Adeno-associated virus vector for treating mucolipidosis type ii

Abstract

To provide an AAV vector that can house GNPTAB together with functional promoter / enhancer sequence and other components of AAV genome while still promoting efficient packaging into a virus particle.SOLUTION: Provided is a recombinant adeno-associated virus (rAAV) vector that includes nucleic acid encoding N-acetylglucosamine-1-phosphoric acid transferase (GNPTAB), and at least one AAV inverted terminal repeat sequence (ITR). In some embodiments, the rAAV vector is included in a rAAV particle, and the particle is contained in a pharmaceutical composition and a kit. The vector, particle, composition and kit are used particularly for treating mucolipidosis type II (ML II) or mucolipidosis type III (ML III) in a mammal.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 267,502, filed December 15, 2015, which is incorporated by reference in its entirety.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following ASCII text file submission are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 159792012640SEQLIST.txt, Data Recorded: December 9, 2016, Size: 21kb).

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

[0004] Mucolipidosis types II and III (ML II; ML III) are autosomal recessive lysosomal storage disorders characterized by deficiencies of UDP-GlcNAc; the lysosomal enzyme; N-acetylglucosamine-1-phosphotransferase (abbreviated as GlcNAc-1-phosphotransferase). GlcNAc-1-phosphotransferase is a lysosomal enzyme that is synthesized from three distinct subunits, α 2 , β 2 and γ 2 It is a hexameric enzyme complex consisting of α 2 Subunits and β 2The subunit is catalytically active and is encoded by a single gene, GNPTAB. Mutations in GNPTAB that result 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 decade of life. In addition, the available treatment options for ML II remain limited. In a minority of patients, only bone marrow transplantation has been attempted. In contrast, mutations in GNPTAB that result in partial reduction of GlcNAc-1-phosphotransferase activity are found in patients with ML III. These patients usually exhibit milder symptoms and slower disease progression, but still exhibit severe health problems such as skeletal abnormalities, developmental delay, and cardiac enlargement. Summary of the Invention [Problem to be solved by the invention]

[0005] The availability of AAV serotypes that exhibit tissue-specific tropism and promote sustained expression of transgenes offers the possibility of AAV-mediated gene therapy for the systemic treatment of lysosomal diseases, including ML II and ML III. Thus, the study of AAV-mediated therapy of ML II / III is important to establish whether this approach represents a potential therapeutic strategy for this devastating disease. However, technical limitations need to be overcome before AAV-mediated therapy of ML II / III can be considered. The coding sequence of GNPTAB (>5.6 kb in humans) is longer than the endogenous AAV genome (~4.7 kb). Therefore, an AAV vector that can accommodate GNPTAB, along with functional promoter / enhancer sequences and other components of the AAV genome, while still promoting efficient packaging into viral particles, would be highly advantageous. [Means for solving the problem]

[0006] The present invention provides 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 (ITR). In some embodiments, the GNPTAB comprises an alpha subunit and a beta subunit. In some embodiments, the GNPTAB is operably linked to a promoter. In some embodiments, the GNPTAB is human GNPTAB. In some embodiments, the GNPTAB comprises 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, the GNPTAB comprises 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 truncated CBA promoter. In some embodiments, the CMV enhancer is a truncated CMV enhancer. In some embodiments, the vector comprises an intron. In some embodiments, the intron is an MVM intron. In some embodiments, the vector comprises a polyadenylation sequence. In some embodiments, the polyadenylation sequence is a bovine growth hormone polyadenylation sequence. In some embodiments, the AAV terminal repeat sequence is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or mouse AAV serotype ITR. In some embodiments, the rAAV vector comprises two ITRs.

[0007] In some aspects, the present invention provides a rAAV particle comprising any one of the rAAV vectors of the above-mentioned embodiments.In some embodiments, the AAV particle comprises 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, capsid of ... In some embodiments, the rAAV particle comprises one or more ITRs from an AAV serotype different from the capsid of the rAAV viral particle. In some embodiments, the rAAV particle comprises an AAV8 capsid and the vector comprises an AAV2 ITR. In some embodiments, the rAAV particle is produced by transfecting a host cell with a nucleic acid encoding a rAAV vector and a nucleic acid encoding AAV rep and cap functions, and providing a nucleic acid encoding AAV helper functions. In some embodiments, the AAV helper functions are provided by transfecting a host cell with a nucleic acid encoding AAV helper functions. In some embodiments, the AAV helper functions are provided by infecting a host cell with an AAV helper virus that provides the AAV helper functions. In some embodiments, the AAV helper virus is an adenovirus, herpes simplex virus, or baculovirus. In some embodiments, the rAAV particle is produced by an AAV producer cell that comprises a nucleic acid encoding a rAAV vector and a nucleic acid encoding AAV rep and cap functions, and provides a nucleic acid encoding AAV helper functions. In some embodiments, the AAV producing cell comprises nucleic acid encoding AAV helper functions.In some embodiments, AAV helper function is provided by infecting AAV production cells with the AAV helper virus that provides AAV helper function.In some embodiments, the AAV helper virus is adenovirus, herpes simplex virus or baculovirus.In some embodiments, the present invention provides pharmaceutical compositions comprising any of the rAAV particles described herein.

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

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

[0010] In some embodiments of the above-described methods, the treatment alleviates one or more symptoms of ML II or ML III, including 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 dysfunction, and / or other conditions. In some embodiments, the treatment is ML The treatment slows the progression of one or more of the symptoms of ML II or ML III, which 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 of alleviating one or more symptoms of ML II or ML III in a mammal, comprising administering to the mammal an effective amount of rAAV particles, the rAAV particles comprising an rAAV vector, the rAAV vector comprising 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, thickening and dysfunction of the mitral valve, constipation or diarrhea. ... The present invention provides a method for delaying the progression of one or more symptoms of ML II or ML III, comprising administering to a mammal an effective amount of an rAAV particle, wherein the rAAV particle comprises an rAAV vector, wherein the rAAV vector comprises a nucleic acid encoding GNPTAB and at least one AAV ITR; and wherein 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.

[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 comprises 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 comprises 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 truncated CBA promoter. In some embodiments, the CMV enhancer is a truncated CMV enhancer. In some embodiments, the vector comprises an intron. In some embodiments, the intron is an MVM intron. In some embodiments, the vector comprises a polyadenylation sequence. In some embodiments, the polyadenylation sequence is a bovine growth hormone polyadenylation sequence. In some embodiments, the AAV terminal repeat sequence is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or mouse AAV serotype ITR. In some embodiments, the rAAV vector comprises two ITRs. In some embodiments, the AAV particles comprise an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, caprine AAV, AAV1 / AAV2 chimera, bovine AAV, murine AAV or rAAV2 / HBoV1 serotype capsid. In some embodiments, the rAAV particles comprise one or more ITRs and a capsid from the same AAV serotype.In some embodiments, the rAAV particle comprises one or more ITRs from an AAV serotype different from the capsid of the rAAV viral particle, hi some embodiments, the rAAV particle comprises an AAV8 capsid and the vector comprises AAV2 ITRs.

[0012] In some of the above embodiments, the rAAV particles encode an rAAV vector. The rAAV particles are produced by transfecting a host cell with a nucleic acid encoding an rAAV vector and a nucleic acid encoding AAV rep and cap functions, and providing a nucleic acid encoding AAV helper functions. In some embodiments, the AAV helper functions are provided by transfecting a host cell with a nucleic acid encoding AAV helper functions. In some embodiments, the 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, a herpes simplex virus, or a baculovirus. In some embodiments, the rAAV particles are produced by an AAV producer cell that includes a nucleic acid encoding an rAAV vector and a nucleic acid encoding AAV rep and cap functions, and provides a nucleic acid encoding AAV helper functions. In some embodiments, the AAV producer cell includes a nucleic acid encoding AAV helper functions. In some embodiments, the AAV helper functions are provided by infecting an AAV producer cell with an AAV helper virus that provides AAV helper functions. In some embodiments, the AAV helper virus is an adenovirus, a herpes simplex virus, or a baculovirus.

[0013] In some embodiments of the above methods, the mammal is a human. In some embodiments, the human is a pediatric subject. In some embodiments, the human is a young adult.

[0014] In some embodiments of the above-mentioned method, the rAAV is administered intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously or intrahepatically. In some embodiments, the rAAV is administered intravenously. In some embodiments, the rAAV is administered to two or more locations. In some embodiments, the administration is repeated. In some embodiments, the rAAV viral particles are in a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier.

[0015] In some aspects, the invention provides for the use of any of the pharmaceutical compositions described herein in the manufacture of a medicament for treating ML II or ML III in a mammal. In some embodiments, the invention provides for the use of any of the pharmaceutical compositions described herein in the manufacture of a medicament for use in any of the methods described herein. In some embodiments, the invention provides for the use of any of the rAAV particles described herein in the manufacture of a medicament for treating ML II or ML III in a mammal. In some embodiments, the invention provides for the use of any of the rAAV particles described herein in the manufacture of a medicament for use in any of the methods described herein. In some embodiments, the invention provides for the use of any of the pharmaceutical compositions described herein for treating ML II or ML III in a mammal. In some embodiments, the invention provides for the use of any of the pharmaceutical compositions described herein for use in any of the methods described herein. In some embodiments, the invention provides for the use of any of the recombinant AAVs described herein for treating ML II or ML III in a mammal. In some embodiments, the invention provides for the use of any of the recombinant AAVs described herein for use in any of the methods described herein. In some embodiments, the invention provides the use of any of the pharmaceutical compositions described herein in the manufacture of a medicament for alleviating one or more symptoms of ML II or ML III in a mammal, or for delaying the progression of one or more symptoms of ML II or ML III in a mammal. In some embodiments, the invention provides the use of any of the rAAV particles ... The present invention provides the use of any of the pharmaceutical compositions described herein for alleviating one or more symptoms of ML II or ML III in a mammal or for delaying the progression of one or more symptoms of ML II or ML III in a mammal. In some embodiments, the present invention provides the use of any of the recombinant AAVs described herein for alleviating one or more symptoms of ML II or ML III in a mammal or for delaying the progression of one or more symptoms of ML II or ML III in a mammal. In some embodiments, 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 airways, frequent respiratory infections, thickening and dysfunction of mitral valve, constipation or diarrhea. In some embodiments, the mammal is a human.

[0016] In some aspects, the present invention provides a kit comprising any rAAV vector described herein, any rAAV particle described herein, 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 described herein, any rAAV particle described herein, or any pharmaceutical composition described herein.In some embodiments, the kit further comprises one or more buffers or pharma- ceutically acceptable excipients.In some embodiments, the kit further comprises instructions for use in treating ML II and / or ML III.

[0017] In some aspects, the present invention provides an animal model for mucolipidosis II (ML II), wherein at least one allele of the N-acetylglucosamine-1-phosphate transferase (GNPTAB) gene comprises a deletion located between exon 12 and exon 20. In some embodiments, at least one allele of the GNPTAB gene comprises 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 a selectable marker. In some embodiments, the selectable 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 animal is immunocompetent or immunodeficient.

[0018] In some aspects, the invention provides a method of 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 GNPTAB gene of the animal. In some embodiments, at least one allele of the GNPTAB gene comprises a deletion spanning exon 12 and exon 20. In some embodiments, the animals are bred to be homozygous for the deletion in the GNPTAB gene. In some embodiments, the animals are bred 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 a selectable marker. In some embodiments, the selectable 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 is a mouse. The strain has a genetic background derived from 129 / Sv and / or C57Bl / 6. In some embodiments, the animals are immunocompetent or immunodeficient.

[0019] In some aspects, the present invention provides a method for evaluating an agent for the treatment of mucolipidosis II (ML II), comprising administering the agent to an animal model described herein, wherein alleviation of one or more symptoms of ML II indicates that the agent provides a beneficial treatment of ML II. In some embodiments, the symptoms of ML II are weight loss, loss of bone density, loss of bone mineral content, 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 agent is a small molecule, a polypeptide, an antibody, a nucleic acid, or a 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] FIG. 1 shows a schematic representation of the mouse GNPTAB gene structure and the genomic insertion site of the gene trapping vector used to generate GNPTAB knockout mice. [Figure 1B] FIG. 1 is a Southern blot showing mouse ES clones used to generate GNPTAB knockout mice. [Diagram 2] Figure 2 shows the growth retardation exhibited 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) Nose-anus 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. [Diagram 3] FIG. 3 shows light microscope images of representative sections of hematoxylin and eosin stained femoral cartilage from wild type (FIG. 3A) and homozygous knockout (FIG. 3B) mice. [Figure 4] Figure 4: Demonstration of autolysosome accumulation in the salivary glands of knockout (KO) mice. (Figure 4A-C) EM shows overview of a salivary acinus from a wild-type mouse, composed of mucosal and serous cells. (Figure 4D) Overview of a KO salivary acinus. The overall structure is disrupted in the KO by the accumulation of numerous large vacuoles. (Figure 4E-F) Vacuoles in (Figure 4D) at higher magnification, surrounded by a single membrane and containing undegraded material. Magnification range is indicated in the box in (Figure 4D). AL, autolysosome; Mu, mucosal cell; N, nucleus; SG, secretory granule. [Diagram 5]Lysosomal enzyme activity in serum from wild-type (open circles) and KO (closed circles) mice, as indicated. Activities of N-acetylglucosaminidase (FIG. 5A), β-hexosaminidase A (FIG. 5B), β-galactosidase (FIG. 5C), and β-glucuronidase (FIG. 5D) are shown. [Figure 6] Figure 6 shows an outline of the experimental timeline for injections. (Figure 6A) Timeline of a long-term treatment study of mice injected with viral vectors at 6 weeks of age. (Figure 6B) The total number of mice (n) injected in each treatment group is shown. [Figure 7A] Schematic diagram of pAAV2 / 8-GNPTAB vector containing mouse GNPTAB cDNA. Mouse GNPTAB cDNA sequence is based on GenBank accession number NM_001004164.2. The nucleotide sequence was codon-optimized for expression in mouse. The amino acid sequence is unchanged. [Figure 7B] FIG. 1 shows a quantitative analysis of livers from AAV-GNPTAB injected KO mice compared to their control littermates. [Figure 8] Figure 8A shows the change in body weight over time from the starting weight for control mice and AAV-GNPTAB-treated KO mice. (Figure 8A) Total body weight of control mice and AAV-GNPTAB-treated KO mice (*p<0.05, Dunnett's multiple comparison test). (Figure 8B) Data is expressed as the amount of weight change over time from the starting weight. [Figure 9] Figure 9 shows the change in body length over time from the starting body length for control mice and AAV-GNPTAB-treated KO mice. (Figure 9A) Data is expressed as the ratio of body length compared before injection and 6 weeks after injection. (Figure 9B) Data is expressed as the ratio of body length compared before injection and 32 weeks after injection. [Figure 10]Histograms showing the levels of bone density before (FIG. 10A), 16 weeks after (FIG. 10B) and 32 weeks after (FIG. 10C) injection. (FIG. 10A) Data from homozygotes and heterozygotes treated with AAV-GNPTAB were compared to those of wild type and heterozygotes (compared to wild type †; p<0.05, ‡; p<0.02, compared to heterozygotes *; p<0.02, **; p<0.002). AAV-GNPTAB treatment resulted in a statistically significant increase in the BMD ratio (post / pre-Tx) in homozygote mice after 16 weeks of treatment (FIG. 10B) and after 32 weeks of treatment (FIG. 10C). (FIG. 10B) After 16 weeks of treatment, GNPTAB null mice treated with AAV-GNPTAB showed a significant increase in the BMD ratio compared to other mice (**; P<0.02). (FIG. 10C) After 32 weeks of treatment, significant differences in BMD ratios were observed in mice treated with GNPTAB (#; p<0.05, **; p<0.02). P values ​​were determined by analysis of two-tailed unpaired t-tests. Data are presented as mean ± SEM. [Figure 11] Histograms showing bone mineral density levels before (FIG. 11A), 16 weeks after (FIG. 11B), and 32 weeks after (FIG. 11C). (FIG. 11A) Data from homozygotes and heterozygotes treated with AAV-GNPTAB were compared to those of wild type and heterozygotes (compared to wild type †; p<0.05, ‡; p<0.02, compared to heterozygotes *; p<0.02, **; p<0.002). AAV-GNPTAB treatment resulted in a statistically significant increase in BMD ratio (post / pre-Tx) in homozygote mice after 16 weeks of treatment (FIG. 11B) and after 32 weeks of treatment (FIG. 11C). (FIG. 11B) After 16 weeks of treatment, GNPTAB null mice treated with AAV-GNPTAB showed a significant increase in BMD ratio compared to other mice (**; P<0.02). (FIG. 11C) After 32 weeks of treatment, significant differences in BMD ratios were observed in mice treated with GNPTAB (#; p<0.05, **; p<0.02). P values ​​were determined by analysis of two-tailed unpaired t-tests. Data are presented as mean ± SEM. [Figure 12]Figure 12A shows histograms showing the levels of percent lean mass before injection (Figure 12A) and the change in percent lean mass 32 weeks after injection (Figure 12B). (Figure 12A) Data from homozygotes and heterozygotes treated with AAV-GNPTAB were compared to those of wild type and heterozygotes (compared to wild type †; p<0.02, compared to heterozygotes *; p<0.05, **; p<0.001). (Figure 12B) After 32 weeks of treatment, no change in % lean mass was observed in mice treated with AAV-GNPTAB. P values ​​were determined by analysis of a two-tailed unpaired t-test. Data are presented as mean ± SEM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0023] In some aspects, 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, the rAAV particles comprising an rAAV vector, the rAAV vector comprising a nucleic acid encoding GNPTAB and at least one AAV ITR. Further provided herein is a method for increasing body size, bone mineral content and / or bone density 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, the rAAV particles comprising an rAAV vector, the rAAV vector comprising a nucleic acid encoding GNPTAB and at least one AAV ITR. In some embodiments, expression of GNPTAB results in an increase in body size, bone mineral content and / or bone density.

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

[0025] I. General techniques The techniques and procedures described or referenced herein generally employ conventional techniques, e.g., those described in Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); Current Protocols in Molecular Biology (eds. FMA Usubel et al., 2003); Methods in Enzymology series (Academic Press, Inc.); PCR 2: A Practical Approach (eds. MJ MacPherson, BD Hames and GR Taylor, 1995); Antibodies, A Laboratory Manual (eds. Harlow and Lane, 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (RIFreshney, 6th ed., J. Wiley and Sons, 2010); Oligonucleotide Synthesis (ed. MJ Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Manual (eds. Notebook (ed. J. E. Elis, Academic Press, 1998); Introduction to Cell and Tissue Culture (JP Mather and PERoberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., J. Wiley and Sons, 1993-8); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (JE Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and Sons, 1998). Sons, 2002); Immunobiology (CA Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty, ed., IRL Press, 1988-1989); Monoclonal Antibodies Antibodies are well understood and commonly used by those skilled in the art, such as using widely used techniques described in: Using Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JBLippincott Company, 2011).

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

[0027] The term "polynucleotide" or "nucleic acid" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, 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 natural nucleotide bases, chemically or biochemically modified nucleotide bases, non-natural nucleotide bases, or derivatized nucleotide bases. The backbone of a nucleic acid may contain sugar and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a nucleic acid may contain polymers of synthetic subunits, such as phosphoramidates, thus forming oligodeoxynucleoside phosphoramidates (P-NH 2) or mixed phosphoramidate-phosphodiester oligomers. In addition, double-stranded nucleic acids can be obtained from the chemical synthesis of single-stranded polynucleotides, either by synthesizing the complementary strands and annealing them under suitable conditions, or by synthesizing the complementary strands de novo using DNA polymerase with suitable primers.

[0028] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, including, but not limited to, peptides, oligopeptides, amino acid dimers, trimers, and multimers. Both full-length proteins and fragments thereof are encompassed by this definition. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, "polypeptide" refers to a protein that includes modifications such as deletions, additions, and substitutions (generally conservative in nature) to the native sequence, so long as the protein maintains a desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutation of the host that produces the protein or by errors due to PCR amplification.

[0029] "Recombinant viral vector" refers to a recombinant polynucleotide vector that includes one or more heterologous sequences (i.e., nucleic acid sequences that are not of viral origin). In the case of a recombinant AAV vector, the recombinant nucleic acid is flanked by at least one, e.g., two, inverted terminal repeats (ITRs).

[0030] A "recombinant AAV vector (rAAV vector)" is a vector that contains at least one, e.g., two rAAV refers to a polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences that are not of AAV origin) flanked by AAV inverted terminal repeats (ITRs). When such rAAV vectors are present in a host cell that is infected with a suitable helper virus (or expresses a suitable helper function) and expresses the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins), they are replicated and packaged in infectious viral particles. When rAAV vectors are incorporated into a larger polynucleotide (e.g., a chromosome or another vector, such as a plasmid used for cloning or transfection), they can be referred to as "pro-vectors," which can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. rAAV vectors can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexes with lipids, encapsulated in liposomes, and in embodiments, encapsidated in viral particles, particularly AAV particles. A rAAV vector can be packaged into an AAV viral capsid to generate a "recombinant adeno-associated viral particle (rAAV particle)."

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

[0032] "Heterologous" means derived from an entity that is genotypically different from the genotype of the entity to which it is compared or the remainder of the entity that is introduced or incorporated. For example, a nucleic acid introduced into a different cell type by genetic engineering techniques is a heterologous nucleic acid (and may encode a heterologous polypeptide when expressed). Similarly, a cellular sequence (e.g., a gene or portion thereof) incorporated into a viral vector is a nucleotide sequence that is heterologous to the vector.

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

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

[0035] The terms "infectious unit (iu)," "infectious particle," or "replication unit" as used with respect to viral titer refer to the number of infectious and replication-competent recombinant AAV vector particles as measured by the infectious center assay, also known as the replication center assay, e.g., as described in McLaughlin et al. (1988) J. Virol., 62:1963-1973.

[0036] The term "transducing units (tu)" as used with respect to viral titer is described in the Examples herein or, for example, in Xiao et al. (1997) Exp. Neurol. Biol., 144:113-124; or Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

[0037] "Inverted terminal repeat" or "ITR" sequences are a term well understood in the art and refer to relatively short inverted sequences found at the ends of viral genomes.

[0038] The term "AAV inverted terminal repeat (ITR)" is well understood in the art and is a sequence of approximately 145 nucleotides present at both ends of a natural single-stranded AAV genome. The outermost 125 nucleotides of the ITR can be in either of two alternate orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain multiple short regions of self-complementarity (designated A, A', B, B', C, C', and D regions), allowing intrastrand base pairing to occur in this part of the ITR.

[0039] "Terminal resolution sequence" or "trs" is a sequence in the D region of AAV ITR that is cleaved by AAV rep protein during viral DNA replication. Mutant terminal resolution sequence is refractory to cleavage by AAV rep protein. "AAV helper function" refers to the function that allows AAV to replicate and be packaged by host cell. AAV helper function can be provided in any of a number of forms, including, but not limited to, helper virus or helper virus genes that assist AAV replication and packaging. Other AAV helper functions, such as genotoxic agents, are known in the art.

[0040] " AAV helper function " refers to the function that allows AAV to replicate and be packaged by host cell. AAV helper function can be provided in any of a number of forms, including but not limited to helper virus or helper virus genes that help AAV replication and packaging. Other AAV helper functions, such as genotoxic substances, are known in the art.

[0041] AAV "helper virus" refers to a virus that allows AAV (which is a defective pablovirus) to replicate and be packaged by a host cell. Many such helper viruses have been identified, including adenoviruses, herpesviruses, poxviruses such as vaccinia, and baculoviruses. Adenoviruses encompass a number of 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 depositories such as the ATCC. Viruses of the herpes family, also available from depositories such as the ATCC, include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and pseudorabies virus (PRV). Baculoviruses available from depositories include Autographa californica nuclear polyhedrosis virus.

[0042] "Percent (%) sequence identity" with respect to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to the amino acid residues or nucleotides in the reference polypeptide or nucleic acid sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity; any conservative substitutions are not considered part of the sequence identity. Alignment for purposes of determining percent amino acid or nucleic acid sequence identity can be accomplished by a variety of techniques within the skill of one of ordinary skill in the art. This can be accomplished in a variety of ways, for example, using publicly available computer software programs, including those described, for example, in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), Supp. 30, section 7.7.18, table 7.7.1, as well as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Potential alignment programs include ALIGN Plus (Scientific and The most commonly used algorithm is the Sigma Algorithm (Educational Software, Pennsylvania). Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms 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 alternatively be said to have or contain a certain % amino acid sequence identity to a given amino acid sequence B) is calculated as follows: the fraction X / Y is multiplied by 100, where X is the number of amino acid residues scored as identical matches by a sequence alignment program in the program's alignment of A with B, and Y is the total number of amino acid residues in B. It is 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 purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to a given nucleic acid sequence D (which can alternatively be said to be a given nucleic acid sequence C having or comprising a certain % nucleic acid sequence identity to a given nucleic acid sequence D) is calculated as follows: the fraction W / Z multiplied by 100, where W is the number of nucleotides scored as identical matches by a sequence alignment program in that program's alignment of C with D, 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, then 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 (eg, a nucleic acid or protein) or cell means that it has been identified and separated and / or recovered from a component of its natural environment.

[0044] An "effective amount" is an amount sufficient to achieve a beneficial or desired result, including a clinical result (e.g., alleviation of symptoms, achievement of a clinical endpoint, etc.). An effective amount can be administered in one or more administrations. In the context of a disease state, an effective amount is an amount sufficient to alleviate, stabilize, or delay the onset of a disease. For example, an effective amount of rAAV particles represents a desired amount of a heterologous nucleic acid, such as a therapeutic polypeptide or therapeutic nucleic acid.

[0045] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, an individual or subject is a human.

[0046] As used herein, "treatment" is an approach to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization of disease state (e.g., not worsening), prevention of disease spread (e.g., metastasis), delay or slowing of disease progression, alleviation or reduction of disease state, and remission (whether partial or complete), regardless of whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to expected survival if not treated.

[0047] "N-acetylglucosamine-1-phosphotransferase (also known as GlcNAc-1-phosphotransferase or GNPTAB)" when used in reference to a gene or coding sequence refers to a polynucleotide sequence that encodes the alpha and beta subunits of the enzyme that catalyzes the chemical reaction responsible for the formation of the lysosomal enzyme N-acetyl-glucosaminyl-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 a polypeptide, "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, CM et al., Invest. Opthamol. Vis. Sci. 2007, 48(11):5221-5228). The complete GlcNAc-1-phosphotransferase enzyme complex consists of the alpha 2 Subunit, β 2 Subunits and γ 2It is known to contain subunits, the alpha and beta subunits of which are necessary 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 the present disclosure. In some embodiments, GNPTAB is variant GNPTAB. In some embodiments, GNPTAB is truncated 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, the variant (e.g., truncated) GNPTAB contains alpha and beta subunits. In some embodiments, variant GNPTAB (e.g., truncated GNPTAB) is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to native GNPTAB. In some embodiments, variant GNPTAB (e.g., truncated GNPTAB) maintains at least about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% activity of native GNPTAB. Examples of human GNPTAB are provided by GenBank Accession Nos. 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 Accession No. NP_001004164. An example of a mouse GNPTAB amino acid sequence is provided by SEQ ID NO:2. Additional examples of GNPTAB are provided by GenBank accession numbers XP_001155334 and XP_509312 (chimpanzee), XP_002687680 and NP_001179157 (cow), XP_416329 (chicken), XP_532667 (dog), XP_001497199 (horse), XP_001079967 and XP_343195 (rat), and NP_001038233 (zebrafish).

[0048] "Mucolipidosis type II" (ML II, ML-II, and ML II are used interchangeably herein) and "Mucolipidosis type III" (ML III, ML-III, and ML III are used interchangeably herein) refer to a class of diseases caused by mutations in the GNPTAB gene. Both diseases are autosomal recessive. However, ML III is typically associated with mutations that cause a milder loss of GNPTAB function compared to ML II. As such, ML II typically results in a more severe disease phenotype than ML III. ML II is also known as I-cell disease. Further description of ML II can be found in OMIM entry #252500. ML III is also known as pseudo-Hurler polydystrophy. Further description of ML III can be found in OMIM entry #252500. Seen in 52600.

[0049] "Chicken β-actin (CBA) promoter" refers to a polynucleotide sequence derived from a chicken β-actin gene (e.g., chicken beta actin represented by GenBank Entrez Gene ID 396526). As used herein, "chicken β-actin promoter" refers to a promoter containing a 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 chosen based on deletion studies showing that sequences upstream of ∼106 can be deleted without significantly affecting promoter activity (Quitschke et al., J. Biol. Chem. 264:9539-9546, 1989).

[0051] Reference herein to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, a statement referring to "about X" includes a statement of "X."

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

[0053] It is understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and / or "consisting essentially of" aspects and embodiments.

[0054] III. Vector In certain aspects, the present invention provides rAAV vectors suitable for use in, e.g., any of the methods, rAAV particles and / or pharmaceutical compositions described herein. For example, in some embodiments, a heterologous nucleic acid (e.g., a polynucleotide sequence encoding a functional GNPTAB polypeptide) is delivered to a subject by a rAAV vector of the present disclosure.

[0055] Certain aspects of the present disclosure relate to the alpha and beta subunits of N-acetylglucosamine-1-phosphate transferase (GNPTAB), such as GNPTAB polypeptide or nucleic acid encoding GNPTAB polypeptide. As known in the art, N-acetylglucosamine-1-phosphate transferase (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 EG432486 or mKIAA1208 in mouse). Examples of GNPTAB genes include, for example, human GNPTAB (e.g., as described in NCBI gene ID number 79158) and mouse GNPTAB (e.g., as described in NCBI gene ID number 432486).

[0056] In some embodiments, the GNPTAB polypeptide is a human GNPTAB polypeptide. Human GNPTAB polypeptide sequences include, but are not limited to, NCBI reference sequence NP_077288. In some embodiments, the GNPTAB polypeptide comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the GNPTAB polypeptide comprises 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, the GNPTAB is a truncated 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, the variant (e.g., truncated) GNPTAB comprises an alpha subunit and a beta subunit. In some embodiments, the GNPTAB polypeptide is a variant GNPTAB polypeptide (e.g., truncated GNPTAB) that maintains at least a portion of the activity of a 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., truncated 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, the heterologous nucleic acid (e.g., a nucleic acid encoding GNPTAB) is operably linked to a promoter. Exemplary promoters include the cytomegalovirus (CMV) immediate early promoter, the RSV LTR, the MoMLV LTR, the phosphoglycerate kinase-1 (PGK) promoter, the simian virus 40 (SV40) promoter and the CK6 promoter, the transthyretin promoter (TTR), the TK promoter, the tetracycline responsive promoter (TRE), the HBV promoter, the hAAT promoter, the LSP promoter, the chimeric liver specific promoter (LSP), the E2F promoter, the telomerase (hTERT) promoter; the cytomegalovirus enhancer / chicken beta actin / rabbit beta-globin promoter (CAG promoter, Niwa et al., Gene, 1991, 108(2):193-9), and the elongation factor 1-alpha promoter (EF1-alpha promoter) (Kim et al., Gene, 1990, 91(2):217-23 and Guo et al., Gene, 1991, 108(2):217-23). Ther., 1996, 3(9):802-10). The promoter is a constitutive promoter, an inducible promoter, or a repressible 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 truncated CBA promoter. In some embodiments, the promoter comprises a truncated CMV enhancer.

[0058] In some embodiments, the vector comprises an intron. For example, in some embodiments, the intron is a chimeric intron derived from chicken beta actin and rabbit beta globin. In some embodiments, the intron is a minute virus of mice (MVM) intron.

[0059] In some embodiments, the vector comprises a polyadenylation (polyA) sequence. The bovine growth hormone (BGH) poly(A) sequence (see, e.g., Accession No. EF592533) Many exemplary polyadenylation sequences are known in the art, such as the SV40 polyadenylation sequence and the HSV TK pA polyadenylation sequence.

[0060] Without wishing to be bound by theory, due to the large size of the GNPTAB coding sequence, it is advantageous to minimize the size of other elements of the rAAV vector (e.g., promoters, enhancers, introns, polyA sequences, 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., 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 the respective promoter sequence. This system can be used to identify a truncated variant promoter that maintains the designed strength (e.g., amount of transcript produced). In some embodiments, the rAAV vector of the present disclosure comprises a modified, truncated and / or truncated CMV enhancer / CBA promoter, e.g., as described herein. Similar methods can be used to identify truncated variant introns that maintain appropriate levels of transcripts, mRNA stability and / or splicing.In some embodiments, the rAAV vector of the present disclosure comprises a truncated intron as described herein.Similar methods can be used to identify truncated variant polyA sequences that maintain appropriate levels of transcripts, mRNA stability and / or polyadenylation.In some embodiments, the rAAV vector of the present disclosure comprises a truncated polyA sequence as described herein.In some embodiments, the GTNAP gene comprises a nucleotide sequence that codes for the amino acid sequence of SEQ ID NO: 1 or 2.

[0061] The present invention contemplates the use of recombinant viral genomes for the introduction of one or more nucleic acid sequences encoding therapeutic polypeptides and / or nucleic acids for packaging into rAAV viral particles. The recombinant viral genome includes any elements for establishing expression of the therapeutic polypeptide and / or nucleic acid, such as promoters, ITRs of the present disclosure, ribosome binding elements, terminators, enhancers, selection markers, introns, polyA signals and / or origins of replication.

[0062] IV. VIRAL PARTICLES AND METHODS FOR PRODUCING VIRAL PARTICLES Certain aspects of the present disclosure relate to rAAV particles, for example, containing the rAAV vector of the present disclosure. In the AAV particle, the nucleic acid is encapsidated into the AAV particle. The AAV particle also comprises capsid protein. In some embodiments, the nucleic acid comprises a control sequence comprising a coding sequence of interest (e.g., a GNPTAB coding sequence) operably linked with the component in the direction of transcription, a transcription start sequence and a transcription termination sequence, thereby forming an expression cassette. The expression cassette is flanked at 5' and 3' ends by at least one functional AAV ITR sequence. By "functional AAV ITR sequence" is meant 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 by reference in their entireties. To practice some embodiments of the invention, a recombinant vector contains at least all of the sequences of AAV essential for encapsidation, and a physical structure for infection by rAAV. AAV ITRs for use in the vectors of the invention may be selected from the group consisting of wild-type nucleotide sequences (e.g., Kotin, Hum. Gene Ther., 1994, 5:793-801). (described in), but may be altered by nucleotide insertions, deletions or substitutions, or the AAV ITRs may be derived from any of several AAV serotypes. Over 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):11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; 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 the present 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, caprine AAV, bovine AAV, or mouse AAV ITR, etc. In some embodiments, the nucleic acid in the AAV includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or mouse AAV ITR, etc.

[0063] In some embodiments, the rAAV particles are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 (e.g., a wild-type AAV6 capsid or a variant AAV6 capsid such as ShH10, as described in U.S. Pre-Grant Publication No. 2012 / 0164106), AAV7, AAV8, AAVrh8, AAVrh8R, AAV9 (e.g., a wild-type AAV9 capsid or a modified AAV9 capsid, as described in U.S. Pre-Grant Publication No. 2013 / 0323226), AAV10, AAVrh10, AAV11, AAV12, a tyrosine capsid mutant, a heparin-binding capsid mutant, an AAV2R471A capsid, an AAVAAV2 / 2-7m8 capsid, an AAV The rAAV particles comprise encapsidated proteins selected from a DJ capsid (e.g., an AAV-DJ / 8 capsid, an AAV-DJ / 9 capsid, or any other capsid described in U.S. Pregrant Publication No. 2012 / 0066783), an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAV1 / AAV2 chimeric capsid, a bovine AAV capsid, a murine AAV capsid, a rAAV2 / HBoV1 capsid, or an AAV capsid described in U.S. Patent No. 8,283,151 or International Publication No. WO / 2003 / 042397. In further embodiments, the rAAV particles comprise 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 a specific target tissue (e.g., diseased tissue). rAAV particles can contain viral proteins and viral nucleic acids of the same serotype or mixed serotypes. For example, rAAV particles contain one or more ITRs and capsid from the same AAV serotype, or rAAV particles contain one or more ITRs from an AAV serotype different from the capsid of the rAAV particle. In certain embodiments, rAAV particles contain AAV8 capsid and one or more (e.g., two) AAV2 ITRs.

[0065] Production of AAV particles Transfection, stable cell line production, and expression of 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 the production of rAAV vectors are known in the art, including infectious hybrid virus production systems, including the rAAV ... In some embodiments, suitable helper virus functions are provided by wild-type or mutant adenoviruses (such as temperature-sensitive adenoviruses), herpes viruses (HSV), baculoviruses, or plasmid constructs that provide helper functions. In some embodiments, the AAV rep and cap gene products are derived from any AAV serotype. Generally, but not necessarily, the AAV rep gene products are of the same serotype as the ITRs of the rAAV vector genome, so long as the rep gene products function to replicate and package the rAAV genome. Suitable media known in the art are used for the production of rAAV vectors. These media include, but are not limited to, media from Hyclone Laboratories and JRH, including modified Eagle's medium (MEM), Dulbecco's modified Eagle's medium (DMEM), 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 media formulations for use in the production of recombinant AAV vectors. In some embodiments, AAV helper functions are provided by adenovirus or HSV.In some embodiments, AAV helper functions are provided by a baculovirus and the host cell is an insect cell (e.g., a Spodoptera frugiperda (Sf9) cell).

[0066] One method for producing rAAV particles is triple transfection method.In brief, the plasmid containing rep gene and capsid gene is transfected (for example, using calcium phosphate method) into cell line (for example, HEK-293 cell) together with helper adenovirus plasmid, and the virus is collected and optionally purified.Thus, in some embodiments, rAAV particles are produced by triple transfection of the nucleic acid encoding rAAV vector, the nucleic acid encoding AAV rep and cap, and the nucleic acid encoding AAV helper virus function into host cell, and the transfection of nucleic acid into host cell produces host cell that has the ability to produce rAAV particles.

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

[0068] In some embodiments, the nucleic acid encoding the AAV rep and cap genes and / or the rAAV genome is stably maintained in the producer cell line. In some embodiments, the nucleic acid encoding the AAV rep and cap genes and / or the rAAV genome is introduced into the cell line on one or more plasmids to generate the producer cell line. In some embodiments, the AAV rep, AAV cap, and the rAAV genome are introduced into the cell on the same plasmid. In other embodiments, the AAV rep, AAV cap, and the rAAV genome are introduced into the cell on different plasmids. In some embodiments, the 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 the cell replicates, or the plasmid is integrated into the cell genome. Various sequences have been identified that allow plasmids to replicate autonomously in cells (e.g., human cells) (see, e.g., Krysan, PJ et al. (1989) Mol. Cell Biol. 9:1026-1033). In some embodiments, the plasmid contains a selectable marker (e.g., an antibiotic resistance marker) that allows for the selection of cells that maintain the plasmid. Selectable markers commonly used in mammalian cells include, but are not limited to, blasticidin, G418, hygromycin B, zeocin, puromycin, and derivatives thereof. Methods for introducing nucleic acids into cells are known in the art and include, but are not limited to, viral transduction, cationic transfection (e.g., using cationic polymers such as DEAE-dextran or cationic lipids such as lipofectamine), calcium phosphate techniques, microinjection, particle bombardment, electroporation, and nanoparticle transfection (for further details see, e.g., Kim, TK and Eberwine, JH (2010) Anal. Bioanal. Chem. 397:3173-3178).

[0069] In some embodiments, the nucleic acid encoding the AAV rep and cap genes and / or the rAAV genome is stably integrated into the genome of the producer cell line. In some embodiments, the nucleic acid encoding the AAV rep and cap genes and / or the rAAV genome is introduced into the cell line on one or more plasmids to generate the producer cell line. In some embodiments, the AAV rep, AAV cap, and rAAV genome are introduced into the cell on the same plasmid. In other embodiments, the AAV The rep, AAV cap, and rAAV genome are introduced into the cell on different plasmids. In some embodiments, the plasmid contains a selective marker (e.g., an antibiotic resistance marker) that allows for the selection of cells that maintain the plasmid. Methods for stably integrating nucleic acids into various host cell lines are known in the art. For example, iterative selection (e.g., by use of a selective marker) is used to select cells that have integrated the nucleic acid containing the selective marker (and the AAV cap and rep genes and / or the rAAV genome). In other embodiments, the nucleic acid is integrated into the cell line in a site-specific manner to generate a production cell line. FLP / FRT (see, e.g., O'Gorman, S. et al. (1991) Science 251:1351-1355), Cre / loxP (see, e.g., Sauer, B. and Henderson, N. (1988) Proc. Natl. Acad. Sci. 85:5166-5170) and other methods are known in the art. Several site-specific recombination systems are known in the art, such as C31-att (see, e.g., Groth, AC et al. (2000) Proc. Natl. Acad. Sci. 97:5995-6000), and phi C31-att (see, e.g., Groth, AC et al. (2000) Proc. Natl. Acad. Sci. 97:5995-6000).

[0070] In some embodiments, the producer cell line is derived from a primate cell line (e.g., a non-human primate cell line such as a Vero cell line or a FRhL-2 cell line). In some embodiments, the cell line is derived from a human cell line. In some embodiments, the producer cell line is derived from HeLa cells, 293 cells, A549 cells, or PERC.6® (Crucell) cells. For example, prior to the introduction and / or stable maintenance / integration of nucleic acid encoding AAV rep and cap genes and / or an oversized rAAV genome into the cell line to generate the producer 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 production cell line is adapted to grow in suspension. As known in the art, anchorage-dependent cells typically cannot grow in suspension without a substrate such as microcarrier beads. Adapting a cell line to grow in suspension includes, for example, growing the cell line in agitated culture using agitation paddles, using culture medium lacking calcium and magnesium ions (and optionally antifoaming agents) to prevent clumping, using culture vessels coated with siliconizing compounds, and selecting cells in culture (rather than in large clumps or on the sides of vessels) at each passage. For further explanation, see, for example, ATCC's Frequently Asked Questions document (available at www.atcc.org / Global / FAQs / 9 / 1 / Adapting%20a%20monolayer%20cell%20line%20to%20suspension-40.aspx) and its citations.

[0072] In some aspects, a method for producing any of the rAAV particles disclosed herein is provided, comprising: (a) culturing a host cell comprising (i) one or more AAV packaging genes, each encoding an AAV replication protein and / or an encapsidation protein; (ii) a rAAV provector comprising a nucleic acid encoding a heterologous nucleic acid as described herein, flanked by at least one AAV ITR, and (iii) an AAV helper function, under conditions in which the rAAV particles are produced; and (b) 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, caprine AAV, bovine AAV, or mouse AAV serotype ITR, etc. For example, in some embodiments, the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In certain embodiments, the nucleic acid in the AAV comprises an AAV2 ITR. In some embodiments, the encapsidation protein is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 In some embodiments, the encapsidation protein is selected from the group consisting of rAAV2 / HBoV1 serotype capsid proteins of capsids of goat AAV, AAV1 / AAV2 chimera, bovine AAV, mouse AAV capsids, or variants thereof. In some embodiments, the encapsidation protein is AAV8 capsid protein. In some embodiments, the rAAV particles contain an AAV8 capsid and a recombinant genome comprising AAV2 ITRs, and a nucleic acid encoding a therapeutic transgene / nucleic acid. The nucleic acid includes a nucleic acid encoding GNPTAB.

[0073] Suitable rAAV production culture media of the invention are supplemented with serum or serum-derived recombinant proteins at levels of 0.5%-20% (V / V or W / V). Alternatively, as known in the art, rAAV vectors are produced in serum-free conditions, also referred to as animal-derived product-free media. Those skilled in the art will appreciate that commercially available or custom 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 production culture.

[0074] rAAV production cultures can be grown under various conditions (such as over a wide range of temperatures and for various lengths of time) appropriate for the particular host cell utilized.As is known in the art, rAAV production cultures include adhesion-dependent cultures that can be cultured in suitable adhesion-dependent vessels, such as roller bottles, hollow fiber filters, microcarriers, and packed bed or fluidized bed bioreactors.rAAV vector production cultures also include suspension-adapted host cells, such as HeLa cells, 293 cells, and SF-9 cells, that can be cultured in various ways, including, for example, in spinner flasks, stirred tank bioreactors, and disposable systems such as Wave bag systems.

[0075] As described more fully in U.S. Patent No. 6,566,118, the rAAV vector particles of the invention are harvested from the rAAV production culture by lysis of the host cells of the production culture, or by harvesting spent medium from the production culture if the cells are cultured under conditions known in the art that cause the rAAV particles to be released from intact cells into the medium. Suitable methods for lysing cells are also known in the art and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals such as detergents and / or proteases.

[0076] In further embodiments, the rAAV particles are purified. The term "purified" as used herein includes preparations of rAAV particles that are free of at least some of the other components that are also present when the rAAV particles occur in nature or when they are first prepared. Thus, for example, isolated rAAV particles are prepared from a feed mixture, such as a culture lysate or production culture supernatant, using a purification technique to enrich it. Enrichment can be measured in a variety of ways, such as, for example, by the proportion of DNase-resistant particles (DRP) or genome copies (gc) present in the solution, or by infectivity, or enrichment can be measured relative to a second potential interfering substance present in the feed mixture, such as, for example, a contaminant, including a production culture contaminant or a process contaminant, including, for example, a helper virus, a medium component, etc.

[0077] In some embodiments, the rAAV production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filters, including, for example, a Millipore Millistak+HC Pod Filter of grade DOHC, a Millipore Millistak+HC Pod Filter of grade A1HC, and a 0.2 μm Filter Opticap XL1O Millipore Express SHC Hydrophilic Membrane filter. Clarification can also be achieved by a variety of 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, the rAAV production culture harvest is further treated with Benzonase® to digest any high molecular weight DNA present in the production culture. In some embodiments, Benzonase® digestion is performed under standard conditions known in the art, e.g., for a period of 30 minutes to several hours, at a temperature ranging from ambient to 37° C., and with a final concentration of 1-2.5 units / ml of Benzonase®.

[0079] rAAV particles can be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) to concentrate rAAV particles; rAAV capture by apatite chromatography; heat inactivation of helper virus; 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 can be used alone, in various combinations, or in different orders. In some embodiments, the method includes all the steps in the order listed 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 No. 6,989,264 and U.S. Patent No. 8,137,948; and International Publication No. WO2010 / 148143.

[0080] V. Treatment Methods Certain aspects of the present disclosure relate to methods of treating mucolipidosis type II and / or mucolipidosis type III, or increasing body size, bone mineral content, or bone density in a mammal having mucolipidosis type II or mucolipidosis type III. These methods are based, in part, on the discovery 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 noted 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 disease caused by mutations in GNPTAB. GNPTAB activity is required to add mannose-6-phosphate to proteins, thereby marking them for transport to lysosomes. In the absence of GNPTAB activity, lysosomal proteins (e.g., lysosomal hydrolases) are instead secreted outside the cell. As a result, substances that are normally broken down in lysosomes, such as glycosaminoglycans, lipids and oligosaccharides, accumulate inside the cell, resulting in the presence of large inclusions. ML II is also referred to as I-cell disease due to the presence of these inclusion cells ("I-cells"), which are identified by microscopy. Symptoms of ML II are often present soon after birth and include skeletal abnormalities, short stature, weak muscle tone, lack of muscle tone (hypotonia), hernias (e.g., distended abdomen, umbilical hernia), hip dislocation and joint deformities, cardiac enlargement, 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, especially speech and motor skills, and cognitive impairment. These symptoms debilitate patients with ML II, who 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 results from a weaker deletion of function in GNPTAB. This leads to mutations that are characterized by a milder disease phenotype. Symptoms of ML III are not fully evident until 3-5 years of age and are highly variable in severity, with some patients living beyond 60 years of age and others not surviving beyond childhood. Symptoms of ML III typically include skeletal abnormalities, short stature, aortic valve disease, corneal opacity, mild organ enlargement, loss of motor function and joint abnormalities. ML III is also referred to as pseudo-Hurler polydystrophy. For a more detailed description, as well as the specific mutations found in ML II and ML III patients, see, for example, Paik, KH et al. (2005) Hum. Mutat. 26(4):308-14.

[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 5-20 times higher in ML II patients than in normal patients. Similarly, in ML III, serum activity of these enzymes is elevated 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 in cultured fibroblasts are absent. 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% N-acetylglucosamine-1-phosphotransferase activity compared to activity from normal patient samples are diagnosed with ML II. In some embodiments, patients whose samples show between 1% and 10% N-acetylglucosamine-1-phosphotransferase activity compared to activity from normal patient samples are diagnosed with ML II.

[0084] In some embodiments, ML II and / or ML III are diagnosed by sequencing the GNPTAB locus for mutations (e.g., the coding sequence, promoter / intron sequence, or any other regulatory sequence). III is also characterized by an increase in urinary polysaccharides and / or urinary glycosaminoglycans, although these symptoms are not specific for ML II and ML III. In some embodiments, ML II and / or ML III are diagnosed prenatally by examining a chorionic sample, e.g., by histology of trophoblasts (see, e.g., Poenaru, L. et al. (1984) Am. J. Hum. Genet. 36(6):1379-85). As described above, in ML II, I cells from a patient 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 the present disclosure) are tested for efficacy of treatment. For example, in some embodiments, treatments for ML II and / or ML III result in increased height, bone mineral content, bone density, and alleviation 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 as described herein. For example, after treating a subject using the methods 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, for example, by one or more clinical parameters, including those described herein. Examples of such tests are known in the art and include objective and subjective tests. These include objective (e.g., subject-reported) measures.

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

[0087] In some embodiments, the activity of one or more lysosomal enzymes, including but 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), is measured in a patient sample (e.g., a serum sample) to monitor the efficacy of the treatment. A decrease in lysosomal enzyme activity in the serum indicates efficacy.

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

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

[0090] The selection of a particular rAAV vector and composition will depend on many different factors, including, but not limited to, the characteristics of the individual's medical history and condition, and the individual being treated. Evaluation of such characteristics and the design of an appropriate treatment regimen is ultimately the responsibility of the prescribing physician.

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

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

[0093] Methods for identifying cells transduced by AAV viral particles are known in the art; for example, immunohistochemistry or the use of markers such as enhanced green fluorescent protein can be used to detect transduction of viral particles; for example, viral particles comprising rAAV capsids having one or more substitutions of amino acids.

[0094] In some embodiments, an effective amount of rAAV particles is administered simultaneously or sequentially to two or more locations. In other embodiments, an effective amount of rAAV particles is administered more than once (e.g., repeatedly) to a single location. In some embodiments, multiple injections of rAAV viral particles are separated by 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, or 24 hours or less.

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

[0096] In some embodiments, the rAAV compositions of the present disclosure are used for administration to humans. In some embodiments, the rAAV compositions of the present disclosure are used for administration to children. Without wishing to be bound by theory, since many of the symptoms of ML II and ML III develop naturally (e.g., growth, limb and joint abnormalities; speech and movement delays), it is particularly advantageous to treat ML II and / or ML III as early in life as possible. In some embodiments, an effective amount of rAAV (in some embodiments, in particle form) 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.

[0097] In some embodiments, the rAAV compositions of the present 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 a patient under the age of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 years of age.

[0098] VI. Kits or Products The rAAV vectors, particles and / or pharmaceutical compositions described herein are contained within a kit or article of manufacture, e.g., designed for use in one of the methods of the 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 fluids (e.g., one, two, three, four or more) suitable for use in the methods of the invention.

[0100] The syringe is any suitable syringe, so long as it can be connected to the cannula for liquid delivery. 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 methods of the present invention include those described herein, such as one or more liquids each containing an effective amount of one or more vectors described herein, and one or more liquids containing one or more therapeutic agents.

[0101] In some embodiments, the kit includes a single liquid (e.g., a pharma- ceutically acceptable liquid containing an effective amount of a vector). In some embodiments, the kit includes two liquids. In some embodiments, the kit includes three liquids. In some embodiments, the kit includes four or more liquids. The 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 transporting the AAV vector compositions of the present disclosure. In some embodiments, the kit includes one or more buffers, such as an aqueous pH buffer. 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 the present 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 separate. 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 administration of the additional therapeutic agent.

[0104] In some embodiments, the kit includes a therapeutic agent for temporarily suppressing the immune system prior to administration of AAV. In some embodiments, the patient is temporarily immunosuppressed immediately before and after virus injection to inhibit T cell responses to AAV particles (see, e.g., Ferreira et al., Hum. Gene Ther. 25:180-188, 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 of the composition of the rAAV particles (e.g., any type of parenteral administration described herein). In some embodiments, the instructions for use include instructions according to 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 the rAAV particles to a mammal (e.g., a human), for example, to treat mucolipidosis type II (ML II) and / or mucolipidosis type III (ML III), to increase body size, to increase bone mineral content, and / or to increase bone density.

[0106] VII. Animal Models The present invention provides an animal model of mucolipidosis II. GNPTAB mutant mice are generated by microinjection of embryonic stem (ES) cell clones into host blastocysts using standard methods. Briefly, targeted disruption of the mouse GNPTAB locus (e.g., deletion of exon 12 to exon 20) is performed by homologous recombination with a replacement vector containing a reporter or selectable marker gene. All offspring were genotyped by polymerase chain reaction analysis on tail snip DNA. All mice used in this study were males identified as wild type (+ / +) mice or heterozygous (+ / -) or homozygous (- / -) mice. In some embodiments, at least one allele of the 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 deletion comprises one or more deletions of 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 a selectable marker. In some embodiments, the selectable marker confers resistance to neomycin. In some embodiments, the animal is a mammal (e.g., rodent, rabbit, cat, dog, pig, monkey). 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 comprises a transgenic 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 .2014, 289(39):26709-21).

[0107] In some aspects, the present invention provides a method for evaluating a drug for the treatment of mucolipidosis II (ML II), comprising administering the drug to an animal model described herein, wherein alleviation of one or more symptoms of ML II indicates that the drug provides a beneficial treatment of ML II. In some embodiments, the symptoms of ML II are weight loss, decreased bone density, decreased bone mineral content, 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 drug is a small molecule, a polypeptide, an antibody, a nucleic acid, or a recombinant viral particle. EXAMPLES

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

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

[0110] method Generation of AAV2 / 8-GNPTAB constructs The coding sequence of mouse GNPTAB was amplified and codon-optimized for expression in mouse by Genescript (Piscataway, NJ, USA). Due to the large size of GNPTAB cDNA and the limited capacity of AAV, the cDNA is not suitable for any available vector. Therefore, a new expression cassette was designed containing a truncated version of the CMV enhancer and chicken beta-actin promoter as well as a very small intron. 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, stored at -70°C, and expressed at 2.5 × 10 12 A concentration of DNase resistant particles / mL was used. Truncated 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 blastocysts using standard methods. Briefly, targeted disruption of the mouse GNPTAB locus (exon 12 to exon 20 were deleted) was performed by homologous recombination with a replacement vector containing the neomycin resistance gene. Mice used in this study were of mixed genetic background (129 / Sv and C57BL / 6). All offspring were genotyped by polymerase chain reaction analysis on tail snip DNA. All mice used in this study were wild type (+ / + ) mice or males identified as heterozygous (+ / -) or homozygous (- / -) mice.

[0112] Mice were housed in groups of 2–5 per cage in colony rooms under a 12-h light / dark cycle. Rodent chow (Harlan Teklad #8604, Madison, WI, USA) and water were available ad libitum. Animal care was performed in accordance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington DC, 1996).

[0113] Vector injection A single bilateral i.v. injection of PBS or AAV-GNPTAB was administered to 6-week-old GNPTAB mice. KO mice. Groups of wild-type or heterozygous mice injected with PBS served as controls. Each mouse injected with AAV-GNPTAB received 3 × 10 11 To reduce or eliminate the immune response, anti-CD40 ligand antibody (MRI) was injected alone according to the protocol by Halbert et al. (1998) J. Virol. 72:9795-9805.

[0114] growth studies All mice were examined weekly by measuring total body weight (g) and body length (nose-to-anus distance, mm) using electronic digital calipers.

[0115] histology Wild-type and GNPTAB mice at 10 months of age - / - Mice were sedated and perfused transcardially with PBS. Following perfusion, femurs were removed, fixed in 4% paraformaldehyde (PFA), decalcified with 0.5M EDTA, and embedded in paraffin. Femoral bone pieces (4 μM) were stained with hematoxylin-eosin. An Aperio ScanScope AT (v101.0) was used for scanning and analysis of slides at 4° C. The tissues were embedded and sectioned for TEM or hematoxylin and eosin (H&E).

[0116] Microstructural analysis Wild-type and KO mice were decapitated, and salivary glands were removed and fixed by immersion in 2.5% glutaraldehyde in PBS overnight at 4°C. The glands were dehydrated and then washed in PBS buffer for an additional 30 min, followed by post-fixation in 1% osmium tetroxide for 1 h at room temperature. Specimens were dehydrated through a graded alcohol series and embedded in Epon812. Semi-thin serial sections were cut at 2.5 μM (Leica ultracut UCT), stained with toluidine blue, and observed under a light microscope. Transmission electron microscopy was performed with a Mirgagni microscope (FEI).

[0117] Lysosomal enzyme assay Blood was drawn, samples were centrifuged (8000xg) for 15 min, and plasma was stored at -70°C. Plasma lysosomal enzyme activities were measured at the Department of Chemical Pathology, Samsung Medical Center, Korea. In ML II, specific GlcNAc-phosphotransferase activity could not be measured. Therefore, the diagnosis was based on elevated plasma lysosomal enzymes.

[0118] DEXA analysis Bone mineral density (BMD), bone mineral content (BMC) and lean mass were measured in anesthetized mice using a pDEXA Sabre X-ray bone densitometer. After anesthesia was administered, the weight of each mouse was recorded and then the mouse was placed in the DEXA scanner. For data analysis, an area including the whole body of the mouse was outlined.

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

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

[0121] result A gene trap method was used to generate GNPTAB KO mice. As illustrated in Figure 1A, mice were generated using homologous recombination with a replacement vector containing a neomycin resistance gene between exon 12 and exon 20. As shown in Figure 1B, the presence of a gene trap in the GNPTAB gene of ES clones was confirmed by Southern blot analysis.

[0122] Comprehensive phenotypic analysis was performed on wild-type, heterozygous and homozygous animals. KO mice were visually distinguishable from wild-type (WT) littermates by their small size. The mean body weight (Fig. 2A) and mean body length (Fig. 2B) were significantly reduced in KO mice. Consistent with their small size, homozygous mice displayed coarse facial features (Fig. 2C).

[0123] As shown in Figure 3A, in normal cartilage, distinct lacunae spaces surrounded the chondrocytes. The cytoplasm contained a single distinct vacuole. In KO mice, femoral chondrocytes were significantly hypertrophied and their enlarged lacunae were completely filled (Figure 3B). The cytoplasm of hypertrophic chondrocytes was distended with abundant microvacuoles containing insufficient amounts of fine granular amphchromatic material. There was less shrinkage of chondrocytes associated with immobilization, filling the enlarged lacunae. This is in contrast to wild-type chondrocytes, which contained a single large distinct vacuole and only partially filled lacunae.

[0124] When examined by light microscopy, the acini of exocrine salivary glands, which are composed of mucous and serous secretory cells, showed extensive vacuolization in KO mice (Gelfman et al., 2007, Invest. Optham. Visual Sci. 48:5221-5228). To gain insight into the underlying pathology, submandibular salivary glands were analyzed by electron microscopy (EM).

[0125] Both mucus- and serous-type secretory cells were readily observed in wild-type mice (Figures 4A-C). In contrast, the overall structure of the submandibular salivary gland of KO mice was highly disrupted, greatly impeding the identification of serous cells in EM sections (Figures 4D-F). Mucus-type secretory cells were filled with large membrane-bound vacuoles containing heterologous material ( Large vacuoles were filled with undegraded cytoplasmic material that had accumulated in the mucosal cells of KO mice and were surrounded by a single membrane. These observations suggest that they may represent autolysosomes formed by fusion of autophagic compartments with lysosomes.

[0126] Patients with ML-II have highly 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 the lysosome. This lack of transport leads to hypersecretion of the enzymes into the blood. Figures 5A-5D show that compared to wild-type mice, KO mice exhibited highly 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 would be expected if GlcNAc-1-phosphotransferase activity is absent in homozygous mice.

[0127] In summary, these experiments demonstrate that GNPTAB KO mice exhibited a distinct phenotype compared to wild-type mice, particularly with respect to growth, salivary gland morphology, and lysosomal enzyme levels. These results indicate that GNPTAB KO mice provide a model system for studying therapeutic treatments for ML-II.

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

[0129] As shown in Figure 6A and Figure 6B, evaluation of all analyses was performed at two time periods after injection: the first started at 16 weeks after injection (at 12 weeks of age) and the second at 32 weeks after injection (at 38 weeks of age). Growth dynamics analyses were added at 6 weeks after injection.

[0130] A plasmid was constructed containing an expression cassette expressing mouse GNPTAB cDNA and BGH polyA signal controlled by the CMV enhancer / CBA promoter (Figure 7A). Full-length GNPTAB was under the control of the truncated 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, neither AAV-GNPTAB mRNA was present in samples from control littermates (Figure 7B).

[0131] As a first test to determine whether AAV-mediated gene transfer affects metabolism in a physiologically relevant manner, 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 was no difference observed between control and AAV-GNPTAB-treated KO mice in terms of weight gain.

[0132] Next, we evaluated the efficacy of attenuating the short stature phenotype in KO mice. In KO mice injected with AAV-GNPTAB, amelioration of short stature was observed after 6 weeks of treatment, showing an increase in body size (Figure 9A, Figure 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 and body composition (as fat mass). Introduction of AAV-GNPTAB induced a relative increase in BMC and BMD in KO mice injected with AAV. Figure 10A-C shows the raw data of BMD obtained before injection (Figure 10A), as well as the relative ratio of BMD compared before and after injection (Figure 10B, Figure 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, the raw bone mineral content (BMC) data showed a strong gene therapy effect (Figure 11A). The ratio data (Figures 11B, 11C) also approached a significant effect on bone growth. These data are also shown in Table 3 below.

[0137] [Table 3]

[0138] Next, analysis of the body's lean content using a DEXA scanner revealed that lean mass was also reduced in KO mice (Figure 12A). As shown in Figure 12B, at 32 weeks post-injection, control mice showed a significant decrease in the percentage of lean mass in comparison with pre-injection data. However, no significant change in lean 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 KO mice failed to grow healthy and had low bone density, as in human ML type II. Using this model, we investigated the potential of gene therapy using AAV vectors for the treatment of ML type II. We found that overexpressed GNPTAB partially protected against the bone growth defect in KO mice. Overall, systemic delivery of GNPTAB by AAV vectors is highly efficient and a promising approach to correct bone pathology in ML type II.

[0141] array Unless otherwise noted, all polypeptide sequences are given N-terminus to C-terminus. Unless otherwise noted, all nucleic acid sequences are shown 5' to 3'. 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. 1. A recombinant adeno-associated virus (rAAV) particle comprising: (a) a vector comprising a nucleic acid encoding N-acetylglucosamine-1-phosphate transferase (GNPTAB) operably linked to a promoter and at least one AAV inverted terminal repeat (ITR), wherein the GNPTAB comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1; and (b) AAV8 capsid; The rAAV particle comprising:

2. The rAAV particle of claim 1 , wherein the GNPTAB comprises an alpha and a beta subunit.

3. The rAAV particle of claim 1 or 2, wherein the GNPTAB is human GNPTAB.

4. The rAAV particle of claim 3, wherein the GNPTAB comprises an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 1, or comprises the amino acid sequence of SEQ ID NO:

1.

5. The rAAV particle of claim 4, wherein the GNPTAB comprises the amino acid sequence of SEQ ID NO:

1.

6. The rAAV particle of any one of claims 1 to 5, wherein the promoter is a CMV enhancer / chicken beta actin (CBA) promoter.

7. A pharmaceutical composition comprising the rAAV particles of any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

8. 10. Use of an effective amount of the rAAV particles of any one of claims 1 to 6 or the pharmaceutical composition of claim 7 for treating mucolipidosis type II (ML II) or mucolipidosis type III (ML III) in a mammal.

9. Use of the rAAV particles of any one of claims 1 to 6 or the pharmaceutical composition of claim 7 in the manufacture of a medicament for treating ML II or ML III in a mammal.

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