Ala mutations at both positions 37 and 40, or substitutions of amino acids other than Lys (K) or Ala (A). In some embodiments, the IGF2 sequences encompassed for use in the rAVV genomes disclosed herein are IGFΔ2-7-K37 or IGFΔ2-7-K40 or IGFΔ1-7-K37 or IGFΔ1-7-K40, which indicate that the IGF2 sequence has a deletion of aa 2-7 or 1-7 and a modification of the Arg (R) residue at position 37 to lysine (i.e., an R37K modification) or an R40K, respectively. In some embodiments, the IGF2 sequences encompassed for use in the rAVV genomes disclosed herein are IGFΔ2-7-K37-K40 or IGFΔ1-7-R37K-R40K, which indicate that the IGF2 sequence has a deletion of residues 2-7 or 1-7 and a modification of the R residues at positions 37 and 40 to lysine (R37K and R40K). In some embodiments, the IGF2 sequence included for use in the rAVV genomes disclosed herein is selected from any of IGFΔ2-7-R37A or IGFΔ2-7-R40A or IGFΔ1-7-R37A or IGFΔ1-7-R40A, IGFΔ2-7-R37A-R40A or IGFΔ1-7-R37A-R40A. Exemplary constructs for IGF2 sequences included for use in the rAVV genomes disclosed herein are disclosed in U.S. Patent Application No. 2012 / 0213762, which is incorporated by reference in its entirety.

[0223] In some embodiments, furin-resistant IGF-2 sequences suitable for the present invention may contain additional mutations, for example, up to 30% or more of the residues of SEQ ID NO:5 may be changed (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, up to 30% or more of the residues may be changed). Thus, a furin-resistant IGF2 mutein suitable for the present invention may have an amino acid sequence that is at least 70%, for example at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, identical to SEQ ID NO:5.

[0224] Furthermore, the use of the IGF2 sequences disclosed herein is also referred to in the art as glycosylation-independent lysosomal targeting (GILT) because M6P as the lysosome-targeting moiety is replaced with the IGF2 sequence. Details of GILT technology are described in U.S. Patent Application Publication Nos. 2003 / 0082176, 2004 / 0006008, 2004 / 0005309, 2003 / 0072761, 2005 / 0281805, and 2005 / 0244400, as well as International Publication Nos. WO 03 / 032913, WO 03 / 032727, WO 02 / 087510, WO 03 / 102583, and WO 2005 / 078077, the disclosures of all of which are incorporated herein by reference.

[0225] D. Spacer and Fusion Junction of the GAA Polypeptide When GAA is expressed as a fusion protein with a secretory signal peptide (e.g., an SS-GAA polypeptide) or a fusion protein with a targeting peptide (i.e., an SS-IGF2-GAA polypeptide double fusion polypeptide), the signal peptide or IGF2 sequence may be fused directly to the GAA polypeptide or separated from the GAA polypeptide by a linker. The amino acid linker (also referred to herein as a "spacer") incorporates one or more amino acids other than those that appear at that position in the native protein. The spacer may generally be designed to be flexible, or may be designed to insert a structure such as an a-helix between the two protein moieties.

[0226] In some embodiments, the spacer or linker may be relatively short, e.g., at least 1, 2, 3, 4, or 5 amino acids, or the sequence Gly-Ala-Pro (SEQ ID NO:31) or Gly-Gly-Gly-Gly-Gly-Pro (SEQ ID NO:32), or may be longer, e.g., 5-10 amino acids or 10-25 amino acids in length. For example, a flexible repeat linker of 3-4 copies of the sequence (GGGGS (SEQ ID NO:33)) and a-helical repeat linker of 2-5 copies of the sequence (EAAAK (SEQ ID NO:34)) have been described (Arai et al. (2004) Proteins: Structure, Function and Bioinformatics 57:829-838).

[0227] The use of another linker, GGGTVGDDDDK (SEQ ID NO:35), has also been reported for IGF2 fusion proteins (DiFalco et al. (1997) Biochem. J. 326:407-413) and is encompassed for use. Linkers incorporating the a-helical portion of a human serum protein may be used to minimize the immunogenicity of the linker region.

[0228] In some embodiments, the spacer is encoded by the nucleic acid ggc gcg ccg (SEQ ID NO:30), which encodes an amino acid spacer comprising the amino acids GAP or Gly-Ala-Pro (SEQ ID NO:31).

[0229] The site of the fusion junction within the GAA polypeptide for fusion with either a signal peptide (to generate an SS-GAA fusion protein) or a targeting peptide (e.g., to generate an SP-IGF2-GAA double fusion polypeptide) should be carefully selected to promote proper folding and activity of each polypeptide within the fusion protein and to prevent premature separation of the signal peptide from the GAA polypeptide.

[0230] In some embodiments, the IGF2 sequence is fused to the GAA polypeptide by a spacer comprising a helical structure. In another specific embodiment, the IGF2 sequence is fused to the GAA polypeptide by a spacer at least 50% identical to the sequence GGGTVGDDDDK (SEQ ID NO:35). In some embodiments of the methods and compositions disclosed herein, the spacer is SEQ ID NO:31 (encoded by the nucleic acid of SEQ ID NO:30). In some embodiments of the methods and compositions disclosed herein, the spacer is selected from any of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35.

[0231] Four exemplary strategies for creating a GF2-GAA fusion protein can be generated as follows: 1. Fusion of IGF2 sequences at the amino terminus of GAA. 2. Insertion of IGF2 sequences between the trefoil domain and the mature region of GAA. 3. Insertion of IGF2 sequences between the mature region of GAA and the C-terminal domain of GAA. 4. Fusion of the IGF2 sequence with the C-terminus of a truncated GAA polypeptide and co-expression of the C-terminal domain.

[0232] For example, a targeting peptide (e.g., an IGF2 sequence) can be fused directly or via a spacer to amino acid 40 or amino acid 70 of GAA, positions that allow protein expression, catalytic activity of the GAA protein, and proper targeting by the IGF2 sequence, as described in the Examples herein. Alternatively, a targeting peptide (e.g., an IGF2 sequence) can be fused at or near the cleavage site that separates the C-terminal domain of GAA from the mature polypeptide. This allows for the synthesis of a GAA protein containing an internal targeting peptide (e.g., an IGF2 sequence), which can be optionally cleaved to release the mature polypeptide or the C-terminal domain from the targeting domain, depending on the location of the cleavage site. Alternatively, the mature polypeptide can be synthesized as a fusion protein at about position 791 without incorporating the C-terminal sequence into the open reading frame of the expression construct.

[0233] To facilitate folding of the IGF2 sequence, GAA amino acid residues adjacent to the fusion junction can be modified. For example, because GAA cystine residues can interfere with proper folding of a targeting peptide (e.g., an IGF2 sequence), the terminal GAA cystine 952 can be deleted or replaced with serine to accommodate a C-terminal targeting peptide (e.g., an IGF2 sequence). The targeting peptide (e.g., an IGF2 sequence) can also be fused just before the final Cys952. The final Cys952 can be mutated to serine, and the penultimate Cys938 can be changed to proline.

[0234] E. CS sequence In some embodiments, the rAAV genome disclosed herein comprises a heterologous nucleic acid sequence that optionally includes a collagen stability sequence (CS or CSS) located 3' to the GAA gene and 5' to the polyA signal. Exemplary collagen stability sequences include: TIFF2025032149000012.tif4128, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Exemplary collagen stability sequences may have the amino acid sequence of PSPLFP (SEQ ID NO:66), or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. CS sequences are disclosed in Holick and Liebhaber, Proc. Nat. Acad. Sci. 94:2410-2414, 1997 (see, e.g., Figure 3, page 5205), which is incorporated herein by reference in its entirety.

[0235] In some embodiments, the rAAV genomes disclosed herein include a heterologous nucleic acid sequence that may optionally include another stability sequence in place of the collagen stability sequence (CS). Other stability sequences are known to those of skill in the art and are encompassed for use in the rAAV genome in place of, or in addition to, the collagen stability sequence disclosed herein.

[0236] F. Promoter In some embodiments, the rAAV genotype includes a promoter to achieve an appropriate level of GAA expression. A suitable promoter can be selected from any of a number of promoters known to those skilled in the art. In some embodiments, the promoter is a cell type-specific promoter. In further embodiments, the promoter is an inducible promoter. In one embodiment, the promoter is located upstream, 5', and operably linked to a heterologous nucleic acid sequence. In some embodiments, the promoter is a hepatocyte-specific promoter, a cardiomyocyte-specific promoter, a neuron-specific promoter, a nerve cell-type specific promoter, a muscle cell-type specific promoter, or another cell type-specific promoter.

[0237] In some embodiments, the constitutive promoter can be selected from a group of constitutive promoters with different strengths and tissue specificities. Some examples of these promoters are shown in Table 4. The rAAV vector genome can generally contain one or more constitutive promoters, such as viral promoters with transcription-promoting activity or promoters derived from mammalian genes. Examples of constitutive viral promoters are the herpes simplex virus (HSV) promoter, the thymidine kinase (TK) promoter, the Rous sarcoma virus (RSV) promoter, the simian virus 40 (SV40) promoter, the mouse mammary tumor virus (MMTV) promoter, the Ad EIA promoter, and the cytomegalovirus (CMV) promoter. Examples of constitutive mammalian promoters include various housekeeping gene promoters, such as those exemplified by the β-actin promoter and the chicken β-actin (CB) promoter, and the CB promoter has been found to be a particularly useful constitutive promoter for expressing GAA.

[0238] In one embodiment, the promoter is a tissue-specific promoter. Examples of tissue-specific promoters that can be used with the rAAV vector genome of the present invention include the creatine kinase promoter, the myogenin promoter, the alpha myosin heavy chain promoter, the myocyte-specific enhancer factor 2 (MEF2) promoter, the myoD enhancer element, albumin, the alpha-1-antitrypsin promoter, and the hepatitis B virus core protein promoter, wherein the hepatitis B virus core protein promoter is specific for hepatocytes.

[0239] In one embodiment, the promoter is an inducible promoter.Examples of suitable inducible promoters include those derived from genes such as cytochrome P450 gene, heat shock protein gene, metallothionein gene and hormone-inducible gene, for example, estrogen gene promoter.Another example of inducible promoter is the tetVP16 promoter, which is responsive to tetracycline.

[0240] Promoters within the rAAV genome disclosed herein include, but are not limited to, neuron-specific promoters such as the synapsin 1 (SYN) promoter; the muscle creatine kinase (MCK) promoter; and the desmin (DES) promoter. In one embodiment, AAV-mediated expression of heterologous nucleic acids (such as human GAA) can be achieved in neurons via the synapsin promoter or in skeletal muscle via the MCK promoter. Other promoters that can be used include the EF, B19p6, CAG, neuron-specific enolase gene promoter; chicken β-actin / CMV hybrid promoter; platelet-derived growth factor gene promoter; bGH, EF1a, CamKIIa, GFAP, RPE, ALB, TBG, MBP, MCK, TNT, aMHC, GFP, RFP, mCherry, CFP, and YFP promoters.

[0241] Table 4: Exemplary promoters TIFF2025032149000013.tif208164

[0242] Liver-specific promoter In some embodiments of the methods and compositions disclosed herein, the promoter is a liver-specific promoter, and can be selected from any liver-specific promoter, including but not limited to, transthyretin promoter (TTR), such as that disclosed in 5,863,541 (TTR promoter), liver-specific promoter (LSP), or LSP promoter (PNAS; 96:3906-3910, 1999, see, for example, page 3906, Materials and Methods, rAAV construction), synthetic liver promoter, and these references are incorporated herein by reference in their entirety. Other liver promoters, such as synthetic liver promoters, can also be used.

[0243] In some embodiments, the TTR promoter is a truncated TTR promoter, e.g., comprising SEQ ID NO:12 or a variant having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% nucleotide sequence identity thereto.

[0244] Other liver-specific promoters include, but are not limited to, promoters for LDL receptor, factor VIII, factor IX, phenylalanine hydroxylase (PAH), ornithine transcarbamylase (OTC), and alpha-1 antitrypsin (hAAT), as well as the HCB promoter. Other liver-specific promoters include the AFP (alpha-fetoprotein) gene promoter and albumin gene promoter as disclosed in EP Patent Publication 0 415 731, the alpha-1 antitrypsin gene promoter as disclosed in Rettenger, Proc. Natl. Acad. Sci. 91 (1994) 1460-1464, the fibrinogen gene promoter, the APO-A1 (apolipoprotein A1) gene promoter, and promoter genes for liver transferases such as SGOT, SGPT, and gamma glutamyltransferase. See also 2001 / 0051611 and PCT Patent Publications WO 90 / 07936 and WO 91 / 02805, which are incorporated by reference in their entireties. In some embodiments, the liver-specific promoter is a recombinant liver-specific promoter, such as those disclosed in US20170326256A1, which is incorporated by reference in its entirety.

[0245] In some embodiments, the liver-specific promoter is a hepatitis B X gene promoter and a hepatitis B core protein promoter. In some embodiments, a liver-specific promoter can be used with its respective enhancer. The enhancer element can be linked to either the 5' or 3' end of the nucleic acid encoding the GAA polypeptide. The hepatitis B X gene promoter and its enhancer can be obtained from the viral genome as a 332 base pair EcoRV-NcoI DNA fragment using the method described in Twu, J Virol. 61 (1987) 3448-3453. The hepatitis B core protein promoter can be obtained from the viral genome as a 584 base pair BamHI-BgIII DNA fragment using the method described in Gerlach, Virol 189 (1992) 59-66. It may be necessary to remove the negative regulatory sequences within the BamHI-BgIII fragment before insertion.

[0246] G. Intron Sequences In some embodiments, the rAAV genotype comprises an intron sequence located 3' to the promoter sequence and 5' to the secretory signal peptide. The intron sequence serves to increase one or more of mRNA stability, mRNA export to the nucleus, and / or expression and / or regulation of the expressed GAA fusion polypeptide (e.g., SS-GAA fusion polypeptide or SS-IGF2-GAA polypeptide).

[0247] In some embodiments, the intron sequence is an MVM intron sequence, such as, but not limited to, the intron sequence of SEQ ID NO:13, or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% nucleotide sequence identity thereto.

[0248] In some embodiments, the intron sequence is, but is not limited to, an HBB2 intron sequence, such as the intron sequence of SEQ ID NO:14, or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% nucleotide sequence identity thereto.

[0249] In some embodiments, the rAAV genotype comprises an intron sequence selected from the group consisting of a human beta globin b2 (or HBB2) intron, a FIX intron, a chicken beta globin intron, and an SV40 intron. In some embodiments, the intron is optionally a modified HBB2 intron (see, e.g., SEQ ID NO: 17 of WO2018046774A1), a modified FIX intron (see, e.g., SEQ ID NO: 19 of WO2018046774A1), or a modified chicken beta globin intron (see, e.g., SEQ ID NO: 21 of WO2018046774A1), or a modified intron such as the modified HBB2 or FIX introns disclosed in WO2015 / 162302, the entire contents of which are incorporated herein by reference.

[0250] H. PolyA In some embodiments, the rAAV vector genome comprises at least one polyA tail located 3' and downstream of a heterologous nucleic acid gene, which in one embodiment encodes a GAA fusion polypeptide (e.g., an SS-GAA fusion polypeptide or an SS-IGF2-GAA polypeptide). In some embodiments, the polyA signal is present 3' to the stability sequence or CS sequence defined herein. Any polyA sequence can be used, including, but not limited to, hGH polyA, synpA polyA, etc. In some embodiments, the polyA is a synthetic polyA sequence. In some embodiments, the rAAV vector genome comprises two polyA tails, for example, an hGH polyA sequence and another polyA sequence, with a spacer nucleic acid sequence located between the two polyA sequences. In some embodiments, the rAAV genome comprises the following elements 3' to the nucleic acid encoding a GAA fusion polypeptide (e.g., an SS-GAA fusion polypeptide or an SS-IGF2-GAA polypeptide) or 3' to the CS sequence: a first polyA sequence, a spacer nucleic acid sequence (100 to 400 bp or about 250 bp), a second polyA sequence, a spacer nucleic acid sequence, and a 3' ITR. In some embodiments, the first and second polyA sequences are hGH polyA sequences; in some embodiments, the first and second polyA sequences are synthetic polyA sequences. In some embodiments, the first polyA sequence is an hGH polyA sequence and the second polyA sequence is a synthetic sequence, or vice versa; i.e., in other embodiments, the first polyA sequence is a synthetic polyA sequence and the second polyA sequence is an hGH polyA sequence. An exemplary polyA sequence is, for example, SEQ ID NO: 15 (hGH polyA sequence), or a polyA nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% nucleotide sequence identity to SEQ ID NO: 15. In some embodiments, hGH polyA sequences encompassed for use are described in Anderson et al. J. Biol. Chem 264(14);8222-8229, 1989 (see, e.g., page 8223, second column, first paragraph), which is incorporated herein by reference in its entirety.

[0251] In some embodiments, the polyA tail can be modified to stabilize the RNA transcript transcribed from the rAAV vector genome, e.g., the transcript of the heterologous gene, which in one embodiment is GAA; in other embodiments, the polyA tail can be modified to contain destabilizing elements.

[0252] In one embodiment, the polyA tail can be modified to become a destabilizing element by altering the length of the polyA tail. In one embodiment, the polyA tail can be extended or shortened. In further embodiments, the 3' untranslated region present between the heterologous gene, in one embodiment, GAA, and the polyA tail can be extended or shortened to alter the expression level of the heterologous gene or to alter the final polypeptide produced. In some embodiments, the 3' untranslated region comprises the GAA 3'UTR (SEQ ID NO:85).

[0253] In another embodiment, the destabilizing element is a microRNA (miRNA) that has the ability to silence (repress translation and promote degradation) the RNA transcript to which the miRNA binds, encoding a heterologous gene. Modulation of the expression of a heterologous gene, in one embodiment, GAA, can be achieved by modifying, adding, or deleting the seed region within the polyA to which the miRNA binds. In one embodiment, adding or deleting the seed region within the polyA tail can increase or decrease the expression of a protein, in one embodiment, GAA, encoded by a heterologous gene in the rAAV vector genome. In a further embodiment, the increase or decrease in expression resulting from the addition or deletion of the seed region depends on the cell type transduced by the AAV containing the rAAV vector genome. For example, a seed region specific to an miRNA that is expressed in muscle cells and cardiac cells but not found in hepatocytes can be used to enable the polypeptide, in one embodiment, GAA, encoded by a heterologous gene to be produced in hepatocytes but not in muscle cells or cardiac cells.

[0254] In another embodiment, the seed region may be modified in the 3' untranslated region located between the heterologous gene and the polyA tail. In a further embodiment, the destabilizing agent may be an siRNA. An siRNA coding region may be included in the rAAV vector genome, generally located downstream, 3', of the polyA tail. In one embodiment, expression of a heterologous gene, in one embodiment, GAA, can be achieved by including an siRNA coding region in the rAAV cassette, for example, downstream, 3', of the polyA tail. In a further embodiment, the promoter driving siRNA expression may be tissue-specific, such that silencing of the siRNA occurs in tissues where expression of the heterologous gene, in one embodiment, GAA, is not desired, and siRNA expression does not occur in tissues where expression of the heterologous gene, in one embodiment, GAA, is desired.

[0255] In all aspects of the methods and compositions disclosed herein, the rAAV genome can also include a stuffer DNA nucleic acid sequence. An exemplary stuffer DNA sequence is SEQ ID NO:71, or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% nucleotide sequence identity thereto. As shown in Figures 8-13 and 14A-14E, the stuffer sequence is, for example, located 3' to the polyA tail and 5' to the '3 ITR sequence. In some embodiments, the stuffer DNA sequence includes a synthetic polyadenylation signal in reverse orientation.

[0256] In some embodiments, a stuffer nucleic acid sequence (also referred to as a "spacer" nucleic acid fragment; see Figures 8-14) may be located between the polyA sequence and the 3' ITR (i.e., the stuffer nucleic acid sequence is located 3' of the polyA sequence and 5' of the 3' ITR) (see, e.g., Figures 8-10). Such stuffer nucleic acid sequences can be about 30 bp, 50 bp, 75 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, or greater than 300 bp. In some embodiments of the methods and compositions disclosed herein, the stuffer nucleic acid fragment is 20-50 bp, 50-100 bp, 100-200 bp, 200-300 bp, 300-500 bp, or any integer between 20-500 bp. Exemplary stuffer (or spacer) nucleic acid sequences include SEQ ID NO:16, SEQ ID NO:71, or SEQ ID NO:78, or a nucleic acid sequence that is at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to SEQ ID NO:16 or SEQ ID NO:71 or SEQ ID NO:78.

[0257] I. AAV ITR The rAAV genomes disclosed herein contain AAV ITRs that have desirable characteristics and can be designed to modulate the activity of, and cellular responses to, vectors into which the ITRs are incorporated. In another embodiment, the AAV ITRs are synthetic AAV ITRs that have desirable characteristics and can be designed to manipulate the activity of, and cellular responses to, vectors containing one or two synthetic ITRs, as set forth in U.S. Patent No. 9,447,433, which is incorporated herein by reference.

[0258] The AAV ITR used in the rAAV genome disclosed herein can be of any serotype suitable for specific applications.In some embodiments, the AAV vector genome is flanked by AAV ITR.In some embodiments, the rAAV vector genome is flanked by AAV ITR, wherein ITR comprises full-length ITR sequence, ITR comprising the sequence comprising the removed CPG island, ITR comprising the sequence comprising the added CPG sequence, truncated ITR sequence, ITR sequence comprising one or more deletions in ITR, ITR sequence comprising one or more additions in ITR, or any combination comprising any part of the above-mentioned ITRs linked together to form hybrid ITR.

[0259] To facilitate long-term expression, in one embodiment, the polynucleotide encoding GAA may be placed between AAV inverted terminal repeats (ITRs) (e.g., a first 5'AAV ITR and a second 3'AAV ITR). AAV ITRs are found at both ends of the WT rAAV vector genome and serve as origins and primers for DNA replication. ITRs are required in cis for AAV DNA replication and for rescue or excision from prokaryotic plasmids. In one embodiment, the AAV ITR sequences contained within the nucleic acid of the rAAV genome may be derived from any AAV serotype (e.g., 1, 2, 3, 3b, 4, 5, 6, 7, 8, 9, and 10) or may be derived from multiple serotypes, for example, may include a combination of portions of two or more AAV serotypes to construct the ITRs. In one embodiment, for use in an rAAV vector comprising an rAAV vector genome, the first and second ITRs should include at least the minimum portions of the WT or modified ITRs required for packaging and replication. In some embodiments, the rAAV vector genome is flanked by AAV ITRs.

[0260] In some embodiments, the rAAV vector genome comprises at least one AAV ITR, wherein the ITR comprises, consists essentially of, or consists of (a) an AAV rep binding element; (b) an AAV terminal resolution sequence; and (c) an AAV RBE (Rep binding element); the ITR does not comprise other AAV ITR sequences. In another embodiment, elements (a), (b), and (c) are derived from an AAV2 ITR, and the ITR does not comprise other AAV2 ITR sequences. In a further embodiment, elements (a), (b), and (c) are derived from any AAV ITR, including, but not limited to, AAV2, AAV8, and AAV9. In some embodiments, the polynucleotide comprises two synthetic ITRs, which may be identical or different.

[0261] In some embodiments, the polynucleotide in the rAAV vector containing the rAAV vector genome contains two ITRs, which may be identical or different.It has been determined that three elements in the ITR are sufficient for ITR function.This minimum functional ITR can be used in all aspects of AAV vector construction and transduction.Additional deletions can define even smaller minimum functional ITRs.Shorter lengths advantageously allow for packaging and transduction of larger transgenic cassettes.

[0262] In another embodiment, each of the elements present in the synthetic ITRs may be the exact sequence present in a naturally occurring AAV ITR (WT sequence), or may differ slightly (e.g., by the addition, deletion, and / or substitution of 1, 2, 3, 4, 5, or more nucleotides) so long as the functionality of the elements of the AAV ITRs continues to function at a level sufficient to not differ substantially from the functionality of those same elements present in a naturally occurring AAV ITR.

[0263] In further embodiments, the rAAV vector containing the rAAV vector genome may contain one or more additional non-AAV cis-elements between the ITRs, such as elements that initiate transcription, elements that mediate enhancer function, elements that enable replication and symmetric distribution during mitosis, or elements that alter the persistence and processing of the transduced genome. Such elements are well known in the art and include, but are not limited to, promoters, enhancers, chromatin-binding sequences, telomere sequences, cis-acting microRNAs (miRNAs), and combinations thereof.

[0264] In another embodiment, the ITRs exhibit modified transcriptional activity compared to naturally occurring ITRs, such as ITR2 from AAV2. The ITR2 sequence is known to have promoter activity in nature. It also has termination activity, essentially similar to a poly(A) sequence. The minimal functional ITRs of the present invention exhibit transcriptional activity, albeit at a reduced level compared to ITR2, as shown in the Examples. Thus, in some embodiments, the ITRs are transcriptionally functional. In other embodiments, the ITRs are transcriptionally defective. In certain embodiments, the ITRs act as transcriptional insulators, for example, preventing transcription of a transgenic cassette present in a vector when the vector is integrated into a host chromosome.

[0265] One aspect of the present invention relates to an rAAV vector genome comprising at least one synthetic AAV ITR in which the nucleotide sequence of one or more transcription factor binding sites within the ITR has been deleted and / or substituted compared to the sequence of a naturally occurring AAV ITR, such as ITR2. In some embodiments, it is a minimal functional ITR in which one or more transcription factor binding sites have been deleted and / or substituted. In some embodiments, at least one transcription factor binding site has been deleted and / or substituted, for example, at least five or more or ten or more transcription factor binding sites, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 transcription factor binding sites.

[0266] In another embodiment, an rAAV vector comprising the rAAV vector genome described herein comprises a polynucleotide comprising at least one synthetic AAV ITR, in which one or more CpG islands (a cytosine base immediately followed by a guanine base (CpG), in such a configuration, cytosine has a tendency to be methylated), which are typically present at or near the transcription start site within the ITR, have been deleted and / or substituted. In one embodiment, deleting or reducing the number of CpG islands can reduce the immunogenicity of the rAAV vector. This is due to the reduced or complete inhibition of TLR-9 binding to the rAAV vector DNA sequence, which occurs in CpG islands. It is also well known that methylation of CpG motifs leads to transcriptional silencing. Removal of CpG motifs within the ITR is expected to result in reduced TLR-9 recognition and / or reduced methylation, and therefore reduced transgene silencing. In some embodiments, it is a minimal functional ITR in which one or more CpG islands have been deleted and / or replaced. In one embodiment, AAV ITR2 is known to contain 16 CpG islands, one or more of which, or all 16 of which can be deleted.

[0267] In some embodiments, at least one CpG motif is deleted and / or substituted, for example, at least 4 or more or 8 or more CpG motifs, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 CpG motifs.The phrase " deleted and / or substituted " as used herein means that one or both nucleotides in CpG motif are deleted, are replaced with different nucleotides, or any combination of deletion and substitution.

[0268] In another embodiment, the synthetic ITR comprises, consists essentially of, or consists of one of the nucleotide sequences listed below: In other embodiments, the synthetic ITR comprises, consists essentially of, or consists of a nucleotide sequence that is at least 80% identical, e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical, to one of the nucleotide sequences listed below. TIFF2025032149000014.tif205160

[0269] In certain embodiments, the rAAV vector genome described herein contains synthetic ITRs that can produce AAV viral particles capable of transducing host cells. Such ITRs can be used, for example, for viral delivery of heterologous nucleic acids. Examples of such ITRs include the above-listed MH-257, MH-258, and MH delta 258.

[0270] In another embodiment, the rAAV vector genome described herein containing synthetic ITRs is unable to produce AAV viral particles. Such ITRs can be used, for example, for non-viral transfer of heterologous nucleic acids. Examples of such ITRs include the MH telomere-1, MH telomere-2, and MH Pol II 258 listed above.

[0271] In a further embodiment, the rAAV vector genome described herein, which includes the synthetic ITRs of the present invention, further includes a second ITR, which may be identical to or different from the first ITR. In one embodiment, the rAAV vector genome further includes a sequence encoding a heterologous nucleic acid, such as a protein or functional RNA. In an additional embodiment, the second ITR cannot be separated by the Rep protein, resulting in double-stranded viral DNA.

[0272] In one embodiment, the rAAV vector genome comprises a polynucleotide comprising the synthetic ITRs of the present invention. In a further embodiment, the viral vector can be a parvovirus vector, e.g., an AAV vector. In another embodiment, the recombinant parvovirus particle (e.g., recombinant AAV particle) comprises the synthetic ITRs.

[0273] Another aspect of the invention relates to a method for increasing the transgenic DNA packaging capacity of an AAV capsid, comprising generating an rAAV vector genome comprising at least one synthetic AAV ITR, wherein the ITR comprises: (a) an AAV rep binding element; (b) an AAV terminal resolution sequence; and (c) an AAV RBE element; and the ITR does not comprise other AAV ITR sequences.

[0274] A further aspect of the present invention relates to a method of altering a cellular response to infection with an rAAV vector genome, comprising generating an rAAV vector genome comprising at least one synthetic ITR, wherein the nucleotide sequences of one or more transcription factor binding sites within the ITR have been deleted and / or substituted, and further wherein the rAAV vector genome comprises at least one synthetic ITR that alters the cellular response to infection.

[0275] An additional aspect of the invention relates to a method of altering a cellular response to infection with an rAAV vector genome, comprising generating an rAAV vector genome comprising at least one synthetic ITR, wherein one or more CpG motifs within the ITR are deleted and / or substituted, and wherein the vector comprising the at least one synthetic ITR results in an altered cellular response to infection.

[0276] III. Vectors and Virions In one embodiment, the rAAV vectors (also referred to as rAAV virions) disclosed herein comprise capsid proteins and an rAAV genome within the capsid proteins. The rAAV capsids of the rAAV virions used to treat Pompe disease are any of those listed in Table 1, or any combination thereof.

[0277] Table 1. AAV serotypes and exemplary published corresponding capsid sequences TIFF2025032149000015.tif122166TIFF2025032149000016.tif245170TIFF2025032 149000017.tif245166TIFF2025032149000018.tif241166TIFF2025032149000019.t if233166TIFF2025032149000020.tif242166TIFF2025032149000021.tif245166TIF F2025032149000022.tif245166TIFF2025032149000023.tif241166TIFF20250321490 00024.tif245166TIFF2025032149000025.tif237166TIFF2025032149000026.tif23 7166TIFF2025032149000027.tif241166TIFF2025032149000028.tif241166TIFF202 5032149000029.tif237166TIFF2025032149000030.tif245166TIFF20250321490000 31.tif245166TIFF2025032149000032.tif241166TIFF2025032149000033.tif228166

[0278] Table 2 lists exemplary chimeric or variant capsid proteins that can be used as AAV capsids in the rAAV vectors described herein, or in any combination with wild-type capsid proteins and / or other chimeric or variant capsid proteins now known or later identified, each of which is incorporated herein. In some embodiments, the rAAV vectors encompassed for use are chimeric vectors, such as those disclosed in US Pat. No. 9,012,224 and US Pat. No. 7,892,809, the entire contents of which are incorporated herein by reference.

[0279] In some embodiments, the rAAV vector is a haploid rAAV vector such as that disclosed in PCT / US18 / 22725, or a polyploid rAAV vector such as that disclosed in PCT / US2018 / 044632 and U.S. Patent Application No. 16 / 151,110, filed July 31, 2018, each of which is incorporated by reference in its entirety. In some embodiments, the rAAV vector is an rAAV3 vector such as that disclosed in 9,012,224 and WO 2017 / 106236, each of which is incorporated by reference in its entirety.

[0280] Table 2: Exemplary chimeric and rAAV variant capsids TIFF2025032149000034.tif178160TIFF2025032149000035.tif123163

[0281] In one embodiment, the rAAV vectors disclosed herein comprise capsid proteins associated with any of the following biological sequence files listed in the file wrappers of patents and published applications issued by the USPTO that describe chimeric or variant capsid proteins that may be incorporated into the AAV capsids of the invention in any combination with wild-type capsid proteins and / or other chimeric or variant capsid proteins now known or later identified (by way of example, 11486254 corresponds to U.S. Patent Application No. 11 / 486,254; the other biological sequence files should be read similarly): 11486254.raw, 11932017.raw, 12172121.raw, 12302206.raw, 12308959 ... 679144.raw, 13036343.raw, 13121532.raw, 13172915.raw, 13583920.raw, 13668120.raw, 13673351.raw, 13 679684.raw, 14006954.raw, 14149953.raw, 14192101.raw, 14194538.raw, 14225821.raw, 14468108.raw, 14 516544.raw, 14603469.raw, 14680836.raw, 14695644.raw, 14878703.raw, 14956934.raw, 15191357.raw, 15 284164.raw, 15368570.raw, 15371188.raw, 15493744.raw, 15503120.raw, 15660906.raw, and 15675677.raw.

[0282] In one embodiment, the AAV capsid proteins and viral capsids of the present invention may be chimeric, in that they may be derived from other viruses, optionally including all or part of capsid subunits derived from other parvoviruses or AAVs, as described, for example, in International Patent Publication WO 00 / 28004, which is incorporated by reference.

[0283] In some embodiments, the rAAV vector genome is single-stranded or monomeric duplex, as described in U.S. Patent No. 8,784,799, incorporated herein.

[0284] In a further embodiment, the AAV capsid proteins and viral capsids of the present invention may be polyploid (also called haploid), in that they may contain different combinations of VP1, VP2, and VP3 AAV serotypes within a single AAV capsid, as described in PCT / US18 / 22725, which is incorporated by reference.

[0285] In one embodiment, the rAAV vector useful in the treatment of Pompe disease disclosed herein is an AAV3b capsid. The AAV3b capsids included for use are described in 2017 / 106236, 9,012,224, and 7,892,809, the entire contents of which are incorporated herein by reference.

[0286] In some embodiments, the AAV3b capsid comprises SEQ ID NO: 44. In one embodiment, an AAV capsid used in the treatment of Pompe disease can be a modified AAV capsid derived in whole or in part from the AAV capsid set forth in SEQ ID NO: 44. In some embodiments, amino acids derived from the AAV3b capsid set forth in SEQ ID NO: 44 can be substituted or have been substituted with amino acids derived from another capsid of a different AAV serotype, where the substituted and / or inserted amino acids can be from any AAV serotype and can include naturally occurring or partially or fully synthetic amino acids.

[0287] In another embodiment, the AAV capsid used in the treatment of Pompe disease is an AAV3b265D capsid. In this specific embodiment, the AAV3b265D capsid comprises a modification of the amino acid sequence of the two-fold axial loop of the AAV3b capsid via replacement of the amino acid G265 of the AAV3b capsid with D265. In some embodiments, the AAV3b265D capsid comprises SEQ ID NO: 46. However, the modified viral capsids of the present invention are not limited to the AAV capsid set forth in SEQ ID NO: 46. In some embodiments, amino acids derived from the AAV3b265D set forth in SEQ ID NO. 46 can be or have been substituted with amino acids derived from a capsid from an AAV of a different serotype, where the substituted and / or inserted amino acids may be derived from any AAV serotype and may include naturally occurring or partially or completely synthetic amino acids.

[0288] In another embodiment, the rAAV vector useful in the treatment of Pompe disease disclosed herein is an AAV3b265D549A capsid. In this specific embodiment, the AAV3b265D549A capsid comprises a modification of the amino acid sequence of the two-fold axial loop of the AAV3b capsid via a replacement of the amino acid G265 of the AAV3b capsid with D265 and a replacement of the amino acid T549 of the AAV3b capsid with A549. In some embodiments, the AAV3b265D549A capsid comprises SEQ ID NO: 50. However, the modified viral capsid of the present invention is not limited to the AAV capsid set forth in SEQ ID NO: 50. In some embodiments, amino acids derived from AAV3b265D549A as set forth in SEQ ID NO:50 may be substituted or replaced with amino acids derived from a capsid derived from an AAV of a different serotype, where the substituted and / or inserted amino acids may be derived from any AAV serotype and may comprise naturally occurring or partially or fully synthetic amino acids. In some embodiments, amino acids derived from AAV3bSASTG (i.e., an AAV3b capsid comprising the Q263A / T265 mutations) may be substituted or replaced with amino acids derived from a capsid derived from an AAV of a different serotype, where the substituted and / or inserted amino acids may be derived from any AAV serotype and may comprise naturally occurring or partially or fully synthetic amino acids.

[0289] In another embodiment, the rAAV vector useful in treating Pompe disease disclosed herein is an AAV3b549A capsid. In this specific embodiment, the AAV3b549A capsid comprises a modification of the amino acid sequence of the two-fold axial loop of the AAV3b capsid via replacement of the amino acid T549 of the AAV3b capsid with A549. In some embodiments, the AAV3b549A capsid comprises SEQ ID NO: 52. However, the modified viral capsids of the present invention are not limited to the AAV capsid set forth in SEQ ID NO: 52. In some embodiments, amino acids derived from the AAV3b549A set forth in SEQ ID NO: 52 can be or have been substituted with amino acids derived from a capsid from an AAV of a different serotype, where the substituted and / or inserted amino acids may be derived from any AAV serotype and may include naturally occurring or partially or fully synthetic amino acids.

[0290] In another embodiment, the rAAV vector useful in treating Pompe disease disclosed herein is an AAV3bQ263Y capsid. In this specific embodiment, the AAV3bQ263Y capsid comprises a modification of the amino acid sequence of the two-fold axial loop of the AAV3b capsid via replacement of amino acid Q263 with Y263 of the AAV3b capsid. In some embodiments, the AAV3b549A capsid comprises SEQ ID NO: 54. However, the modified viral capsids of the present invention are not limited to the AAV capsid set forth in SEQ ID NO: 54. In some embodiments, amino acids derived from the AAV3bQ263Y set forth in SEQ ID NO: 54 can be or have been substituted with amino acids derived from a capsid from an AAV of a different serotype, where the substituted and / or inserted amino acids may be derived from any AAV serotype and may include naturally occurring or partially or completely synthetic amino acids.

[0291] In another embodiment, the rAAV vectors useful in treating Pompe disease disclosed herein are of the AAV3bSASTG serotype or comprise an AAV3bSASTG capsid. In this specific embodiment, the AAV3bSASTG capsid comprises amino acid sequence modifications to include the SASTG mutation; specifically, the AAV3b capsid is modified to resemble the AAV2 Q263A / T265 subvariant (see Messina EL, et al., Adeno-associated viral vectors based on serotype 3b use components of the fibroblast growth factor receptor signaling complex for efficient transduction. Hum. Gene Ther. 2012 Oct:23(10):1031-4; Piacentino III, Valentino, et al., "X-linked inhibitor of apoptosis protein-mediated attenuation of apoptosis, using a novel cardiac-enhanced adeno-associated viral vector." Human gene therapy, both of which are incorporated by reference in their entireties. 23.6(2012):635-646) by introducing these modifications into analogous positions within the AAV3b capsid. Thus, in some embodiments, the rAAV vectors useful in treating Pompe disease disclosed herein are of the AAV3bSASTG serotype or comprise an AAV3bSASTG capsid, including an AAV3b Q263A / T265 capsid. In some embodiments, amino acids from AAV3bSASTG can be substituted or have been substituted with amino acids from a capsid derived from an AAV of a different serotype, where the substituted and / or inserted amino acids can be from any AAV serotype and can include naturally occurring or partially or fully synthetic amino acids.

[0292] To facilitate cell introduction, the rAAV vector genome useful in the present invention is a recombinant nucleic acid construct containing (1) a heterologous sequence to be expressed (in one embodiment, a polynucleotide encoding a GAA polypeptide), and (2) viral sequence elements that facilitate the integration and expression of the heterologous gene. The viral sequence elements may include sequences of the AAV vector genome required in cis for DNA replication and packaging into AAV capsids (e.g., functional ITRs). In one embodiment, the heterologous gene encodes GAA, which is useful for correcting GAA deficiency in patients with Pompe disease. In one embodiment, such a rAAV vector genome may also contain a marker or reporter gene. In one embodiment, the rAAV vector genome has one or more of the AAV3b wild-type (WT) cis genes replaced or deleted in whole or in part, but may retain functional flanking ITR sequences.

[0293] In one embodiment, the rAAV vector disclosed herein useful in the treatment of Pompe disease comprises a rAAV genome disclosed herein encapsulated by an AAV3b capsid. In some embodiments, the rAAV vectors disclosed herein useful in treating Pompe disease comprise a rAAV genome disclosed herein encapsulated by any AAV3b capsid selected from an AAV3b capsid (SEQ ID NO:44); an AAV3b265D capsid (SEQ ID NO:46), an AAV3b ST(S663V+T492V) capsid (SEQ ID NO:48), an AAV3b265D549A capsid (SEQ ID NO:50); an AAV3b549A capsid (SEQ ID NO:52); an AAV3bQ263Y capsid (SEQ ID NO:54), or an AAV3bSASTG (i.e., Q263A / T265) capsid.

[0294] In some embodiments of the methods and compositions disclosed herein, the rAAV vectors disclosed herein are selected from the group consisting of SEQ ID NO:57 (AAT-V43M-wtGAA(delta 1-69 aa)); SEQ ID NO:58 (ratFN1-IGF2V43M-wtGAA(delta 1-69 aa)); SEQ ID NO:59 (hFN1-IGF2V43M-wtGAA(delta 1-69 aa)); SEQ ID NO:60 (ATT-IGF2Δ2-7-wtGAA(delta 1-69)); SEQ ID NO:61 (FN1 rat-IGFΔ2-7-wtGAA(delta 1-69)); SEQ ID NO: The nucleic acid sequence includes any of NO:62 (hFN1-IGFΔ2-7-wtGAA(Delta 1-69)), or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity thereto. In some embodiments of the methods and compositions disclosed herein, the rAAV vector is selected from the group consisting of AAT_hIGF2-V43M_wtGAA_del1-69_stuffer.V02 (SEQ ID NO:79); FIB rat_hIGF2-V43M_wtGAA_del1-69_stuffer.V02 (SEQ ID NO:80); FIB hum_hIGF2-V43M_wtGAA_del1-69_stuffer.V02 (SEQ ID NO:81); AAT_GILT_wtGAA_del1-69_stuffer.V02 (SEQ ID NO:82); FIB rat_GILT_wtGAA_del1-69_stuffer.V02 (SEQ ID NO:83); FIB hum_GILT_wtGAA_del1-69_stuffer.V02 (SEQ ID NO:84); NO:84), or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity thereto.

[0295] IV. Optimized rAAV Vector Genome In some embodiments of the methods and compositions disclosed herein, the optimized rAAV vector genome is generated from any of the elements disclosed herein and includes, in any combination, a nucleic acid sequence encoding a promoter, ITRs, a polyA tail, elements that can increase or decrease expression of a heterologous gene, and in one embodiment, a nucleic acid sequence that is codon-optimized for expression of the GAA protein in vivo (i.e., coGAA), and optionally includes one or more elements for reducing immunogenicity. Such optimized rAAV vector genomes can be used with any AAV capsid that has tropism for the tissues and cells in which the rAAV vector genome is to be transduced and expressed.

[0296] AAV3b capsid modification In some embodiments of the methods and compositions disclosed herein, the AAV3b capsid for use in the rAAV vectors disclosed herein is an AAV3b capsid (SEQ ID NO:44); AAV3b265D capsid (SEQ ID NO:46), AAV3b ST(S663V+T492V) capsid (SEQ ID NO:48), AAV3b265D549A capsid (SEQ ID NO:50); AAV3b549A capsid (SEQ ID NO:52); AAV3bQ263Y capsid (SEQ ID NO:54), or a capsid similar to those described in Nienaber et al., Hum. Gen Ther., 2012, 23(10); 1031-42 and Piacentino III, Valentino, et al., "X-linked inhibitor of apoptosis protein-mediated attenuation of apoptosis, using a novel cardiac-enhanced adeno-associated viral vector." Human gene therapy 23.6(2012):635-646 (both of which are incorporated by reference in their entireties), the AAV3b capsid may have, for example, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, about 75% to about 99%, about 80% to about 99%, about 85% to about 99%, about 90% to about 99%, about 95% to about 99%, about 75% to about 97%, about 80% to about 97%, about 85% to about 97%, about 90% to about 97%, or about 95% to about 97% amino acid identity with any of the AAV3bSASTG capsids (i.e., AAV3b capsids containing the Q263A / T265 mutations) disclosed inIn yet another aspect of this embodiment, the AAV derived from AAV3b is an AAV3b capsid (SEQ ID NO: 44); AAV3b265D capsid (SEQ ID NO: 46), AAV3b ST(S663V+T492V) capsid (SEQ ID NO: 48), AAV3b265D549A capsid (SEQ ID NO: 50); AAV3b549A capsid (SEQ ID NO: 52); AAV3bQ263Y capsid (SEQ ID NO: 54), or the capsids described in Nienaber et al., Hum. Gen Ther., 2012, 23(10); 1031-42 and Piacentino III, Valentino, et al., "X-linked inhibitor of apoptosis protein-mediated attenuation of apoptosis, using a novel cardiac-enhanced adeno-associated viral vector." Human gene therapy 23.6(2012):635-646 (i.e., AAV3b capsids containing the Q263A / T265 mutation), for example, in the range of about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, about 75% to about 99%, about 80% to about 99%, about 85% to about 99%, about 90% to about 99%, about 95% to about 99%, about 75% to about 97%, about 80% to about 97%, about 85% to about 97%, about 90% to about 97%, or about 95% to about 97% amino acid identity, yet the capsid is still an AAV protein having functional activity.

[0297] In some embodiments of the methods and compositions disclosed herein, the AAV serotype (e.g., AAV3b) comprises a SASTG mutation as described in Messina EL, et al., Adeno-associated viral vectors based on serotype 3b use components of the fibroblast growth factor receptor signaling complex for efficient transduction. Hum. Gene Ther. 2012 Oct:23(10):1031-42, which is incorporated herein by reference in its entirety.

[0298] In some embodiments of the methods and compositions disclosed herein, the AAV3b capsid for use in the rAAV vectors disclosed herein is, for example, an AAV3b capsid (SEQ ID NO:44); an AAV3b265D capsid (SEQ ID NO:46), an AAV3b ST(S663V+T492V) capsid (SEQ ID NO:48), an AAV3b265D549A capsid (SEQ ID NO:50); an AAV3b549A capsid (SEQ ID NO:52); an AAV3bQ263Y capsid (SEQ ID NO:54), or (Nienaber et al., Hum. Gen. a deletion, addition, and / or substitution of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 consecutive amino acids compared to the amino acid sequence of an AAV3bSASTG capsid (i.e., an AAV3b capsid containing the Q263A / T265 mutations) (as disclosed in Ther., 2012, 23(10); 1031-42); or an AAV3b capsid (SEQ ID NO: 44); an AAV3b265D capsid (SEQ ID NO: 46), an AAV3b ST(S663V+T492V) capsid (SEQ ID NO: 48), an AAV3b265D549A capsid (SEQ ID NO: 50); an AAV3b549A capsid (SEQ ID NO: 51), NO:52); AAV3bQ263Y capsid (SEQ ID NO:54), AAV3bSASTG capsid (i.e., AAV3b capsid containing the Q263A / T265 mutations) (as disclosed in Nienaber et al., Hum. Gen Ther, 2012, 23(10); 1031-42), or an AAV3bSASTG capsid (i.e., an AAV3b capsid containing the Q263A / T265 mutations) that have deletions, additions, and / or substitutions of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 consecutive amino acids.In yet another embodiment, the AAV3b capsid for use in the rAAV vectors disclosed herein is, for example, an AAV3b capsid (SEQ ID NO:44); an AAV3b265D capsid (SEQ ID NO:46), an AAV3b ST(S663V+T492V) capsid (SEQ ID NO:48), an AAV3b265D549A capsid (SEQ ID NO:50); an AAV3b549A capsid (SEQ ID NO:52); an AAV3bQ263Y capsid (SEQ ID NO:54), or (Nienaber et al., Hum. Gen. a deletion, addition, and / or substitution of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 consecutive amino acids compared to the amino acid sequence of an AAV3bSASTG capsid (i.e., an AAV3b capsid containing the Q263A / T265 mutations) (as disclosed in Ther., 2012, 23(10); 1031-42); or an AAV3b capsid (SEQ ID NO: 44); an AAV3b265D capsid (SEQ ID NO: 46), an AAV3b ST(S663V+T492V) capsid (SEQ ID NO: 48), an AAV3b265D549A capsid (SEQ ID NO: 50); an AAV3b549A capsid (SEQ ID NO: 51), NO:52); AAV3bQ263Y capsid (SEQ ID NO:54), AAV3bSASTG capsid (i.e., an AAV3b capsid containing the Q263A / T265 mutations) (as disclosed in Nienaber et al., Hum. Gen Ther, 2012, 23(10); 1031-42), while still having functional activity.

[0299] In some embodiments of the methods and compositions disclosed herein, the AAV3b capsid for use in the rAAV vectors disclosed herein can be, for example, an AAV3b capsid (SEQ ID NO:44); AAV3b265D capsid (SEQ ID NO:46), AAV3b ST(S663V+T492V) capsid (SEQ ID NO:48), AAV3b265D549A capsid (SEQ ID NO:50); AAV3b549A capsid (SEQ ID NO:52); AAV3bQ263Y capsid (SEQ ID NO:54), (Nienaber et al., Hum. Gen. Ther., 2012, 23(10); 1031-42) with the amino acid sequence of an AAV3bSASTG capsid (i.e., an AAV3b capsid containing the Q263A / T265 mutation) in the range of about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, about 75% to about 99%, about 80% to about 99%, about 85% to about 99%, about 90% to about 99%, about 95% to about 99%, about 75% to about 97%, about 80% to about 97%, about 85% to about 97%, about 90% to about 97%, or about 95% to about 97%.In further embodiments, the AAV3b capsid for use in the rAAV vectors disclosed herein can be, for example, an AAV3b capsid (SEQ ID NO:44); AAV3b265D capsid (SEQ ID NO:46), AAV3b ST(S663V+T492V) capsid (SEQ ID NO:48), AAV3b265D549A capsid (SEQ ID NO:50); AAV3b549A capsid (SEQ ID NO:52); AAV3bQ263Y capsid (SEQ ID NO:54), (Nienaber et al., Hum. Gen. Ther., 2012, 23(10); 1031-42), but still has functional activity.

[0300] V. Treatment Methods A. Pompe disease Pompe disease is a rare genetic disorder caused by a deficiency of acid alpha-glucosidase (GAA), an enzyme required to break down glycogen, a storage form of sugar used for energy. Pompe disease is also known as glycogen storage disease type II, GSD II, glycogen storage disease type II, acid maltase deficiency, alpha-1,4-glucosidase deficiency, diffuse glycogenic cardiac hypertrophy, and generalized glycogen storage disease cardiac. Glycogen accumulation causes progressive muscle weakness throughout the body (myopathy), affecting various body systems, particularly the heart, skeletal muscle, liver, respiratory, and nervous systems.

[0301] The major clinical manifestations of Pompe disease vary widely depending on the age of disease onset and residual GAA activity. Residual GAA activity correlates with the amount and tissue distribution of glycogen stores and with the severity of the disease. Infantile-onset Pompe disease (<1% of normal GAA activity) is the most severe form and is characterized by hypotonia, generalized muscle weakness, and hypertrophic cardiomyopathy, as well as massive glycogen accumulation in the heart and other muscle tissues. Death typically occurs within the first year of life due to cardiopulmonary failure. Hirschhorn et al. (2001) "Glycogen Storage Disease Type II: Acid Alpha-glucosidase (Acid Maltase) Deficiency," in Scriver et al., eds., The Metabolic and Molecular Basis of Inherited Disease, 8th Ed., New York: McGraw-Hill, 3389-3420. Juvenile-onset (1-10% of normal GAA activity) and adult-onset (10-40% of normal GAA activity) Pompe disease are clinically more heterogeneous, with greater variability in age of onset, clinical manifestations, and disease progression. Juvenile-onset and adult-onset Pompe disease are generally characterized by the absence of severe cardiac involvement, a later age of onset, and slower disease progression, but the resulting involvement of respiratory or limb muscles results in significant morbidity and mortality. Life expectancy is variable, but death generally occurs due to respiratory failure. Hirschhorn et al. (2001) "Glycogen Storage Disease Type II: Acid Alpha-glucosidase (Acid Maltase) Deficiency," in Scriver et al., eds., The Metabolic and Molecular Basis of Inherited Disease, 8th Ed., New York: McGraw-Hill, 3389-3420.

[0302] In any embodiment of the methods and compositions disclosed herein, suitable GAA enzymes for treating Pompe disease include wild-type human GAA, or fragments or sequence variants thereof that retain the ability to cleave the α1-4 linkage of linear oligosaccharides. In some embodiments of the methods and compositions disclosed herein, the GAA protein is encoded by a wild-type GAA nucleic acid sequence, for example, SEQ ID NO:11 or SEQ ID NO:72. In some embodiments of the methods and compositions disclosed herein, the GAA protein is encoded by a GAA nucleic acid sequence that has been codon-optimized for one or more of the following: (1) enhanced in vivo expression, (2) reduced CpG islands, or (3) reduced innate immune response. In some embodiments of the methods and compositions disclosed herein, the GAA protein is encoded by any nucleic acid sequence selected from codon-optimized GAA nucleic acid sequences, such as SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76, or any nucleic acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76.

[0303] In some embodiments of the methods and compositions disclosed herein, the rAAV vector described herein transduces the liver of a subject and secretes the hGAA polypeptide into the blood perfusing the patient's tissue, where it is taken up by cells with the aid of the fused IGF2 sequence and transported to lysosomes, where the enzyme acts to eliminate material accumulated in the lysosomes due to the enzyme deficiency. For lysosomal enzyme replacement therapy to be effective, the therapeutic enzyme must be delivered to the lysosomes of the appropriate cells in the tissue where the storage defect is manifested.

[0304] The terms "cation-independent mannose-6-phosphate receptor (CI-MPR)," "M6P / IGF-II receptor," "CI-MPR / IGF-II receptor," "IGF-II receptor," or "IGF2 receptor," or their abbreviations, are used interchangeably herein and refer to a cellular receptor that binds both M6P and IGF-II.

[0305] B. Modulation of GAA levels in cells ex vivo The nucleic acids, vectors, and virions described herein can be used to modulate intracellular GAA levels. The method includes administering to cells a composition containing a nucleic acid comprising a polynucleotide encoding GAA located between two AAV ITRs. The cells can be derived from any animal to which the nucleic acids of the present invention can be administered. Mammalian cells derived from subjects with GAA deficiency (e.g., humans, dogs, cats, pigs, sheep, mice, rats, rabbits, cows, goats, etc.) are typical target cells for use in the present invention. In some embodiments, the cells are hepatocytes or myocardial cells, e.g., myocardiocytes.

[0306] In one embodiment, ex vivo delivery of cells transduced by rAAV vectors is disclosed herein. In a further embodiment, ex vivo gene delivery can be used to transplant cells transduced by the rAAV vectors disclosed herein back into a host. In a further embodiment, ex vivo stem cell (e.g., mesenchymal stem cell) therapy can be used to transplant cells transduced by the rAAV vectors disclosed herein back into a host. In another embodiment, a suitable ex vivo protocol can include several steps.

[0307] In some embodiments, a segment of target tissue (e.g., muscle, liver tissue) can be collected from a subject, and the rAAV vector described herein can be used to transduce a nucleic acid encoding GAA into the host's cells. These genetically modified cells can then be transplanted back into the host. Several approaches can be used to reintroduce cells into the host, including intravenous injection, intraperitoneal injection, subcutaneous injection, or in situ injection into the target tissue. Microencapsulation of modified ex vivo cells transduced or infected with the rAAV vector described herein is another technique that can be used in the present invention. Autologous and allogeneic cell transplantation can be used in accordance with the present invention.

[0308] In yet another embodiment, disclosed herein is a method for treating a GAA deficiency in a subject, comprising administering to the subject a therapeutically effective amount of cells expressing GAA as disclosed herein in a pharmaceutically acceptable carrier. In some embodiments, the subject is a human.

[0309] C. Increasing GAA Activity in a Subject The nucleic acids, vectors, and virions described herein can be used to modulate the level of functional GAA polypeptide in a subject, such as a human subject, or a subject with or at risk of having Pompe disease. The method includes administering to the subject a composition comprising an rAAV vector comprising the rAAV genome described herein, which comprises a heterologous nucleic acid encoding GAA located between two AAV ITRs, wherein hGAA is linked to a signal peptide described herein and optionally linked to an IGF-2 sequence disclosed herein. The subject can be any animal, and suitable subjects include mammals (e.g., humans, dogs, cats, pigs, sheep, mice, rats, rabbits, cows, goats, etc.). The methods and compositions of the present invention are particularly applicable to GAA-deficient human subjects.

[0310] Furthermore, the nucleic acids, vectors, and virions described herein can be administered to animals, including humans, by any suitable method and in any suitable formulation. For example, in any embodiment of the methods and compositions disclosed herein, the rAAV vectors or rAAV genomes disclosed herein may be introduced directly into an animal through, for example, oral, rectal, transmucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or intraembryonic), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [e.g., administration to skeletal muscle, diaphragm muscle, and / or cardiac muscle], intradermal, intrapleural, intracerebral, and intra-articular), topical (e.g., both cutaneous and mucosal surfaces, e.g., respiratory tract surfaces, and transdermal administration), intralymphatic, etc., administration, by direct injection into a tissue or organ (e.g., into the liver, skeletal muscle, cardiac muscle, diaphragm muscle, or brain), or other parenteral routes, depending on the desired route of administration and the tissue being targeted.

[0311] In any embodiment of the methods and compositions disclosed herein, administration to skeletal muscles according to the present invention includes, but is not limited to, administration to skeletal muscles of the limbs (e.g., upper arm, forearm, thigh, and / or lower leg), back, neck, head (e.g., tongue), chest, abdomen, pelvis / perineum, and / or digits. Suitable skeletal muscles include the abductor digiti minimi (of the hand), abductor hallucis (of the foot), abductor hallucis (of the digit), abductor ossis metatarsi (of the fifth metatarsal), and the skeletal muscles of the pelvis / perineum. quinti), abductor pollicis brevis, abductor pollicis longus, adductor brevis, adductor hallucis (digital) adductor, adductor longus, adductor magnus, adductor pollicis, elbow muscle, anterior scalene muscle, genu muscles, biceps brachii, biceps femoris, brachialis, brachialis, brachioradialis, buccinator, coracobrachialis, corrugator supercilii, deltoid, depressor anguli oris, depressor labii inferioris, Digastric, dorsal interosseous muscle (of the hand), dorsal interosseous muscle (of the foot), extensor carpi radialis brevis, extensor carpi radialis longus, extensor carpi ulnaris, extensor digitorum minimi, extensor digitorum brevis, extensor digitorum longus, extensor hallucis brevis, extensor hallucis longus, extensor index muscle, extensor pollicis brevis, extensor pollicis longus, flexor carpi radialis Muscles: Flexor carpi ulnaris, Flexor digitorum minimi brevis (of the hand), Flexor digitorum brevis (of the foot), Flexor digitorum brevis, Flexor digitorum longus, Flexor digitorum profundus, Flexor digitorum superficialis, Flexor hallucis brevis, Flexor hallucis longus, Flexor pollicis brevis, Flexor pollicis longus, Frontalis, Gastrocnemius, Geniohyoid, Gluteus maximus, Gluteus medius, Gluteus minimus, Gracilis, Iliocostalis cervix, Iliocostalis lumborum, Iliocostalis thoracic, Iliacus, Gemini inferior, Inferior oblique, Inferior rectus, Infraspinatus, Interspinatus, Intertransversus, Lateral pterygoid, Lateral rectus, Latissimus dorsi, Levator angle oris, Levator labii superioris, Levator ala labii naris superioris, Levator palpebrae superioris, Levator scapulae, Rotator longus rotator), longissimus capitis, longissimus cervix, longissimus thoracis, longus capitis, longus colli, lumbrical muscles (of the hand), lumbrical muscles (of the foot), masseter, medial pterygoid, medial rectus, scalene medialis, multifidus, mylohyoid, oblique capitis inferior, oblique capitis superior, obturator externus, obturator internus, occipitalis, omohyoid, opponens digiti minimi, opponens pollicis, orbicularis oculi, orbicularis oris, palmar interosseous muscles, palmaris brevis, palmaris longus, pubococcus, pectoralis major, pectoralis minor, peroneus brevis , peroneus longus, third peroneus, piriformis, plantar interosseus, plantaris, platysma, popliteus, posterior scalene, pronator quadratus, pronator teres, psoas major, quadratus femoris, quadratus plantaris, rectus capitis anterior, rectus capitis lateralis, rectus capitis posterior major, rectus capitis posterior minor, rectus femoris, rhomboid major, rhomboid minor, sartorius, scalene minimus, semimembranosus, semispinalis capitis, semispinalis cervix, semispinalis thoracic, semitendinosus, serratus anterior, short rotatorrotator, soleus, spinalis capitis, spinalis cervicis, thoracics, splenius capitis, splenius cervicis, sternocleidomastoid, sternohyoid, sternothyroid, stylohyoid, subclavius, subscapularis, superior gemellus, superior oblique, superior rectus, supinator, supraspinatus, temporalis, tensor fascia lata, teres major, teres minor, thoracis, thyrohyoid, tibialis anterior, tibialis posterior, trapezius, triceps brachii, vastus intermedius, vastus lateralis, vastus medialis, zygomaticus major, and zygomaticus minor, as well as any other suitable skeletal muscles known in the art.

[0312] In any embodiment of the methods and compositions disclosed herein, administration to the myocardium includes administration to the left atrium, right atrium, left ventricle, right ventricle, and / or septum. The viral vector and / or capsid can be delivered to the myocardium by intravenous administration, intra-arterial administration, e.g., intra-aortic administration, direct cardiac injection (e.g., into the left atrium, right atrium, left ventricle, right ventricle), and / or coronary perfusion.

[0313] In any embodiment of the methods and compositions disclosed herein, administration to the diaphragm muscle can be by any suitable method, including intravenous administration, intraarterial administration, and / or intraperitoneal administration.

[0314] In any embodiment of the methods and compositions disclosed herein, the rAAV vectors and / or rAAV genomes disclosed herein are administered to cells of the skeletal muscle, liver, diaphragm, ribs, and / or cardiac muscle of a subject. For example, a conventional syringe and needle can be used to inject a suspension of rAAV virions into an animal. Parenteral administration of the rAAV vectors and / or rAAV genomes by injection can be performed, for example, by bolus injection or continuous infusion. The formulations for injection can be presented in unit dosage form, for example, in ampoules or multi-dose containers with added preservatives. The compositions can take the form of a suspension, solution, or emulsion in an oily or aqueous medium, and can contain pharmaceutical formulation agents such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the rAAV vectors and / or rAAV genomes disclosed herein can be in powder form (e.g., lyophilized) for reconstitution with an appropriate medium, for example, sterile pyrogen-free water, before use.

[0315] In specific embodiments, multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., or more administrations) can be used to achieve a desired level of gene expression over a period of time, such as hours, days, weeks, months, years, etc. Dosing can be a single dosage or can be cumulative (continuous dosing), and can be readily determined by one of skill in the art. For example, treatment of a disease or disorder can include a single administration of an effective dose of a pharmaceutical composition viral vector disclosed herein. Alternatively, treatment of a disease or disorder can include multiple administrations of an effective dose of a viral vector administered over a range of time periods, such as once daily, twice daily, three times daily, every 2-3 days, or once weekly.

[0316] The timing of administration can vary from individual to individual, depending on factors such as the severity of the individual's symptoms.For example, an effective dose of the viral vector disclosed herein can be administered to an individual once every six months indefinitely or until the individual no longer requires treatment.Those skilled in the art will recognize that during the course of treatment, the individual's condition can be monitored, and the effective amount of the viral vector disclosed herein that is administered can be adjusted accordingly.

[0317] Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquid prior to injection, or emulsions. Alternatively, the viral vectors and / or viral capsids of the present invention can be administered locally rather than systemically, for example, in a depot or sustained-release formulation. Furthermore, viral vectors and / or viral capsids can be delivered attached to a surgically implantable matrix (e.g., as described in U.S. Patent Application Publication No. US-2004-0013645-A1). The viral vectors and / or viral capsids disclosed herein can be administered to a subject's lungs by any suitable means, optionally by administering an aerosol suspension of respirable particles composed of the viral vectors and / or viral capsids, which are inhaled by the subject. The respirable particles can be in liquid or solid form. As known to those skilled in the art, aerosols of liquid particles containing viral vectors and / or viral capsids can be generated by any suitable means, such as a pressure-driven aerosol nebulizer or an ultrasonic nebulizer. See, e.g., U.S. Patent No. 4,501,729. Aerosols of solid particles containing viral vectors and / or capsids can similarly be generated by any solid particle pharmaceutical aerosol generator using techniques known in the pharmaceutical arts.

[0318] In some embodiments, the rAAV vectors and / or rAAV genomes disclosed herein can be formulated with a solvent, emulsion, or other diluent in an amount sufficient to dissolve the rAAV vectors disclosed herein. In other aspects of this embodiment, the rAAV vectors and / or rAAV genomes disclosed herein may be formulated in an amount of solvent, emulsion, or other diluent, e.g., less than about 90% (v / v), less than about 80% (v / v), less than about 70% (v / v), less than about 65% (v / v), less than about 60% (v / v), less than about 55% (v / v), less than about 50% (v / v), less than about 45% (v / v), less than about 40% (v / v), less than about 35% (v / v), less than about 30% (v / v), less than about 25% (v / v), less than about 20% (v / v), less than about 15% (v / v), less than about 10% (v / v), less than about 5% (v / v), or less than about 1% (v / v).In other aspects, the rAAV vectors and / or rAAV genomes disclosed herein are, for example, about 1% (v / v) to 90% (v / v), about 1% (v / v) to 70% (v / v), about 1% (v / v) to 60% (v / v), about 1% (v / v) to 50% (v / v), about 1% (v / v) to 40% (v / v), about 1% (v / v) to 30% (v / v), or the like. (v / v), approximately 1%(v / v)~20%(v / v), approximately 1%(v / v)~10%(v / v), approximately 2%(v / v)~50%(v / v), approximately 2%(v / v)~40%(v / v), approximately 2% (v / v) ~ 30% (v / v), approximately 2% (v / v) ~ 20% (v / v), approximately 2% (v / v) ~ 10% (v / v), approximately 4% (v / v) ~ 50% (v / v), Approx. 4%(v / v)~40%(v / v), Approx. 4%(v / v)~30%(v / v), Approx. 4%(v / v)~20%(v / v), Approx. 4%(v / v)~10%(v / v), Approx. 6% (v / v) ~ 50% (v / v), approximately 6% (v / v) ~ 40% (v / v), approximately 6% (v / v) ~ 30% (v / v), approximately 6% (v / v) ~ 20% (v / v), approximately 6% (v / The composition may contain a solvent, emulsion, or other diluent in an amount ranging from about 8% (v / v) to 10% (v / v), about 8% (v / v) to 50% (v / v), about 8% (v / v) to 40% (v / v), about 8% (v / v) to 30% (v / v), about 8% (v / v) to 20% (v / v), about 8% (v / v) to 15% (v / v), or about 8% (v / v) to 12% (v / v).

[0319] In any embodiment of the methods and compositions disclosed herein, the rAAV vectors and / or rAAV genomes disclosed herein encapsulated by any AAV3b capsid of any serotype, for example, but not limited to, selected from: an AAV3b capsid (SEQ ID NO:44); an AAV3b265D capsid (SEQ ID NO:46), an AAV3b ST(S663V+T492V) capsid (SEQ ID NO:48), an AAV3b265D549A capsid (SEQ ID NO:50); an AAV3b549A capsid (SEQ ID NO:52); an AAV3bQ263Y capsid (SEQ ID NO:54), or an AAV3bSASTG capsid (i.e., an AAV3b capsid containing the Q263A / T265 mutations), may comprise a therapeutically effective amount of a therapeutic compound. In one embodiment, as used herein, without limitation, the term "effective amount" is synonymous with "therapeutically effective amount," "effective dose," or "therapeutically effective dose." In one embodiment, the effectiveness of the therapeutic compounds disclosed herein for treating Pompe disease can be determined by observing an improvement in an individual based on, without limitation, one or more clinical symptoms and / or physiological indicators associated with Pompe disease. In one embodiment, an improvement in symptoms associated with Pompe disease can be indicated by a reduced need for concomitant treatment.

[0320] Exemplary modes of administration include oral, rectal, transmucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or intraembryonic), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [e.g., administration to skeletal muscle, diaphragm muscle, and / or cardiac muscle], intradermal, intrapleural, intracerebral, and intraarticular), topical (e.g., both skin and mucosal surfaces, e.g., respiratory tract surfaces, and transdermal administration), intralymphatic, etc., including direct injection into a tissue or organ (e.g., into the liver, skeletal muscle, cardiac muscle, diaphragm muscle, or brain). Administration may also be to a tumor (e.g., at or near a tumor or lymph node). The most appropriate route in a given case will depend on the nature and severity of the condition being treated and / or prevented and the nature of the specific vector used.

[0321] To facilitate delivery of the rAAV vector and / or rAAV genome disclosed herein, it can be mixed with a carrier or excipient.Carriers and excipients that can be used include saline (particularly sterilized pyrogen-free saline), salt buffer (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohol, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol.USP-grade carriers and excipients are particularly useful for delivering virions to human subjects.

[0322] In addition to the formulations previously described, the rAAV vectors and / or rAAV genomes disclosed herein can be formulated as depot preparations. Such long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or by IM injection. Thus, for example, the rAAV vectors and / or rAAV genomes disclosed herein can be formulated with suitable polymeric or hydrophobic materials (e.g., as emulsions with acceptable oils), or as ion exchange resins, or as poorly soluble derivatives.

[0323] In any embodiment of the methods and compositions disclosed herein, the method relates to the treatment of Pompe disease caused by a deficiency of GAA in a subject, wherein an rAAV vector and / or rAAV genome disclosed herein is administered to a patient suffering from Pompe disease, and following administration, GAA is secreted from cells in the liver and there is uptake of the secreted GAA by cells in skeletal muscle tissue, cardiac muscle tissue, diaphragm muscle tissue, or a combination thereof, wherein the uptake of the secreted GAA results in a decrease in lysosomal glycogen storage in the tissue. In some embodiments, the rAAV vectors and / or rAAV genomes disclosed herein are encapsulated by a capsid, e.g., any AAV3b capsid selected from an AAV3b capsid (SEQ ID NO:44); an AAV3b265D capsid (SEQ ID NO:46), an AAV3b ST(S663V+T492V) capsid (SEQ ID NO:48), an AAV3b265D549A capsid (SEQ ID NO:50); an AAV3b549A capsid (SEQ ID NO:52); or an AAV3bQ263Y capsid (SEQ ID NO:54).

[0324] In a specific embodiment, at least about 10 per dose 2 ~about 10 8 cells or at least about 10 3 ~about 10 6 The cells are administered in a pharmaceutically acceptable carrier. In further embodiments, the dosage of the viral vector and / or capsid administered to a subject can be routinely determined depending on the mode of administration, the disease or condition to be treated and / or prevented, the condition of the individual subject, the specific viral vector or capsid, the nucleic acid to be delivered, etc. Exemplary doses to achieve a therapeutic effect are at least about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 3, 10 14 , 10 15 Transducing units, optionally about 10 8 ~10 13 The titer of transducing units.

[0325] In another aspect, disclosed herein is a method for administering a nucleic acid encoding GAA to a cell, comprising contacting the cell with an rAAV vector and / or rAAV genome disclosed herein under conditions such that the nucleic acid is introduced into the cell and expressed to produce GAA. In some embodiments, the cell is a cultured cell. In some embodiments, the cell is an in vivo cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the method for administering a nucleic acid encoding GAA to a cell further comprises collecting the GAA secreted into the cell culture medium.

[0326] D. Improving motor neuron function in mammals In any embodiment of the methods and compositions disclosed herein, the rAAV vectors and / or rAAV genomes disclosed herein are useful in compositions and methods for increasing phrenic nerve activity in mammals with Pompe disease and / or insufficient GAA levels. For example, the rAAV vectors and / or rAAV genomes disclosed herein, e.g., rAAV vectors and / or rAAV genomes encapsulated by a capsid, e.g., any AAV3b capsid selected from the group consisting of an AAV3b capsid (SEQ ID NO:44); an AAV3b265D capsid (SEQ ID NO:46), an AAV3b ST(S663V+T492V) capsid (SEQ ID NO:48), an AAV3b265D549A capsid (SEQ ID NO:50); an AAV3b549A capsid (SEQ ID NO:52); and an AAV3bQ263Y capsid (SEQ ID NO:54), can be administered to the central nervous system (e.g., a neuron). In another embodiment, retrograde transport of the rAAV vectors and / or rAAV genomes disclosed herein encoding GAA from the diaphragm (or other muscles) to the phrenic nerve or other motor neurons can result in biochemical and physiological correction of Pompe disease. These same principles can be applied to other neurodegenerative diseases.

[0327] In one embodiment, the rAAV is of any serotype listed in Table 1, e.g., AAV8 or AAV3, or AAV3b (including, but not limited to, AAV3b serotypes AAV3b265D, AAV3b265D549A, AAV3b549A, AAV3bQ263Y, AAV3bSASTG (i.e., AAV3b capsid containing the Q263A / T265 mutation) serotypes). The GAA construct can reduce weakness in a patient's lower extremities, e.g., legs, trunk, and / or arms, by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% in a patient afflicted with Pompe disease, compared to a patient not receiving the same treatment. In other aspects of this embodiment, any serotype of AAV GAA reduces weakness in a patient's lower extremities, e.g., legs, trunk, and / or arms, in a patient with Pompe disease, compared to a patient not receiving the same treatment, for example, by about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about The concentration can be reduced by 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

[0328] In any embodiment of the methods and compositions disclosed herein, rAAV of any serotype set forth in Table 1, for example, AAV8 or AAV3b disclosed herein (including, but not limited to, AAV3b serotypes AAV3b265D, AAV3b265D549A, AAV3b549A, AAV3bQ263Y, and AAV3bSASTG (i.e., AAV3b capsids containing the Q263A / T265 mutations)). The GAA construct can reduce one or more of the following in patients with Pompe disease, for example, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, compared to patients not receiving the same treatment: shortness of breath, movement disorder, lung infection, severe curvature of the spine, sleep dyspnea, hepatomegaly, tongue enlargement, and / or joint contractures. In other aspects of the methods and compositions of this embodiment, the AAV3bQ263Y disclosed herein can be used in combination with other AAV3bQ263Y constructs. GAA is used to improve one or more of the following in patients with Pompe disease, compared to patients not receiving the same treatment: for example, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90% %, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70% reduction in: shortness of breath, movement disorders, lung infections, severe curvature of the spine, sleep dyspnea, hepatomegaly, tongue enlargement, and / or joint contractures.

[0329] In any embodiment of the methods and compositions disclosed herein, the rAAV vectors and / or rAAV genomes disclosed herein of any serotype disclosed herein can reduce one or more of the following in patients with Pompe disease, for example, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, compared to patients not receiving the same treatment: shortness of breath, impaired movement, lung infections, severe curvature of the spine, sleep dyspnea, hepatomegaly, tongue enlargement, and / or joint contractures. In other aspects of this embodiment, the rAAV vectors and / or rAAV genomes disclosed herein of any serotype enhance one or more of the following in patients with Pompe disease compared to patients not receiving the same treatment, for example, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 9 ... The following symptoms may be reduced by about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%: shortness of breath, movement disorders, lung infections, severe curvature of the spine, sleep dyspnea, hepatomegaly, tongue enlargement, and / or joint contractures.

[0330] In any of the embodiments of the methods and compositions disclosed herein, the symptoms associated with Pompe disease are reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, and the severity of the symptoms associated with Pompe disease is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In another embodiment, the symptoms associated with Pompe disease are between about 10% and about 100%, between about 20% and about 100%, between about 30% and about 100%, between about 40% and about 100%, between about 50% and about 100%, between about 60% and about 100%, between about 70% and about 100%, between about 80% and about 100%, between about 10% and about 90%, between about 20% and about 90%, between about 30% and about 90%, between about 40% and about 90%, It decreases by about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

[0331] In one embodiment, adverse effects associated with Pompe disease are reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, and the severity of adverse effects associated with Pompe disease is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In another embodiment, the adverse effects associated with Pompe disease are between about 10% and about 100%, between about 20% and about 100%, between about 30% and about 100%, between about 40% and about 100%, between about 50% and about 100%, between about 60% and about 100%, between about 70% and about 100%, between about 80% and about 100%, between about 10% and about 90%, between about 20% and about 90%, between about 30% and about 90%, between about 40% and about 90% , about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

[0332] D. Mouse Model E. Immunosuppression In any embodiment of the methods and compositions disclosed herein, the subject receiving the rAAV vector or rAAV genome disclosed herein is administered an immunosuppressant. Various methods are known to result in immunosuppression of the immune response of patients receiving AAV. Methods known in the art include administering immunosuppressants, such as proteasome inhibitors, to patients. For example, one such proteasome inhibitor known in the art is bortezomib, as disclosed in U.S. Patent No. 9,169,492 and U.S. Patent Application No. 15 / 796,137, both of which are incorporated herein by reference. In another embodiment, the immunosuppressant can be an antibody, such as a polyclonal, monoclonal, scfv, or other antibody-derived molecule that can suppress the immune response, for example, through the elimination or suppression of antibody-producing cells. In a further embodiment, the immunosuppressive element can be a short hairpin RNA (shRNA). In such an embodiment, the coding region of the shRNA is included in the rAAV cassette and is generally located downstream, 3', of the polyA tail. shRNAs can be targeted to reduce or eliminate the expression of immune stimulators such as cytokines, growth factors (including transforming growth factors β1 and β2, TNF, and others publicly known).

[0333] V. Administration The dosage of the rAAV vector or rAAV genome disclosed herein to be administered to a subject can be routinely determined depending on the mode of administration, the disease or condition to be treated and / or prevented, the condition of the individual subject, the particular viral vector or capsid, and the nucleic acid to be delivered, etc. Exemplary doses to achieve a therapeutic effect are at least about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 Transducing units, optionally about 108 ~about 10 13 The titer of transducing units.

[0334] In further embodiments, administration of the rAAV vector or rAAV genome disclosed herein to a subject results in the production of a GAA protein having a circulating half-life of 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, or more.

[0335] In one embodiment, the duration of administration of an rAAV vector or rAAV genome disclosed herein to a subject is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more. In further embodiments, the period of time for which administration is discontinued is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.

[0336] In another embodiment, administration of an rAAV vector or rAAV genome disclosed herein for the treatment of Pompe disease results in a weight gain of, e.g., at least 0.5 pounds, at least 1 pound, at least 1.5 pounds, at least 2 pounds, at least 2.5 pounds, at least 3 pounds, at least 3.5 pounds, at least 4 pounds, at least 4.5 pounds, at least 5 pounds, at least 5.5 pounds, at least 6 pounds, at least 6.5 pounds, at least 7 pounds, at least 7.5 pounds, at least 8 pounds, at least 8.5 pounds, at least 9 pounds, at least 9.5 pounds, at least 10 pounds, at least 10.5 pounds, at least 11 pounds, at least 11.5 pounds, at least 12 pounds, at least 12.5 pounds, at least 13 pounds, at least 13.5 pounds, at least 14 pounds, at least 14.5 pounds, at least 15 pounds, at least 20 pounds, at least 25 pounds, at least 30 pounds, or at least 50 pounds. In another embodiment, an AAV GAA of any serotype disclosed herein for the treatment of Pompe disease results in a weight gain of, e.g., 0.5 lbs to 50 lbs, 0.5 lbs to 30 lbs, 0.5 lbs to 25 lbs, 0.5 lbs to 20 lbs, 0.5 lbs to 15 lbs, 0.5 lbs to 10 lbs, 0.5 lbs to 7.5 lbs, 0.5 lbs to 5 lbs, 1 lb to 15 lbs, 1 lb to 10 lbs, 1 lb to 7.5 lbs, 1 lb to 5 lbs, 2 lbs to 10 lbs, 2 lbs to 7.5 lbs.

[0337] All aspects of the compositions and methods of the presently disclosed technology can be defined in one or more of the following numbered sections: 1. Within its genome (a) 5' and 3' AAV inverted terminal repeats (ITRs), and (b) a heterologous nucleic acid sequence encoding a fusion polypeptide comprising a secretory signal peptide and an alpha-glucosidase (GAA) polypeptide, the heterologous nucleic acid sequence being located between the 5'ITR and the 3'ITR, the heterologous nucleic acid being operably linked to a promoter; A recombinant adeno-associated virus (AAV) vector comprising: 2. The recombinant AAV vector of item 1, wherein the heterologous nucleic acid sequence encoding the fusion polypeptide further comprises an IGF-2 sequence located between the secretory signal peptide and the alpha-glucosidase (GAA) polypeptide. 3. The AAV genome, in the 5' to 3' direction, (a) 5'ITR, (b) promoter sequence; (c) intron sequences; (d) a nucleic acid encoding a secretory signal peptide; (e) a nucleic acid encoding an IGF-2 sequence; (f) a nucleic acid encoding an alpha-glucosidase (GAA) polypeptide; (g) a polyA sequence, and (h) 3' ITR 3. The recombinant AAV vector of item 1 or 2, comprising: 4. The recombinant AAV vector of any of items 1 to 3, wherein the secretory signal peptide is selected from the group consisting of an AAT signal peptide, a fibronectin signal peptide (FN), a GAA signal peptide, and an active fragment thereof having secretory signal activity. 5. The recombinant AAV vector of any of items 1 to 3, wherein the IGF-2 leader sequence binds to the human cation-independent mannose-6-phosphate receptor (CI-MPR) or the IGF-2 receptor. 6. The recombinant AAV vector of items 1 to 5, wherein the IGF-2 sequence comprises SEQ ID NO:5 or at least one amino modification in SEQ ID NO:5 that binds to the IGF-2 receptor. 7. The recombinant AAV vector of any of items 1 to 6, wherein the at least one amino acid modification in SEQ ID NO:5 is a V43M amino acid modification (SEQ ID NO:8 or SEQ ID NO:9), or Δ2-7 (SEQ ID NO:6), or Δ1-7 (SEQ ID NO:7). 8. The recombinant AAV vector of any of items 1 to 7, wherein the promoter is constitutive, cell-specific, or inducible. 9. The recombinant AAV vector of any of items 1 to 8, wherein the promoter is a liver-specific promoter. 10. The recombinant AAV vector of any of items 1 to 9, wherein the liver-specific promoter is selected from the group consisting of a transthyretin promoter (TTR), an LSP promoter (LSP), and a synthetic liver-specific promoter. 11. The recombinant AAV vector of any of items 1 to 10, wherein the nucleic acid sequence encodes a wild-type GAA polypeptide or a modified GAA polypeptide. 12. The recombinant AAV vector of any of items 1 to 11, wherein the nucleic acid sequence encoding the GAA polypeptide is a human GAA gene or a human codon-optimized GAA gene (coGAA) or a modified GAA nucleic acid sequence. 13. The recombinant AAV vector of any of items 1 to 12, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized for enhanced expression in vivo. 14. The recombinant AAV vector of any of items 1 to 13, wherein the nucleic acid sequence encoding the GAA polypeptide has been codon-optimized to reduce CpG islands. 15. The recombinant AAV vector of any of items 1 to 14, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized to reduce the innate immune response. 16. The recombinant AAV vector of any of items 1 to 15, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized to reduce CpG islands and reduce the innate immune response. 17. The recombinant AAV vector of any of items 1 to 16, wherein the encoded fusion polypeptide further comprises a spacer comprising a nucleotide sequence for at least one amino acid located amino-terminal to the GAA polypeptide and C-terminal to the IGF-2 sequence. 18. The recombinant AAV vector of any of items 1 to 7, further comprising a nucleic acid encoding a spacer of at least one amino acid located between the nucleic acid encoding the IGF-2 sequence and the nucleic acid encoding the GAA polypeptide. 19. The recombinant AAV vector of any of items 1 to 8, further comprising at least one polyA sequence located 3' to the nucleic acid encoding the GAA gene and 5' to the 3' ITR sequence. 20. The recombinant AAV vector of any of items 1 to 19, wherein the heterologous nucleic acid sequence further comprises a collagen stability (CS) sequence located 3' to the nucleic acid encoding the GAA polypeptide and 5' to the 3' ITR sequence. 21. The recombinant AAV vector of any of items 1 to 20, further comprising a nucleic acid encoding a collagen stability (CS) sequence located between the nucleic acid encoding the GAA polypeptide and the polyA sequence. 22. The recombinant AAV vector of any of items 1 to 21, further comprising an intron sequence located 5' to the sequence encoding the secretory signal peptide and 3' to the promoter. 23. The recombinant AAV vector of any of items 1 to 22, wherein the intron sequence comprises an MVM sequence or an HBB2 sequence, wherein the MVN sequence comprises the nucleic acid sequence of SEQ ID NO:13 or a nucleic acid sequence having at least about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99% sequence identity to SEQ ID NO:13, and the HBB2 sequence comprises the nucleic acid sequence of SEQ ID NO:14 or a nucleic acid sequence having at least about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99% sequence identity to SEQ ID NO:14. 24. The recombinant AAV vector of any of items 1 to 23, wherein the ITRs comprise an insertion, deletion, or substitution. 25. The recombinant AAV vector of any of items 1 to 24, wherein one or more CpG islands within the ITRs have been removed. 26. The recombinant AAV vector of any of items 1 to 25, wherein the secretory signal peptide is fibronectin signal peptide (FN1) or an active fragment thereof having secretory signal activity (e.g., the FN1 signal peptide has any of the sequence of SEQ ID NOs:18-21, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any of SEQ ID NOs:18-21), and the heterologous nucleic acid sequence encodes an IGF-2 sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or an IGF2 peptide having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs:5-9. 27. The recombinant AAV vector of any of items 1 to 3, wherein the encoded secretory signal peptide is an AAT signal peptide or an active fragment thereof having secretory signal activity (e.g., the AAT signal peptide has the sequence of SEQ ID NO:17 or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:17), and the heterologous nucleic acid sequence encodes an IGF-2 sequence selected from any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or an IGF2 peptide having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs:5-9. 28. The recombinant AAV vector of any of items 1 to 27, wherein the IGF-2 sequence is SEQ ID NO:8 or SEQ ID NO:9, or an IGF2 peptide having at least about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99% sequence identity to SEQ ID NO:8 or SEQ ID NO:9. 29. The recombinant AAV vector of any of items 1 to 28, which is a chimeric AAV vector, a haploid AAV vector, a hybrid AAV vector, or a polyploid AAV vector. 30. The recombinant AAV vector of any of items 1 to 29, comprising a capsid protein selected from any AAV serotype in the group consisting of those listed in Table 1, and any combination thereof. 31. The recombinant AAV vector of any of items 1 to 30, wherein the serotype is AAV3b. 32. The recombinant AAV vector of any of items 1 to 31, wherein the AAV3b serotype comprises one or more mutations in the capsid protein selected from 265D, 549A, or Q263Y. 33. The recombinant AAV vector of any of items 1 to 32, wherein the AAV3b serotype is selected from any of AAV3b265D, AAV3b265D549A, AAV3b549A, AAV3bQ263Y, or AAV3bSASTG. 34. Within its genome (a) 5' and 3' AAV inverted terminal repeats (ITRs), and (b) a heterologous nucleic acid sequence encoding a fusion polypeptide comprising an alpha-glucosidase (GAA) polypeptide, the heterologous nucleic acid sequence being located between the 5'ITR and the 3'ITR, the heterologous nucleic acid being operably linked to a liver-specific promoter; Including, containing the capsid protein of the AAV3b serotype, Recombinant adeno-associated virus (AAV) vectors. 35. The recombinant AAV vector of item 34, wherein the fusion polypeptide further comprises a secretory signal peptide located N-terminally of the GAA polypeptide. 36. The recombinant AAV vector of item 34 or 35, wherein the heterologous nucleic acid sequence encoding the fusion polypeptide further comprises an IGF-2 sequence located between the secretory signal peptide and the alpha-glucosidase (GAA) polypeptide. 37. The AAV genome, in the 5' to 3' direction, (a) 5'ITR, (b) a liver-specific promoter sequence; (c) intron sequences; (d) a nucleic acid encoding a secretory signal peptide; (e) a nucleic acid encoding an IGF-2 sequence; (f) a nucleic acid encoding an alpha-glucosidase (GAA) polypeptide; (g) a polyA sequence, and (h) 3' ITR 35. The recombinant AAV vector of item 34, comprising: 38. The recombinant AAV vector of any of items 34 to 37, wherein the secretory signal peptide is selected from the group consisting of an AAT signal peptide, a fibronectin signal peptide (FN), a GAA signal peptide, and an active fragment thereof having secretory signal activity. 39. The recombinant AAV vector of any of items 34 to 38, wherein the IGF-2 leader sequence binds to the human cation-independent mannose-6-phosphate receptor (CI-MPR) or the IGF-2 receptor. 40. The recombinant AAV vector of any of items 34 to 39, wherein the IGF-2 sequence comprises SEQ ID NO:5 or SEQ ID NO:5 with at least one amino acid modification that affects binding to the IGF-2 receptor. 41. The recombinant AAV vector of item 40, wherein the at least one amino acid modification in SEQ ID NO:5 is a V43M amino acid modification (SEQ ID NO:8 or SEQ ID NO:9), or Δ2-7 (SEQ ID NO:6), or Δ1-7 (SEQ ID NO:7). 42. The recombinant AAV vector of any of items 34 to 41, wherein the liver-specific promoter is selected from the group consisting of a transthyretin promoter (TTR), an LSP promoter (LSP), and a synthetic liver-specific promoter. 43. The recombinant AAV vector of any of items 34 to 42, wherein the nucleic acid sequence encodes a wild-type GAA polypeptide or a modified GAA polypeptide. 44. The recombinant AAV vector of any of items 34 to 43, wherein the nucleic acid sequence encoding the GAA polypeptide is a human GAA gene, a human codon-optimized GAA gene (coGAA), or a modified GAA nucleic acid sequence. 45. The recombinant AAV vector of any of items 34 to 44, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized for enhanced in vivo expression. 46. ​​The recombinant AAV vector of any of items 34 to 44, wherein the nucleic acid sequence encoding the GAA polypeptide has been codon-optimized to reduce CpG islands. 47. The recombinant AAV vector of any of items 34 to 44, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized to reduce the innate immune response. 48. The recombinant AAV vector of any of items 34 to 44, wherein the nucleic acid sequence encoding the GAA polypeptide has been codon-optimized to reduce CpG islands and lower the innate immune response. 49. The recombinant AAV vector of any of items 34 to 49, wherein the intron sequence comprises an MVM sequence or an HBB2 sequence, wherein the MVN sequence comprises the nucleic acid sequence of SEQ ID NO:13 or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:13, and the HBB2 sequence comprises the nucleic acid sequence of SEQ ID NO:14 or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:14. 50. The recombinant AAV vector of any of items 34 to 49, wherein the ITRs comprise an insertion, deletion, or substitution. 51. The recombinant AAV vector of item 40, wherein one or more CpG islands within the ITRs have been removed. 52. The recombinant AAV vector of any of items 34 to 49, wherein the secretory signal peptide is a fibronectin signal peptide (FN1) or an active fragment thereof having secretory signal activity (e.g., the FN1 signal peptide has any of the sequences of SEQ ID NOs:18-21, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs:18-21), and the heterologous nucleic acid sequence encodes an IGF-2 sequence selected from any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or an IGF2 peptide having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs:5-9. 53. The recombinant AAV vector of any of items 34 to 49, wherein the encoded secretory signal peptide is an AAT signal peptide or an active fragment thereof having secretory signal activity (e.g., the AAT signal peptide has the sequence of SEQ ID NO: 17, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 17), and the heterologous nucleic acid sequence encodes an IGF-2 sequence selected from any of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or an IGF2 peptide having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs: 5 to 9. 54. The recombinant AAV vector of any of items 34 to 49, wherein the IGF-2 sequence is SEQ ID NO:8 or SEQ ID NO:9, or an IGF2 peptide having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:8 or SEQ ID NO:9. 55. A pharmaceutical composition comprising any of the recombinant AAV vectors described above and a pharmaceutically acceptable carrier. 56. A liver-specific promoter operably linked to a nucleic acid sequence, the nucleic acid sequence comprising, in the following order: a nucleic acid encoding a secretory signal peptide; a nucleic acid encoding an IGF-2 sequence; and a nucleic acid encoding a GAA polypeptide. A nucleic acid sequence comprising: 57. (a) 5' and 3' AAV inverted terminal repeat (ITR) nucleic acid sequences, and (b) a heterologous nucleic acid sequence encoding a fusion polypeptide comprising a secretory signal peptide and an alpha-glucosidase (GAA) polypeptide, the heterologous nucleic acid sequence being located between a 5'ITR sequence and a 3'ITR sequence, the heterologous nucleic acid being operably linked to a promoter; A nucleic acid sequence for a recombinant adeno-associated virus (rAAV) vector genome, comprising: 58. The nucleic acid sequence of item 56 or 57, wherein the heterologous nucleic acid sequence encoding the fusion polypeptide further comprises an IGF-2 sequence located between the secretory signal peptide and the alpha-glucosidase (GAA) polypeptide. 59. The nucleic acid sequence of item 56 or 58, wherein the nucleic acid encoding the secretion signal is selected from any of SEQ ID NOs: 17, 22-26, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any of SEQ ID NOs: 17 or 22-26. 60. The nucleic acid sequence of any of items 56 to 59, wherein the nucleic acid encoding the IGF-2 sequence is selected from any of SEQ ID NO:2 (IGF2-Δ2-7), SEQ ID NO:3 (IGF2-Δ1-7), or SEQ ID NO:4 (IGF2 V43M), or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any of SEQ ID NO:2, 3, or 4. 61. The nucleic acid sequence of any of items 56 or 60, wherein the nucleic acid sequence encoding the GAA polypeptide is a human GAA gene or a human codon-optimized GAA gene (coGAA) or a modified GAA nucleic acid sequence. 62. The nucleic acid sequence of any of items 56 to 61, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized for enhanced expression in vivo. 63. The nucleic acid sequence of any of items 56 to 62, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized to reduce CpG islands. 64. The nucleic acid sequence of any of items 56 to 63, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized to reduce the innate immune response. 65. The nucleic acid sequence of any of items 56 to 64, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized to reduce CpG islands and reduce the innate immune response. 66. The nucleic acid sequence of any of items 56 to 65, wherein the nucleic acid encoding the GAA polypeptide is selected from any of SEQ ID NO:11 (full-length hGAA), SEQ ID NO:55 (Dwight cDNA), SEQ ID NO:56 (hGAAΔ1-66), or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any of SEQ ID NO:11, 55, or 56. 67. The nucleic acid sequence of item 56 or 57, wherein the nucleic acid encoding the GAA polypeptide is selected from any of SEQ ID NO:74 (codon optimized 1), SEQ ID NO:75 (codon optimized 2), and SEQ ID NO:76 (codon optimized 3), or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any of SEQ ID NO:74, 75, or 76. 68. SEQ ID NO:57 (AAT-V43M-wtGAA(delta 1-69aa)); SEQ ID NO:58 (rat FN1-IGF2V43M-wtGAA(delta 1-69aa)); SEQ ID NO:59 (hFN1-IGF2V43M-wtGAA(delta 1-69aa)); SEQ ID NO:60 (ATT-IGF2Δ2-7-wtGAA(delta 1-69)); SEQ ID NO:61 (FN1 rat-IGFΔ2-7-wtGAA(delta 1-69)); SEQ ID NO:62 (hFN1-IGFΔ2-7-wtGAA(delta 1-69)), SEQ ID NO:79 (AAT_hIGF2-V43M_wtGAA_del1-69_stuffer.V02); SEQ ID SEQ ID NO:80 (FIB rat_hIGF2-V43M_wtGAA_del1-69_stuffer.V02); SEQ ID NO:81 (FIB hum_hIGF2-V43M_wtGAA_del1-69_stuffer.V02); SEQ ID NO:82 (AAT_GILT_wtGAA_del1-69_stuffer.V02); SEQ ID NO:83 (FIB rat_GILT_wtGAA_del1-69_stuffer.V02); SEQ ID NO:84 (FIB hum_GILT_wtGAA_del1-69_stuffer.V02), or SEQ ID The nucleic acid sequence of item 56 or 57, selected from any of the nucleic acid sequences having at least 80%, 85%, 90%, 95%, or 98% identity to NOs: 57, 58, 59, 60, 61, 62, 79, 80, 81, 82, 83, or 84. 69. A method for treating a subject having glycogen storage disease type II (GSD II, Pompe disease, acid maltase deficiency) or a deficiency of alpha-glucosidase (GAA) polypeptide, comprising administering to the subject any of the recombinant AAV vectors or rAAV genomes or nucleic acid sequences of any of items 1 to 58. 70. The method of item 69, wherein the GAA polypeptide is secreted from the liver of the subject and there is uptake of the secreted GAA by skeletal muscle tissue, cardiac muscle tissue, diaphragm muscle tissue, or a combination thereof, wherein the uptake of the secreted GAA results in a reduction in lysosomal glycogen storage in the tissue. 71. The method of any of items 69 to 70, wherein the administration to the subject is selected from intramuscular administration, subcutaneous administration, intraspinal administration, intracisternal administration, intrathecal administration, and intravenous administration. 72. A cell containing the nucleic acid sequence of any one of items 56 to 68. 73. Any of the cells of items 72 to 73, which are human cells. 74. The cell of any one of items 72 to 73, which is a non-human mammalian cell. 75. Any cell of items 72-73 that is an insect cell. 76. A cell containing the recombinant AAV vector of any one of items 1 to 54. 77. A host animal containing the recombinant AAV vector of any one of items 1 to 54. 78. The host animal of item 78, which is a mammal. 79. The host animal of item 78 or 79, which is a non-human mammal. 80. The host animal of item 78 is a human. 81. The pharmaceutical composition of item 55 for use in any of the methods of items 69 to 71. 82. A host animal containing cells according to any of items 72 to 75. 83. A host animal containing the recombinant AAV vector of any one of items 1 to 54. 84. The host animal of item 78, which is a mammal. 85. The host animal of item 78 or 79, which is a non-human mammal. 86. The host animal of item 78 is a human. [Example]

[0338] The following non-limiting examples are provided solely for illustrative purposes to facilitate a more complete understanding of the representative embodiments currently contemplated. These examples are intended to be merely a subset of all possible situations in which AAV virions and rAAV vectors can be used. Thus, these examples should not be construed as limiting any of the embodiments described herein, such as those relating to AAV virions and rAAV vectors and / or their methods and uses. Ultimately, AAV virions and vectors can be used in virtually any situation in which gene delivery is desired.

[0339] Example 1: Construction of the rAAV genome A number of rAAV genomes were constructed using the Gibson cloning methodology. The following rAAV genomes were generated: SEQ ID NO: 57 (AAT-V43M-wtGAA (delta 1-69 aa)); SEQ ID NO: 58 (rat FN1-IGF2V43M-wtGAA (delta 1-69 aa)); SEQ ID NO: 59 (hFN1-IGF2V43M-wtGAA (delta 1-69 aa)); SEQ ID NO: 60 (ATT-IGF2Δ2-7-wtGAA (delta 1-69)); SEQ ID NO: 61 (FN1 rat-IGFΔ2-7-wtGAA (delta 1-69)); SEQ ID NO: 62 (hFN1-IGFΔ2-7-wtGAA (delta 1-69)).

[0340] Gibson cloning involves cloning blocks (e.g., three blocks) of nucleic acid sequences together. The general protocol is as follows: combine the following reagents into a one-tube reaction: (i) Gibson Assembly Master Mix (exonuclease, DNA polymerase, DNA ligase, buffer), (ii) DNA inserts (Blocks 1-3) with 15-25 bp homologous ends (see Figure 7), and (iii) a linearized DNA backbone with 15-25 bp ends homologous to the outermost DNA inserts (see Figure 7). The reaction is incubated at 50°C for 15-60 minutes. The reaction mixture is transformed into competent cells and plated on kanamycin agar plates. Minipreps of fully assembled plasmid DNA are screened via restriction digestion and / or colony PCR analysis and confirmed by DNA sequencing. Confirmed clones are expanded for maxiprep generation and transiently transfected into suspension HEK293 cells with adenovirus helper, XX680 Kan, and the appropriate Rep / Cap helper for rAAV generation.

[0341] Figures 8-13 show the cloning of nucleic acid blocks to generate exemplary rAAV genomes. For example, Figure 8 shows the generation of an rAAV genome containing AAT-V43M-wtGAA (delta 1-69 aa); Figure 9 shows the generation of an rAAV genome containing rat FN1-IGF2V43M-wtGAA (delta 1-69 aa); Figure 10 shows the generation of an rAAV genome containing hFN1-IGF2V43M-wtGAA (delta 1-69 aa); Figure 11 shows the generation of an rAAV genome containing ATT-IGF2Δ2-7-wtGAA (delta 1-69); Figure 12 shows the generation of an rAAV genome containing FN1 rat-IGFΔ2-7-wtGAA (delta 1-69), and Figure 13 shows the generation of an rAAV genome containing hFN1-IGFΔ2-7-wtGAA (delta 1-69).

[0342] While Figures 8-13 show that wtGAA(Δ1-69) is an exemplary GAA enzyme, this nucleic acid sequence can be readily replaced by one skilled in the art with a codon-optimized nucleic acid sequence for enhanced in vivo expression, reduced immune response, and / or reduced CpG islands. Also shown in the cloning blocks illustrated in Figures 8-13 is the generation of an rAAV genome with a 3-amino acid (3 aa) spacer nucleic acid sequence located 3' to the nucleic acid sequence encoding the IGF(V42M) or IGFΔ2-7 targeting peptide and 5' to the nucleic acid encoding the GAA enzyme, and a stuffer nucleic acid sequence located 3' to the polyA sequence and 5' to the 3' ITR sequence (referred to as the "spacer" sequence in Figures 8-10).

[0343] Example 2: Generation of rAAV Vectors The rAAV Pro10 cell line was used to package the rAAV genome into capsids to generate rAAV vectors. For proof-of-principle purposes of rAAV vector construction only, the capsid used was an AAV3b capsid.

[0344] Generation of rAAV Pro10 cell line: A triple transfection method was used to generate rAAV in suspension HEK293 cells, which can be scaled up to generate clinical-grade vectors. Alternatively, different plasmids can be used, such as (1) pXX680-ad helper, and (2) pXR3 Rep and Cap, and (3) a transgene plasmid (ITR-transgene-ITR).

[0345] The rAAV genome produced in Example 1 is used to produce rAAV vectors using the Pro10 cell line, as described in U.S. Patent No. 9,441,206, the entire contents of which are incorporated herein by reference. Specifically, rAAV vectors or rAAV virions are produced using a method comprising: (a) providing HEK293 cells (e.g., ATTC No. PTA 13274) with an AAV expression system; (b) culturing the cells under conditions in which AAV particles are produced; and (c) optionally isolating the AAV particles. The triple transfection ratio of plasmids and transfection cocktail volume can be optimized by varying the plasmid ratios of XX680, AAV rep / cap helper, and TR plasmids to determine the optimal plasmid ratio for rAAV vector production.

[0346] In some cases, cells are cultured in suspension under conditions that allow AAV particles to be produced. In another embodiment, cells are cultured under animal-component-free conditions. The animal-component-free medium can be any animal-component-free medium (e.g., serum-free medium) compatible with HEK293 cells. Examples include, but are not limited to, SFM4Transfx-293 (Hyclone), Ex-Cell 293 (JRH Biosciences), LC-SFM (Invitrogen), and Pro293-S (Lonza). Conditions sufficient for AAV particle replication and packaging can be, for example, the presence of AAV sequences (e.g., AAV rep and AAV cap sequences) sufficient for replication of the rAAV genome described herein and encapsulation by the AAV capsid, as well as helper sequences derived from adenovirus and / or herpesvirus.

[0347] Example 3: Evaluation of rAAV vectors Whole blood clearance. Figure 1 shows a 3 x 10 12Results are shown from an experiment in which 3 kg of seronegative male macaques were intravenously injected with 1000 μg / kg of different AAV serotypes (AAV3b, AAV3ST, AAV8, and AAV9). The macaques were sacrificed 60 days after administration of the different AAV serotypes. When vector genomes were searched for in whole blood, the results showed that AAV3b was cleared within 1 week and was undetectable at the time of sacrifice, whereas AAV8 and AAV9 were still detectable in whole blood when the macaques were sacrificed.

[0348] Liver-specific vector potency: Figure 2 shows a 3 x 10 12 Figure 2 shows results from an experiment in which different AAV serotypes (AAV3b, AAV3ST, AAV8, and AAV9) were intravenously injected into 3 kg seronegative male macaques at 1000 ng / kg. The macaques were sacrificed 60 days after administration of the different AAV serotypes. Vector genomes were quantified in each of the three liver lobes from each macaque. The limit of quantification was 0.002 ng / dg. Based on the results presented in Figure 2, AAV3b was found to be a potent liver vector. AAV3b was more liver-specific than AAV8 and was cleared from the blood more rapidly than AAV9. The AAV3ST variant did not provide a significant beneficial effect.

[0349] Example 4: Measurement of GAA secretion into the supernatant and GAA uptake assay Measurement of GAA in the supernatant Therefore, the rAAV genome produced in Example 1 is tested for secretion of GAA polypeptide into the supernatant. Measurement of GAA in the supernatant can be assessed using 4-methylumbelliferyl-α-D-glucoside (4-MU) substrate (4-MU assay), as described by Kikuchi et al. (Kikuchi, Tateki, et al., "Clinical and metabolic correction of Pompe disease by enzyme therapy in acid maltase-deficient quail," The Journal of Clinical Investigation 101.4 (1998):827-833).

[0350] Briefly, HEK293 cells can be transfected with rAAV genome SEQ ID NO: 57 (AAT-V43M-wtGAA (delta 1-69 aa)); SEQ ID NO: 58 (rat FN1-IGF2V43M-wtGAA (delta 1-69 aa)); SEQ ID NO: 59 (hFN1-IGF2V43M-wtGAA (delta 1-69 aa)); SEQ ID NO: 60 (ATT-IGF2Δ2-7-wtGAA (delta 1-69)); SEQ ID NO: 61 (FN1 rat-IGFΔ2-7-wtGAA (delta 1-69)); SEQ ID NO: 62 (hFN1-IGFΔ2-7-wtGAA (delta 1-69)). GAA activity is measured over 24 hours as a percentage of initial activity (t=0). Samples were assayed for GAA enzyme activity based on the hydrolysis of the fluorogenic substrate 4-MU-α-glucose at 0, 3, 6, and 24 hours. GAA activity was expressed as a % of initial activity, i.e., residual activity.

[0351] Alternatively, after harvesting, the culture supernatant was partially purified by HIC chromatography. All samples were treated with PNGase before electrophoresis. Expression of GAA polypeptide by cells can be assessed using SDS-PAGE and immunoblotting.

[0352] GAA uptake assay and measurement of GAA uptake in tissues The rAAV genomes generated in Examples 1 and 2 are then tested for retention of cellular uptake activity. For example, HEK293 cells can be transfected with the rAAV genomes SEQ ID NO: 57 (AAT-V43M-wtGAA(delta 1-69 aa)); SEQ ID NO: 58 (rat FN1-IGF2V43M-wtGAA(delta 1-69 aa)); SEQ ID NO: 59 (hFN1-IGF2V43M-wtGAA(delta 1-69 aa)); SEQ ID NO: 60 (ATT-IGF2Δ2-7-wtGAA(delta 1-69)); SEQ ID NO: 61 (FN1 rat-IGFΔ2-7-wtGAA(delta 1-69)); SEQ ID NO: 62 (hFN1-IGFΔ2-7-wtGAA(delta 1-69)).

[0353] The 4-MU assay (described above) for assessing rhGAA uptake into mammalian cells is described in U.S. Patent Application No. US2009 / 0117091A1, the entire contents of which are incorporated herein by reference. The rAAV vectors or rAAV genomes produced in Examples 1 and 2 were incubated in a 20 μl reaction mixture containing 123 mM sodium acetate, pH 4.0, containing 10 mM 4-methylumbelliferyl α-D-glucosidase substrate (Sigma, Catalog No. M-9766). The reaction was incubated at 37° C. for 1 hour and terminated with 200 μl of buffer containing 267 mM sodium carbonate, 427 mM glycine, pH 10.7. Fluorescence was measured in a 96-well microtiter plate with an excitation wavelength of 355 nm and a filter wavelength of 460 nm and compared to a standard curve derived from 4-methylumbelliferone (Sigma, Catalog No. M1381). One GAA 4 MU unit is defined as 1 nmole of 4-methylumbelliferone hydrolysis per hour. The specific activity of exemplary rAAV genomes in fibroblasts was evaluated, such as SEQ ID NO: 57 (AAT-V43M-wtGAA (delta 1-69 aa)); SEQ ID NO: 58 (rat FN1-IGF2V43M-wtGAA (delta 1-69 aa)); SEQ ID NO: 59 (hFN1-IGF2V43M-wtGAA (delta 1-69 aa)); SEQ ID NO: 60 (ATT-IGF2Δ2-7-wtGAA (delta 1-69)); SEQ ID NO: 61 (FN1 rat-IGFΔ2-7-wtGAA (delta 1-69)); SEQ ID NO: 62 (hFN1-IGFΔ2-7-wtGAA (delta 1-69)). The enzymatic activity of the IGF2-GAA fusion polypeptide and / or SS-IGF2-GAA double fusion polypeptide is assessed and compared to untagged GAA (wtGAA).

[0354] Cell-based uptake assays can also be performed to demonstrate the ability of IGF2-tagged or untagged GAA to enter target cells. Rat L6 myoblasts are seeded in 24-well plates at a density of 1 x 10 cells per well 24 hours before uptake. At the start of the experiment, the medium is removed from the cells and replaced with 0.5 ml of uptake medium containing the rAAV vector produced in Examples 1 and 2. To demonstrate the specificity of uptake, some wells also contained the competitor M6P (5 mM final concentration) and / or IGF-2 (18 μg / ml final concentration). After 18 hours, the medium is aspirated from the cells, and the cells are washed four times with PBS. The cells are then lysed with 200 μl of CelLytic M™ lysis buffer. The lysates are assayed for GAA activity using 4 MU substrate as described above. Protein is determined using the Pierce BCA™ protein assay kit.

[0355] Typical uptake experiments are performed in CHO cells, although other cell lines and myoblast cell lines may be used. Uptake of GAA polypeptides into rat L6 myoblasts is expected to be virtually unaffected by the addition of large molar excesses of M6P, but uptake is expected to be significantly abolished by excess IGF-2. In contrast, uptake of wtGAA is expected to be significantly abolished by the addition of excess M6P, but virtually unaffected by competition with IGF2. Furthermore, uptake of IGF2V43M-wtGAA and IGF delta 2-7 wtGAA is expected to be significantly unaffected by excess IGF-2.

[0356] Example 5 Half-life of GAA in rat L6 myoblasts Uptake experiments were performed in rat L6 myoblasts using the rAAV vectors prepared in Examples 1 and 2, as previously described (see Examples 3 and 4). After 18 hours, the medium was aspirated from the rAAV vector-transfected cells, and the cells were washed four times with PBS. At this time point, duplicate wells were lysed (time point 0), and the lysates were frozen at -80°C. Thereafter, duplicate wells were lysed daily and stored for analysis. After 14 days, all lysates were assayed for GAA activity to assess half-life and whether the IGF-2-tagged GAA enzyme persisted in cells with kinetics similar to that of untagged GAA.

[0357] Example 6: Processing of GAA after uptake Mammalian GAA typically undergoes sequential proteolytic processing in lysosomes, as described by Moreland et al. (2005) J. Biol. Chem., 280:6780-6791 and references contained therein. The processed protein yields a pattern of 70 kDa, 20 kDa, and 10 kDa peptides, as well as several smaller peptides. To determine whether the IGF2-GAA fusion polypeptide and / or SS-IGF2-GAA double fusion polypeptide undergoes processing similar to that of untagged GAA, aliquots of lysates from the above uptake experiments were analyzed by Western blot using a monoclonal antibody that recognizes the 70 kDa IGF-2 peptide and a larger intermediate with an IGF-2 tag. The similar profiles of the polypeptides identified in this experiment indicate that after entry into cells, the IGF-2 sequence is lost and the IGF-2-GAA polypeptide undergoes processing similar to untagged GAA, demonstrating that the IGF-2 sequence has little or no effect on the behavior of GAA inside the cell.

[0358] Example 7: Pharmacokinetics The pharmacokinetics of IGF2-GAA fusion polypeptide and / or SS-IGF2-GAA double fusion polypeptide produced by rAAV vector can be measured in wild-type 129 mice. 129 mice are injected with the rAAV vector produced in Examples 1 and 2. Serum samples are collected before injection and 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, 4 hours, and 8 hours after injection. The animals are then sacrificed. The serum samples are assayed by quantitative Western blot. To determine whether IGF-2 fused with GAA polypeptide is removed from circulation too rapidly, the half-life of GAA derived from the rAAV vector expressing IGF2-GAA fusion polypeptide or SS-IGF2-GAA double fusion polypeptide is evaluated.

[0359] Example 8: Tissue half-life of GAA The purpose of this experiment was to determine the rate at which IGF2-GAA fusion polypeptides or SS-IGF2-GAA dual fusion polypeptides expressed from rAAV vectors lose GAA activity after reaching target tissues. In the Pompe mouse model, MYOZYME® appears to have a tissue half-life of approximately 6-7 days in various muscle tissues (Application Number 125141 / 0 to the Center for Drug Evaluation and Research and Center for Biologics Evaluation and Research, Pharmacology Reviews).

[0360] The rAAV vectors produced in Examples 1 and 2 were injected into the jugular vein of Pompe mice (Pompe mouse model 6neo / 6neo, described in Raben (1998) JBC, 273:19086-19092, the disclosure of which is incorporated herein by reference). The mice were then sacrificed 1, 5, 10, and 15 days after injection. Tissue samples were homogenized and GAA activity was measured according to standard procedures. From the decay curves in different tissues (e.g., quadriceps tissue; heart tissue; diaphragm tissue; and liver tissue), the tissue half-lives of GAA activity from the IGF2-GAA fusion polypeptide and / or SS-IGF2-GAA double fusion polypeptide and untagged GAA were calculated, and the half-lives in each tissue were calculated. To determine whether IGF2-GAA fusion polypeptides and / or SS-IGF2-GAA double fusion polypeptides expressed from the rAAV vectors described herein appear to persist with similar kinetics to untagged GAA in Pompe mouse cells, this can be compared to their half-lives in rat L6 myoblasts. Furthermore, knowledge of the decay kinetics of IGF2-GAA fusion polypeptides and / or SS-IGF2-GAA double fusion polypeptides can aid in the design of appropriate dosing intervals.

[0361] Example 9: Uptake of IGF2-GAA fusion polypeptide and / or SS-IGF2-GAA double fusion polypeptide into lysosomes of C2C12 mouse myoblasts C2C12 mouse myoblasts grown on polylysine-coated slides (BD Biosciences) were transduced with the rAAV vectors prepared in Examples 1 and 2. After washing, the cells were incubated in growth medium for 1 hour, then washed four times with D-PBS, and fixed with methanol at room temperature for 15 minutes. The following incubations were all performed at room temperature, with three washes with D-PBS between each. The slides were permeabilized with 0.1% Triton X-100 for 15 minutes and then blocked with blocking buffer (10% heat-inactivated horse serum (Invitrogen) in D-PBS). The slides were incubated with the primary mouse monoclonal anti-GAA antibody 3A6-1F2 (1:5,000 in blocking buffer), followed by a secondary rabbit anti-mouse IgG antibody conjugated with AF594 (Invitrogen A11032, 1:200 in blocking buffer). The slides were then incubated with FITC-conjugated rat anti-mouse LAMP-1 (BD Pharmingen 553793, 1:50 in blocking buffer). The slides were mounted with mounting solution containing DAPI (Invitrogen) and viewed under a Nikon Eclipse 80i microscope equipped with fluorescein isothiocyanate, Texas Red, and DAPI filters (Chroma Technology). Images were captured using a photometric Cascade camera controlled by MetaMorph software (Universal Imaging) and merged using Photoshop software (Adobe). Colocalization of the signal detected by the anti-GAA antibody with that ...

Claims

1. Within that genome (a) the 5' and 3' AAV inverted terminal repeats (ITRs); and (b) a heterologous nucleic acid sequence encoding a fusion polypeptide comprising a secretory signal peptide and an alpha-glucosidase (GAA) polypeptide, the heterologous nucleic acid being located between the 5'ITR and the 3'ITR, the heterologous nucleic acid being operably linked to a promoter; A recombinant adeno-associated virus (AAV) vector comprising:

2. The recombinant AAV vector of claim 1, wherein the heterologous nucleic acid sequence encoding a fusion polypeptide further comprises an IGF-2 sequence located between the secretory signal peptide and the alpha glucosidase (GAA) polypeptide.

3. The genome of the AAV is, in a 5' to 3' direction, (a) 5′ITR, (b) a promoter sequence; (c) intron sequences, (d) a nucleic acid encoding a secretory signal peptide; (e) a nucleic acid encoding an IGF-2 sequence; (f) a nucleic acid encoding an alpha-glucosidase (GAA) polypeptide; (g) a polyA sequence, and (h) 3′ ITR The recombinant AAV vector of claim 2, comprising:

4. The recombinant AAV vector of any one of claims 1 to 3, wherein the secretory signal peptide is selected from an AAT signal peptide, a fibronectin signal peptide (FN), a GAA signal peptide, or an active fragment thereof having secretory signal activity.

5. The recombinant AAV vector of any one of claims 1 to 3, wherein the IGF-2 leader sequence binds to the human cation-independent mannose-6-phosphate receptor (CI-MPR) or the IGF-2 receptor.

6. The recombinant AAV vector of claim 5, wherein the IGF-2 sequence comprises SEQ ID NO:5 or at least one amino modification in SEQ ID NO:5 that binds to the IGF-2 receptor.

7. The recombinant AAV vector of claim 6, wherein the at least one amino modification in SEQ ID NO:5 is a V43M amino acid modification (SEQ ID NO:8 or SEQ ID NO:9) or Δ2-7 (SEQ ID NO:6) or Δ1-7 (SEQ ID NO:7).

8. The recombinant AAV vector of any one of claims 1 to 3, wherein the promoter is constitutive, cell-specific, or inducible.

9. The recombinant AAV vector of any one of claims 1 to 3, wherein the promoter is a liver-specific promoter.

10. The recombinant AAV vector of claim 9, wherein the liver-specific promoter is selected from the group consisting of a transthyretin promoter (TTR), an LSP promoter (LSP), and a synthetic liver-specific promoter.

11. The recombinant AAV vector of claim 1 or 2, wherein the nucleic acid sequence encodes a wild-type GAA polypeptide or a modified GAA polypeptide.

12. The recombinant AAV vector of any one of claims 1 to 3, wherein the nucleic acid sequence encoding the GAA polypeptide is a human GAA gene or a human codon-optimized GAA gene (coGAA) or a modified GAA nucleic acid sequence.

13. The recombinant AAV vector of any one of claims 1 to 3 or claim 12, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized for enhanced expression in vivo.

14. The recombinant AAV vector of any one of claims 1 to 3 or claim 12, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized to reduce CpG islands.

15. The recombinant AAV vector of any one of claims 1 to 3 or claim 12, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized to reduce the innate immune response.

16. The recombinant AAV vector of any one of claims 1 to 3 or claim 12, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized to reduce CpG islands and lower the innate immune response.

17. The recombinant AAV vector of any one of claims 1 to 3, wherein the encoded fusion polypeptide further comprises a spacer comprising a nucleotide sequence for at least one amino acid located amino-terminal to the GAA polypeptide and C-terminal to the IGF-2 sequence.

18. The recombinant AAV vector of claim 3, further comprising a nucleic acid encoding a spacer of at least one amino acid located between the nucleic acid encoding the IGF-2 sequence and the nucleic acid encoding the GAA polypeptide.

19. The recombinant AAV vector of any one of claims 1 to 3, further comprising at least one polyA sequence located 3' to the nucleic acid encoding the GAA gene and 5' to the 3' ITR sequence.

20. The recombinant AAV vector of any one of claims 1 to 3, wherein the heterologous nucleic acid sequence further comprises a collagen stability (CS) sequence located 3' to the nucleic acid encoding the GAA polypeptide and 5' to the 3' ITR sequence.

21. The recombinant AAV vector of claim 3 or 20, further comprising a nucleic acid encoding a collagen stability (CS) sequence located between the nucleic acid encoding the GAA polypeptide and the polyA sequence.

22. The recombinant AAV vector of any one of claims 1 to 3, further comprising an intron sequence located 5' to the sequence encoding the secretory signal peptide and 3' to the promoter.

23. The recombinant AAV vector of claim 22, wherein the intron sequence comprises an MVM sequence or an HBB2 sequence.

24. The recombinant AAV vector of any one of claims 1 to 3, wherein the ITR sequences comprise an insertion, deletion, or substitution.

25. The recombinant AAV vector of claim 24, wherein one or more CpG islands within the ITRs are removed.

26. The recombinant AAV vector of any one of claims 1 to 3, wherein the secretory signal peptide is a fibronectin signal peptide (FN1) or an active fragment thereof having secretory signal activity, and the IGF-2 sequence is selected from any one of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:

9.

27. The recombinant AAV vector of any one of claims 1 to 3, wherein the encoded secretory signal peptide is an AAT signal peptide, or an active fragment thereof having secretory signal activity, and the IGF-2 sequence is selected from any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:

9.

28. The recombinant AAV vector of claim 26 or 27, wherein the IGF-2 sequence is SEQ ID NO:8 or SEQ ID NO:

9.

29. The recombinant AAV vector of any one of claims 1 to 3, which is a chimeric AAV vector, a haploid AAV vector, a hybrid AAV vector, or a polyploid AAV vector.

30. The recombinant AAV vector of any one of claims 1 to 3, comprising a capsid protein selected from any AAV serotype in the group consisting of those listed in Table 1, and any combination thereof.

31. The recombinant AAV vector of claim 30, wherein the serotype is AAV3b.

32. The recombinant AAV vector of claim 31, wherein the AAV3b serotype comprises one or more mutations in the capsid protein selected from 265D, 549A, or Q263Y.

33. The recombinant AAV vector of claim 31, wherein the AAV3b serotype is selected from any of AAV3b265D, AAV3b265D549A, AAV3b549A, or AAV3bQ263Y, or AAV3bSASTG.

34. Within that genome (a) the 5' and 3' AAV inverted terminal repeats (ITRs); and (b) a heterologous nucleic acid sequence encoding a fusion polypeptide comprising an alpha-glucosidase (GAA) polypeptide, the heterologous nucleic acid being located between the 5'ITR and the 3'ITR, the heterologous nucleic acid being operably linked to a liver-specific promoter; Including, Containing the capsid protein of the AAV3b serotype, Recombinant adeno-associated virus (AAV) vectors.

35. The recombinant AAV vector of claim 34, wherein the fusion polypeptide further comprises a secretory signal peptide located N-terminally of the GAA polypeptide.

36. The recombinant AAV vector of claim 34, wherein the heterologous nucleic acid sequence encoding a fusion polypeptide further comprises an IGF-2 sequence located between the secretory signal peptide and the alpha glucosidase (GAA) polypeptide.

37. The genome of the AAV is, in a 5' to 3' direction, (a) 5′ITR, (b) a liver-specific promoter sequence; (c) intron sequences, (d) a nucleic acid encoding a secretory signal peptide; (e) a nucleic acid encoding an IGF-2 sequence; (f) a nucleic acid encoding an alpha-glucosidase (GAA) polypeptide; (g) a polyA sequence, and (h) 3′ ITR The recombinant AAV vector of claim 34, comprising:

38. The recombinant AAV vector of any one of claims 34 to 37, wherein the secretory signal peptide is selected from an AAT signal peptide, a fibronectin signal peptide (FN), a GAA signal peptide, or an active fragment thereof having secretory signal activity.

39. The recombinant AAV vector of any one of claims 34 to 37, wherein the IGF-2 leader sequence binds to the human cation-independent mannose-6-phosphate receptor (CI-MPR) or the IGF-2 receptor.

40. The recombinant AAV vector of claim 39, wherein the IGF-2 sequence comprises SEQ ID NO:5 or comprises at least one amino modification in SEQ ID NO:5 that affects binding to the IGF-2 receptor.

41. The recombinant AAV vector of claim 40, wherein the at least one amino modification in SEQ ID NO:5 is a V43M amino acid modification (SEQ ID NO:8 or SEQ ID NO:9) or Δ2-7 (SEQ ID NO:6) or Δ1-7 (SEQ ID NO:7).

42. The recombinant AAV vector of any one of claims 34 to 41, wherein the liver-specific promoter is selected from the group consisting of a transthyretin promoter (TTR), an LSP promoter (LSP), and a synthetic liver-specific promoter.

43. The recombinant AAV vector of any one of claims 34 to 42, wherein the nucleic acid sequence encodes a wild-type GAA polypeptide or a modified GAA polypeptide.

44. The recombinant AAV vector of any one of claims 34 to 43, wherein the nucleic acid sequence encoding the GAA polypeptide is a human GAA gene or a human codon-optimized GAA gene (coGAA) or a modified GAA nucleic acid sequence.

45. The recombinant AAV vector of any one of claims 34 to 44, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized for enhanced expression in vivo.

46. The recombinant AAV vector of any one of claims 34 to 44, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized to reduce CpG islands.

47. The recombinant AAV vector of any one of claims 34 to 44, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized to reduce the innate immune response.

48. The recombinant AAV vector of any one of claims 34 to 44, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized to reduce CpG islands and reduce innate immune responses.

49. The recombinant AAV vector of any one of claims 34 to 49, wherein the intron sequence comprises an MVM sequence or an HBB2 sequence.

50. The recombinant AAV vector of any one of claims 34 to 49, wherein the ITRs comprise an insertion, deletion, or substitution.

51. The recombinant AAV vector of claim 40, wherein one or more CpG islands within the ITRs are removed.

52. The recombinant AAV vector of any one of claims 34 to 49, wherein the secretory signal peptide is a fibronectin signal peptide (FN1) or an active fragment thereof having secretory signal activity, and the IGF-2 sequence is selected from any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:

9.

53. The recombinant AAV vector of any one of claims 34 to 49, wherein the encoded secretory signal peptide is an AAT signal peptide, or an active fragment thereof having secretory signal activity, and the IGF-2 sequence is selected from any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:

9.

54. The recombinant AAV vector of any one of claims 34 to 49, wherein the IGF-2 sequence is SEQ ID NO:8 or SEQ ID NO:

9.

55. A pharmaceutical composition comprising a recombinant AAV vector according to any one of the preceding claims and a pharma- ceutically acceptable carrier.

56. A liver-specific promoter operably linked to a nucleic acid sequence, the nucleic acid sequence comprising, in the following order: a nucleic acid encoding a secretory signal peptide; a nucleic acid encoding an IGF-2 sequence; and a nucleic acid encoding a GAA polypeptide. A nucleic acid sequence comprising:

57. (a) 5' and 3' AAV inverted terminal repeat (ITR) nucleic acid sequences; and (b) a heterologous nucleic acid sequence encoding a fusion polypeptide comprising a secretory signal peptide and an alpha-glucosidase (GAA) polypeptide, the heterologous nucleic acid sequence being located between a 5'ITR sequence and a 3'ITR sequence, the heterologous nucleic acid being operably linked to a promoter; A nucleic acid sequence for a recombinant adeno-associated virus (rAAV) vector genome, comprising:

58. 58. The nucleic acid sequence of claim 57, wherein the heterologous nucleic acid sequence encoding a fusion polypeptide further comprises an IGF-2 sequence located between the secretory signal peptide and the alpha glucosidase (GAA) polypeptide.

59. 58. The nucleic acid sequence of claim 56 or 57, wherein the nucleic acid encoding the secretion signal is selected from any of SEQ ID NOs: 17, 22-26, or a nucleic acid having at least about 85% sequence identity thereto.

60. 60. The nucleic acid sequence of claim 59, wherein the nucleic acid encoding the IGF-2 sequence is selected from SEQ ID NO:2 (IGF2-A2-7), SEQ ID NO:3 (IGF2-A1-7), or SEQ ID NO:4 (IGF2 V43M), or a nucleic acid having at least about 85% sequence identity thereto.

61. 58. The nucleic acid sequence of claim 56 or 57, wherein the nucleic acid sequence encoding the GAA polypeptide is a human GAA gene or a human codon-optimized GAA gene (coGAA) or a modified GAA nucleic acid sequence.

62. 58. The nucleic acid sequence of claim 56 or 57, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized for enhanced expression in vivo.

63. 58. The nucleic acid sequence of claim 56 or 57, wherein the nucleic acid sequence encoding the GAA polypeptide has been codon-optimized to reduce CpG islands.

64. 58. The nucleic acid sequence of claim 56 or 57, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized to reduce the innate immune response.

65. 58. The nucleic acid sequence of claim 56 or 57, wherein the nucleic acid sequence encoding the GAA polypeptide is codon-optimized to reduce CpG islands and reduce the innate immune response.

66. 58. The nucleic acid sequence of claim 56 or 57, wherein the nucleic acid encoding the GAA polypeptide is selected from SEQ ID NO:11 (full length hGAA), SEQ ID NO:55 (Dwight cDNA), or SEQ ID NO 56 (hGAAΔ1-66).

67. The nucleic acid sequence of claim 56 or 57, wherein the nucleic acid encoding the GAA polypeptide is selected from any of SEQ ID NO:74 (codon optimization 1), SEQ ID NO:75 (codon optimization 2), and SEQ ID NO:76 (codon optimization 3).

68. SEQ ID NO:57 (AAT-V43M-wtGAA(delta 1-69aa)); SEQ ID NO:58 (rat FN1-IGF2V43M-wtGAA(delta 1-69aa)); SEQ ID NO:59 (hFN1-IGF2V43M-wtGAA(delta 1-69aa)); SEQ ID NO:60 (ATT-IGF2Δ2-7-wtGAA(delta 1-69)); SEQ ID NO:61 (FN1 rat-IGFΔ2-7-wtGAA(delta 1-69)); SEQ ID NO:62 (hFN1-IGFΔ2-7-wtGAA(delta 1-69)); SEQ ID NO:79 (AAT_hIGF2-V43M_wtGAA_del1-69_stuffer.V02); SEQ ID 58. The nucleic acid sequence of claim 56 or 57, selected from SEQ ID NO:80 (FIBrat_hIGF2-V43M_wtGAA_del1-69_stuffer.V02); SEQ ID NO:81 (FIBhum_hIGF2-V43M_wtGAA_del1-69_stuffer.V02); SEQ ID NO:82 (AAT_GILT_wtGAA_del1-69_stuffer.V02); SEQ ID NO:83 (FIBrat_GILT_wtGAA_del1-69_stuffer.V02); SEQ ID NO:84 (FIBhum_GILT_wtGAA_del1-69_stuffer.V02), or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity thereto.

69. A method for treating a subject having glycogen storage disease type II (GSD II, Pompe disease, acid maltase deficiency) or a deficiency of an alpha-glucosidase (GAA) polypeptide, comprising administering to the subject a recombinant AAV vector or rAAV genome or nucleic acid sequence according to any one of the preceding claims.

70. The method of claim 69, wherein the GAA polypeptide is secreted from the subject's liver and there is uptake of the secreted GAA by skeletal muscle tissue, cardiac muscle tissue, diaphragm muscle tissue, or a combination thereof, wherein the uptake of the secreted GAA results in a reduction in lysosomal glycogen storage in the tissue.

71. 70. The method of claim 69, wherein said administration to said subject is selected from intramuscular administration, subcutaneous administration, intraspinal administration, intracisternal administration, intrathecal administration, and intravenous administration.

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