Adeno-associated virus vectors for treating mucolipidosis type II

Abstract

rAAV vectors for treating mucolipidosis type II and / or type III are provided. [Solution] A recombinant adeno-associated virus (rAAV) vector is provided, which comprises a nucleic acid encoding the alpha and beta subunits of N-acetylglucosamine-1-phosphate transferase (GNPTAB) and at least one AAV inverted terminal repeat (ITR).

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 62 / 267,502, filed on December 15, 2015, which is hereby incorporated by reference in its entirety.

[0002] Submission of Sequence Listing in ASCII Text File The following content of the submission in ASCII text file is hereby incorporated by reference in its entirety: Sequence Listing in Computer Readable Format (CRF) (filename: 159792012640SEQLIST.txt, data record date: December 9, 2016, size: 21 kb).

[0003] The present invention relates to rAAV vectors, particles, compositions, and methods, kits and uses related thereto for treating mucolipidosis type II and / or type III.

Background Art

[0004] Mucolipidosis types II and III (ML II; ML III) are autosomal recessive lysosomal storage disorders characterized by a deficiency in UDP-GlcNAc; a lysosomal enzyme; N-acetylglucosamine-1-phosphotransferase (abbreviated as GlcNAc-1-phosphotransferase). GlcNAc-1-phosphotransferase is a hexameric enzyme complex consisting of three distinct subunits: α2, β2, and γ2. The α2 and β2 subunits are catalytically active and are encoded by a single gene, GNPTAB. Mutations in GNPTAB resulting in complete loss of enzyme activity are found in patients with ML II. Mucolipidosis II is highly progressive, and patients rarely survive beyond the first 10 years of life. In addition, treatment options available for ML II remain limited. In a small number of patients, only bone marrow transplantation has been attempted. In contrast, mutations in GNPTAB resulting in a partial reduction in GlcNAc-1 phosphotransferase activity are found in patients with ML III. These patients typically exhibit milder symptoms and slower disease progression, but still present with serious health problems such as skeletal abnormalities, growth retardation, and cardiomegaly. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The availability of AAV serotypes that exhibit tissue-specific tropism and promote sustained expression of transgenes offers the potential for AAV-mediated gene therapy for the systemic treatment of lysosomal storage diseases, including ML II and ML III. Therefore, research into AAV-mediated therapy for ML II / III is crucial to establish whether this approach represents a potential therapeutic strategy for this devastating disease. However, before considering AAV-mediated therapy for ML II / III, technical limitations must be overcome. The coding sequence of GNPTAB (over 5.6 kb in humans) is longer than the endogenous AAV genome (approximately 4.7 kb). Therefore, AAV vectors capable of accommodating GNPTAB, along with functional promoter / enhancer sequences and other components of the AAV genome, while still facilitating efficient packaging within viral particles, are highly advantageous. [Means for solving the problem]

[0006] The present invention relates to a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid encoding N-acetylglucosamine-1-phosphate transferase (GNPTAB) and at least one AAV inverted terminal repeat sequence (ITR) of recombinant adeno-associated virus (rAAV). The AAV vector is provided. In some embodiments, GNPTAB includes an alpha subunit and a beta subunit. In some embodiments, GNPTAB is operably linked to a promoter. In some embodiments, GNPTAB is human GNPTAB. In some embodiments, GNPTAB includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, GNPTAB includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the promoter is a CMV enhancer / chicken beta-actin (CBA) promoter. In some embodiments, the CBA promoter is a modified CBA promoter. In some embodiments, the modified CBA promoter is a cleaved CBA promoter. In some embodiments, the CMV enhancer is a truncated CMV enhancer. In some embodiments, the vector includes an intron. In some embodiments, the intron is an MVM intron. In some embodiments, the vector includes a polyadenylated sequence. In some embodiments, the polyadenylated sequence is a bovine growth hormone polyadenylated sequence. In some embodiments, the AAV terminal repeat sequence is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotype ITR. In some embodiments, the rAAV vector contains two ITRs.

[0007] In some embodiments, the present invention provides rAAV particles comprising an rAAV vector from any one of the embodiments described above. In some embodiments, the AAV particles comprise AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1 / AAV2 chimera, bovine AAV, mouse AAV, or rAAV2 / HBoV1 serotype capsids. In some embodiments, the rAAV particles comprise one or more ITRs and capsids derived from the same AAV serotype. In some embodiments, rAAV particles contain one or more ITRs derived from an AAV serotype different from the capsid of the rAAV virus particle. In some embodiments, rAAV particles contain an AAV8 capsid, and the vector contains an AAV2 ITR. In some embodiments, rAAV particles are produced by transfecting a host cell with nucleic acids encoding an rAAV vector, as well as nucleic acids encoding AAV rep and cap functions, and by providing nucleic acids encoding AAV helper functions. In some embodiments, AAV helper functions are provided by transfecting a host cell with nucleic acids encoding AAV helper functions. In some embodiments, AAV helper functions are provided by infecting a host cell with an AAV helper virus that provides AAV helper functions. In some embodiments, the AAV helper virus is an adenovirus, herpes simplex virus, or baculovirus. In some embodiments, rAAV particles are produced by an AAV-producing cell containing nucleic acids encoding an rAAV vector, as well as nucleic acids encoding AAV rep and cap functions, and by providing nucleic acids encoding AAV helper functions. In some embodiments, AAV-producing cells contain nucleic acids that encode AAV helper functions.In some embodiments, the AAV helper function is provided by infecting AAV-producing cells with an AAV helper virus that provides the AAV helper function. In some embodiments, the AAV helper virus is an adenovirus, herpes simplex virus, or baculovirus. In some embodiments, the present invention provides a pharmaceutical composition comprising any of the rAAV particles described herein.

[0008] In some embodiments, the present invention provides a method for treating mucolipidosis type II (ML II) or mucolipidosis type III (ML III) in a mammal, comprising administering an effective amount of either rAAV particles or a pharmaceutical composition described herein to the mammal.

[0009] In some embodiments, the present invention provides a method for treating mucolipidosis type II (ML II) or mucolipidosis type III (ML III) in a mammal, comprising administering an effective amount of rAAV particles to the mammal, wherein the rAAV particles comprise an rAAV vector, and the rAAV vector comprises a nucleic acid encoding N-acetylglucosamine-1-phosphate transferase (GNPTAB) and at least one AAV ITR. In some embodiments, the present invention provides a method for maintaining or increasing the body size of a mammal having mucolipidosis type II (ML II) or mucolipidosis type III (ML III), comprising administering an effective amount of rAAV particles to the mammal, wherein the rAAV particles comprise an rAAV vector, and the rAAV vector comprises a nucleic acid encoding N-acetylglucosamine-1-phosphate transferase (GNPTAB) and at least one AAV ITR, wherein the expression of GNPTAB results in maintaining or increasing body weight and / or maintaining or increasing height. In some embodiments, the present invention provides a method for preventing a decrease in body size in a mammal having mucolipidosis type II (ML II) or mucolipidosis type III (ML III), comprising administering an effective amount of rAAV particles to the mammal, wherein the rAAV particles comprise an rAAV vector, the rAAV vector comprises a nucleic acid encoding N-acetylglucosamine-1-phosphate transferase (GNPTAB) and at least one AAV ITR, and the expression of GNPTAB prevents weight loss. In some embodiments, the present invention provides a method for maintaining or increasing bone mineral density in a mammal having mucolipidosis type II (ML II) or mucolipidosis type III (ML III), comprising administering an effective amount of rAAV particles to the mammal, wherein the rAAV particles comprise an rAAV vector, the rAAV vector comprises a nucleic acid encoding GNPTAB and at least one AAV ITR, and the expression of GNPTAB results in the maintenance or increase of bone mineral density.In some embodiments, the present invention provides a method for preventing bone mineral density loss in mammals having mucolipidosis type II (ML II) or mucolipidosis type III (ML III), comprising administering an effective amount of rAAV particles to the mammal, wherein the rAAV particles comprise an rAAV vector, the rAAV vector comprises a nucleic acid encoding GNPTAB and at least one AAV ITR, and the expression of GNPTAB prevents bone mineral density loss. In some embodiments, the present invention provides a method for maintaining or increasing bone density in mammals having mucolipidosis type II (ML II) or mucolipidosis type III (ML III), comprising administering an effective amount of rAAV particles to the mammal, wherein the rAAV particles comprise an rAAV vector, the rAAV vector comprises a nucleic acid encoding GNPTAB and at least one AAV ITR, and the expression of GNPTAB results in the maintenance or increase of bone density. In some embodiments, the present invention provides a method for preventing bone density loss in mammals having mucolipidosis type II (ML II) or mucolipidosis type III (ML III), comprising administering an effective amount of rAAV particles to the mammal, wherein the rAAV particles comprise an rAAV vector, the rAAV vector comprises a nucleic acid encoding GNPTAB and at least one AAV ITR, and the expression of GNPTAB prevents bone density loss.

[0010] In some embodiments of the method described above, the treatment alleviates one or more symptoms of ML II or ML III, which include skeletal abnormalities, cognitive impairment, delayed development of gross and fine motor skills, hearing loss, lack of muscle tone, distended abdomen, umbilical hernia, progressive mucosal thickening of the airways, frequent respiratory infections, and mitral valve Thickening and dysfunction of the cervix, constipation or diarrhea. In some embodiments, treatment involves ML It slows the progression of one or more symptoms of ML II or ML III, which include skeletal abnormalities, cognitive impairment, delayed development of gross and fine motor skills, hearing loss, lack of muscle tone, distended abdomen, umbilical hernia, progressive mucosal thickening of the airways, frequent respiratory infections, thickening and dysfunction of the mitral valve, constipation or diarrhea. In some embodiments, the present invention provides a method for alleviating one or more symptoms of ML II or ML III in mammals, comprising administering an effective amount of rAAV particles to a mammal, wherein the rAAV particles comprise an rAAV vector, the rAAV vector comprises a nucleic acid encoding GNPTAB and at least one AAV ITR; the one or more symptoms of ML II or ML III are skeletal abnormalities, cognitive impairment, delayed development of gross and fine motor skills, hearing loss, lack of muscle tone, distended abdomen, umbilical hernia, progressive mucosal thickening of the airways, frequent respiratory infections, mitral valve thickening and dysfunction, constipation or diarrhea. In some embodiments, the present invention provides a method for alleviating one or more symptoms of ML II or ML III in mammals. A method for delaying the progression of one or more symptoms of ML II or ML III, comprising administering an effective amount of rAAV particles to a mammal, wherein the rAAV particles comprise an rAAV vector, the rAAV vector comprises a nucleic acid encoding GNPTAB and at least one AAV ITR; the method provides that one or more symptoms of ML II or ML III are skeletal abnormalities, cognitive impairment, delayed development of gross and fine motor skills, hearing loss, lack of muscle tone, distended abdomen, umbilical hernia, progressive mucosal thickening of the airways, frequent respiratory infections, mitral valve thickening and dysfunction, constipation or diarrhea.

[0011] In some embodiments of the above-described method, GNPTAB is operably linked to a promoter. In some embodiments, GNPTAB is human GNPTAB. In some embodiments, GNPTAB contains an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, GNPTAB contains the amino acid sequence of SEQ ID NO: 1. In some embodiments, the promoter is a CMV enhancer / chicken beta-actin (CBA) promoter. In some embodiments, the CBA promoter is a modified CBA promoter. In some embodiments, the modified CBA promoter is a cleaved CBA promoter. In some embodiments, the CMV enhancer is a truncated CMV enhancer. In some embodiments, the vector contains an intron. In some embodiments, the intron is an MVM intron. In some embodiments, the vector contains a polyadenylated sequence. In some embodiments, the polyadenylated sequence is a bovine growth hormone polyadenylated sequence. In some embodiments, the AAV terminal repeat sequence is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotype ITR. In some embodiments, the rAAV vector contains two ITRs. In some embodiments, the AAV particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1 / AAV2 chimera, bovine AAV, mouse AAV, or rAAV2 / HBoV1 serotype capsids. In some embodiments, the rAAV particles include one or more ITRs and capsids derived from the same AAV serotype.In some embodiments, the rAAV particle contains one or more ITRs derived from a different AAV serotype than the capsid of the rAAV virus particle. In some embodiments, the rAAV particle contains an AAV8 capsid, and the vector contains an AAV2 ITR.

[0012] In some embodiments of the above-described set, the rAAV particles coat the rAAV vector. AAV is produced by transfecting a host cell with nucleic acids encoding the AAV vector, as well as nucleic acids encoding the AAV rep and cap functions, and by providing nucleic acids encoding the AAV helper function. In some embodiments, the AAV helper function is provided by transfecting a host cell with nucleic acids encoding the AAV helper function. In some embodiments, the AAV helper function is provided by infecting a host cell with an AAV helper virus that provides the AAV helper function. In some embodiments, the AAV helper virus is an adenovirus, herpes simplex virus, or baculovirus. In some embodiments, rAAV particles are produced by an AAV-producing cell containing nucleic acids encoding the rAAV vector, as well as nucleic acids encoding the AAV rep and cap functions, and provide nucleic acids encoding the AAV helper function. In some embodiments, the AAV-producing cell contains nucleic acids encoding the AAV helper function. In some embodiments, the AAV helper function is provided by infecting an AAV-producing cell with an AAV helper virus that provides the AAV helper function. In some embodiments, the AAV helper virus is an adenovirus, herpes simplex virus, or baculovirus.

[0013] In some embodiments of the method described above, the mammal is a human. In some embodiments, the human is a child. In some embodiments, the human is a young adult.

[0014] In some embodiments of the methods described above, rAAV is administered intravenously, intraperitoneally, intra-arterially, intramuscularly, subcutaneously, or intrahepatically. In some embodiments, rAAV is administered intravenously. In some embodiments, rAAV is administered at two or more locations. In some embodiments, administration is repeated. In some embodiments, rAAV virus particles are present in a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0015] In some embodiments, the present invention provides the use of any of the pharmaceutical compositions described herein in the manufacture of a pharmaceutical for the treatment of ML II or ML III in mammals. In some embodiments, the present invention provides the use of any of the pharmaceutical compositions described herein in the manufacture of a pharmaceutical for use in any of the methods described herein. In some embodiments, the present invention provides the use of any of the rAAV particles described herein in the manufacture of a pharmaceutical for the treatment of ML II or ML III in mammals. In some embodiments, the present invention provides the use of any of the rAAV particles described herein in the manufacture of a pharmaceutical for use in any of the methods described herein. In some embodiments, the present invention provides the use of any of the pharmaceutical compositions described herein for the treatment of ML II or ML III in mammals. In some embodiments, the present invention provides the use of any of the pharmaceutical compositions described herein for use in any of the methods described herein. In some embodiments, the present invention provides the use of any of the recombinant AAVs described herein for the treatment of ML II or ML III in mammals. In some embodiments, the present invention provides the use of any of the recombinant AAVs described herein for use in any of the methods described herein. In some embodiments, the present invention provides the use of any of the pharmaceutical compositions described herein in the manufacture of a pharmacopoeia for alleviating one or more symptoms of ML II or ML III in mammals, or for delaying the progression of one or more symptoms of ML II or ML III in mammals. In some embodiments, the present invention provides the use of any of the rAAV particles described herein in the manufacture of a pharmacopoeia for alleviating one or more symptoms of ML II or ML III in mammals, or for delaying the progression of one or more symptoms of ML II or ML III in mammals. In some embodiments, the present invention The present invention provides the use of any of the pharmaceutical compositions described herein to alleviate one or more symptoms of ML II or ML III in mammals, or to delay the progression of one or more symptoms of ML II or ML III in mammals. In some embodiments, the present invention provides the use of any of the recombinant AAVs described herein to alleviate one or more symptoms of ML II or ML III in mammals, or to delay the progression of one or more symptoms of ML II or ML III in mammals. In some embodiments, one or more symptoms of ML II or ML III are skeletal abnormalities, cognitive impairment, delayed development of gross and fine motor skills, hearing loss, lack of muscle tone, distended abdomen, umbilical hernia, progressive mucosal thickening of the airways, frequent respiratory infections, mitral valve thickening and dysfunction, constipation or diarrhea. In some embodiments, the mammal is a human.

[0016] In some embodiments, the present invention provides a kit comprising any rAAV vector, any rAAV particle, or any pharmaceutical composition described herein. In some embodiments, the kit is for treating ML II or ML III according to any method described herein. In some embodiments, the kit comprises any rAAV vector, any rAAV particle, or any pharmaceutical composition described herein. In some embodiments, the kit further comprises one or more buffers or pharmaceutically acceptable excipients. In some embodiments, the kit further comprises instructions for use in the treatment of ML II and / or ML III.

[0017] In some embodiments, the present invention provides an animal model of mucolipidosis II (ML II) in which at least one allele of the N-acetylglucosamine-1-phosphatetransferase (GNPTAB) gene contains a deletion located between exon 12 and exon 20. In some embodiments, at least one allele of the GNPTAB gene contains a deletion spanning exon 12 and exon 20. In some embodiments, the animal is homozygous for the deletion in the GNPTAB gene. In some embodiments, the animal is heterozygous for the deletion in the GNPTAB gene. In some embodiments, a portion of the GNPTAB gene is replaced by a gene encoding a reporter and / or selective marker. In some embodiments, the selective marker confers resistance to neomycin. In some embodiments, the animal is a mammal. In some embodiments, the mammal is a rodent. In some embodiments, the rodent is a mouse. In some embodiments, the mouse has a genetic background derived from 129 / Sv and / or C57Bl / 6. In some embodiments, the animals are either immunocompetent or immunodeficient.

[0018] In some embodiments, the present invention provides a method for generating an animal model of mucolipidosis II (ML II), comprising introducing a deletion between exon 12 and exon 20 in at least one allele of the animal's GNPTAB gene. In some embodiments, at least one allele of the GNPTAB gene contains a deletion spanning exon 12 and exon 20. In some embodiments, the animals are crossed to be homozygous for the deletion in the GNPTAB gene. In some embodiments, the animals are crossed to be heterozygous for the deletion in the GNPTAB gene. In some embodiments, a portion of the GNPTAB gene is replaced by a gene encoding a reporter and / or selective marker. In some embodiments, the selective marker confers resistance to neomycin. In some embodiments, the animals are mammals. In some embodiments, the mammals are rodents. In some embodiments, the rodents are mice. In some embodiments, the mice The animals have a genetic background derived from 129 / Sv and / or C57Bl / 6. In some embodiments, the animals are immunocompetent or immunodeficient.

[0019] In some embodiments, the present invention provides a method for evaluating agents for the treatment of mucolipidosis II (ML II), comprising administering the agent to an animal model described herein, wherein the relief of one or more symptoms of ML II indicates that the agent provides a beneficial treatment for ML II. In some embodiments, the symptoms of ML II are weight loss, decreased bone density, decreased bone mineral density, skeletal abnormalities, cognitive impairment, delayed development of gross and fine motor skills, hearing loss, lack of muscle tone, distended abdomen, umbilical hernia, progressive mucosal thickening of the airways, frequent respiratory infections, mitral valve thickening and dysfunction, constipation or diarrhea. In some embodiments, the agent is a small molecule, polypeptide, antibody, nucleic acid or recombinant viral particle.

[0020] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

Brief Description of the Drawings

[0021] [Figure 1A] It is a figure showing a schematic diagram of the genomic insertion site of a gene trapping vector used to generate the mouse GNPTAB gene structure and GNPTAB knockout mice. [Figure 1B] It is a figure showing a Southern blot of mouse ES clones used to generate GNPTAB knockout mice. [Figure 2] It is a figure showing the growth retardation presented in GNPTAB knockout mice. (Figure 2A) Body weights of 6-week-old wild-type (+ / +), heterozygous (+ / -), and homozygous (- / -) mice (***; p < 0.0001; Bonferroni's multiple comparison test). (Figure 2B) Nasal tip - anal length (mm) of 6-week-old wild-type, heterozygous, and homozygous mice (*; p < 0.02; Bonferroni's multiple comparison test). (Figure 2C) Gross morphology of wild-type and homozygous mice. [Figure 3] It is a figure showing optical microscope images of representative sections of hematoxylin and eosin-stained femoral cartilage from wild-type mice (Figure 3A) and homozygous knockout mice (Figure 3B). [Figure 4] It is a figure demonstrating the accumulation of autolysosomes in the salivary glands of knockout (KO) mice. (Figures 4A - 4C) EM shows an overview of a salivary gland acinus of a wild-type mouse, composed of mucous cells and serous cells. (Figure 4D) Overview of a KO salivary gland acinus. The overall structure is disrupted in KO by the accumulation of a very large number of vacuoles. (Figures 4E - 4F) Vacuoles in (Figure 4D) at high magnification, surrounded by a single membrane and containing undigested material. The magnification range is shown in the box of (Figure 4D). AL, autolysosome; Mu, mucous cell; N, nucleus; SG, secretory granule. [Figure 5]As shown, this figure illustrates the lysosomal enzyme activity in the serum of wild-type mice (white circles) and knockout mice (black circles). It shows the activity of N-acetylglucosaminidase (Figure 5A), β-hexosaminidase A (Figure 5B), β-galactosidase (Figure 5C), and β-glucuronidase (Figure 5D). [Figure 6] This figure shows an overview of the schedule for the injection experiment. (Figure 6A) Schedule for the long-term treatment study of mice injected with a viral vector at 6 weeks of age. (Figure 6B) Shows the total number of mice (n) injected in each treatment group. [Figure 7A] This figure shows a schematic diagram of a pAAV2 / 8-GNPTAB vector containing mouse GNPTAB cDNA. The mouse GNPTAB cDNA sequence is based on GenBank acceptance number NM_001004164.2. The nucleotide sequence has been codon-optimized for expression in mouse. The amino acid sequence is unchanged. [Figure 7B] This figure shows a quantitative analysis of livers from knockout mice injected with AAV-GNPTAB, compared to their control littermates. [Figure 8] This figure shows the change in body weight over time from baseline for control mice and AAV-GNPTAB-treated knockout mice. (Figure 8A) Total body weight of control mice and AAV-GNPTAB-treated knockout mice (*p<0.05, Dunnett's multiple comparison test). (Figure 8B) Data are expressed as the amount of change in weight over time from baseline. [Figure 9] This figure shows the change in body length over time from baseline for control mice and AAV-GNPTAB-treated knockout mice. (Figure 9A) Data are expressed as the ratio of body length before injection and 6 weeks after injection. (Figure 9B) Data are expressed as the ratio of body length before injection and 32 weeks after injection. [Figure 10]This figure shows histograms of bone mineral density levels before injection (Figure 10A), 16 weeks after injection (Figure 10B), and 32 weeks after injection (Figure 10C). (Figure 10A) Data from homozygous and heterozygous mice treated with AAV-GNPTAB were compared with wild-type and heterozygous mice (compared to wild-type†; p<0.05, ‡; p<0.02; compared to heterozygous*; p<0.02, **; p<0.002). AAV-GNPTAB treatment resulted in a statistically significant increase in the BMD ratio (post / pre-treatment Tx) in homozygous mice 16 weeks after treatment (Figure 10B) and 32 weeks after treatment (Figure 10C). (Figure 10B) 16 weeks after treatment, GNPTAB null mice treated with AAV-GNPTAB showed a significantly greater increase in the BMD ratio than other mice (**; P<0.02). (Figure 10C) 32 weeks after treatment, a significant difference in BMD ratio was observed in mice treated with GNPTAB (#; p<0.05, **; p<0.02). P-values ​​were determined by analysis of two-sided independent t-tests. Data are shown as mean ± SEM. [Figure 11] This figure shows histograms of bone mineral density levels before injection (Figure 11A), 16 weeks after injection (Figure 11B), and 32 weeks after injection (Figure 11C). (Figure 11A) Data from homozygous and heterozygous mice treated with AAV-GNPTAB were compared with wild-type and heterozygous mice (compared to wild-type†; p<0.05, ‡; p<0.02; compared to heterozygous*; p<0.02, **; p<0.002). AAV-GNPTAB treatment resulted in a statistically significant increase in the BMD ratio (post / pre-treatment Tx) in homozygous mice 16 weeks after treatment (Figure 11B) and 32 weeks after treatment (Figure 11C). (Figure 11B) 16 weeks after treatment, GNPTAB null mice treated with AAV-GNPTAB showed a significant increase in the BMD ratio compared to other mice (**; P<0.02). (Figure 11C) 32 weeks after treatment, a significant difference in BMD ratio was observed in mice treated with GNPTAB (#; p<0.05, **; p<0.02). P-values ​​were determined by analysis of two-sided independent t-tests. Data are shown as mean ± SEM. [Figure 12]Figure 12A shows histograms of the percentage lean body mass level before injection and the change in percentage lean body mass 32 weeks after injection (Figure 12B). (Figure 12A) Data from homozygous and heterozygous mice treated with AAV-GNPTAB were compared with wild-type and heterozygous mice (compared to wild-type†; p<0.02, compared to heterozygous*; p<0.05, **; p<0.001). (Figure 12B) After 32 weeks of treatment, no change in percentage lean body mass was observed in mice treated with AAV-GNPTAB. P values ​​were determined by analysis of two-sided independent t-tests. Data are shown as mean ± SEM. [Modes for carrying out the invention]

[0022] In some embodiments, the present invention provides a recombinant adeno-associated virus (rAAV) vector comprising nucleic acids encoding the alpha and beta subunits (GNPTAB) of N-acetylglucosamine-1-phosphate transferase, and at least one AAV inverted terminal repeat (ITR). Further herein, rAAV particles comprising the rAAV vector of the present disclosure and pharmaceutical compositions comprising the rAAV particles of the present disclosure are provided.

[0023] In some embodiments, the present invention further provides a method for treating mucolipidosis type II (ML II) or mucolipidosis type III (ML III) in a mammal, comprising administering an effective amount of rAAV particles to the mammal, wherein the rAAV particles comprise an rAAV vector, and the rAAV vector comprises a nucleic acid encoding GNPTAB and at least one AAV ITR. The present invention further provides a method for increasing body size, bone mineral density and / or bone density in a mammal having mucolipidosis type II (ML II) or mucolipidosis type III (ML III), comprising administering an effective amount of rAAV particles to the mammal, wherein the rAAV particles comprise an rAAV vector, and the rAAV vector comprises a nucleic acid encoding GNPTAB and at least one AAV ITR. In some embodiments, GNPTAB expression results in an increase in body size, bone mineral density and / or bone density.

[0024] In some embodiments, the present invention further provides uses and / or kits for treating ML II or ML III using, for example, the rAAV vectors, rAAV particles, or pharmaceutical compositions of the present disclosure.

[0025] I. General techniques The techniques and procedures described or referenced herein are generally based on conventional methods, such as Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); Current Protocols in Molecular Biology (FMAusubel et al., eds., 2003); Methods in Enzymology series (Academic Press, Inc.); PCR 2: A Practical Approach (MJ MacPherson, B.D. Hames and GR. Taylor, eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (RI Freshney, 6th edition, J. Wiley and Sons, 2010); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by JECellis, Academic Press, 1998); Introduction to Cell and Tissue Culture (JP Mather and PER Oberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., J. Wiley and Sons, 1993-98); Handbook of Experimental Immunology (DM Weir and C.C. Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells (J. Mmiller and M. P Calos, eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Oligan et al., eds., 1991); Short Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and Sons, 2002); Immunobiology (CA Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty, ed., IRL Press, 1988-1989); Monoclonal Antibo The methods described in *dies: A Practical Approach* (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); *Using Antibodies: A Laboratory Manual* (edited by E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); *The Antibodies* (edited by M. Zanetti and JDCapra, Harwood Academic Publishers, 1995); and *Cancer: Principles and Practice of Oncology* (edited by V. DeVita et al., JBLippincott Company, 2011) are well understood and commonly used by those skilled in the art.

[0026] II. Definition When used herein, "vector" refers to a recombinant plasmid or virus containing nucleic acid to be delivered to a host cell, either in vitro or in vivo.

[0027] As used herein, the terms “polynucleotide” or “nucleic acid” refer to polymeric forms of nucleotides of any length, which are either ribonucleotides or deoxyribonucleotides. Therefore, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other naturally occurring nucleotide bases, chemically or biochemically modified nucleotide bases, unnatural nucleotide bases, or derivatized nucleotide bases. The nucleic acid backbone may contain sugars and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugars or phosphate groups. Alternatively, the nucleic acid backbone may contain polymers of synthetic subunits such as phosphoramidates, and thus may be oligodeoxynucleoside phosphoramidates (P-NH2) or mixed phosphoramidate-phosphodiester oligomers. In addition, double-stranded nucleic acids can be obtained from the chemical synthesis products of single-stranded polynucleotides by either synthesizing complementary strands and annealing these strands under appropriate conditions, or by de novo synthesis of complementary strands using DNA polymerase with appropriate primers.

[0028] The terms “polypeptide” and “protein” are used interchangeably to refer to polymers of amino acid residues, and are not limited to those of the shortest length. Such polymers of amino acid residues may contain native or non-native amino acid residues, and these include, but are not limited to, peptides, oligopeptides, amino acid dimers, trimers, and polymers. Both full-length proteins and their fragments are encompassed in this definition. These terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, and phosphorylation. Furthermore, for the purposes of this invention, “polypeptide” refers to a protein that includes modifications such as deletions, additions, and substitutions (generally of a conserved nature) to its native sequence, as long as the protein maintains the desired activity. These modifications may be intentional, such as those resulting from site-directed mutagenesis, or accidental, such as those resulting from mutations in the host producing the protein or errors resulting from PCR amplification.

[0029] A "recombinant viral vector" refers to a recombinant polynucleotide vector that contains one or more heterogeneous sequences (i.e., nucleic acid sequences that are not of viral origin). In the case of a recombinant AAV vector, the recombinant nucleic acid has at least one, for example, two, inverted terminal repeats (ITRs) adjacent to it.

[0030] A recombinant AAV vector (rAAV vector) contains at least one, for example, two This refers to a polynucleotide vector containing one or more heterogeneous sequences (i.e., nucleic acid sequences not of AAV origin) adjacent to an AAV inverted terminal repeat (ITR). Such rAAV vectors, when present in host cells infected with a suitable helper virus (or expressing a suitable helper function), as well as host cells expressing the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins), are replicated and packaged in infectious viral particles. When an rAAV vector is incorporated into a larger polynucleotide (e.g., a chromosome, or another vector such as a plasmid used for cloning or transfection), the rAAV vector may be referred to as a “provector,” which can be “rescued” by replication and capsidation in the presence of AAV packaging and suitable helper functions. rAAV vectors can be any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexes with lipids, encapsulated in liposomes, and, in embodiments, capsidized in viral particles, particularly AAV particles. rAAV vectors can be packaged into AAV virus capsids to generate "recombinant adeno-associated virus particles (rAAV particles)."

[0031] "rAAV virus" or "rAAV virus particle" refers to a viral particle consisting of at least one AAV capsid protein and a capsidized rAAV vector genome.

[0032] "Heterogeneous" means that the genotype of the entity being compared or the rest of the entity being introduced or incorporated originates from an entity with a different genotype. For example, nucleic acids introduced into a different cell type by genetic engineering techniques are heterogeneous nucleic acids (and may encode heterogeneous polypeptides if expressed). Similarly, a cell sequence (e.g., a gene or a portion thereof) incorporated into a viral vector is a heterogeneous nucleotide sequence relative to that vector.

[0033] The term "transgene" refers to a nucleic acid that can be introduced into a cell, transcribed into RNA, and, in some cases, translated and / or expressed under appropriate conditions. In some embodiments, the transgene confers a desired characteristic to the introduced cell or otherwise produces a desired therapeutic or diagnostic outcome. In another embodiment, the transgene is transcribed into a molecule that mediates RNA interference, such as siRNA.

[0034] The terms “genomic particles (gp),” “genomic equivalents,” or “genomic copies” used in relation to viral titer refer to the number of virions containing recombinant AAV DNA genome, regardless of infectivity or functionality. The number of genomic particles in a particular vector preparation can be measured by the examples described herein or by procedures such as those described, for example, Clark et al. (1999) Hum. Gene Ther., 10: pp. 1031-1039; Veldwijk et al. (2002) Mol. Ther., 6: pp. 272-278.

[0035] The terms “infectious units (iu),” “infectious particles,” or “replication units” used in relation to viral titer refer to the number of infectious and replication-compatible recombinant AAV vector particles measured by an infectious center assay, also known as a replication center assay, as described, for example, by McLaughlin et al. (1988) J. Virol., 62: pp. 1963–1973.

[0036] The term “transduction unit (tu)” as used in relation to viral titer is described in the examples herein or, for example, in Xiao et al. (1997) Exp. Neur This refers to the number of infectious recombinant AAV vector particles that produce a functional transgene product, as measured in a functional assay, such as those described in obiol., 144: pp. 113-124; or in Fisher et al. (1996) J. Virol., 70: pp. 520-532 (LFU assay).

[0037] An "inverted terminal repeat" or "ITR" sequence is a well-understood term in the field, referring to a relatively short, inverted sequence found at the end of a viral genome.

[0038] The term "AAV inverted terminal repeat (ITR)," a term well understood in the art, is a sequence of approximately 145 nucleotides present at both ends of a natural single-stranded AAV genome. The outermost 125 nucleotides of an ITR can exist in one of two alternating orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several self-complementary short regions (indicated as regions A, A', B, B', C, C', and D), and intra-strand base pairing can occur in this portion of the ITR.

[0039] A "terminal resolution sequence" or "trs" is a sequence within the D region of the AAV ITR that is cleaved by the AAV rep protein during viral DNA replication. A variant terminal resolution sequence is refractory to cleavage by the AAV rep protein. "AAV helper function" refers to a function that enables AAV to be replicated and packaged by a host cell. AAV helper function is provided in one of many forms, including but not limited to helper viruses or helper virus genes that assist in AAV replication and packaging. Other AAV helper functions, such as genotoxic substances, are known in the art.

[0040] "AAV helper function" refers to a function that enables AAV to be replicated and packaged by a host cell. AAV helper function is provided in one of many forms, including but not limited to helper viruses or helper virus genes that assist in AAV replication and packaging. Other AAV helper functions, such as genotoxic substances, are known in the art.

[0041] A "helper virus" for AAV refers to a virus that enables AAV (which is a deficient Pablo virus) to replicate and package by a host cell. Many such helper viruses have been identified, including adenoviruses, herpesviruses, poxviruses, such as vaccinia, and baculoviruses. Adenoviruses encompass numerous different subgroups, but adenovirus type 5 (Ad5) of subgroup C is the most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and available from contract laboratories such as ATCC. Herpes family viruses, also available from contract laboratories such as ATCC, include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and pseudorabies virus (PRV). An example of a baculovirus available from contract laboratories is Autographa California nuclear polyhedrosis virus.

[0042] The “sequence identity percentage (%)” for a reference polypeptide sequence or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to those in the reference polypeptide sequence or nucleic acid sequence, after the sequences have been aligned and gaps introduced as necessary to achieve the maximum sequence identity percentage. Any conservative substitutions are not considered part of the sequence identity. Alignment for the purpose of determining the sequence identity percentage of amino acids or nucleic acids is within the scope of the skills of those skilled in the art. This can be achieved by means of, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., ed., 1987), Supp. 30, Section 7.7.18, Table 7.7.1, as well as by using publicly available computer software programs, including BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Potential alignment programs include ALIGN Plus (Scientific and This is Educational Software (Pennsylvania). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. For the purposes of this specification, the amino acid sequence identity % of a given amino acid sequence A to a given amino acid sequence B (which can be rephrased as a given amino acid sequence A having or containing a certain amino acid sequence identity % to a given amino acid sequence B) is calculated as follows: multiply fraction X / Y by 100, where X is the number of amino acid residues scored by a sequence alignment program as identical matches in the alignment of A and B in that program, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity % of A to B will not be equal to the amino acid sequence identity % of B to A. For the purposes of this specification, the nucleic acid sequence identity % of a given nucleic acid sequence C to a given nucleic acid sequence D (which can be rephrased as a given nucleic acid sequence C having or containing a certain nucleic acid sequence identity % to a given nucleic acid sequence D) is calculated as follows: multiply the fraction W / Z by 100, where W is the number of nucleotides scored by a sequence alignment program as identical in the alignment of C and D in that program, and Z is the total number of nucleotides in D. It is understood that if the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the nucleic acid sequence identity % of C to D will not be equal to the nucleic acid sequence identity % of D to C.

[0043] An "isolated" molecule (e.g., nucleic acid or protein) or cell means that it has been identified and separated from its natural environment and / or recovered.

[0044] An "effective dose" is the amount sufficient to achieve a beneficial or desired outcome, including clinical outcomes (e.g., symptom relief, achievement of clinical endpoints). An effective dose can be administered in one or more doses. In terms of disease conditions, an effective dose is the amount sufficient to alleviate, stabilize, or delay the onset of the disease. For example, an effective dose of rAAV particles represents a desired amount of heterogeneous nucleic acid, such as a therapeutic polypeptide or therapeutic nucleic acid.

[0045] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0046] As used herein, “treatment” is an approach to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction of disease severity, stabilization of the disease state (e.g., no worsening), prevention of disease spread (e.g., metastasis), delay or slowing of disease progression, relief or reduction of the disease state, and remission (whether partial or complete), whether detectable or undetectable. “Treatment” may also mean extending survival compared to survival predicted without treatment.

[0047] When used in reference to a gene or coding sequence, “N-acetylglucosamine-1-phosphotransferase (also known as GlcNAc-1-phosphotransferase or GNPTAB)” refers to the polynucleotide sequence encoding the alpha and beta subunits of an enzyme that catalyzes the chemical reactions involved in the formation of the lysosomal enzymes N-acetylglucosaminyl-phospho-D-mannose and UMP from UDP-N-acetyl-D-glucosamine and the lysosomal enzyme D-mannose (EC code 2.7.8.17). When used in reference to polypeptides, “N-acetylglucosamine-1-phosphotransferase (aka GlcNAc-1-phosphotransferase or GNPTAB)” refers to the alpha and beta subunits of the aforementioned enzyme (Kudo, M. et al., J Biol Chem. 2005, 280(43):36141-9; Gelfman, C. et al., Invest. Opthamol. Vis. Sci. 2007, 48(11):5221-5228). The complete GlcNAc-1-phosphotransferase enzyme complex is known to contain an α2 subunit, a β2 subunit, and a γ2 subunit, the alpha and beta subunits being required for enzymatic activity. Any enzyme known or predicted to catalyze the reaction described in EC code 2.7.8.17 and / or perform the molecular function described in GO term GO:0003976 is GNPTAB of this disclosure. In some embodiments, GNPTAB is variant GNPTAB. In some embodiments, GNPTAB is cleaved GNPTAB. In some embodiments, the nucleic acid encoding GNPTAB is about 4.7 kb. In some embodiments, the nucleic acid encoding GNPTAB is less than about 4.7 kb. In some embodiments, variant (e.g., cleaved) GNPTAB includes an alpha subunit and a beta subunit.In some embodiments, variant GNPTAB (e.g., cleaved GNPTAB) is identical to natural GNPTAB by at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, variant GNPTAB (e.g., cleaved GNPTAB) retains the activity of natural GNPTAB by at least about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%. Examples of human GNPTAB are provided by GenBank acceptance numbers NP_077288.2 and NM_024312.4. An example of a human GNPTAB amino acid sequence is provided by SEQ ID NO: 1. An example of mouse GNPTAB is provided by GenBank acceptance number NP_001004164. An example of a mouse GNPTAB amino acid sequence is provided by SEQ ID NO: 2. An example of GNPTAB additions is provided by GenBank acceptance numbers XP_001155334 and XP_509312 (chimpanzees), XP_002687680 and NP_001179157 (cattle), XP_416329 (chickens), XP_532667 (dogs), XP_001497199 (horses), XP_001079967 and XP_343195 (rats), and NP_001038233 (zebrafish).

[0048] Mucolipidosis type II (the terms ML II, ML-II, and ML type II are used interchangeably herein) and mucolipidosis type III (the terms ML III, ML-III, and ML type III are used interchangeably herein) refer to a class of diseases caused by mutations in the GNPTAB gene. Both diseases are autosomal recessive genetic disorders. However, ML III is typically associated with mutations that result in a milder loss of GNPTAB function compared to ML II. Therefore, ML II typically results in a more severe disease phenotype than ML III. ML II is also known as an I-cell disorder. A further description of ML II can be found in OMIM entry #252500. ML III is also known as pseudo-Hurler polydystrophy. A further description of ML III can be found in OMIM entry #252500. It can be seen at 52600.

[0049] The term "chicken β-actin (CBA) promoter" refers to a polynucleotide sequence derived from the chicken β-actin gene (e.g., chicken beta-actin represented by GenBank Entrez Gene ID396526). As used herein, the term "chicken β-actin promoter" refers to a promoter containing the cytomegalovirus (CMV) early enhancer element, the promoter and first exon and intron of the chicken β-actin gene, and the splice acceptor of the rabbit beta-globin gene, such as the sequence described in Miyazaki, J. et al., (1989) Gene 79(2):269-77. As used herein, the term "CAG promoter" is used interchangeably. As used herein, the term "CMV early enhancer / chicken beta-actin (CAG) promoter" is used interchangeably.

[0050] The truncated chicken beta-actin promoter was selected based on deletion studies showing that deletion upstream of sequence ~106 can be performed without significantly affecting promoter activity (Quitschke et al., J. Biol. Chem. 264: pp. 9539-9546, 1989).

[0051] References of “about” a value or parameter in this specification include (and describe) embodiments relating to that value or parameter itself. For example, a statement referring to “about X” includes a statement of “X”.

[0052] When used herein, the singular articles "a," "an," and "the" include plural references unless otherwise indicated.

[0053] The aspects and embodiments of the present invention described herein are understood to include “including,” “consisting of,” and / or “essentially consisting of.”

[0054] III. Vectors In certain embodiments, the present invention provides rAAV vectors suitable for use in any of the methods, rAAV particles, and / or pharmaceutical compositions described herein, for example. For example, in some embodiments, heterogeneous nucleic acids (e.g., polynucleotide sequences encoding functional GNPTAB polypeptides) are delivered to a target by the rAAV vectors of the present disclosure.

[0055] Certain aspects of this disclosure relate to the alpha and beta subunits (GNPTAB) of N-acetylglucosamine-1-phosphatetransferase, e.g., GNPTAB polypeptide or nucleic acid encoding the GNPTAB polypeptide. As is known in the art, the N-acetylglucosamine-1-phosphatetransferase (also known as N-acetylglucosamine-1-phosphotransferase) enzyme comprises two alpha subunits, two beta subunits, and two gamma subunits. The alpha and beta subunits are encoded by the GNPTAB gene (also known as GNPTA, I-cell disease, or ICD, or mouse EG432486 or mKIAA1208). Examples of GNPTAB genes include, for example, human GNPTAB (e.g., described with NCBI gene ID number 79158) and mouse GNPTAB (e.g., described with NCBI gene ID number 432486).

[0056] In some embodiments, the GNPTAB polypeptide is a human GNPTAB polypeptide. Examples of human GNPTAB polypeptide sequences include, but are not limited to, NCBI reference sequence number NP_077288. In some embodiments, the GNPTAB polypeptide contains the amino acid sequence of SEQ ID NO: 1. In some embodiments, the GNPTAB polypeptide contains an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, GNPTAB is a cleaved form of GNPTAB. In some embodiments, the nucleic acid encoding GNPTAB is about 4.7 kb. In some embodiments, the nucleic acid encoding GNPTAB is less than approximately 4.7 kb. In some embodiments, the variant (e.g., cleaved) GNPTAB comprises an alpha subunit and a beta subunit. In some embodiments, the GNPTAB polypeptide is a variant GNPTAB polypeptide (e.g., cleaved GNPTAB) that maintains at least a portion of the activity of the wild-type GNPTAB polypeptide (e.g., at least about 5%, 10%, 25%, 50%, 75%, or 100% of the activity of wild-type GNPTAB). In some embodiments, the variant GNPTAB polypeptide (e.g., cleaved GNPTAB) has higher activity compared to the wild-type GNPTAB polypeptide (e.g., at least about 125%, 150%, 200%, 300%, or 500% higher activity compared to wild-type GNPTAB).

[0057] In some embodiments, heterologous nucleic acids (e.g., nucleic acids encoding GNPTAB) are operably linked to promoters. Exemplary promoters include the cytomegalovirus (CMV) initial promoter, RSV LTR, MoMLV LTR, phosphoglycerate kinase-1 (PGK) promoter, Simianvirus 40 (SV40) promoter and CK6 promoter, trans tyretin promoter (TTR), TK promoter, tetracycline-responsive promoter (TRE), HBV promoter, hAAT promoter, LSP promoter, chimeric liver-specific promoter (LSP), E2F promoter, telomerase (hTERT) promoter; cytomegalovirus enhancer / chicken beta-actin / rabbit β-globin promoter (CAG promoter, Niwa et al., Gene, 1991, 108(2):193-199) and elongation factor 1-alpha promoter (EFl-alpha promoter) (Kim et al., Gene, 1990, 91(2):217-23 and Guo et al., Gene). Examples include, but are not limited to, Ther., 1996, 3(9):802-810. The promoter is a constitutive promoter, an inductive promoter, or an inhibitory promoter. In some embodiments, the promoter comprises a human β-glucuronidase promoter or a cytomegalovirus (CMV) enhancer linked to a chicken β-actin (CBA) promoter. In some embodiments, the promoter comprises a modified CBA promoter or a cleaved CBA promoter. In some embodiments, the promoter comprises a truncated CMV enhancer.

[0058] In some embodiments, the vector includes introns. For example, in some embodiments, the introns are chimeric introns derived from chicken beta-actin and rabbit beta-globin. In some embodiments, the introns are mouse microviral (MVM) introns.

[0059] In some embodiments, the vector includes a polyadenylated (polyA) sequence, such as the bovine growth hormone (BGH) poly(A) sequence (see, for example, acceptance number EF592533). Numerous examples of polyadenylated sequences are known in the art, such as the SV40 polyadenylated sequence and the HSV TK pA polyadenylated sequence.

[0060] While we do not wish to be bound by theory, the large size of the GNPTAB coding sequence makes it advantageous to minimize the size of other elements of the rAAV vector (e.g., promoter, enhancer, intron, polyA sequence, etc.). In some embodiments, truncated variants of any of the promoters described herein are used in the rAAV vector. Methods for generating truncated variants of promoters (e.g., the promoters listed above) are known in the art. For example, a promoter of interest can be mutated by introducing one or more nucleotide deletions and / or substitutions into the promoter sequence, and such variant promoter sequences can be individually cloned into a vector containing a reporter construct under the control of each promoter sequence. This system can be used to identify truncated variant promoters that maintain the desired strength (e.g., amount of transcript produced). In some embodiments, the rAAV vectors of this disclosure include modified, cleaved, and / or truncated CMV enhancer / CBA promoters, such as those described herein. Similar methods can be used to identify truncated variant introns that adequately maintain transcript, mRNA stability, and / or splicing levels. In some embodiments, the rAAV vectors of this disclosure contain truncated introns as described herein. Similar methods can be used to identify truncated variant poly-A sequences that adequately maintain transcript, mRNA stability, and / or polyadenylation levels. In some embodiments, the rAAV vectors of this disclosure contain truncated poly-A sequences as described herein. In some embodiments, the GTNAP gene contains a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 or 2.

[0061] The present invention considers the use of recombinant viral genomes for introducing nucleic acids to be packaged into one or more nucleic acid sequences encoding therapeutic polypeptides and / or rAAV viral particles. The recombinant viral genome includes any elements for establishing the expression of therapeutic polypeptides and / or nucleic acids, such as promoters, ITRs as disclosed herein, ribosome-binding elements, terminators, enhancers, selection markers, introns, poly(A) signals and / or replication start sites.

[0062] IV. Virus particles and methods for producing virus particles Certain aspects of this disclosure relate, for example, to rAAV particles containing the rAAV vector of this disclosure. In AAV particles, nucleic acids are capsidized to the AAV particles. The AAV particles also contain a capsid protein. In some embodiments, the nucleic acid includes a regulatory sequence comprising a coding sequence of interest (e.g., a GNPTAB coding sequence), a transcription start sequence, and a transcription termination sequence operably linked to the components in the direction of transcription, thereby forming an expression cassette. The expression cassette is flanked at its 5' and 3' ends by at least one functional AAV ITR sequence. "Functional AAV ITR sequence" means that the ITR sequence functions as intended for the rescue, replication, and packaging of AAV virions. See Davidson et al., PNAS, 2000, 97(7)3428-32; Passini et al., J. Virol., 2003, 77(12):7034-40; and Pechan et al., Gene Ther., 2009, 16:10-16, all of which are incorporated herein by reference in their entirety. To carry out certain aspects of the present invention, the recombinant vector comprises at least all of the AAV sequence essential for capsidization and the physical structure for infection by rAAV. The AAV ITR for use in the vector of the present invention is the wild-type nucleotide sequence (e.g., Kotin, Hum. Gene Ther., 1994, 5:793-801). It is not necessary to have the AAV serotypes listed in [reference], but rather they can be modified by nucleotide insertion, deletion, or substitution, or the AAV ITR may be derived from any of several AAV serotypes. More than 40 AAV serotypes are currently known, and new serotypes and variants of existing serotypes continue to be identified. See Gao et al., PNAS, 2002, 99(18):11854-56; Gao et al., PNAS, 2003, 100(10):6081-66; and Bossis et al., J. Virol., 2003, 77(12):6799-810. The use of any AAV serotype is considered to be within the scope of this invention. In some embodiments, the rAAV vector is a vector derived from an AAV serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV ITR. In some embodiments, the nucleic acids in the AAV include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV ITR.

[0063] In some embodiments, rAAV particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 (e.g., wild-type AAV6 capsid, or variant AAV6 capsids such as ShH10, as described in U.S. Pregrant Publication No. 2012 / 0164106), AAV7, AAV8, AAVrh8, AAVrh8R, AAV9 (e.g., wild-type AAV9 capsid, or modified AAV9 capsids, as described in U.S. Pregrant Publication No. 2013 / 0323226), AAV10, AAVrh10, AAV11, AAV12, tyrosine capsid variants, heparin-binding capsid variants, AAV2R471A capsid, AAVAAV2 / 2-7m8 capsid, and AAV The AAV particles contain capsidized proteins selected from DJ capsids (e.g., AAV-DJ / 8 capsid, AAV-DJ / 9 capsid, or any other capsid described in U.S. Pre-Grant Publication No. 2012 / 0066783), AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2 / HBoV1 capsid, or AAV capsids described in U.S. Patent No. 8,283,151 or International Publication No. WO / 2003 / 042397. In a further embodiment, the rAAV particles contain capsid proteins of AAV serotypes of clades A-F.

[0064] Different AAV serotypes are used to optimize the transduction of specific target cells or to target specific cell types within specific target tissues (e.g., diseased tissues). rAAV particles may contain viral proteins and viral nucleic acids of the same or mixed serotypes. For example, an rAAV particle may contain one or more ITRs and capsids derived from the same AAV serotype, or an rAAV particle may contain one or more ITRs derived from a different AAV serotype than the capsid of the rAAV particle. In certain embodiments, an rAAV particle may contain an AAV8 capsid and one or more (e.g., two) AAV2 ITRs.

[0065] Production of AAV particles Transfection, stable cell line production, and adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, JE et al. (1997) J. Virology 71(11):8780-8789), and baculovirus-AAV hybrids (Urabe, M et al., (2002) Human Gene Therapy 13(16):1935-1943; Kotin, R. (2011) H Numerous methods for producing rAAV vectors, including infectious hybrid virus production systems (including um Mol Genet.20(R1):pp. R2-R6), are known in the art. An rAAV-producing culture for the production of rAAV virus particles requires all of the following: 1) suitable host cells, 2) suitable helper virus function, 3) AAV rep gene and cap gene and gene product; 4) nucleic acid (such as therapeutic nucleic acid) with at least one AAV ITR sequence adjacent to it (e.g., an AAV genome encoding GNPTAB); and 5) suitable culture medium and culture components to support rAAV production. In some embodiments, suitable host cells are primate host cells. In some embodiments, suitable host cells are human-derived cell lines such as HeLa cells, A549 cells, 293 cells, or Perc.6 cells. In some embodiments, appropriate helper virus function is provided by wild-type adenovirus or mutant adenovirus (such as temperature-sensitive adenovirus), herpesvirus (HSV), baculovirus, or plasmid construct that provides helper function. In some embodiments, the AAV rep gene product and cap gene product are derived from any AAV serotype. While not mandatory, generally, the AAV rep gene product is of the same serotype as the ITR of the rAAV vector genome, insofar as the rep gene product functions to replicate and package the rAAV genome. Appropriate media known in the art are used for the production of the rAAV vector. These media include, but are not limited to, modified Eagle medium (MEM), Dulbecco's modified Eagle medium (DMEM), media from Hyclone Laboratories and JRH, custom formulations such as those described in U.S. Patent No. 6,566,118, and Sf-900 II SFM medium described in U.S. Patent No. 6,723,551, each of which is incorporated herein by reference in its entirety, particularly with respect to custom medium formulations for use in the production of recombinant AAV vectors. In some embodiments, the AAV helper function is provided by adenovirus or HSV.In some embodiments, the AAV helper function is provided by a baculovirus, and the host cell is an insect cell (e.g., Spodoptera frugiperda (Sf9) cell).

[0066] One method for producing rAAV particles is triple transfection. Briefly, a plasmid containing the rep gene and capsid gene is transfected into a cell line (e.g., HEK-293 cells) together with a helper adenovirus plasmid (e.g., using the calcium phosphate method), the virus is recovered, and optionally purified. Thus, in some embodiments, rAAV particles are produced by triple transfection into a host cell of nucleic acids encoding the rAAV vector, nucleic acids encoding AAV rep and cap, and nucleic acids encoding AAV helper virus function, and the transfection of nucleic acids into a host cell generates a host cell capable of producing rAAV particles.

[0067] In some embodiments, rAAV particles are produced by a cell line production method (see Martin et al. (2013) Human Gene Therapy Methods 24:253-269; U.S. Pre-grant Publication No. 2004 / 0224411; and Liu, XL et al. (1999) Gene Ther. 6:293-299). Briefly, a cell line (e.g., HeLa cell line, 293 cell line, A549 cell line, or Perc. 6 cell line) is stably transfected with a plasmid containing a vector genome (e.g., GNPTAB) containing a rep gene, a capsid gene, and a promoter-heterogeneous nucleic acid sequence. The cell line is screened to select a lead clone for rAAV production, which is then deployed in a production bioreactor and infected with a helper virus (e.g., adenovirus or HSV) to initiate rAAV production. Next, the virus is collected and the adenovirus is inactivated (e.g., by heat) and / or removed. Then, the rAAV particles are purified. Thus, in some embodiments, the rAAV particles were produced by a production cell line containing one or more nucleic acids encoding the rAAV vector, nucleic acids encoding AAV rep and cap, and nucleic acids encoding AAV helper virus function. As described herein, the production cell line method is advantageous for the production of rAAV particles with oversized genomes compared to the triple transfection method.

[0068] In some embodiments, the nucleic acids encoding the AAV rep and cap genes, and / or the rAAV genome, are stably maintained in the producing cell line. In some embodiments, the nucleic acids encoding the AAV rep and cap genes, and / or the rAAV genome, are introduced into a cell line in one or more plasmids to generate a producing cell line. In some embodiments, the AAV rep, AAV cap, and rAAV genome are introduced into cells in the same plasmid. In other embodiments, the AAV rep, AAV cap, and rAAV genome are introduced into cells in different plasmids. In some embodiments, a cell line stably transfected with the plasmid maintains the plasmid over multiple cell line passages (e.g., 5, 10, 20, 30, 40, 50, or more than 50 cell passages). For example, the plasmid is replicated as a cell replica, or the plasmid is incorporated into the cell genome. Various sequences have been identified that enable plasmids to autonomously replicate in cells (e.g., human cells) (see, e.g., Krysan, PJ et al. (1989) Mol. Cell Biol. 9: pp. 1026-1033). In some embodiments, plasmids contain selective markers (e.g., antibiotic resistance markers) that allow for the selection of cells that maintain the plasmid. Selective markers commonly used in mammalian cells include, but are not limited to, blastosidine, G418, hygromycin B, zeocin, puromycin, and their derivatives. Methods for introducing nucleic acids into cells are known in the art and are not limited to, but include, viral transduction, cationic transfection (e.g., using cationic polymers such as DEAE-dextran or cationic lipids such as lipofectamine), calcium phosphate methods, microinjection, particulate guns, electroporation, and nanoparticle transfection (for further details, see, for example, Kim, TK and Eberwine, JH (2010) Anal. Bioanal. Chem. 397: pp. 3173-3178).

[0069] In some embodiments, the nucleic acids encoding the AAV rep and cap genes, and / or the rAAV genome, are stably integrated into the genome of the producing cell line. In some embodiments, the nucleic acids encoding the AAV rep and cap genes, and / or the rAAV genome, are introduced into a cell line in one or more plasmids to generate a producing cell line. In some embodiments, the AAV rep, AAV cap, and rAAV genome are introduced into the cell in the same plasmid. In other embodiments, AAV The rep, AAV cap, and rAAV genomes are introduced into cells using different plasmids. In some embodiments, the plasmids contain selective markers (e.g., antibiotic resistance markers) that allow for the selection of cells that retain the plasmid. Methods for stably incorporating nucleic acids into various host cell lines are known in the art. For example, repeated selection (e.g., by the use of selective markers) is used to select cells into which nucleic acids containing selective markers (and AAV cap and rep genes, as well as / or rAAV genomes) are incorporated. In other embodiments, nucleic acids are incorporated into cell lines in a site-specific manner to generate productive cell lines. Examples include FLP / FRT (e.g., see O'Gorman, S. et al. (1991) Science 251:1351-1355) and Cre / loxP (e.g., Sauer, B. and Henderson, N. (1988) Proc. Natl. Acad. Sci. 85:5166-5170). Several site-specific recombinant systems are known in the art, such as (see page 5995-6000) and phi C31-att (see, for example, Groth, AC et al. (2000) Proc. Natl. Acad. Sci. 97: pp. 5995-6000).

[0070] In some embodiments, the producing cell line is derived from a primate cell line (e.g., a non-human primate cell line such as the Vero cell line or FRhL-2 cell line). In some embodiments, the cell line is derived from a human cell line. In some embodiments, the producing cell line is derived from HeLa cells, 293 cells, A549 cells, or PERC.6® (Crucell) cells. For example, prior to the introduction of AAV rep and cap genes and / or nucleic acids encoding an oversized rAAV genome into the cell line and / or stable maintenance / incorporation for generating the producing cell line, the cell line is a HeLa cell line, a 293 cell line, an A549 cell line, or a PERC.6® (Crucell) cell line, or a derivative thereof.

[0071] In some embodiments, the resulting cell lines are adapted for growth in suspension. As is known in the art, anchorage-dependent cells typically cannot grow in suspension without a substrate such as microcarrier beads. Adapting a cell line for growth in suspension includes, for example, growing the cell line in agitated culture using a stirring paddle, using a culture medium lacking calcium and magnesium ions (and optionally an antifoaming agent) to prevent agglomeration, using a culture vessel coated with a silicon-treated compound, and selecting cells in the culture at each passage (rather than in large agglomerations or on the sides of the vessel). For further explanation, see, for example, the ATCC Frequently Asked Questions document (available at www.atcc.org / Global / FAQs / 9 / 1 / Adapting%20a%20monolayer%20cell%20line%20to%20suspension-40.aspx) and its references.

[0072] In some embodiments, a method is provided for producing any of the rAAV particles disclosed herein, comprising (a) culturing a host cell having AAV helper function, under conditions in which rAAV particles are produced, (i) one or more AAV package genes each encoding an AAV replication protein and / or capsidization protein; (ii) an rAAV probe vector comprising a nucleic acid encoding a heterologous nucleic acid as described herein, with at least one AAV ITR adjacent to it; and (iii) recovering the rAAV particles produced by the host cell. In some embodiments, the at least one AAV ITR is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotype ITR, etc. For example, in some embodiments, the AAV serotypes are AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In certain embodiments, the nucleic acids in the AAV include AAV2 ITR. In some embodiments, the capsidized proteins are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 The group consists of N587A, AAV2 E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1 / AAV2 chimera, bovine AAV, mouse AAV capsid rAAV2 / HBoV1 serotype capsid proteins, or variants thereof. In some embodiments, the capsidized protein is the AAV8 capsid protein. In some embodiments, the rAAV particle comprises a recombinant genome including the AAV8 capsid and AAV2 ITR, as well as a nucleus encoding a therapeutic transgene / nucleic acid. It contains an acid (for example, a nucleic acid that codes for GNPTAB).

[0073] A suitable rAAV-producing culture medium of the present invention is supplemented with serum or serum-derived recombinant protein at a level of 0.5% to 20% (V / V or W / V). Alternatively, as is known in the art, rAAV vectors are produced under serum-free conditions, which are also referred to as animal-derived product-free media. Those skilled in the art will understand that commercially available or custom-made media designed to support the production of rAAV vectors are also supplemented with one or more cell culture components known in the art, including but not limited to glucose, vitamins, amino acids and / or growth factors, to increase the titer of rAAV in the producing culture.

[0074] rAAV-producing cultures can be grown under a variety of conditions suitable for utilizing specific host cells (such as a wide temperature range and varying lengths of time). As is known in the art, rAAV-producing cultures include adhesion-dependent cultures that can be cultured in suitable adhesion-dependent containers such as roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluid-bed bioreactors. rAAV vector-producing cultures also include suspension-compatible host cells such as HeLa cells, 293 cells, and SF-9 cells, which can be cultured in a variety of ways, including disposable systems such as stirred flasks, stirred-tank bioreactors, and WaveBag systems.

[0075] As fully described in U.S. Patent No. 6,566,118, the rAAV vector particles of the present invention are harvested from rAAV-producing cultures by lysing the host cells of the producing culture, or by harvesting the spent medium from the producing culture, provided that the cells are cultured under conditions known in the art such that the cells release rAAV particles into the medium from intact cells. Suitable methods for lysing cells are also known in the art and include, for example, multiple freeze / thaw cycles, sonication, microdissolution, and treatment with chemicals such as surfactants and / or proteases.

[0076] In further embodiments, rAAV particles are purified. As used herein, the term “purified” includes preparations of rAAV particles that do not contain at least some of the other components that would be present if the rAAV particles were naturally occurring or if they were initially prepared. Thus, for example, isolated rAAV particles are prepared from a raw material mixture, such as a culture lysate or a productive culture supernatant, by using a purification technique to concentrate it. Concentration can be measured in various ways, such as by the proportion of DNase-resistant particles (DRPs) or genome copies (gc) present in the solution, or by infectivity, or the concentration can be measured in relation to contaminants, such as productive culture contaminants or process contaminants, including second potential interfering substances present in the raw material mixture, such as helper viruses, culture medium components, etc.

[0077] In some embodiments, rAAV-producing culture samples are clarified to remove host cell debris. In some embodiments, the producing culture samples are clarified by filtration through a series of depth filters, including, for example, a Grade DOHC Millipore Millistak+HC Pod Filter, a Grade A1HC Millipore Millistak+HC Pod Filter, and a 0.2 μm Filter Opticap XL1O Millipore Express SHC Hydrophilic Membrane filter. Clarification can also be achieved by various other standard techniques known in the art, such as centrifugation or filtration through any cellulose acetate filter with a pore size of 0.2 μm or larger known in the art.

[0078] In some embodiments, rAAV-producing culture samples are further treated with Benzonase® to digest any high molecular weight DNA present in the culture. In some embodiments, Benzonase® digestion is carried out under standard conditions known in the art, for example, for 30 minutes to several hours, at a temperature ranging from ambient temperature to 37°C, and containing Benzonase® at a final concentration of 1 to 2.5 units / ml.

[0079] rAAV particles can be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anion exchange filtration; tangent flow filtration (TFF) for concentration of rAAV particles; rAAV capture by apatite chromatography; thermal inactivation of helper viruses; rAAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and rAAV capture by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. These steps may be used individually, in various combinations, or in different orders. In some embodiments, the method includes all steps in the order described below. Methods for purifying rAAV particles can be found, for example, in Xiao et al. (1998) Journal of Virology 72:2224-2232; U.S. Patent Nos. 6,989,264 and 8,137,948; and in International Publication No. WO2010 / 148143.

[0080] V. Treatment Methods Certain aspects of this disclosure relate to methods for treating mucolipidosis type II and / or mucolipidosis type III, or methods for increasing body size, bone mineral density, or bone density in mammals having mucolipidosis type II or mucolipidosis type III. These methods are, in part, based on the findings described herein that AAV-mediated expression of GNPTAB can alleviate symptoms of ML II, such as impaired bone growth, in a mouse disease model. As stated above, both diseases are caused by loss-of-function mutations in the GNPTAB gene, which encodes the alpha and beta subunits of the catalytic GlcNAc-1 phosphotransferase.

[0081] ML II is known as an autosomal recessive genetic disorder caused by mutations in GNPTAB. GNPTAB activity is required to attach mannose-6-phosphate to proteins, thereby marking them for transport to lysosomes. In the absence of GNPTAB activity, lysosomal proteins (e.g., lysosomal hydrolase) are instead secreted extracellularly. As a result, substances that are normally disrupted in lysosomes, such as glycosaminoglycans, lipids, and oligosaccharides, accumulate in the cell, leading to the presence of large inclusion bodies. ML II is also called I-cell disease due to the presence of these inclusion body cells ("I cells"), which are identified by microscopy. Symptoms of ML II are often present immediately after birth and include skeletal abnormalities, short stature, weak muscle tone, lack of muscle tone (hypotension), hernias (e.g., distended abdomen, umbilical hernia), hip dislocation and joint deformities, cardiomegaly, mitral valve thickening and dysfunction, progressive mucosal thickening of the airways, coarse and / or noisy breathing, frequent respiratory infections, constipation, diarrhea, delayed development of gross and fine motor skills, hearing loss, and growth retardation, particularly in vocal and motor skills, and cognitive impairment. These symptoms debilitate patients with ML II, and they are typically unable to walk independently and do not survive beyond infancy.

[0082] Like ML II, ML III is known in the art as an autosomal recessive genetic disorder caused by mutations in GNPTAB. However, compared to ML II, ML III typically exhibits a weaker loss of function in GNPTAB. This leads to mutations, which are characterized by a milder disease phenotype. The symptoms of ML III are not fully apparent until 3-5 years of age, and the severity is quite variable; some patients live beyond 60 years of age, while others do not survive beyond childhood. Typical symptoms of ML III include skeletal abnormalities, short stature, aortic valve disease, corneal opacity, mild organ dilation, loss of motor function, and joint abnormalities. ML III is also called pseudo-Hurler polydystrophy. For a more detailed explanation, as well as specific mutations found in patients with ML II and ML III, see, for example, Paik, KH et al. (2005) Hum. Mutat. 26(4):308-314.

[0083] Various diagnostic tests for ML II and ML III are known in the art. In some embodiments, ML II and / or ML III are diagnosed by measuring the activity of one or more lysosomal enzymes in serum samples or other patient samples (e.g., skin samples or cultured fibroblasts). The activity of certain lysosomal enzymes in serum is 5 to 20 times higher in ML II patients than in normal patients. Similarly, in ML III, the serum activity of these enzymes increases up to 10 times. These enzymes include, but are not limited to, beta-D-hexosaminidase (EC code 3.2.1.52), beta-D-glucuronidase (EC code 3.2.1.31), beta-D-galactosidase (EC code 3.2.1.23), alpha-L-fucosidase (EC code 3.2.1.51), and alpha-D-mannosidase (EC code 3.2.1.24). In contrast, these activities are deficient in cultured fibroblasts. In some embodiments, N-acetylglucosamine-1-phosphotransferase activity is measured to diagnose ML II or ML III. In some embodiments, patients whose samples show less than 1% of N-acetylglucosamine-1-phosphotransferase activity compared to the activity from samples of normal patients are diagnosed with ML II. In some embodiments, patients whose samples show between 1% and 10% of N-acetylglucosamine-1-phosphotransferase activity compared to the activity from samples of normal patients are diagnosed with ML II.

[0084] In some embodiments, ML II and / or ML III are diagnosed by sequencing the GNPTAB locus for the mutation (e.g., coding sequence, promoter / intron sequence, or any other regulatory sequence). ML III is also characterized by increased urinary polysaccharides and / or urinary glycosaminoglycans, although these symptoms are not specific to ML II and ML III. In some embodiments, ML II and / or ML III are diagnosed prenatally by examining a chorionic sample, for example, by histological examination of a trophoblast (see, e.g., Poenaru, L. et al. (1984) Am.J.Hum.Genet. 36(6):1379-85). As mentioned above, in ML II, I cells from a patient's sample (e.g., fibroblasts) are also identified by microscopy.

[0085] In some embodiments, treatments for ML II and / or ML III (e.g., gene therapy vectors of this disclosure) are tested for therapeutic efficacy. For example, in some embodiments, treatments for ML II and / or ML III result in increased height, increased bone mineral density, and relief of one or more symptoms of ML II and / or ML III as described herein. The efficacy of rAAV administration can be monitored by several diagnostic criteria described herein. For example, after treating a subject using the method of the present invention, the subject is evaluated for improvement and / or stabilization and / or delay in the progression of one or more signs or symptoms of the disease state by one or more clinical parameters, including, for example, those described herein. Examples of such tests are known in the art and include objective and subjective tests. This includes measurements (e.g., those reported by the subject).

[0086] In some embodiments, GNPTAB expression is measured to monitor the efficacy of a treatment. Measuring GNPTAB expression refers to measuring the expression of GNPTAB mRNA and / or protein. Various methods for measuring mRNA and / or protein expression are known in the art and are not limited to, but include qPCR, Northern blotting, RNA-seq, semi-quantitative PCR, Western blotting, mass spectrometry, and ELISA.

[0087] In some embodiments, but not limited to, the activity of one or more lysosomal enzymes, including beta-D-hexosaminidase (EC code 3.2.1.52), beta-D-glucuronidase (EC code 3.2.1.31), beta-D-galactosidase (EC code 3.2.1.23), alpha-L-fucosidase (EC code 3.2.1.51), and alpha-D-mannosidase (EC code 3.2.1.24), is measured in a patient sample (e.g., serum sample) to monitor the efficacy of the treatment. A decrease in serum lysosomal enzyme activity indicates efficacy.

[0088] In some embodiments, improvement in joint or limb function is an efficacy. In some embodiments, improvement in vocalization is an efficacy. In some embodiments, improvement in motor function is an efficacy. In some embodiments, increased growth rate (e.g., increased body length) is an efficacy. In some embodiments, bone density, bone mineral content, and / or growth (e.g., body length) are evaluated to monitor the efficacy of the treatment, as described in the examples herein. Methods for monitoring body length are well known to those skilled in the art. Tests for monitoring bone density and bone mineral content (e.g., densitometry tests) are also well known to those skilled in the art (e.g., as described herein) and are not limited to, but include dual-energy X-ray absorptiometry (DEXA) scans, peripheral dual-energy X-ray absorptiometry (P-DEXA) scans, dual-photon absorption (DPA) and computed tomography (CT) scans.

[0089] In some embodiments, the treatment for ML II and / or ML III (e.g., the gene therapy vectors of this disclosure) is tested in animal models. Animal models for ML II and ML III are known in the art. In some embodiments, the treatment for ML II and / or ML III is tested in mouse models, such as the recombinant mouse models described in the examples herein. In some embodiments, the treatment for ML II is tested in cat models for ML II (see Mazrier, H. et al. (2003) J.Hered. 94(5): pp. 363-73 or Bosshard, NU et al. (1996) Vet. Pathol. 33(1): pp. 1-13 for further explanation).

[0090] The selection of specific rAAV vectors and compositions depends on many different factors, including, but not limited to, the individual's medical history and condition characteristics, as well as the individual being treated. Ultimately, it is the prescribing physician's responsibility to evaluate these characteristics and design an appropriate treatment regimen.

[0091] In some embodiments, the present invention provides a method for treating ML II and / or ML III by administering an effective dose of the rAAV particles of this disclosure. The rAAV is administered to a specific tissue of interest or systemically. In some embodiments, an effective dose of rAAV is administered parenterally. Parenteral administration routes include, but are not limited to, intravenous, intraperitoneal, intraosseous, intraarterial, intracerebral, intramuscular, intrathecal, subcutaneous, intraventricular, and intrahepatic. In some embodiments, an effective dose of rAAV is administered in a single dose. It is administered via a route. In some embodiments, an effective dose of rAAV is administered through a combination of two or more administration routes. In some embodiments, an effective dose of rAAV is administered at one site. In other embodiments, an effective dose of rAAV is administered at two or more sites.

[0092] An effective amount of rAAV (in some embodiments, in the form of particles) is administered depending on the therapeutic objective. For example, if the desired therapeutic effect can be achieved with a low percentage of transduction, then the therapeutic objective is usually to achieve or exceed this level of transduction. In some cases, this level of transduction can be achieved by transducing only about 1-5% of target cells of the desired tissue type, in some embodiments, at least about 20% of cells of the desired tissue type, in some embodiments, at least about 50% of cells of the desired tissue type, in some embodiments, at least about 80% of cells of the desired tissue type, in some embodiments, at least about 95% of cells of the desired tissue type, and in some embodiments, at least about 99% of cells of the desired tissue type. The rAAV composition is administered in one or more doses, either during the same procedure or with intervals of several days, weeks, months, or years. One or more of the administration routes described herein may be used. In some embodiments, multiple vectors are used to treat humans.

[0093] Methods for identifying cells transduced by AAV virus particles are known in the art; for example, immunohistochemical tests or the use of markers such as highly sensitive green fluorescent protein can be used to detect transduction of viral particles; for example, viral particles containing rAAV capsids having one or more amino acid substitutions.

[0094] In some embodiments, an effective dose of rAAV particles is administered simultaneously or sequentially to two or more locations. In other embodiments, an effective dose of rAAV particles is administered two or more times (e.g., repeatedly) to a single location. In some embodiments, multiple injections of rAAV virus particles are spaced out by no more than 1, 2, 3, 4, 5, 6, 9, 12, or 24 hours.

[0095] An effective amount of rAAV (in some embodiments, in the form of particles) is administered depending on the therapeutic objective. For example, if the desired therapeutic effect can be achieved with a low percentage of transduction, then the therapeutic objective is usually to achieve or exceed this level of transduction. In some cases, this level of transduction can be achieved by transducing only about 1-5% of target cells, at least about 20% of cells of the desired tissue type in some embodiments, at least about 50% of cells of the desired tissue type in some embodiments, at least about 80% of cells of the desired tissue type in some embodiments, at least about 95% of cells of the desired tissue type in some embodiments, and at least about 99% of cells of the desired tissue type in some embodiments. The rAAV composition is administered in one or more doses, either during the same procedure or with intervals of several days, weeks, months, or years. In some embodiments, multiple vectors are used to treat mammals (e.g., humans).

[0096] In some embodiments, the rAAV compositions of this disclosure are used for administration to humans. In some embodiments, the rAAV compositions of this disclosure are used for administration to children. While we do not wish to be bound by theory, since many of the symptoms of ML II and ML III develop spontaneously (e.g., growth, limb and joint abnormalities; speech and motor delays), it is particularly advantageous to treat ML II and / or ML III as early as possible during life. In some embodiments, an effective amount of rAAV is (in some embodiments, In particle form, it is administered to patients under 1 month, under 2 months, under 3 months, under 4 months, under 5 months, under 6 months, under 7 months, under 8 months, under 9 months, under 10 months, under 11 months, under 1 year, under 13 months, under 14 months, under 15 months, under 16 months, under 17 months, under 18 months, under 19 months, under 20 months, under 21 months, under 22 months, under 2 years, or under 3 years of age.

[0097] In some embodiments, the rAAV compositions of this disclosure are used for administration to young adults. In some embodiments, an effective amount of rAAV (in some embodiments, in the form of particles) is administered to patients under 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 years of age.

[0098] VI. Kit or Product The rAAV vectors, particles, and / or pharmaceutical compositions described herein are contained in a kit or product designed for use in one of the methods of the present invention described herein.

[0099] Generally, the system includes a cannula, one or more syringes (e.g., one, two, three, four or more), and one or more liquids (e.g., one, two, three, four or more), which are suitable for use in the method of the present invention.

[0100] The syringe is any suitable syringe, as long as it can be connected to a cannula for the delivery of liquid. In some embodiments, the system has one syringe. In some embodiments, the system has two syringes. In some embodiments, the system has three syringes. In some embodiments, the system has four or more syringes. Suitable liquids for use in the method of the present invention include those described herein, for example, one or more liquids each containing an effective amount of one or more vectors as described herein, and one or more liquids containing one or more therapeutic agents.

[0101] In some embodiments, the kit comprises a single liquid (e.g., a pharmaceutically acceptable liquid containing an effective amount of vector). In some embodiments, the kit comprises two liquids. In some embodiments, the kit comprises three liquids. In some embodiments, the kit comprises four or more liquids. Examples of liquids include diluents, buffers, excipients, or any other liquids described herein or known in the art that are suitable for delivery, dilution, stabilization, buffering, or otherwise transport of the AAV vector composition of this disclosure. In some embodiments, the kit comprises one or more buffers, e.g., pH-buffered aqueous solutions. Examples of buffers include, but are not limited to, phosphate buffers, citrate buffers, Tris buffers, HEPES buffers, and other organic acid buffers.

[0102] In some embodiments, the kit includes a container. Suitable containers include, for example, vials, bags, syringes, and bottles. The container is made of one or more materials, such as glass, metal, or plastic. In some embodiments, the container is used to hold the rAAV composition of the present disclosure. In some embodiments, the container also holds liquids and / or other therapeutic agents.

[0103] In some embodiments, the kit includes an additional therapeutic agent along with the rAAV composition of this disclosure. In some embodiments, the rAAV composition and the additional therapeutic agent are mixed. In some embodiments, the rAAV composition and the additional therapeutic agent are kept separately. In some embodiments, the rAAV composition and the additional therapeutic agent are in the same container. In some embodiments, the rAAV composition and the additional therapeutic agent are in different containers. In some embodiments, the rAAV composition and the additional therapeutic agent are administered simultaneously. In some embodiments, the rAAV composition and the additional therapeutic agent are administered on the same day. In some embodiments, the rAAV composition is administered within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, or 6 months of the administration of the additional therapeutic agent.

[0104] In some embodiments, the kit includes therapeutic agents to transiently suppress the immune system before administration of AAV. In some embodiments, the patient's immune system is transiently suppressed immediately before and after viral injection to inhibit the T-cell response to AAV particles (see, e.g., Ferreira et al., Hum. Gene Ther. 25: pp. 180-188, 2014). In some embodiments, the kit further provides cyclosporine, mycophenolate mofetil, and / or methylprednisolone.

[0105] The rAAV particles and / or compositions of the present invention are further packaged in a kit, including instructions for use. In some embodiments, the kit further includes a device for delivery (e.g., parenteral administration of any kind described herein) of the rAAV particle composition. In some embodiments, the instructions for use include instructions by one of the methods described herein. In some embodiments, the instructions are printed on a label provided (e.g., affixed) to the container. In some embodiments, the instructions for use include instructions for administering an effective amount of rAAV particles to a mammal (e.g., human) for, for example, to treat mucolipidosis type II (ML II) and / or mucolipidosis type III (ML III), to increase body size, to increase bone mineral density, and / or to increase bone density.

[0106] VII. Animal Models This invention provides an animal model of mucolipidosis II. GNPTAB mutant mice are generated by microinjection of embryonic stem (ES) cell clones into host blast cysts using standard methods. Briefly, disruption of the target of the mouse GNPTAB locus (e.g., deletion of exons 12-20) is performed by homologous recombination with a substitution vector containing a reporter or selective marker gene. All offspring were genotyped by polymerase chain reaction analysis of tail snip DNA. All mice used in this study were males identified as wild-type (+ / +) mice, heterozygous (+ / -) mice, or homozygous (- / -) mice. In some embodiments, at least one allele of the GNPTAB gene contains a deletion located between exons 12 and 20. In some embodiments, at least one allele of the GNPTAB gene contains a deletion spanning exons 12 and 20. In some embodiments, the deletion includes deletion of one or more exons 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, a portion of the GNPTAB gene is replaced by a gene encoding a reporter and / or selective marker. In some embodiments, the selective marker confers resistance to neomycin. In some embodiments, the animal is a mammal (e.g., rodents, rabbits, cats, dogs, pigs, monkeys). In some embodiments, the mammal is a rodent (e.g., mouse, rat, hamster, guinea pig). In some embodiments, the animal is immunocompetent or immunodeficient. In some embodiments, the animal model includes a genetically modified animal. Other mouse models of ML II are described by Gelfman et al. (Invest. Optham. Visual Sci. 2007, 48:5221-5228) and Paton, L. et al. (J Biol Chem). Provided by 2014, 289(39):26709-21).

[0107] In some embodiments, the present invention provides a method for evaluating agents for the treatment of mucolipidosis II (ML II), comprising administering the agent to an animal model described herein, wherein the alleviation of one or more symptoms of ML II indicates that the agent provides a beneficial treatment for ML II. In some embodiments, the symptoms of ML II are weight loss, decreased bone density, decreased bone mineral density, skeletal abnormalities, cognitive impairment, delayed development of gross and fine motor skills, hearing loss, lack of muscle tone, distended abdomen, umbilical hernia, progressive mucosal thickening of the airways, frequent respiratory infections, mitral valve thickening and dysfunction, constipation and / or diarrhea. In some embodiments, the agent is a small molecule, polypeptide, antibody, nucleic acid or recombinant viral particle. [Examples]

[0108] The present invention will be more fully understood by reference to the following embodiments. However, these should not be construed as limiting the scope of the invention. It should be understood that the embodiments and examples described herein are for illustrative purposes only and suggest various modifications or changes to those skilled in the art in light of them, and that such modifications or changes should be included within the spirit and scope of this application and the appended claims.

[0109] Example 1: Generation and characterization of GNPTAB knockout mice Mucolipidosis type II (ML type II or ML-II, also known as II-cell disease) ML (OMIM entry #252500) is an autosomal recessive lysosomal storage disorder caused by a deficiency of N-acetylglucosamine-1-phosphotransferase (GNPTAB). The presence of numerous inclusion bodies in the cytoplasm of fibroblasts, the absence of mucopolysaccharidosis, increased serum lysosomal enzyme activity, and decreased GlcNAc-phosphotransferase activity are characteristic features of this disease. To study the pathology of ML type II, GNPTAB knockout (KO) mice were developed and characterized.

[0110] method Generation of the AAV2 / 8-GNPTAB construct The coding sequence for mouse GNPTAB was amplified and codon-optimized for expression in mice using Genescript (Piscataway, NJ, USA). Due to the large size of GNPTAB cDNA and the limited capacity of AAV, the cDNA was unsuitable for any available vector. Therefore, a novel expression cassette containing a truncated CMV enhancer and chicken beta-actin promoter, as well as very small introns, was designed. Expression of full-length GNPTAB mRNA was confirmed by transient infection of HEK293 cells in vitro, followed by quantitative RT-PCR. This AAV2 / 8-GNPTAB was purified and stored at -70°C, and 2.5 × 10⁶ units were stored. 12 The study used concentrations of DNase-resistant particles / mL. Shortened enhancer and promoter sequences were used for these studies.

[0111] animal GNPTAB mutant mice were generated by microinjection of embryonic stem (ES) cell clones into host blast cysts using standard methods. Briefly, target disruption of the mouse GNPTAB locus (deletion of exons 12-20) was performed by homologous recombination with a substitution vector containing the neomycin resistance gene. The mice used in this study had a mixed genetic background (129 / Sv and C57BL / 6). All offspring were genotyped by polymerase chain reaction analysis of tail snip DNA. All mice used in this study were wild-type (+ / + The mice were identified as males, either as heterozygous (+ / -) mice or homozygous (- / -) mice.

[0112] Mice were housed in colony rooms under a 12-hour light-dark cycle, in groups of 2-5 mice per cage. Rodent feed (Harlan Teklad #8604, Madison, WI, USA) and water were freely available. Animal care was carried out in accordance with the guidelines described in the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington DC, 1996).

[0113] Vector injection A single bilateral IV injection of PBS or AAV-GNPTAB is administered to 6-week-old GNPTAB The study was performed on knockout mice. A control group consisted of wild-type or heterozygous mice injected with PBS. Each mouse injected with AAV-GNPTAB measured 3 × 10⁻¹⁴ units. 11 Vector particles were administered. To reduce or eliminate the immune response, anti-CD40 ligand antibody (MRI) was injected alone, according to the protocol of Harlbert et al. (1998) J. Virol. 72: pp. 9795-9805.

[0114] growth studies All mice were examined weekly by measuring their total body weight (g) and body length (nasal-to-anal distance, mm) using an electronic digital caliper.

[0115] histology 10-month-old wild-type mice and GNPTAB - / - Mice were sedated and perfused via the heart with PBS. Following perfusion, the femur was removed, fixed in 4% paraformaldehyde (PFA), decalcified with 0.5 M EDTA, and embedded in paraffin. Femoral fragments (4 μM) were stained with hematoxylin-eosin. Aperio ScanScope AT (v101.0) was used for scanning and analysis of slides at 4°C. This tissue was embedded and cut for TEM or hematoxylin and eosin (H&E).

[0116] Microstructural analysis Wild-type and knockout mice were decapitated, their salivary glands removed, and fixed by immersion in glutaraldehyde in 2.5% PBS overnight at 4°C. The salivary glands were dehydrated, then washed in PBS buffer for a further 30 minutes, and subsequently post-fixed in 1% osmium tetroxide at room temperature for 1 hour. The specimens were dehydrated through a stepwise alcohol series and embedded in Epon812. Semi-thin serial sections were cut to 2.5 μM (Leica ultracut UCT), stained with toluidine blue, and observed under a light microscope. Transmission electron microscopy was performed using a Mirgagni microscope (FEI).

[0117] Lysosomal Enzyme assay Blood was extracted, the sample was centrifuged for 15 minutes (8000xg), and the plasma was stored at -70°C. Plasma lysosomal enzyme activity was measured at the Department of Chemical Pathology, Samsung Medical Center, South Korea. Specific GlcNAc-phosphotransferase activity could not be measured in ML II. Therefore, the diagnosis was based on elevated plasma lysosomal enzymes.

[0118] DEXA analysis Bone mineral density (BMD), bone mineral content (BMC), and lean body mass were measured in anesthetized mice using a pDEXA SabreX bone densitometer. After administering anesthesia, the weight of each mouse was recorded, and then the mouse was placed on a DEXA scanner. For data analysis, contours of the entire body region of the mouse were drawn.

[0119] Real-time PCR Real-time polymerase chain reaction was performed, and GNPTAB mRNA levels were quantified using the ABI PRISM 7900HT system and TaqMan gene expression assay (Applied Biosystems, Foster City, CA). mRNA levels are expressed in relation to GAPDH levels. 2-ΔΔ The data was analyzed using the CT method and SDS2.3 software (Applied Biosystems).

[0120] statistical analysis GraphPad Prism Version 5 was used for statistical analysis and the creation of figures and tables. Significant differences were determined by independent Student's t-test and one-way ANOVA. Unless otherwise specified, all data are expressed as mean ± SEM.

[0121] result GNPTAB knockout mice were generated using a gene trapping method. As explained in Figure 1A, mice were generated using homologous recombination with a substitution vector containing the neomycin resistance gene between exons 12 and 20. As shown in Figure 1B, the presence of gene traps in the GNPTAB gene of the ES clones was confirmed by Southern blot analysis.

[0122] Comprehensive phenotypic analyses were performed on wild-type, heterozygous, and homozygous mice. KO mice were visually distinguishable from wild-type (WT) littermates due to their smaller size. Average body weight (Figure 2A) and average body length (Figure 2B) were significantly reduced in KO mice. Consistent with their smaller size, homozygous mice exhibited a coarser facial morphology (Figure 2C).

[0123] As shown in Figure 3A, in normal cartilage, distinct foveal spaces surrounded the chondrocytes. The cytoplasm contained a single, distinct vacuole. In KO mice, femoral chondrocytes were significantly hypertrophied, and their enlarged foves were completely filled (Figure 3B). The cytoplasm of the hypertrophied chondrocytes was swollen with abundant micro-vacuoles containing insufficient amounts of granular amphichromatic material. There was little contraction of the chondrocytes associated with fixation, and they filled their enlarged foves. This is in contrast to wild-type chondrocytes, which contained a single, large, distinct vacuole and whose foves were only partially filled.

[0124] Examination with a light microscope revealed that the acini of the exocrine salivary glands, which consist of mucinous and serous secretory cells, showed extensive vacuolation in KO mice (Gelfman et al., 2007, Invest. Optham. Visual Sci. 48: pp. 5221-5228). To gain insight into the underlying pathological condition, the submandibular salivary glands were analyzed using an electron microscope (EM).

[0125] Both myxoid and serous secretory cells were readily observed in wild-type mice (Figures 4A–4C). In contrast, the overall structure of the submandibular salivary glands in KO mice was severely disrupted, greatly hindering the identification of serous cells in EM sections (Figures 4D–4F). Myxoid secretory cells were filled with large membrane-bound vacuoles containing heterologous material. (Figure 4F, Figure 4F). The large vacuoles were filled with undegraded cytoplasm accumulated in the mucosal cells of KO mice and surrounded by a single membrane. These observations suggest that these may represent autolysosomes formed by the fusion of an autophagy compartment with a lysosome.

[0126] Patients with ML-II have significantly elevated levels of lysosomal enzymes in their serum because they are unable to synthesize the mannose-6-phosphate recognition marker essential for the precise targeting of these enzymes to lysosomes. This lack of transport results in excessive secretion of enzymes into the blood. Figures 5A–5D show that KO mice, compared to wild-type mice, exhibited significantly increased levels of lysosomal enzymes such as N-acetylglucosaminidase (Figure 5A), β-hexosaminidase A (Figure 5B), β-galactosidase (Figure 5C), and β-glucuronidase (Figure 5D). Consistent with observations in humans, this phenotype is expected when GlcNAc-1-phosphotransferase activity is absent in homozygous mice.

[0127] In summary, these experiments demonstrate that GNPTAB knockout mice exhibit distinct phenotypes compared to wild-type mice, particularly in terms of growth, salivary gland morphology, and lysosomal enzyme levels. These results suggest that GNPTAB knockout mice can serve as a model system for studying therapeutic treatments for ML-II.

[0128] Example 2: Evaluation of AAV-mediated administration of GNPTAB in GNPTAB KO mice Since patients with ML-II exhibit growth retardation, the primary objective of this study was to evaluate the efficacy of AAV-mediated GNPTAB administration in promoting growth in the ML-II model. Therefore, the phenotypes of wild-type mice, GNPTAB heterozygous mice, and GNPTAB knockout mice were analyzed compared to GNPTAB knockout mice injected with AAV-GNPTAB.

[0129] As shown in Figures 6A and 6B, all analyses were evaluated at two time points after injection: the first at 16 weeks post-injection (at 12 weeks of age), and the second at 32 weeks post-injection (at 38 weeks of age). Growth analysis was added at 6 weeks post-injection.

[0130] Plasmids containing expression cassettes expressing mouse GNPTAB cDNA and BGH polyA signaling controlled by a CMV enhancer / CBA promoter were constructed (Figure 7A). Full-length GNPTAB was controlled under the shortened CMV enhancer / CBACBA promoter described herein. Quantitative real-time PCR analysis of livers from GNPTAB KO mice injected with AAV2 / 8-GNPTAB showed specific and high expression of AAV-GNPTAB. As expected, no AAV-GNPTAB mRNA was present in samples from control littermates (Figure 7B).

[0131] As an initial test to determine whether AAV-mediated gene transfer affects metabolism in a physiologically relevant way, weight gain was monitored for 32 weeks after injection. As shown in Figure 8A, no effect of AAV-GNPTAB treatment was observed in KO mice. Figure 8B shows that there were no observed differences in weight gain between control and AAV-GNPTAB-treated KO mice.

[0132] Next, we evaluated the effectiveness of reducing the stature phenotype in KO mice. In KO mice injected with AAV-GNPTAB, improvement in stature was observed 6 weeks after treatment, and an increase in body size was seen (Figures 9A, 9B, and Table 1 below).

[0133] [Table 1]

[0134] Next, bone mineral density (BMD), bone mineral content (BMC), and body composition were measured. DEXA is an X-ray-based imaging technique for determining bone mineral content and body composition (as body fat mass). The introduction of AAV-GNPTAB induced a relative increase in BMC and BMD in AAV-injected KO mice. Figures 10A–10C show the raw BMD data obtained before injection (Figure 10A), as well as the relative ratio of BMD before and after injection (Figures 10B, 10C). These data are also shown in Table 2 below. These results demonstrate a significant effect of gene transfer on bone mineral density.

[0135] [Table 2]

[0136] Similarly, raw bone mineral density (BMC) data showed a strong gene therapy effect (Figure 11A). Ratio data (Figures 11B and 11C) were also close to a significant effect on bone growth. These data are also shown in Table 3 below.

[0137] [Table 3]

[0138] Next, analysis of lean body mass content using a DEXA scanner revealed that lean body mass also decreased in KO mice (Figure 12A). As shown in Figure 12B, at 32 weeks post-injection, control mice showed a significant decrease in percentage lean body mass compared to pre-injection data. However, no significant change in lean body mass was observed in AAV-GNPTAB-treated KO mice. These data are also shown in Table 4 below.

[0139] [Table 4]

[0140] In summary, GNPTAB knockout mice, like those in human ML type II, were unable to grow healthily and had low bone density. This model was used to investigate the potential of gene therapy using AAV vectors for the treatment of ML type II. Overexpression of GNPTAB was found to partially protect against bone growth disorders in knockout mice. Overall, systemic delivery of GNPTAB via AAV vectors is highly efficient and represents a promising approach for correcting bone pathology in ML type II.

[0141] array Unless otherwise noted, all polypeptide sequences are indicated from the N-terminus to the C-terminus. Unless otherwise noted, all nucleic acid sequences are shown in 5'-3' format. GNPTAB Human GNPTAB protein sequence (SEQ ID NO: 1) MLFKLLQRQTYTCLSHRYGLYVCFLGVVVTIVSAFQFGEVVLEWSRDQYHVLFDSYRDNIAGKSFQNRLC LPMPIDVVYTWVNGTDLELLKELQQVREQMEEEQKAMREILGKNTTEPTKKSEKQLECLLTHCIKVPPMLV LDPALPANITLKDLPSLYPSFHSASDIFNVAKPKNPSTNVSVVVFDSTKDVEDAHSGLLKGNSRQTVWRG YLTTDKEVPGLVLMQDLAFLSGFPPTFKETNQLKTKLPENLSSKVKLLQLYSEASVALLKLNNPKDFQEL NKQTKKNMTIDGKELTISPAYLLWDLSAISQSKQDEDISASRFEDNEELRYSLRSIERHAPWVRNIFIVT NGQIPSWLNLDNPRVTIVTHQDVFRNLSHLPTFSSPAIESHIHRIEGLSQKFIYLNDDVMFGKDVWPDDF YSHSKGQKVYLTWPVPNCAEGCPGSWIKDGYCDKACNNSACDWDGGDCSGNSGGSRYIAGGGGTGSIGVG QPWQFGGGINSVSYCNQGCANSWLADKFCDQACNVLSCGFDAGDCGQDHFHELYKVILLPNQTHYIIPKG ECLPYFSFAEVAKRGVEGAYSDNPIIRHASIANKWKTIHLIMHSGMNATTIHFNLTFQNTNDEEFKMQIT VEVDTREGPKLNSTAQKGYENLVSPITLLPEAEILFEDIPKEKRFPKFKRHDVNSTRRAQEEVKIPLVNI SLLPKDAQLSLNTLDLQLEHGDITLKGYNLSKSALLRSFLMNSQHAKIKNQAIITDETNDSLVAPQEKQV HKSILPNSLGVSERLQRLTFPAVSVKVNGHDQGQNPPLDLETTARFRVETHTQKTIGGNVTKEKPPSLIV PLESQMTKEKKITGKEKENSRMEENAENHIGVTEVLLGRKLQHYTDSYLGFLPWEKKKYFQDLLDEEESL KTQLAYFTDSKNTGRQLKDTFADSLRYVNKILNSKFGFTSRKVPAHMPHMIDRIVMQELQDMFPEEFDKT SFHKVRHSEDMQFAFSYFYYLMSAVQPLNISQVFDEVDTDQSGVLSDREIRTLATRIHELPLSLQDLTGL EHMLINCSKMLPADITQLNNIPPTQESYYDPNLPPVTKSLVTNCKPVTDKIHKAYKDKNKYRFEIMGEEE IAFKMIRTNVSHVVGQLDDIRKNPRKFVCLNDNIDHNHKDAQTVKAVLRDFYESMFPIPSQFELPREYRN RFLHMHELQEWRAYRDKLKFWTHCVLATLIMFTIFSFFAEQLIALKRKIFPRRRRIHKEASPNRIRV Mouse GNPTAB protein sequence (SEQ ID NO: 2) MLLKLLQRQTYTCLSHRYGLYVCFVGVVVTIVSAFQFGEVVLEWSRDQYHVLFDSYRDNIAGKSFQNRLC LPMPIDVVYTWVNGTDLELLKELQQVREHMEEEQRAMRETLGKNTTEPTKKSEKQLECLLTHCIKVPPMLV LDPPLPANCTLKDLPTLYPSFHAASDMFNVAKPKNPSTNVSVVVFDTTKDVEDAHAGPFKGGSKQMVWRA YLTTDKEAPGLVLMQGLAFLSGFPPTFKETSQLKTKLPEKLSSKIKLLRLYSEASVALLKLNNPKGFQEL NKQTKKNMTIDGKELTISPAYLLWDLSAISQSKQDEDVSASRFEDNEELRYSLRSIERHAPWVRNIFIVT NGQIPSWLNLDNPRVTIVTHQDIFQNLSHLPTFSSPAIESHIHRIEGLSQKFIYLNDDVMFGKDVWPDDF YSHSKGQKVYLTWPVPNCAEGCPGSWIKDGYCDKACNNSACDWDGGDCSGNTAGNRFVAGGGGTGNIGAG QHWQFGGGINTISYCNQGCANSWLADKFCDQACNVLSCGFDAGDCGQDHFHELYKVTLLPNQTHYVVPKG EYLSYFSFANIARRGVEGTYSDNPIIRHASIANKWKTIHLIMHSGMNATTIYFNLTLQNANDEEFKIQIA VEVDTREAPKLNSTTQKAYESLVSPVTPLPQADVPFEDVPKEKRFPKIRRHDVNATGRFQEEVKIPRVNI SLLPKEAQVRLSNLDLQLERGDITLKGYNLSKSALLRSFLGNSLDTKIKPQARTDETKGNLEVPQENPSH RRPHGFAGEHRSERWTAPAETVTVKGRDHALNPPPVLETNARLAQPTLGVTVSKENLSPLIVPPESHLPK EEESDRAEGNAVPVKELVPGRRLQQNYPGFLPWEKKKYFQDLLDEEESLKTQLAYFTDSKHTGRQLKDTF ADSLRYVNKILNSKFGFTSRKVPAHMPHMIDRIVMQELQDMFPEEFDKTSFHKVRHSEDMQFAFSYFYYL MSAVQPLNISQVFHEVDTDQSGVLSDREIRTLATRIHDLPLSLQDLTGLEHMLINCSKMLPANITQLNNI PPTQEAYYDPNLPPVTKSLVTNCKPVTDKIHKAYKDKNKYRFEIMGEEEIAFKMIRTNVSHVVGQLDDIR KNPRKFVCLNDNIDHNHKDARTVKAVLRDFYESMFPIPSQFELPREYRNRFLHMHELQEWRAYRDKLKFW THCVLATLIIFTIFSFFAEQIIALKRKIFPRRRIHKEASPDRIRV

Claims

1. Recombinant adeno-associated virus (rAAV) particles, (a) A vector comprising a nucleic acid encoding N-acetylglucosamine-1-phosphate transferase (GNPTAB) operably linked to a CMV enhancer and a chicken beta-actin (CBA) promoter, and at least one AAV2 inverted terminal repeat (ITR), wherein the GNPTAB comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1; and (b) AAV8 capsid The rAAV particles, including the rAAV particles.

2. The GNPTAB comprises an alpha subunit and a beta subunit, according to claim 1. The rAAV particles described.

3. The rAAV particle according to claim 1, wherein the GNPTAB is human GNPTAB.

4. The rAAV particle according to claim 1, wherein the GNPTAB contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, or contains the amino acid sequence of SEQ ID NO:

1.

5. The vector is an rAAV particle according to claim 1, comprising an intron.

6. The rAAV particle according to claim 5, wherein the intron is an MVM intron.

7. The vector is an rAAV particle according to claim 1, comprising a polyadenylated sequence.

8. The polyadenylated sequence is a bovine growth hormone polyadenylated sequence, as described in claim 7. The rAAV particle.

9. The vector is an rAAV particle according to any one of claims 1 to 8, comprising two ITRs.

10. A pharmaceutical composition comprising rAAV particles according to any one of claims 1 to 9.

11. The pharmaceutical composition according to claim 10, for use in treating mucolipidosis type II (ML II) or mucolipidosis type III (ML III) in mammals.

12. The pharmaceutical composition according to claim 11, wherein the treatment alleviates or delays the progression of one or more symptoms of ML II or ML III, where one or more symptoms of ML II or ML III are skeletal abnormalities, cognitive impairment, delayed development of gross and fine motor skills, hearing loss, lack of muscle tone, distended abdomen, umbilical hernia, progressive mucosal thickening of the airway, respiratory infection, mitral valve thickening and dysfunction, constipation or diarrhea.

13. A pharmaceutical composition according to claim 10, for use in maintaining, preventing the decrease of, or increasing body size, bone mineral content, or bone density in a mammal having mucolipidosis type II (ML II) or mucolipidosis type III (ML III), wherein the rAAV particles comprise an rAAV vector, the rAAV vector comprises a nucleic acid encoding N-acetylglucosamine-1-phosphate transferase (GNPTAB) and at least one AAV ITR, the expression of which results in maintaining, preventing the decrease of, or increasing body weight; maintaining or increasing height; and / or maintaining, preventing the decrease of, or increasing bone mineral content or bone density.

14. A pharmaceutical composition according to claim 10, for use in a mammal to alleviate or delay the progression of one or more symptoms of ML II or ML III, wherein the rAAV particles comprise an rAAV vector, the rAAV vector comprises a nucleic acid encoding GNPTAB and at least one AAV ITR; and the one or more symptoms of ML II or ML III are skeletal abnormalities, cognitive impairment, delayed development of gross and fine motor skills, hearing loss, lack of muscle tone, distended abdomen, umbilical hernia, progressive mucosal thickening of the airway, respiratory infection, mitral valve thickening and dysfunction, constipation or diarrhea.

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