Methods for generating, characterizing, and using Pompe disease mouse models

Abstract

Disclosed herein are transgenic non-human animal models of Pompe disease, methods for their production, and methods for their use. Also disclosed herein are nucleic acid molecules useful for producing non-human animal models of Pompe disease. In some embodiments, provided herein are transgenic non-human animal models comprising a nucleic acid sequence of an acid alpha-glucosidase (GAA) gene, or a fragment thereof, comprising a mutation, wherein the mutation causes defective splicing of pre-mRNA transcribed from the nucleic acid sequence, and the nucleic acid sequence comprising the mutation would encode a polypeptide with GAA activity if the nucleic acid sequence did not comprise the mutation. In some embodiments, the mutation is a TG mutation. In some embodiments, the mutation is an IVS1-13T-G mutation.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 377,516, filed September 28, 2022, which is incorporated by reference in its entirety.

[0002] Related Information The contents of any patents, patent applications, and references cited throughout this specification are hereby incorporated by reference in their entirety. Reference to sequence listings submitted electronically via EFS-WEB

[0003] The contents of the electronically submitted Sequence Listing filed herewith (Name: 4140_0610001_SequenceListing_ST26.xml, Size: 122,920 bytes, Creation Date: September 5, 2023) are incorporated herein by reference in their entirety.

[0004] The present disclosure relates to the field of medicine, including inherited genetic disorders and diseases, and more particularly to methods for generating and using non-human animal models for investigating the etiology and treatment of inherited genetic disorders and diseases. [Background technology]

[0005] Pompe disease (glycogen storage disorder type II, OMIM#232300) is an inherited autosomal metabolic disorder caused by a deficiency of acid alpha-glucosidase (GAA), which acts in lysosomes and is involved in the breakdown of glycogen into glucose (Hirschhorn R, Reuser AJJ. Glycogen storage disease type II: Acid alpha-glucosidase (acid maltase) deficiency. In: Scriver CR, Beaudet AL, Sly WS, et al. (Eds.) The Metabolic & Molecular Bases of Inherited Disease, McGraw-Hill, New York, 2001:3389-420; van der Ploeg AT, Reuser AJ. Pompe's disease. Lancet 2008;372:1342-53; Toscano A, Musumeci O. Pathophysiological mechanisms in glycogenosis type II. II. In: Filosto M, Toscano A, Padovani A, editors. Advances in Diagnosis and Management of Glycogenosis II. New York: Nova Science Publisher Inc; 2012:17-21).

[0006] Glycogen accumulates in lysosomes and the cytoplasm, leading to tissue damage both directly and by affecting different downstream metabolic pathways, including the autophagy process. Cardiac and skeletal muscle are the primary tissues involved, but GAA deficiency is ubiquitous, and Pompe disease is considered a multisystem disorder (Chan J, et al., The emerging phenotype of late-onset Pompe disease: A systematic literature review. Mol Genet Metab 2017;120:163-72; Montagnese F, et al. Clinical and molecular aspects of 30 patients with late-onset Pompe disease (LOPD): unusual features and response to treatment. J Neurol 2015;262:968-78; van Capelle CI, et al. Childhood Pompe disease: clinical spectrum and genotype in 31 patients. Orphanet J Rare Dis 2016;11:65).

[0007] Pompe disease is a progressive disorder and, based on age at onset, it can occur as a severe infantile form (IOPD), presenting with cardiac hypertrophy, respiratory insufficiency and distension, or as a more benign and more heterogeneous late-onset form (LOPD), with involvement of respiratory and skeletal muscles (van der Ploeg AT, Reuser AJ. Pompe's disease. Lancet 2008;372:1342-53).

[0008] In LOPD, the first clinical symptoms may be proximal muscle weakness or other complaints such as exercise intolerance, muscle pain, or isolated hyperCKemia. Clinical symptoms are similar to those of other inherited or acquired muscle disorders, such as limb-girdle muscular dystrophy (LGMD), other muscle glycogen storage diseases, and inflammatory myopathies (Preisler N, et al. Late-onset Pompe disease is prevalent in unclassified limb-girdle muscular dystrophies. Mol Genet Metab 2013;110:287-9; Savarese M, et al. The genetic basis of undiagnosed muscular dystrophies and myopathies: Results from 504 patients. Neurology 2016;87:71-6).

[0009] Multiple mutations in the acid maltase gene have been shown to cause Pompe disease. In LOPD, the most common mutation is the IVS1 splice site mutation (IVS1-13T-G; 606800.0006), which can exist in either heterozygosity or homozygosity (e.g., Montalvo, A.L.E., et al., Mutation profile of the GAA gene in 40 Italian patients with late-onset glycogen storage disease type II. Hum. Mutat. 27:999-1006, 2006; Herbert, M., et al., Early-onset symptoms and clinical course of Pompe disease associated with the c.-32-13T-G variant. Molec. Genet. Metab. 126:106-116, 2019).

[0010] Several animal models exist that mimic the phenotypic signs of Pompe disease. For example, acid maltase-deficient Japanese quail exhibit progressive myopathy and are unable to lift their wings, fly, or right themselves from a supine position in the flip test (Kikuchi, T, et al., Clinical and metabolic correction of Pompe disease by enzyme therapy in acid maltase-deficient quail. J. Clin. Invest. 101:827-833, 1998). In mice in which the GAA gene was disrupted by gene targeting in embryonic stem cells, homozygosity for the knockout was associated with a lack of enzyme activity and accumulation of glycogen in cardiac and skeletal muscle lysosomes by 3 weeks of age, which gradually increased thereafter (Raben, N, et al., Targeted disruption of the acid alpha-glucosidase gene in mice causes an illness with critical features of both infantile and adult human glycogen storage disease type II. J. Biol. Chem. 273:19086-19092, 1998). Gaa null mice also exhibited increased glycogen levels in cervical motor neurons and larger neuronal cell body sizes in phrenic neurons, and had reduced ventilation during resting breathing and hypercapnic challenge compared to wild-type mice, indicating respiratory failure (DeRuisseau, LR, et al., Neural deficits contribute to respiratory insufficiency in Pompe disease. Proc. Nat. Acad. Sci. 106:9419-9424, 2009).

[0011] All currently available mouse models of Pompe disease contain a null mutation in the GAA gene (Gaa tm1Vdp / Gaa tm1Vdp、Bijvoet AG、van de Kamp EH,et al.,Generalized glycogen storage and cardiomegaly in a knockout mouse model of Pompe disease,Hum Mol Genet,1998,7(1)53-62、Gaa tm1Rabn / Gaa tm1Rabn 、Raben N,et al.,Targeted disruption of the acid alpha-glucosidase gene in mice causes an illness with critical features of both infantile and adult human glycogen storage disease type II,J Biol Chem 1998 273(30)19086-92、およびRaben N,et al.,Modulation of disease severity in mice with targeted disruption of the acid alpha-glucosidase gene,Neuromuscul Disord 2000 10(4-5)283-91、Gaa tm1Rabn / Gaa tm1Rabn Tg(CMV-GAA*P545L)#Kjv / 0;Khanna R,et al.,The pharmacological chaperone AT2220 increases the specific activity and lysosomal delivery of mutant acid alpha-glucosidase,and promotes glycogen reduction in a transgenic mouse model of Pompe disease.PLoS One.2014;9(7):e102092、Gaa tm2Rabn / Gaa tm2Rabn, Raben N, et al., Modulation of disease severity in mice with targeted disruption of the acid alpha-glucosidase gene. Neuromuscul Disord. 2000 Jun;10(4-5):283-91, Gaa tm1.1Rabn / Gaatm1.1Rabn , Raben N, et al., Targeted disruption of the acid alpha-glucosidase gene in mice causes an illness with critical features of both infantile and adult human glycogen storage disease type II. J Biol Chem. 1998 Jul 24;273(30):19086-92), none of which have the most common IVS1-13T-G mutation.

[0012] Although these models are useful for investigating several potential therapies for Pompe disease, none of them are suitable for investigating potential therapies that specifically target the highly prevalent IVS1-13T-G mutation, such as gene therapy that targets specific mutations.

[0013] For over a decade, enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) has been the only specific treatment for the disease. Although several studies have demonstrated the efficacy of ERT, primarily in IOPD, early initiation of ERT has become essential to avoid irreversible muscle damage (Angelini C, et al., Observational clinical study in juvenile-adult glycogenosis type 2 patients undergoing enzyme replacement therapy for up to 4 years. J Neurol 2012;259:952-8; Chien YH, et al., Pompe disease: early diagnosis and early treatment make a difference. Pediatr Neonatol 2013;54:219-27). Therefore, there is a need for an animal model of Pompe disease that is suitable for investigating alternative therapies, including gene therapy specifically targeting the highly prevalent IVS1-13T-G mutation. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Hirschhorn R, Reuser AJJ. Glycogen storage disease type II: Acid alpha-glucosidase (acid maltase) deficiency. In: Scriver CR, Beaudet AL, Sly WS, et al. (Eds.) The Metabolic & Molecular Bases of Inherited Disease, McGraw-Hill, New York, 2001: 3389-420 [Non-patent document 2] van der Ploeg AT,Reuser AJ.Pompe's disease.Lancet 2008;372:1342-53、Toscano A,Musumeci O.Pathophysiological mechanisms in glycogenosis type II.In:Filosto M,Toscano A,Padovani A,editors.Advances in Diagnosis and Management of Glycogenosis II.New York:Nova Science Publisher Inc;2012:17-21

Outdoor Tools3

Outdoor Tools 4

Direct Environment 5

Outdoor Configuration6

Direct Environment 7

Outdoor Track 8

Outdoor Tools9

Outdoor Tools 10

Outdoor Content11

Outdoor Tools 12

Outdoor Content13

Outdoor Tools 14

Outdoor Tools 15

Outdoor Content 16

[0015] In some aspects, provided herein are transgenic non-human animal models comprising a nucleic acid sequence of an acid alpha-glucosidase (GAA) gene, or a fragment thereof, comprising a mutation, wherein the mutation causes defective splicing of pre-mRNA transcribed from the nucleic acid sequence, and the nucleic acid sequence comprising the mutation would encode a polypeptide having GAA activity if the nucleic acid sequence did not comprise the mutation. In some aspects, the mutation is a TG mutation. In some aspects, the mutation is an IVS1-13T-G mutation.

[0016] In some embodiments, the GAA gene, or a fragment thereof, containing the mutation, is transcribed into a pre-mRNA. In some embodiments, the pre-mRNA transcribed from the GAA gene, or a fragment thereof, containing the mutation, is processed by splicing into a mature mRNA.

[0017] In some embodiments, mature mRNA derived from pre-mRNA transcribed from a GAA gene, or a fragment thereof, containing a mutation differs from mature mRNA derived from pre-mRNA transcribed from a GAA gene, or a fragment thereof, that does not contain the mutation. In some embodiments, the mutation weakens the splice acceptor of GAA exon 2. In some embodiments, the mutation results in skipping of exon 2. In some embodiments, mature mRNA derived from pre-mRNA transcribed from a GAA gene, or a fragment thereof, that contains a mutation does not contain exon 2.

[0018] In some embodiments, mature mRNA derived from pre-mRNA transcribed from a GAA gene, or fragment thereof, containing a mutation is translated into a polypeptide with reduced GAA activity compared to a polypeptide translated from mature mRNA transcribed from a GAA gene, or fragment thereof, that does not contain a mutation. In some embodiments, mature mRNA derived from pre-mRNA transcribed from a GAA gene, or fragment thereof, that contains a mutation is translated into a polypeptide that does not have GAA activity.

[0019] In some aspects, the non-human animal model is a model of Pompe disease. In some aspects, the non-human animal model is a model of late-onset Pompe disease.

[0020] In some embodiments, the nucleic acid sequence of the GAA gene, or a fragment thereof, including the mutation, comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 or 2.

[0021] In some embodiments, the nucleic acid sequence of the GAA gene, or a fragment thereof, comprising the mutation is inserted into the Rosa26 locus. In some embodiments, the nucleic acid sequence of the GAA gene, or a fragment thereof, comprising the mutation is inserted into the endogenous GAA locus.

[0022] In some embodiments, the nucleic acid sequence of the GAA gene, or a fragment thereof, containing a mutation, is operably linked to a heterologous promoter. In some embodiments, the heterologous promoter is selected from the group consisting of the CMV early enhancer / chicken beta actin (CBA) promoter, the CAG promoter, CMV, EF1α, EF1α with a CMV enhancer, the CMV promoter with a CMV enhancer (CMVe / p), and the CMV promoter with an SV40 intron. In some embodiments, the heterologous promoter is the CAG promoter. In some embodiments, the heterologous promoter comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:3.

[0023] In some embodiments, the nucleic acid sequence of the GAA gene, or a fragment thereof, comprising the mutation is operably linked to a heterologous polyadenylation signal. In some embodiments, the heterologous polyadenylation signal is the rGB-pA polyadenylation signal. In some embodiments, the polyadenylation signal comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:4.

[0024] In some embodiments, the transgenic non-human animal model is generated by an RNA-guided CRISPR-Cas nuclease system.

[0025] In some embodiments, the transgenic non-human animal model is a mouse. In some embodiments, the mouse is a C57BL / 6 mouse.

[0026] In some aspects, the acid alpha-glucosidase (GAA) gene is a human acid alpha-glucosidase (GAA) gene.

[0027] In some embodiments, at least one copy of the acid alpha-glucosidase gene endogenous to the non-human animal model is present in the genome of the transgenic non-human animal model. In some embodiments, all copies of the acid alpha-glucosidase gene endogenous to the non-human animal model are present in the genome of the transgenic non-human animal model. In some embodiments, at least one copy of the acid alpha-glucosidase gene endogenous to the non-human animal model is absent from the genome of the transgenic non-human animal model. In some embodiments, all copies of the acid alpha-glucosidase gene endogenous to the non-human animal model are absent from the genome of the transgenic non-human animal model.

[0028] In some aspects, provided herein are recombinant nucleic acid molecules comprising a nucleic acid sequence of an acid alpha-glucosidase (GAA) gene, or a fragment thereof, comprising a 5' homology arm, a polyadenylation signal, and a mutation that causes defective splicing of a pre-mRNA transcribed from the nucleic acid sequence, wherein the nucleic acid sequence comprising the mutation would encode a polypeptide having GAA activity if the nucleic acid sequence did not comprise the mutation, promoter, or 3' homology arm.

[0029] In some embodiments, the recombinant nucleic acid molecule further comprises a Neo (neomycin) resistance gene, an Amp (ampicillin) resistance gene.

[0030] In some embodiments, the nucleic acid sequence of the acid alpha-glucosidase (GAA) gene, or a fragment thereof, including the mutation, comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:1 or 2.

[0031] In some embodiments, the promoter is selected from the group consisting of a CMV early enhancer / chicken beta actin (CBA) promoter, a CAG promoter, CMV, EF1α, EF1α with a CMV enhancer, a CMV promoter with a CMV enhancer (CMVe / p), or a CMV promoter with an SV40 intron. In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:3.

[0032] In some embodiments, the polyadenylation signal is an rGB-pA polyadenylation signal. In some embodiments, the polyadenylation signal comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:4.

[0033] In some embodiments, the homology arms comprise a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a region of the Rosa26 locus in the mouse genome. In some embodiments, the homology arms comprise a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a region of the GAA locus in the mouse genome.

[0034] In some aspects, the acid alpha-glucosidase (GAA) gene is a human acid alpha-glucosidase (GAA) gene.

[0035] In some aspects, provided herein are methods of generating a transgenic mouse comprising delivering to a cell a recombinant nucleic acid molecule of the present disclosure.

[0036] In some embodiments, the cell is mouse embryonic stem cell or one-cell mouse embryo.In some embodiments, the method for generating transgenic mouse, comprising delivering the recombinant nucleic acid molecule of the present disclosure to cell, further comprises delivering sgRNA and Cas9 nuclease to cell.In some embodiments, the delivered sgRNA targets the locus in mouse cell genome.

[0037] In some aspects, the nucleic acid sequence of the acid alpha-glucosidase (GAA) gene, or a fragment thereof, including the polyadenylation signal, mutations, and promoter contained in the recombinant nucleic acid molecule of the present disclosure is stably integrated into a locus in the mouse genome.

[0038] In some aspects, the nucleic acid sequence of the acid alpha-glucosidase (GAA) gene, or a fragment thereof, including a polyadenylation signal, a mutation, a promoter, a Neo (neomycin) resistance gene, and an Amp (ampicillin) resistance gene contained in the recombinant nucleic acid molecule of the present disclosure are stably integrated into a locus in the mouse genome.

[0039] In some embodiments, the locus is the Rosa26 locus or the GAA locus.

[0040] In some embodiments, the cell contains at least one copy of the acid alpha-glucosidase gene endogenous to the cell. In some embodiments, the cell contains all copies of the acid alpha-glucosidase gene endogenous to the cell. In some embodiments, the cell lacks at least one copy of the acid alpha-glucosidase gene endogenous to the cell. In some embodiments, the cell lacks all copies of the acid alpha-glucosidase gene endogenous to the cell.

[0041] In some aspects, provided herein are methods for testing splice regulators, comprising: (a) administering a splice regulator to a transgenic non-human animal model of the present disclosure; (b) obtaining a test sample from the non-human animal model; and (c) assaying for the presence of (i) mature mRNA derived from pre-mRNA transcribed from a GAA gene, or a fragment thereof, comprising a mutation, and / or (ii) mature mRNA derived from pre-mRNA transcribed from a GAA gene, or a fragment thereof, that does not comprise a mutation.

[0042] In some aspects, the splice regulator is a small molecule or an antisense oligonucleotide. In some aspects, the splice regulator is administered to a transgenic non-human animal model. In some aspects, the splice regulator is administered to a cell, tissue, or organ derived from a transgenic non-human animal model.

[0043] In some embodiments, the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or fragment thereof, comprising the mutation, does not differ from the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or fragment thereof, not comprising the mutation, after administration of a splice regulator. In some embodiments, the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or fragment thereof, comprising the mutation, comprises exon 2, after administration of a splice regulator. In some embodiments, the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or fragment thereof, comprising the mutation, is translated into a polypeptide that has the GAA activity of a polypeptide translated from the mature mRNA transcribed from the GAA gene, or fragment thereof, not comprising the mutation, after administration of a splice regulator.

[0044] In some embodiments, mature mRNA derived from pre-mRNA transcribed from a GAA gene, or fragment thereof, comprising a mutation, differs from mature mRNA derived from pre-mRNA transcribed from a GAA gene, or fragment thereof, not comprising the mutation, after administration of a splice regulator. In some embodiments, mature mRNA derived from pre-mRNA transcribed from a GAA gene, or fragment thereof, comprising the mutation, does not comprise exon 2, after administration of a splice regulator. In some embodiments, mature mRNA derived from pre-mRNA transcribed from a GAA gene, or fragment thereof, comprising the mutation, is translated into a polypeptide with reduced GAA activity, compared to a polypeptide translated from mature mRNA transcribed from a GAA gene, or fragment thereof, not comprising the mutation, after administration of a splice regulator.

[0045] In some embodiments, the method of the present disclosure comprises extracting mRNA from cells, tissues, or organs derived from a transgenic non-human animal model before and after administration of a splice regulator. In some embodiments, the method of the present disclosure comprises reverse transcribing the extracted mRNA into cDNA. In some embodiments, the cDNA is amplified by PCR comprising a first pair of primers capable of amplifying exon junctions unaffected by mutations and a second pair of primers capable of amplifying exon junctions affected by mutations. In some embodiments, the cDNA is amplified by PCR comprising a pair of primers capable of amplifying cDNA derived from mRNA comprising exon junctions unaffected by mutations and cDNA derived from mRNA comprising exon junctions affected by mutations.

[0046] In some embodiments, the primer comprises a nucleotide sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 11-12.

[0047] In some aspects, provided herein are methods of generating a transgenic mouse, comprising mating a first transgenic mouse generated by the methods disclosed herein with a second transgenic mouse that lacks all copies of the mouse GAA gene.

[0048] In some embodiments, provided herein are methods for testing splice regulators, including: (a) administering a splice regulator to a transgenic non-human animal model disclosed herein, wherein all copies of the acid alpha-glucosidase gene endogenous to the non-human animal model are absent from the genome of the transgenic non-human animal model; (b) obtaining a test sample from the non-human animal model; and (c) assaying for the presence of (i) a protein product translated from a mature mRNA derived from a pre-mRNA transcribed from a GAA gene, or a fragment thereof, containing a mutation, and / or (ii) a protein product translated from a mature mRNA derived from a pre-mRNA transcribed from a GAA gene, or a fragment thereof, that does not contain a mutation. In some embodiments, the splice regulator is a small molecule. In some embodiments, the splice regulator is an antisense oligonucleotide. In some embodiments, the splice regulator is administered to a transgenic non-human animal model. In some embodiments, the splice regulator is administered to a cell, tissue, or organ derived from the transgenic non-human animal model.

[0049] In some embodiments, the protein product translated from the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or fragment thereof, comprising the mutation, does not differ from the protein product translated from the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or fragment thereof, not comprising the mutation, after administration of a splice regulator. In some embodiments, the protein product translated from the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or fragment thereof, not comprising the mutation, after administration of a splice regulator, comprises the amino acid sequence encoded by exon 2. In some embodiments, the protein product translated from the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or fragment thereof, not comprising the mutation, has the GAA activity of the polypeptide translated from the mature mRNA transcribed from the GAA gene, or fragment thereof, not comprising the mutation, after administration of a splice regulator.

[0050] In some embodiments, the protein product translated from a mature mRNA derived from a pre-mRNA transcribed from a GAA gene, or a fragment thereof, that contains a mutation differs from the protein product translated from a mature mRNA derived from a pre-mRNA transcribed from a GAA gene, or a fragment thereof, that does not contain the mutation, after administration of a splice regulator. In some embodiments, the protein product translated from a mature mRNA derived from a pre-mRNA transcribed from a GAA gene, or a fragment thereof, that contains the mutation does not contain the amino acid sequence encoded by exon 2, after administration of a splice regulator. In some embodiments, the protein product translated from a mature mRNA derived from a pre-mRNA transcribed from a GAA gene, or a fragment thereof, that contains the mutation has reduced GAA activity compared to a polypeptide translated from a mature mRNA transcribed from a GAA gene, or a fragment thereof, that does not contain the mutation, after administration of a splice regulator.

[0051] In some embodiments, the protein content from cells, tissues, or organs from transgenic non-human animal models before and after administration of splice regulators. In some embodiments, the protein content is analyzed by Western blot assay. In some embodiments, the Western blot assay comprises anti-GAA antibody. [Brief explanation of the drawings]

[0052] [Figure 1] Figure 1 shows a schematic diagram of the strategy used to generate the LOPD mice of the present disclosure. The entire genomic sequence of the human acid alpha-glucosidase (GAA) gene carrying the IVS1 mutation was inserted into the mouse Rosa26 locus by CRISPR / Cas9 genome engineering. The inserted sequence further contains a CAG promoter and a rBG pA polyadenylation signal.

[0053] [Figure 2]Figure 2 shows a schematic diagram of the restriction enzyme map of the cloning vector, which contains, from 5' to 3', the 5' Rosa26 homology arm, rGB pA, the entire genomic sequence of the human acid alpha-glucosidase (GAA) gene carrying the IVS1 mutation, the CAG promoter, the 3' Rosa26 homology arm, a Neo (neomycin) cassette, and an Amp (ampicillin) resistance gene.

[0054] [Figure 3] FIG. 3 shows gel electrophoresis of the indicated restriction enzyme digests demonstrating correct insertion of the insert into the cloning vector.

[0055] [Figure 4A] Figure 4A shows the genotyping strategy for detection of the human GAA gene at the Rosa26 locus within the genome of F1 mice originating from a single F0 founder.

[0056] [Figure 4B] Figure 4B shows gel electrophoresis of PCR reactions performed for genotyping to detect the human GAA gene at the Rosa26 locus in the genome of the founder F0 knock-in mice. Genotyping analysis demonstrated the integration of the human GAA gene at the Rosa26 locus in the genome of F1 mice originating from a single F0 founder.

[0057] [Figure 5A] Figure 5A shows the genotyping strategy for detection of cloning vectors (random integration) into the genome of founder F0 knock-in mice.

[0058] [Figure 5B] Figure 5B shows gel electrophoresis of PCR reactions performed for genotyping to detect (random integration) of the cloning vector into the genome of the founder F0 knock-in mice. Genotyping analysis showed the absence of random integration of the cloning vector into the genome of the founder F0 knock-in mice.

[0059] [Figure 6A] Figures 6A-C show the results of Sanger sequencing of the human GAA gene amplified by PCR from genomic DNA of F0 knock-in mice, which confirmed the correct orientation of both the 5' homologous arm and the 3' Rosa26 homologous arm, as well as the presence of the IVS1 mutation. [Figure 6B] Same as above. [Figure 6C] Same as above.

[0060] [Figure 7A] Figure 7A shows the results of multiplex qPCR analysis performed on genomic DNA from F2 mice to determine the genomic copy number of the human GAA gene. All three analyzed mice showed a single copy of GAA inserted into their genome.

[0061] [Figure 7B] FIG. 7B shows the results of qPCR analysis performed on genomic DNA from F2 mice to determine the relative expression levels of the three GAA alternative splice forms (TV1, TV2, and TV3).

[0062] [Figure 8A] Figure 8A shows the results of qPCR analysis performed on genomic DNA of F2 mice to determine the relative expression levels of the human and mouse GAA genes in different tissues.

[0063] [Figure 8B] Figure 8B shows the results of qPCR analysis performed on genomic cDNA derived from RNA extracted from LOPD mouse quadriceps muscles of F2 mice to determine the relative amounts of correctly spliced ​​GAA RNA (exon 1-2 junction) and total GAA RNA (exon 6-7 junction). The results are consistent with missplicing at the exon 1-2 junction.

[0064] [Figure 9]Figure 9 shows the results of end-point RT-PCR targeting the exon 1-5 region performed on RNA extracted from LOPD mice (LOPD mice), which shows a mis-splicing pattern similar to that observed in LOPD patient cells. Also shown is end-point RT-PCR targeting the exon 1-5 region performed on RNA extracted from healthy myotubes (healthy myotubes) and LOPD patient cells (LOPD myotubes).

[0065] [Figure 10A] Figure 10A shows MiSeq analysis of the RT-PCR amplified pool of exons 1–5, showing several major GAA slice variants in the quadriceps muscle of LOPD mice.

[0066] [Figure 10B] FIG. 10B shows a schematic illustrating the similarity between the major GAA splice variants observed in LOPD mouse quadriceps and those observed in LOPD patient cells.

[0067] [Figure 11A] Figures 11A–F show histocytological analyses (PAS, Figures 11A–C, and H&E, Figures 11D–F) of quadriceps (Figures 11D–F) and diaphragm (Figures 11A–C) muscles dissected from GAALOPD(IVS1)+ / − Gaa+ / + (Figures 11B–E), Gaa− / − (JAX 004154) (Figures 11A–D), and wild-type (Figures 11C–F) mice. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above. [Figure 11E] Same as above. [Figure 11F] Same as above.

[0068] [Figure 12] FIG. 12 shows MiSeq analysis of RT-PCR amplified pools of exons 1-5 from GAALOPD (IVS1)+ / − Gaa+ / + mice treated with PPMO1 or sterile saline.

[0069] [Figure 13A] 13A-B show the results of qPCR analysis of amplified pools obtained from GAALOPD(IVS1)+ / −Gaa+ / + mice treated with PPMO2, PPMO3, NTC, or saline at the indicated doses.

[0070] Figure 13A shows the % of correctly spliced ​​GAA. Figure 13B shows the % of mis-spliced ​​SV1, 2, SV5, and SV3, 7 splice variants, as indicated. Data represent mean ± SE. Figure 13B shows a schematic diagram of SV1, SV2, SV53, SV5, and SV7. [Figure 13B] Same as above. [Figure 13C] Same as above.

[0071] [Figure 14A] Figures 14A-B show the results of qPCR analysis of GAA expression in RNA pools derived from GAALOPD(IVS1)+ / − Gaa+ / + mice treated with PPMO at the indicated doses in a dose-response experiment for PPMO1-5 (Figure 14A) or PPMO2 (Figure 14B), as indicated. Data represent the mean ± SE. [Figure 14B] Same as above.

[0072] [Figure 15] Figure 15 shows quantification of GAA protein expression by Western blot in fully humanized GAALOPD (IVS1) + / − Gaa − / − mice treated with PPMO2 and control (NTC and sterile saline-treated) animals. Data represent mean ± SE. DETAILED DESCRIPTION OF THE INVENTION

[0073] 1.Definition In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly stated herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0074] It should be noted that, as used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of any named or enumerated components; for example, a "nucleic acid sequence" is understood to represent one or more nucleic acid sequences unless otherwise specified. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0075] Furthermore, when used herein, "and / or" is to be understood as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or C; A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0076] Wherever an embodiment is described herein with the term "comprising," it is understood that otherwise similar embodiments described by "consisting of" and / or "consisting essentially of" are also provided.

[0077] The term "about" refers to a value that falls within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within or more than 1 standard deviation, according to practice in the art. Alternatively, "about" can mean a range of up to 10% or 20% (i.e., ±10% or ±20%). For example, about 3 mg can include any number between 2.7 mg and 3.3 mg (10%) or between 2.4 mg and 3.6 mg (20%). Furthermore, particularly with respect to biological systems or processes, the term can mean a value that is up to an order of magnitude or up to 5 times greater. When specific values ​​are provided in this application and claims, unless otherwise specified, the meaning of "about" should be assumed to be within an acceptable error range for that particular value.

[0078] The term "at least" before a value or series of values ​​is understood to include the value adjacent to the term "at least" and all subsequent values ​​(numbers, integers, or fractions) that can be logically included, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When "at least" appears before a series of numerical values ​​or ranges, it is understood that "at least" can modify each of the numerical values ​​in the series or range. "At least" is also not limited to integers, regardless of the number of significant digits (e.g., "at least 5%" includes 5.0%, 5.1%, and 5.18%).

[0079] As used herein, "less than" or "below" is understood as the value adjacent to the phrase and the logically lower value (number, integer, or fraction) such that logically zero from the context. When "less than" appears before a series of values ​​or ranges, it is understood that the "less than" can modify each of the series of values ​​or ranges.

[0080] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and fractions thereof (such as integer tenths and hundredths), where appropriate.

[0081] As used herein, the term "derived from" refers to a component that is isolated from or made using a particular molecule or organism, or information (e.g., an amino acid sequence or a nucleic acid sequence) from a particular molecule or organism.

[0082] As used herein, the term "test sample" refers to a non-human animal model in its entirety or any part derived therefrom (eg, organs, tissues, cells, or any combination thereof).

[0083] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably in this application. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA (e.g., messenger RNA (mRNA), plasmid DNA (pDNA), or complementary DNA (cDNA)). As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide" are defined as commonly understood by those skilled in the art as a molecule comprising two or more covalently linked nucleosides. Such covalently linked nucleosides may also be referred to as a nucleic acid molecule or oligomer. Polynucleotides can be produced recombinantly, enzymatically, or synthetically, for example, by solid-phase chemical synthesis followed by purification. When referring to a polynucleotide or nucleic acid sequence, reference is made to the sequence or order of the nucleobase moieties of the covalently linked nucleotides or nucleosides, or modifications thereof. An "isolated" nucleic acid or polynucleotide refers to a nucleic acid molecule, DNA, or RNA, that has been removed from its natural environment. An isolated polynucleotide includes a recombinant polynucleotide maintained in a heterologous host cell or a polynucleotide purified (partially or substantially) in solution. An isolated RNA molecule includes an in vivo or in vitro RNA transcript of a polynucleotide. An isolated polynucleotide or nucleic acid further includes such molecules produced synthetically. Furthermore, a polynucleotide or nucleic acid may be or include regulatory elements such as a promoter, a ribosome binding site, or a transcription terminator (e.g., a polyadenylation signal). The nucleic acid may be contained in a vector.

[0084] As used herein, the terms "ASO" and "antisense oligomer" are used interchangeably and refer to a polynucleotide containing nucleotides that hybridize to a target nucleic acid molecule (e.g., pre-mRNA) sequence by Watson-Crick base pairing or wobble base pairing (GU).

[0085] As used herein, the term "splice switch oligonucleotide," or "SSO," refers to an antisense reagent (e.g., and antisense oligomer) that regulates splicing by binding splice sites and / or splicing regulatory sequences and competing with or interacting with cis- and trans-acting factors for their targets. SSOs can restore aberrant splicing, alter the relative expression of existing mRNAs, or produce novel splice variants that are not normally expressed.

[0086] As used herein, the term "specifically hybridize" refers to the ability of a molecule (e.g., an antisense oligomer such as an SSO) to hybridize to one nucleic acid sequence (e.g., a splice site and / or splicing control sequence) with greater affinity than it hybridizes to another nucleic acid sequence. Antisense oligonucleotides can specifically hybridize to more than one target sequence.

[0087] As used herein, the term "modulate" or "modulation" refers to the change in the quantity or quality of function or activity compared to the function or activity before modulation.For example, modulation includes either an increase (stimulation or induction) or decrease (inhibition or reduction) of gene expression.As another example, modulation of expression can include perturbation of the splice site selection of pre-mRNA processing, resulting in a change in the amount of specific splice variants present compared to the unperturbed condition.

[0088] As used herein, "variant" refers to alternative molecules (e.g., alternative RNA transcripts) that can be coded and / or produced from a single genomic region of DNA.Variants include, but are not limited to, pre-mRNA variants, which are transcripts produced from the same genomic DNA and contain both intronic and exon sequences that differ from other transcripts produced from the same genomic DNA at either their start or stop positions.Variants also include, but are not limited to, "mature mRNA variants", which are mRNA molecules that originate from the same genomic region of DNA, may originate from the same or different pre-mRNAs, and are characterized by having different splice junctions or alternative start and stop codons.

[0089] As used herein, the term "nucleotide" refers to a monomeric unit of nucleic acid polymers (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). Naturally occurring nucleotides are composed of three subunit molecules: a nucleobase, a five-carbon sugar (ribose or deoxyribose), and a phosphate group consisting of one to three phosphates. As used herein, the term "nucleobase," also known as a "nitrogenous base" or "base," refers to a biological compound that forms a nucleoside, which, in turn, is the building block of a nucleotide. A naturally occurring "nucleoside" comprises a nucleobase and a five-carbon sugar. The nucleotides, nucleosides, nucleobases, sugar moieties, and phosphate groups may be naturally occurring or modified. The term "naturally occurring nucleotide" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" includes nucleotides with modified or substituted sugar groups, modified nucleobases, modified phosphate groups, and / or modified backbones.

[0090] Nucleobase can be any naturally occurring nucleobase such as adenine, guanine, cytosine, thymine and uracil, or any synthetic or modified nucleobase that can hydrogen bond with the nucleobase present in target pre-mRNA.Examples of modified nucleobase include but are not limited to hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine and 5-hydroxymethylcytosine.

[0091] As used herein, the terms "backbone" and "backbone structure" refer to the bond between nucleic acid monomers. In naturally occurring oligonucleotides, the backbone comprises 3'-5' phosphodiester bonds that link the sugar moieties of the oligomer. The backbone structure can comprise, for example, phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoramidate, etc. For example, LaPlanche et al. Nucleic Acids Res.14:9081(1986); Stec et al.J.Am.Chem.Soc.106:6077(1984), Stein et al.Nucleic Acids Res.16:3209(1988), Zon et al.Anti Cancer Drug Design 6:539(1991), Zon et al. See al.Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)), Stec et al. US Pat. No. 5,151,510, Uhlmann and Peyman Chemical Reviews 90:543 (1990). The backbone structure may contain peptide bonds rather than phosphorus bonds, for example, in peptide nucleic acids (PNAs), or linking groups including carbamate groups, amide groups, and linear and cyclic hydrocarbon groups. The backbone modification may be phosphothioate or phosphoramidate bonds.

[0092] The sugar moiety may comprise a ribose or deoxyribose found in naturally occurring nucleotides, or a sugar analog containing a modified sugar moiety or morpholine ring. Non-limiting examples of modified sugar moieties include 2'-substituted sugars such as 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F;N3'→P5' phosphoramidate, 2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, carbamate-modified sugars, and bicyclic modified sugars. The sugar moiety modification may also include an extra bridge bond, such as in locked nucleic acids (LNAs). The sugar analog may contain a morpholine ring, such as phosphorodiamidate morpholino (PMO). The sugar moiety may include a ribofuranyl or 2'deoxyribofuranyl modification. The sugar moiety may include a 2',4'-constrained 2'O-methyloxyethyl (cMOE) modification. The sugar moiety may include a cEt 2',4'-constrained 2'-O-ethyl BNA modification. The sugar moiety may include a tricycloDNA (tcDNA) modification. The sugar moiety may include an ethylene nucleic acid (ENA) modification. The sugar moiety may include an MCE modification. Modifications are described in the literature, for example, by Jarver, et al., 2014, "A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications," Nucleic Acid Therapeutics 24(1):37-47, which is incorporated herein by reference.

[0093] As used herein, the term "vector" includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, retroviral vectors, viral vectors such as adenoviral vectors or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs). Such vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for expression of an operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify a specific desired DNA fragment and may contain specific functional sequences necessary for the insertion and / or expression of the desired DNA fragment. A "vector" can be any vehicle for cloning and / or transferring a nucleic acid into a host cell, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, or virion. The term "vector" includes both viral and non-viral vehicles for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. In some embodiments, insertion of a polynucleotide into an appropriate vector can be achieved by ligating the appropriate polynucleotide fragment into a selected vector, which may or may not have complementary cohesive termini. Vectors can be engineered to encode a selectable marker or reporter that provides for selection or identification of cells that have incorporated the vector. Expression of the selectable marker or reporter allows for identification and / or selection of host cells that have incorporated and expressed other coding regions contained in the vector.Examples of selectable marker genes described in the literature include genes that confer resistance to neomycin, ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc., as well as genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, etc. Examples of reporters described in the literature include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selectable markers can also be considered reporters.

[0094] As used herein, the term "RNA" refers to a nucleic acid molecule containing ribonucleotide residues. In preferred embodiments, RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of a bD-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such modifications may refer to internal RNA nucleotides or the addition of non-nucleotide material to the end(s) of the RNA. As used herein, it is also contemplated that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. In the present disclosure, these modified RNAs are considered analogs of naturally occurring RNA. As used herein, the term "mRNA" refers to a single-stranded RNA that encodes the amino acid sequence of one or more peptides (e.g., oligopeptides or polypeptides) or proteins. As used herein, the term "mRNA" includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. An mRNA molecule may also contain a 5' untranslated region (5'-UTR) and / or a 3' untranslated region (3'-UTR). In some embodiments, RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, mRNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid containing deoxyribonucleotides.

[0095] As used herein, the term "expression" refers to the process by which a gene produces a biochemical, e.g., a polypeptide. The process includes any indication of the functional presence of a gene in a cell, including, but not limited to, gene knock-in and both transient and stable expression. This may include, but is not limited to, transcription of a gene into messenger RNA (mRNA) and translation of such mRNA into polypeptide(s). Expression of a gene produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide translated from an mRNA transcript. Gene products described herein further include mRNAs that have been processed by post-transcriptional modifications, e.g., capping, splicing, and / or polyadenylation, or peptides that have post-translational modifications, e.g., methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage.

[0096] As used herein, the term "polypeptide" is intended to encompass the singular form "polypeptide" as well as the plural form "polypeptides" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain(s) of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain(s) of two or more amino acids are included in the definition of "polypeptide," and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be produced in any manner, including by chemical synthesis.

[0097] The polypeptides disclosed herein can be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids in size. Polypeptides can have a defined three-dimensional structure, but do not necessarily have such a structure. Polypeptides that have a defined three-dimensional structure are said to be folded, while polypeptides that do not have a defined three-dimensional structure, rather, can adopt a number of different conformations and are said to be unfolded.

[0098] As used herein, the term "coding sequence" or "encoding" a sequence refers to a specific molecule, a nucleic acid, that is transcribed (in the case of DNA) or translated (in the case of mRNA) into a polypeptide in vitro or in vivo when operably linked to appropriate control sequences, such as a promoter. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. While a "stop codon" (e.g., TAG, TGA, or TAA) is not translated into an amino acid and is considered part of the coding region, any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. Coding sequences include, but are not limited to, cDNA from prokaryotic or eukaryotic organisms, genomic DNA sequences from prokaryotic or eukaryotic organisms, and synthetic DNA sequences. A transcription termination sequence is typically located 3' to the coding sequence.

[0099] As used herein, the term "exon" refers to a coding section of a DNA molecule or an RNA molecule transcribed from a DNA molecule that is translated into a protein. Exons may be separated by intervening sections of DNA known as "introns," which do not encode proteins. Therefore, as used herein, the term "intron" refers to a segment of nucleic acid that is transcribed and present in the pre-mRNA but is excised by the splicing mechanism and therefore not present in the mature mRNA transcript. After transcription, the new, immature strand of messenger RNA, called "pre-mRNA," may contain both introns and exons. These pre-mRNA molecules undergo a modification process in the nucleus called splicing, during which non-coding introns are excised, leaving only the coding exons in the "mature mRNA." Splicing produces a mature messenger RNA molecule, which is then translated into a protein. The term "first exon" refers to a coding sequence or sequence of nucleic acid that encodes a polypeptide or polypeptide region, and the term "second exon" refers to a different sequence of nucleic acid that encodes a different, second coding sequence or second polypeptide region. If two exons are separated in the pre-mRNA by an intervening intron, the splicing machinery operates to remove the intervening intron and join the two exons in the mature mRNA.

[0100] The term "polyadenylation signal" refers to a nucleic acid sequence present in an RNA transcript that enables the transcript to be polyadenylated in the presence of the enzyme polyadenylation transferase.

[0101] The term "promoter" refers to a minimal sequence sufficient to direct transcription, preferably in eukaryotic cells. A promoter is intended as a DNA region that binds RNA polymerase and directs the enzyme to transcribe an operably linked DNA sequence. A DNA sequence is operably linked to a promoter if the promoter is capable of effecting transcription of that DNA sequence. Promoters for use in the present invention include viral promoters, mammalian promoters, and yeast promoters that confer high levels of expression, such as the CMV early enhancer / chicken β-actin (CAG) promoter, or mammalian cytomegalovirus or CMV promoters. The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, results in the production of the gene product in the cell under most or all physiological conditions of the cell. The term "inducible" promoter means that, when operably linked to a polynucleotide encoding a specific gene product, the promoter essentially results in the production of the gene in the cell only when the corresponding inducer is present in the cell. As used herein, the term "regulatory sequence" refers to a nucleic acid sequence capable of regulating the expression of a gene operably linked to said regulatory sequence; non-limiting examples of regulatory sequences are enhancers (DNA sequences that increase the transcription level of an operably linked gene) and silencer-enhancers (DNA sequences that decrease the transcription level of an operably linked gene).

[0102] As used herein, the term "splicing" refers to the process by which introns are removed from a primary transcript (pre-mRNA) and exons are joined to form a mature mRNA. Introns are removed by cleavage at conserved sequences called "splice sites" or "splice sites." These sites are located at the 5' and 3' ends of the intron. Most commonly, the removed RNA sequence begins with the dinucleotide GU at its 5' end and ends with AG at its 3' end. These consensus sequences are known to be important because altering one of the conserved nucleotides can result in the inhibition of splicing. Another important sequence occurs at the branch site, located anywhere from 18 to 40 nucleotides upstream from the 3' end of the intron. The branch site always contains an adenine but is otherwise loosely conserved. A typical sequence is YNYYRAY, where Y represents a pyrimidine, N represents any nucleotide, R represents any purine, and A represents an adenine. Rarely, splice site sequences beginning with the dinucleotide AU and ending with AC are found, and these are spliced ​​via a similar mechanism.

[0103] Splicing occurs in several steps and is catalyzed by small nuclear ribonucleoproteins (snRNPs, commonly pronounced "snap"). First, the pre-mRNA is cleaved at the 5' end of the intron after the attachment of a snRNP called U1 to a complementary sequence within the intron. The cleaved end then attaches to a downstream conserved branch site region through pairing of guanine and adenine nucleotides from the 5' end and branch site, respectively, to form a loop structure known as a lariat. The bond between the guanine and adenine bases occurs through a chemical reaction known as transesterification, in which the hydroxyl (OH) group on the adenine carbon atom attacks the bond of the guanine nucleotide at the splice site. Thus, the guanine residue is cleaved from the RNA strand and forms a new bond with adenine.

[0104] Next, snRNPs U2 and U4 / U6 appear to contribute to positioning the 5' end and proximal branch site. With the involvement of U5, the 3' end of the intron is brought into proximity, cleaved, and joined to the 5' end. This process occurs by transesterification, in which an OH group at the 3' end of the exon attacks the phosphodiester bond at the 3' splice site. Adjacent exons are covalently linked, and the resulting lariat is released upon binding of U2, U5, and U6. In addition to consensus sequences at their splice sites, eukaryotic genes with long introns also contain exon splicing enhancers (ESEs). These sequences, which help position the splicing apparatus, are found in the exons of genes and bind proteins that help recruit the splicing machinery to the correct site. While most splicing occurs between exons on a single RNA transcript, trans-splicing occasionally occurs, in which exons on different pre-mRNAs are ligated together.

[0105] The splicing process occurs in a cellular system called the spliceosome, where snRNPs are found along with additional proteins. Primary spliceosomes are among the most abundant structures in cells, and secondary spliceosomes have recently been identified that process a small category of introns. These introns are called U12-type introns because they depend on the action of a snRNP called U12 (the more common introns mentioned above are called U2-type introns). Although the role of U12-type introns has yet to be defined, their persistence throughout evolution and conservation among homologous genes in widely divergent species suggests an important functional basis.

[0106] As used herein, the term "alternative splicing" refers to a deviation from constitutive splicing, in which introns are removed and exons are present in the order they appear in a gene. In alternative splicing, certain exons are skipped, resulting in various forms of mature mRNA from a single pre-RNA transcript. Weaker splicing signals at alternative splice sites, shorter exon lengths, or higher sequence conservation surrounding orthologous alternative exons ultimately affect the exons included in the mature mRNA. Three possible mechanisms have been proposed for the origin of alternative splicing: exon shuffling, exonization of transposable elements, and constitutively spliced ​​exons. Alternative splicing explains the discrepancy between the number of protein-coding genes in humans (approximately 25,000) and the >90,000 different proteins actually produced.

[0107] The terms "operably linked," "operably inserted," "operably positioned," "under control," or "under transcriptional control" mean that a promoter is in the correct location and orientation relative to a nucleic acid to control RNA polymerase initiation and expression of a gene. In some aspects, the term "operably linked" means that a DNA sequence and a regulatory sequence are connected in a way that allows for gene expression when the appropriate molecule (e.g., a transcriptional activator protein) is bound to the regulatory sequence. In some aspects, the term "operably inserted" means that a DNA of interest that is introduced into a cell is positioned adjacent to a DNA sequence that directs the transcription and translation of the introduced DNA (i.e., promotes the production of a polypeptide encoded by the DNA of interest, for example).

[0108] As used herein, the term "recombinant DNA / RNA technology" refers to the manipulation of nucleic acid sequences outside of an organism. This technology includes, but is not limited to, the manipulation of nucleic acid sequences (e.g., coding sequences, regulatory elements (e.g., promoters, enhancers, silencers, termination sequences), linkers (e.g., spacers, internal ribosome entry sites, cleavage sites) from various sources), the insertion of nucleic acid sequences from various sources into appropriate vectors (e.g., delivery vectors, expression vectors, integration vectors), modifying or altering nucleotide sequences (e.g., by mutagenesis, insertion of modified nucleotides, 5'-capping, polyadenylation), and synthesizing artificial nucleotide sequences. Nucleic acid sequences can be manipulated outside of an organism using various techniques described in the literature (e.g., molecular cloning, polymerase chain reaction (PCR), digestion with restriction enzymes, in vitro ligation, mutagenesis, site-directed mutagenesis, transformation or transduction of prokaryotic and eukaryotic cells, in vitro DNA / RNA synthesis, in vitro RNA-5'-capping, in vitro RNA-polyadenylation, complementary DNA (cDNA) synthesis, nucleic acid isolation, etc.) (e.g., Green & Sambrook Molecular Cloning: A Laboratory Manual, volumes 1-3, 4 th (See edition).

[0109] As used herein, the term "recombinant" refers to any nucleic acid (e.g., DNA or RNA), peptide (e.g., oligopeptide, polypeptide, or protein), cell, or organism that is produced by combining genetic material from two or more different sources. For example, a "recombinant DNA" molecule is a DNA molecule that is derived from one organism and inserted into a host organism to produce a new gene combination. For example, a "recombinant RNA" molecule (e.g., a recombinant mRNA molecule) is an RNA molecule that is derived from one organism and inserted into a host organism to result in expression of a desired gene product in the host cell.

[0110] As used herein, the term "transgene" or "Tg" refers to genetic material (e.g., a gene) that has been artificially inserted into or will be inserted into the genome of an animal. The source from which the transgene originates may be any source; for example, the transgene may be derived from any living organism, e.g., an animal, or may be artificially synthesized by any of the techniques described in the literature. The transgene may be manipulated or modified through any of the various techniques described in the literature that can be used to manipulate nucleic acid sequences outside of an organism. For example, a transgene may be isolated from the genome of an organism, manipulated outside the organism to introduce a desired mutation, and then introduced into the genome of another organism. The coding region of the transgene may be operably linked to a promoter or one or more regulatory sequences capable of directing / regulating the expression of the transgene in the transgenic organism. The transgene may exist as an extrachromosomal element within the cells of the transgenic organism or may be stably integrated into the genome of the cells of the transgenic organism. A transgene contained in the genome of a germ cell of a transgenic organism can be transmitted to the offspring of the transgenic organism. The transgene contained in the genome of the somatic cell of a transgenic organism cannot be transmitted to the offspring of the transgenic organism.The transgene can be randomly integrated into the genome of a transgenic organism or into a specific locus of the genome of a transgenic organism.As used herein, the term "locus" refers to the physical location or position of a specific DNA sequence, for example, a gene, in a genome.

[0111] A non-human organism (eg, a prokaryote or eukaryote) that contains a transgene (eg, one or more transgenes) is defined as a non-human "transgenic organism."

[0112] As used herein, a "transgenic organism" (e.g., a mouse) refers to any organism that has been genetically modified. For example, a transgenic organism is an organism whose genome has been genetically modified to contain one or more transgenes and / or to remove or inactivate (in whole or in part) one or more specific genes. A transgenic organism whose genome has been genetically modified to contain a transgene (e.g., a specific locus, a "targeted mutant") is also called a knock-in organism (e.g., a knock-in mouse). A transgenic organism whose genome has been genetically modified to achieve complete loss or inactivation of a gene is also called a knock-out organism or a null organism (e.g., a knock-out mouse or a null mouse). A transgenic organism whose genome has been genetically modified to achieve partial loss or inactivation of a gene is also called a knock-down organism (e.g., a knock-down mouse).

[0113] The term "transgenic organism" also encompasses "conditional transgenic organisms," in which genetic modification can occur upon satisfaction of certain conditions, such as exposure of the animal to a substance that promotes genetic modification, introduction of an enzyme that promotes genetic modification (e.g., Cre in a Cre-lox system), or other conditions that direct genetic modification at any time after fertilization or after birth. A "transgenic organism" may, for example, be a non-human animal, e.g., a non-human mammal such as a mouse.

[0114] Transgenic organisms can also be produced by replicating parent transgenic organisms, for example, by mating parent transgenic organisms carrying one or more transgenes in their germline cell genomes, by deleting (completely or partially) one or more genes in their germline cell genomes, or by having one or more (completely or partially) inactivated genes in their germline cell genomes.

[0115] Organisms can be genetically engineered to produce transgenic organisms by any of the techniques described in the literature. For example, organisms such as mice can be genetically engineered by retroviral infection of mouse embryos, by microinjection of foreign DNA into one-cell mouse embryos, or by genetic manipulation of mouse embryonic stem cells. Organisms can also be genetically engineered using a number of genome editing techniques, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or the RNA-guided CRISPR-Cas nuclease system. ZFNs and TALENs use a strategy of tethering an endonuclease catalytic domain to a modular DNA-binding protein to induce targeted DNA double-strand breaks (DSBs) at specific genomic loci. The CRISPR-Cas nuclease system is based on the use of Cas9 nuclease, which is guided by a small RNA to target DNA via Watson-Crick base pairing.

[0116] As used herein, the term "mutation" refers to any change in the DNA sequence of an organism. A mutation can refer to the substitution of one or more bases in a DNA sequence with one or more different bases (base substitution, e.g., TG mutation). A mutation can also refer to the deletion or insertion of one or more bases in a DNA sequence. Mutations can result from errors in DNA replication during cell division, exposure to mutagens, viral infection, or can be artificially introduced into a gene by any of the different techniques described in the literature (e.g., homologous recombination or site-directed mutagenesis). Germline mutations can be transmitted to offspring, while somatic mutations cannot.

[0117] CRISPR-Cas is a microbial adaptive immune system that uses RNA-guided nucleases to cleave foreign genetic elements. Three types of CRISPR systems (I-III) have been identified across a wide range of bacterial and archaeal hosts, each containing a cluster of CRISPR-associated (Cas) genes, a non-coding RNA, and a characteristic array of repetitive elements (direct repeats). These repeats are spaced by short, variable sequences derived from the exogenous DNA target, known as protospacers, which together comprise the CRISPR RNA (crRNA) array. Within the DNA target, each protospacer is always associated with a protospacer adjacent motif (PAM), which can vary depending on the specific CRISPR system.

[0118] The type II CRISPR system is one of the best characterized, consisting of the nuclease Cas9, a crRNA array encoding guide RNAs, and a necessary auxiliary trans-activating crRNA (tracrRNA) that facilitates processing of the crRNA array into individual units. Each crRNA unit then contains a 20-nt guide sequence and partial direct repeats, the former of which directs Cas9 to a 20-bp DNA target via Watson-Crick base pairing.

[0119] The RNA-guided nuclease function of CRISPR-Cas can be reconstituted in mammalian cells through heterologous expression of human codon-optimized Cas9 and the necessary RNA components. Furthermore, crRNA and tracrRNA can be fused together to create chimeric single-target guide RNAs (sgRNAs). Thus, by altering the 20-nt guide sequence within the sgRNA, Cas9 can be reoriented toward almost any target of interest in close proximity to the PAM sequence.

[0120] Cas9 then generates sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs). Upon cleavage by Cas9, the target locus typically undergoes one of two major pathways for DNA damage repair: error-prone non-homologous end joining (NHEJ) or high-fidelity homology-directed repair (HDR), both of which can be used to achieve the desired editing results. In the absence of a repair template, the DSB is religated through the NHEJ process, leaving a scar in the form of an insertion / deletion (indel) mutation. NHEJ can be used to mediate gene knockouts because indels occurring within coding exons can result in frameshift mutations and premature stop codons. Multiple DSBs can be additionally used to mediate larger deletions within the genome. HDR is an alternative major DNA repair pathway. While HDR typically occurs at a lower and substantially more variable frequency than NHEJ, it can be used to generate precise and defined modifications at the target locus in the presence of an exogenously introduced repair template. The repair template can be either a traditional double-stranded DNA targeting construct with homologous arms flanking the insertion sequence, or in the form of a single-stranded DNA oligonucleotide (ssODN). The latter provides an effective and simple method for making small edits to the genome, such as introducing single-base mutations to probe for causative genetic mutations. Unlike NHEJ, HDR is generally only active in dividing cells, and its efficiency can vary greatly depending on the cell type and condition, as well as the genomic locus and repair template.

[0121] As used herein, the term "cell" or "cells" refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell(s). As used herein, the scope of the term also encompasses progeny that may or may not actually be identical to the parent cell, since certain modifications may occur in successive generations due to either mutation or environmental influences.

[0122] As used herein, the terms "acid alpha-glucosidase," "alpha-glucosidase," "acid alpha-1,4-glucosidase," or "acid maltase" (GAA) refer to a polypeptide with enzyme activity involved in the breakdown of glycogen. GAA can hydrolyze glycogen to glucose (i.e., GAA activity). As used herein, the term "GAA gene" refers to any nucleic acid sequence encoding a polypeptide (e.g., an enzyme) with GAA activity (i.e., the ability to participate in the breakdown (hydrolysis) of glycogen to glucose). For example, in humans, the GAA gene is found on the long arm of chromosome 17 (17q25.2-q25.3) and consists of 20 exons. GAA pre-RNA is subject to alternative splicing, which generates mature RNAs containing different exon combinations. The IVS1-13T-G (c.-32-13T>G) mutation weakens the splice acceptor of GAA exon 2, resulting in splicing defects and exon 2 skipping. The splicing defect and skipping of exon 2 results in low levels of active enzyme (12% of normal) resulting from leakage of normally spliced ​​mRNA.

[0123] As used herein, the term "GAA splice variant" refers to any nucleic acid sequence (relative to the wild-type GAA gene) that contains a mutation that causes defective splicing of the pre-mRNA transcribed from the nucleic acid sequence, and the nucleic acid sequence that contains the mutation encodes a polypeptide (e.g., an enzyme) that has the GAA activity (i.e., the ability to participate in the breakdown (hydrolysis) of glycogen to glucose) that the nucleic acid sequence does not contain. A nucleic acid sequence containing the sequence of the human GAA gene that contains the IVS1-13T-G (c.-32-13T>G) mutation is an example of a "GAA splice variant." As used herein, the term "defective splicing" refers to any form of splicing that is not physiological. For example, any mature mRNA that presents an exon-intron combination not found under physiological conditions is considered to be derived from defective splicing.

[0124] As used herein, the term "administration" refers to the administration of a composition or substance to a subject or system. Administration to an animal subject (e.g., a human) may be by any appropriate route. "Administering" refers to the physical introduction of a composition or substance, which may contain a therapeutic agent, into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Examples of administration routes include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral administration and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. Administration may also be via a parenteral route, for example, orally. Other parenteral routes include topical, epidermal, or mucosal routes of administration, such as intranasal, intravaginal, rectal, intraperitoneal, sublingual, or topical. Administration can also be, for example, single, multiple, and / or over one or more extended periods.

[0125] As used herein, the terms "treat," "treated," and "treating" refer to both therapeutic and prophylactic treatment or preventative measures, where the objective is to reverse, alleviate, ameliorate, relieve, inhibit, or slow the progression, occurrence, severity, or recurrence of an undesired symptom, complication, condition, biochemical manifestation of a disorder, or disease, or to obtain a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, a decrease in the extent of a condition, disorder, or disease, a stabilized (i.e., not worsening) state of a condition, disorder, or disease, a delay in the onset or a slowing of the onset of a condition, disorder, or disease, an improvement (whether partial or total) in a condition, disorder, or disease state, whether detectable or undetectable, an improvement in at least one measurable physical parameter, not necessarily discernible by the patient, or an enhancement or amelioration of a condition, disorder, or disease. In some embodiments, treatment involves eliciting a clinically significant response without excessive levels of side effects. In some embodiments, treatment involves prolonging survival compared to expected survival if not receiving treatment. As used herein, the term "amelioration" or "ameliorating" refers to a reduction in the severity of at least one indicator of a condition or disease. As used herein, the term "preventing" or "prevention" refers to delaying or postponing the onset, occurrence, or progression of a condition or disease over a period of time, including weeks, months, or years. As used herein, the term "prophylactic" (e.g., "prophylactic agent," "prophylactic treatment," "prophylactically effective amount") refers to any complete or partial prevention of a disease or its symptoms, and / or may be therapeutic in terms of a partial or complete cure of the disease and / or side effects and / or symptoms resulting from the disease.

[0126] As used herein, the term "gene therapy" refers to the administration of a nucleic acid sequence (e.g., a polynucleotide comprising a promoter operably linked to a nucleic acid encoding an immunomodulatory protein (e.g., a cytokine or subunit thereof) or a functional fragment thereof disclosed herein) to an individual's cells and / or tissues to treat a disease, reduce the symptoms of a disease, or reduce the likelihood of a disease. Gene therapy also includes the administration of splice switch oligonucleotides (SSOs). As used herein, the term "splice switch oligonucleotide" refers to a short, synthetic, antisense, modified nucleic acid that base pairs with a pre-mRNA and interferes with the splicing of a transcript by blocking / promoting RNA-RNA base pairing or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Splice switch oligonucleotides can be used to prevent aberrant splicing and enhance normal processing of the transcript. An exogenous molecule or sequence is understood to be a molecule or sequence that does not normally occur in the treated cell, tissue, and / or individual. Both acquired and congenital diseases are amenable to gene therapy.

[0127] As used herein, the term "subject" refers to any living organism to which a composition or substance (e.g., a nucleotide molecule) can be administered, for example, for experimental, diagnostic, preventative, and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). A subject can be a human or animal seeking or in need of treatment, in need of treatment, undergoing treatment, undergoing treatment, or in the future undergoing treatment, or being cared for by a trained professional for a particular disease or condition.

[0128] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 5th ed., 2013, Academic Press, and the Oxford Dictionary of Biochemistry and Molecular Biology, 2006, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0129] Units, prefixes, and symbols are denoted in their International System of Units (SI) recognized form. Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not intended to limit the various aspects of this disclosure, which may be incorporated by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0130] Various aspects of the invention are described in further detail in the following sections.

[0131] 2. Genetically engineered non-human animal models of Pompe disease The present invention provides methods for generating a non-human animal model of Pompe disease (PD) by introducing a nucleic acid sequence of a GAA splice variant (also referred to herein as the mutGAA gene), or a fragment thereof, into the genome of the non-human animal model. In some embodiments, the GAA splice variant, or a fragment thereof, comprises a TG mutation. In some embodiments, the GAA splice variant, or a fragment thereof, comprises a Pompe disease-associated IVS1-13T-G mutation (i.e., "GAA-IVS1-13T-G"). In some embodiments, the nucleic acid sequence of the GAA splice variant, or a fragment thereof, is comprised in a transgene. In some embodiments, the nucleic acid sequence of the GAA splice variant, or a fragment thereof, is comprised in a transgene contained in the genome of the non-human animal model. In some embodiments, the non-human animal model of Pompe disease is a transgenic non-human animal model comprising a transgene comprising a "GAA splice variant." In some embodiments, the non-human animal model of Pompe disease is a transgenic non-human animal model comprising a transgene comprising a TG mutation. In some embodiments, the non-human animal model of Pompe disease is a transgenic non-human animal model comprising a transgene comprising the IVS1-13T-G mutation. In some embodiments, the GAA gene is a human GAA gene, and the "GAA splice variant" is a human GAA splice variant. In some embodiments, the "GAA splice variant" or a fragment thereof comprises a nucleotide sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 or 2.

[0132] In some embodiments, the genome of the transgenic Pompe disease non-human animal model comprises a single copy of a GAA splice variant, or a fragment thereof. In some embodiments, the genome of the transgenic Pompe disease non-human animal model comprises more than one copy of a GAA splice variant, or a fragment thereof. In some embodiments, the genome of the transgenic Pompe disease non-human animal model comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 copies of a GAA splice variant, or a fragment thereof. In some embodiments, the GAA splice variant, or a fragment thereof, comprises a TG mutation. In some embodiments, the GAA splice variant, or a fragment thereof, comprises an IVS1-13T-G mutation. In some embodiments, the GAA splice variant, or a fragment thereof, is present in the transgenic Pompe disease non-human animal model as an extrachromosomal element (e.g., a minichromosome, a bacterial artificial chromosome (BAC), or a yeast artificial chromosome (YAC)). In some embodiments, the exogenous nucleic acid sequence is stably integrated into the genome of the transgenic non-human Pompe disease animal model. In some embodiments, the GAA splice variant, or a fragment thereof, is stably integrated into the genome of the transgenic non-human Pompe disease animal model. In some embodiments, the GAA splice variant, or a fragment thereof, is stably integrated into one chromosome of the transgenic non-human Pompe disease animal model. In some embodiments, the GAA splice variant, or a fragment thereof, is stably integrated into more than one chromosome of the transgenic non-human Pompe disease animal model. In some embodiments, the GAA splice variant, or a fragment thereof, is stably integrated into an autosome or a sex chromosome of the transgenic non-human Pompe disease animal model. In some embodiments, the GAA splice variant, or a fragment thereof, is stably integrated into one homologous chromosome of the transgenic non-human Pompe disease animal model. In some embodiments, the GAA splice variant, or a fragment thereof, is stably integrated into both homologous chromosomes of the transgenic non-human Pompe disease animal model.

[0133] In some embodiments, the GAA splice variant, or a fragment thereof, is randomly integrated into the genome of a non-human Pompe disease animal model. In some embodiments, the GAA splice variant, or a fragment thereof, is integrated into a specific locus of the genome of a non-human Pompe disease animal model. In some embodiments, the GAA splice variant, or a fragment thereof, is integrated into the ROSA26 locus of the genome of a non-human Pompe disease animal model. In some embodiments, the GAA splice variant, or a fragment thereof, integrated into the ROSA26 locus of the genome of a non-human Pompe disease animal model does not disrupt expression of the endogenous GAA gene. In some embodiments, the GAA splice variant, or a fragment thereof, is integrated into the endogenous GAA locus of the genome of a non-human Pompe disease animal model. In some embodiments, the GAA splice variant, or a fragment thereof, integrated into the endogenous GAA locus of the genome of a non-human Pompe disease animal model disrupts expression of the endogenous GAA gene. In some embodiments, the GAA splice variant, or a fragment thereof, integrated into the endogenous GAA locus of the Pompe disease non-human animal model genome does not disrupt expression of the endogenous GAA gene.

[0134] As used herein, the term "Rosa26 locus" or "Gt(ROSA)26Sor" refers to a specific genetic site (i.e., a locus) located on mouse chromosome 6 that encodes a long non-coding RNA (lncRNA) under the control of a constitutive promoter. The Rosa26 locus is ubiquitously expressed in mouse embryos and is a widely used site for the integration of transgenes and reporter constructs, making it an ideal location for targeted knock-in. In some embodiments, the GAA splice variant is integrated into a genomic safe harbor locus other than Rosa26. As used herein, the term genomic safe harbor (GSH) locus refers to a site in the genome that can accommodate the integration of new genetic material in a manner that ensures that newly inserted genetic elements (i) function predictably and (ii) do not cause changes in the host genome that pose a risk to the host cell or organism. Non-limiting examples of GSH loci are the Rosa26 locus, Polr2a locus, MYH9 locus, and the Hipp11 intergenic region.

[0135] In some embodiments, the genome of the non-human animal model (e.g., the genome of a mouse model) comprises at least one copy of an acid alpha-glucosidase gene endogenous to the non-human animal model (i.e., at least one copy of an acid alpha-glucosidase gene endogenous to the non-human animal model is present in the genome of the transgenic non-human animal model). In some embodiments, the genome of the non-human animal model (e.g., the genome of a mouse model) comprises all copies (e.g., two copies) of an acid alpha-glucosidase gene endogenous to the non-human animal model (i.e., all copies of an acid alpha-glucosidase gene endogenous to the non-human animal model are present in the genome of the transgenic non-human animal model). In some embodiments, the genome of the non-human animal model (e.g., the genome of a mouse model) lacks at least one copy of an acid alpha-glucosidase gene endogenous to the non-human animal model (i.e., at least one copy of an acid alpha-glucosidase gene endogenous to the non-human animal model is absent in the genome of the transgenic non-human animal model). In some embodiments, the genome of the non-human animal model (e.g., the genome of the mouse model) lacks all copies (e.g., two copies) of the acid alpha-glucosidase gene endogenous to the non-human animal model (i.e., all copies of the acid alpha-glucosidase gene endogenous to the non-human animal model are not present in the genome of the transgenic non-human animal model).

[0136] In some embodiments, the GAA splice variant, or a fragment thereof, is contained in the genome of somatic cells of a non-human animal model of Pompe disease. In some embodiments, the GAA splice variant, or a fragment thereof, is contained in the genome of germ cells of a non-human animal model of Pompe disease. In some embodiments, the GAA splice variant, or a fragment thereof, is contained in the genome of somatic cells and germ cells of a non-human animal model of Pompe disease. In some embodiments, the GAA splice variant, or a fragment thereof, is not transmitted to offspring. In some embodiments, the GAA splice variant, or a fragment thereof, is transmitted to offspring.

[0137] In some embodiments, the non-human animal model is a vertebrate, such as a mammal. This embodiment is not limited to any one species of animal, but provides any suitable non-human species. For example, in certain embodiments, the animal is a non-human mammal, such as a cow, pig, goat, horse, or rodent (such as a rat, mouse, or hamster). In some embodiments, the animal is a rodent, such as a rat, mouse, or hamster. In certain embodiments, the animal is a mouse. For example, transgenic mice can be generated as described and exemplified herein. Mouse strains that can be used to generate transgenic mice include, but are not limited to, CD-1® nude mice, CD-1 mice, NU / NU mice, BALB / C nude mice, BALB / C mice, NIH-III mice, SCID™ mice, outbred SCID™ mice, SCID™ beige mice, C3H mice, C57BL / 6 mice, DBA / 2 mice, FVB mice, CB17 mice, 129 mice, SJL mice, B6C3F1 mice, BDF1 mice, CDF1 mice, CB6F1 mice, CF-1 mice, Swiss Webster mice, SKH1 mice, PGP mice, and B6SJL mice, and various substrains within each mouse (e.g., J or N substrains) can also be used. Furthermore, mice derived from any crossbreeding (e.g., inbreeding, intercross breeding, cross breeding, or backcross breeding) of any mouse strain can be used. As used herein, the term "inbreeding" refers to the mating of closely related individuals or individuals with closely similar genetic makeup. As used herein, the term "intercross breeding" refers to mating from parents of different breeds or species. As used herein, the term "cross" refers to mating purebred parents of two different breeds, varieties, or populations, often with the intention of producing offspring that share the pedigree traits of both parents. As used herein, the term "backcross breeding" refers to mating cross-cross progeny of a bidirectional backcross to one parent breed.In some embodiments, the non-human animal model is a C57BL / 6 mouse.

[0138] During the initial construction of non-human transgenic animals (i.e., non-human animal models), "chimeras" or "chimeric animals" are generated in which only a subset of cells have an altered genome (e.g., a genome containing a transgene). Chimeras are primarily used in breeding to generate transgenic animals with germline transmission, i.e., transgenic animals with an exogenous nucleic acid sequence stably integrated into the genome of their germ cells. Animals with heterozygous germline alterations are generated by breeding chimeras. Male and female heterozygotes with germline transmission are then bred to generate homozygous transgenic animals. Transgenic animals can also be bred with animals of different genetic backgrounds (e.g., xenograft animal models, various disease animal models, or transgenic animals with different transgenes) to generate transgenic animals with specific genetic backgrounds. Thus, in some embodiments, the transgenic animal is a chimeric transgenic animal. In certain embodiments, the transgenic animal is a heterozygous transgenic animal. In other embodiments, the transgenic animal is a homozygous transgenic animal. In yet other embodiments, the transgenic animals are homozygous or heterozygous transgenic animals with a particular genetic background (e.g., xenograft animal models, various disease animal models, or transgenic animals with different transgenes).

[0139] In some embodiments, the non-human transgenic animal model of Pompe disease is a transgenic mouse that comprises a single copy of a GAA splicing variant. In some embodiments, the GAA splicing variant is stably integrated into the Rosa26 locus of the transgenic mouse genome. In some embodiments, the transgenic mouse is a C57BL / 6 mouse. In some embodiments, the GAA splicing variant comprises the IVS1-13T-G mutation.

[0140] In some embodiments, the GAA splice variant is expressed in a transgenic mouse with an expression pattern similar to that of mouse GAA in a mouse. In some embodiments, the GAA splice variant is expressed in a transgenic mouse with an expression pattern similar to that of mouse GAA in a human. In some embodiments, the expression level of the GAA splice variant in a transgenic mouse is similar to that of mouse GAA in a mouse. In some embodiments, the expression level of the GAA splice variant G in a transgenic mouse is similar to that of human GAA in a human. In some embodiments, the expression level of the GAA splice variant in a transgenic mouse is different from that of mouse GAA in a mouse. In some embodiments, the expression level of the GAA splice variant in a transgenic mouse is different from that of human GAA in a human.

[0141] In some embodiments, the level of GAA splice variant expression is determined directly or indirectly by the copy number of the GAA splice variant, the genomic site at which the GAA splice variant is integrated, and / or the promoter and / or regulatory region operably linked to the GAA splice variant.

[0142] The expression patterns of GAA splicing variants or mouse GAA (e.g., transgenic PD mice), as well as the expression patterns of GAA in isolated human cells, can be assayed by methods described in the literature, including, but not limited to, in situ hybridization or immunohistochemical staining (HM). The expression levels of GAA splicing variants or mouse GAA (e.g., transgenic PD mice), as well as the expression levels of GAA in isolated human cells, can be measured by methods described in the literature, including, but not limited to, Northern blot, Western blot, RT-PCR, or quantitative RT-PCR. The expression levels of various housekeeping genes (e.g., Hprt, GADPH, β-actin, ubiquitin, or hsp90) can be measured using similar methods. Relative gene expression levels normalized based on the expression levels of housekeeping genes from the same sample can be compared.

[0143] In some embodiments, GAA splice variant gene is expressed in the cell of non-human Pompe disease animal model.In some embodiments, GAA splice variant gene is transcribed into pre-mRNA in the cell of non-human Pompe disease animal model.In some embodiments, pre-mRNA is processed into mature mRNA in the cell of non-human Pompe disease animal model.In some embodiments, mature mRNA is translated into polypeptide in the cell of non-human animal model.

[0144] In some embodiments, the mutation contained in the GAA splice variant gene causes defective splicing. In some embodiments, the pre-mRNA transcribed from the GAA splice variant is processed by splicing in a different manner compared to the way the pre-mRNA transcribed from the GAA gene without the mutation is processed. In some embodiments, the polypeptide translated from the mature mRNA transcribed from the GAA splice variant gene has reduced GAA activity compared to the polypeptide translated from the mature mRNA transcribed from the GAA gene without the mutation. In some embodiments, the polypeptide translated from the mature mRNA transcribed from the GAA splice variant gene does not have GAA activity.

[0145] 3. Generation of Genetically Engineered Non-human Animal Models of Pompe Disease In certain embodiments, the transgenic non-human animal model of Pompe disease of the present disclosure is generated by introducing a GAA splice variant, or a fragment thereof, into the germline of a non-human animal (e.g., a mouse). In some embodiments, the transgenic animal is generated by introducing a GAA splice variant, or a fragment thereof, into the germline of a mouse.

[0146] In some embodiments, the GAA splice variant is derived from the wild-type GAA gene (i.e., wt-GAA). In some embodiments, a mutation (e.g., a TG mutation such as an IVS1-13T-G mutation) may be introduced into the wild-type GAA gene by any one of the techniques described in the literature. For example, the mutation can be introduced into the wild-type GAA gene by site-directed mutagenesis (SDM) or by homologous recombination (HR). The desired mutation can be introduced into the wt-GAA nucleotide sequence at any time before, simultaneously with, or after introduction of the transgene into the non-human animal model to obtain the GAA splice variant. In some embodiments, the GAA splice variant is not derived from the wild-type GAA gene.

[0147] The wt-GAA or GAA splicing variant (e.g., containing a TG mutation such as the IVS1-13T-G mutation) may be of natural or artificial origin. In some embodiments, the wt-GAA or GAA splicing variant is artificially synthesized by any of the techniques described in the literature. In some embodiments, the wt-GAA or GAA splicing variant is derived from a non-human animal. Examples of non-human animals that can be used in this embodiment are not limited to any one species of animal, but any suitable non-human species is provided. For example, in certain embodiments, the animal is a non-human mammal, such as a cow, pig, goat, horse, rodent (such as a rat, mouse, or hamster). In some embodiments, the wt-GAA or GAA splicing variant is derived from a human. In some embodiments, the wt-GAA or GAA splicing variant is derived from an organ derived from a non-human animal or a human. In some embodiments, the wt-GAA or GAA splicing variant is derived from a tissue derived from a non-human animal or a human. In some embodiments, the wt-GAA or GAA splicing variant is derived from a cell derived from a non-human animal or human. In some embodiments, the non-human animal or human does not have a mutation at the GAA locus. In some embodiments, the non-human animal or human has a mutation at the GAA locus. In some embodiments, the non-human animal or human has a TG mutation at the GAA locus. In some embodiments, the non-human animal or human has an IVS1-13T-G mutation at the GAA locus.

[0148] In some embodiments, the wt-GAA, or GAA splice variant, may be genomic DNA, complementary DNA (cDNA), a hybrid sequence, a synthetic sequence, or a semi-synthetic sequence.

[0149] In some embodiments, wt-GAA or GAA splicing variants can be derived from a genomic library. As used herein, the term "genomic library" refers to a collection of total genomic DNA from a single organism. The DNA is stored in a collection of identical vectors, each containing a different DNA insert. The vectors in a genomic library can be any type of vector, and non-limiting examples of vectors that can be used in a genomic library include plasmids, phage lambda, cosmids, bacteriophage P1 vectors, P1 artificial chromosomes, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), etc. The genomic library can be screened to select vectors containing the nucleic acid of interest by any of the methods described in the literature.

[0150] In some embodiments, the wild-type GAA gene contained in a vector (e.g., a BAC derived from a genomic library) can be modified to contain a mutation (e.g., a TG mutation such as the IVS1-13T-G mutation) by any one of the methods described in the literature (e.g., SDM, or HR).

[0151] Any nucleotide sequence desired to be operably linked to a GAA splice variant can be operably linked to a GAA splice variant by any one of the techniques described in the literature. Alternatively, any nucleotide sequence desired to be operably linked to a GAA splice variant can be operably linked to wt-GAA, and a mutation to obtain the GAA splice variant can then be introduced into the wt-GAA nucleotide sequence at any time, before, simultaneously with, or after introduction of the transgene into the non-human animal model.

[0152] In some embodiments, the GAA splice variant comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 or 2.

[0153] In some embodiments, the GAA splice variant, or wt-GAA, can be cloned (or subcloned, if contained in a vector, e.g., a genomic library vector) into any vector containing any sequence desired to be operably linked to the GAA splice variant, or wt-GAA. For example, the GAA splice variant, or wt-GAA, can be cloned (or subcloned) into a vector containing a particular promoter, a particular regulatory element (e.g., an enhancer), a particular polyadenylation signal, and / or any other particular nucleic acid sequence desired to be operably linked to the GAA splice variant, or wt-GAA.

[0154] Molecular cloning techniques are described in the literature (see, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989)).

[0155] In some embodiments, the GAA splice variant, or wt-GAA, is operably linked to a promoter and / or control region (e.g., in a recombinant nucleic acid molecule). In some embodiments, the GAA splice variant, or wt-GAA, is operably linked to an endogenous promoter and / or control region, or an exogenous promoter and / or control region. In some embodiments, the promoter and / or control region are homologous (e.g., a mouse promoter and / or control region of a transgenic mouse). In some embodiments, the promoter is homologous (e.g., a mouse promoter of a transgenic mouse). In some embodiments, the control region is homologous (e.g., a mouse control region of a transgenic mouse). In some embodiments, the promoter and control region are homologous (e.g., a mouse promoter and / or control region of a transgenic mouse). In some embodiments, the promoter is a homologous mouse GAA or Rosa26 promoter. In some embodiments, the control region is a homologous mouse GAA or Rosa26 control region. In some embodiments, the promoter and control region are a homologous mouse GAA or Rosa26 promoter and control region. In some embodiments, the promoter and / or control region are heterologous (e.g., a non-mouse eukaryotic (e.g., human), bacterial, or viral promoter and / or control region in a transgenic mouse). In some embodiments, the promoter is heterologous (e.g., a non-mouse eukaryotic (e.g., human), bacterial, or viral promoter in a transgenic mouse). In some embodiments, the control region is heterologous (e.g., a non-mouse eukaryotic (e.g., human), bacterial, or viral control region in a transgenic mouse). In some embodiments, the promoter and control region are heterologous (e.g., a non-mouse eukaryotic (e.g., human), bacterial, or viral promoter and / or control region in a transgenic mouse). In some embodiments, the promoter is a heterologous human promoter. In some embodiments, the heterologous human promoter is a GAA human promoter.In some embodiments, the regulatory region is a heterologous human regulatory region. In some embodiments, the heterologous human regulatory region is a GAA human regulatory region.

[0156] Additional non-limiting examples of heterologous promoters that can be used in the present invention include the CMV early enhancer / chicken β-actin (CBA) promoter, the CAG promoter, CMV, EF1α, EF1α with a CMV enhancer, the CMV promoter with a CMV enhancer (CMVe / p), and the CMV promoter with an SV40 intron. The promoter can be constitutive or inducible (e.g., inducible or repressible). The promoter can also be a tissue-specific or stage-specific promoter that directs the expression of a GAA splice variant or wt-GAA to a specific tissue or a specific developmental stage. In a preferred embodiment, the GAA splice variant or wt-GAA is operably linked to a heterologous promoter. In a more preferred embodiment, the heterologous promoter is a CAG promoter. In an even more preferred embodiment, the heterologous promoter is a CAG promoter comprising a nucleotide sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:3.

[0157] The regulatory region can be used to regulate (e.g., increase or decrease) the expression level of a GAA splice variant or to direct expression of a GAA splice variant to a particular tissue or at a particular developmental stage. In some embodiments, the regulatory region increases expression of a GAA splice variant. In some embodiments, the regulatory region decreases expression of a GAA splice variant. In some embodiments, the regulatory region increases or decreases expression of a GAA splice variant in a particular tissue or at a particular developmental stage.

[0158] In some embodiments, the GAA splicing variant or wt-GAA is operably linked to a polyadenylation signal (e.g., in a recombinant nucleic acid molecule). The polyadenylation signal sequence can be selected from any of a variety of polyadenylation signal sequences described in the literature. In some embodiments, the polyadenylation signal is a homologous polyadenylation signal. In a more preferred embodiment, the polyadenylation signal sequence is rBG pA. In an even more preferred embodiment, the polyadenylation signal sequence is rBG pA, which comprises a nucleotide sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:4.

[0159] In a preferred embodiment, the GAA splice variant or wt-GAA is cloned into a vector comprising a CAG promoter and rBG pA. In an even more preferred embodiment, the GAA splice variant or wt-GAA is cloned into a vector comprising a CAG promoter comprising a nucleotide sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:3, and an rBG pA comprising a nucleotide sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:4.

[0160] In a preferred embodiment, GAA-IVS1-13T-G is cloned into a PUC57 vector using seamless cloning enzyme C115 (Vazyme) (PUC57-hGAA-IVS1-13T-G). In an even more preferred embodiment, PUC57-hGAA-IVS1-13T-G comprises a nucleotide sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 13.

[0161] In some embodiments, the GAA splice variant, or wt-GAA, optionally operably linked to any promoter, and / or regulatory element, and / or polyadenylation signal, is linked to a nucleic acid sequence comprising homologous arms (e.g., in a recombinant nucleic acid molecule). As used herein, the term "homologous arms" refers to nucleic acid sequences that are identical (fully or partially) to nucleic acid sequences contained in the genome of an organism into which it is desired to insert a transgene, and that are designed to allow specific alignment of the sequence comprising the transgene to the desired genomic sequence (locus) in the organism's genome.

[0162] In a preferred embodiment, GAA-IVS1-13T-G operably linked to a CAG promoter and rBG pA is cloned into the Rosa26 locus. In an even more preferred embodiment, GAA-IVS1-13T-G operably linked to a CAG promoter and rBG pA is cloned in reverse orientation into intron 1 of the Rosa26 locus. Sequences of the Rosa26 locus adjacent to GAA-IVS1-13T-G operably linked to a CAG promoter and rBG pA can be used as homologous arms.

[0163] In some embodiments, the GAA splice variant, or wt-GAA, operably linked to any promoter, and / or regulatory elements, and / or polyadenylation signal, and optionally linked to a nucleic acid sequence comprising homology arms, is amplified by any one of the techniques described in the literature (e.g., PCR) to obtain a sufficient amount of genetic material to genetically engineer an organism.

[0164] In an even more preferred embodiment, GAA-IVS1-13T-G operably linked to the CAG promoter and rBG pA is cloned in reverse orientation into intron 1 of the Rosa26 locus and amplified by PCR. In an even more preferred embodiment, PCR amplification is performed by using primers annealing to Rosa26 homology arms. In some embodiments, the Rosa26 homology arms comprise a nucleotide sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14 or 15. In some embodiments, primers with Rosa26 homology arms comprise a nucleotide sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 16-17.

[0165] In some embodiments, the GAA splice variant, or wt-GAA, optionally operably linked to a nucleic acid sequence comprising any promoter, and / or regulatory elements, and / or polyadenylation signal, and / or homology arms, is used to produce transgenic animals by any one of the techniques described in the literature.

[0166] In some embodiments, a GAA splice variant or wt-GAA, optionally operably linked to a nucleic acid sequence comprising any promoter, and / or regulatory elements (e.g., enhancers), and / or polyadenylation signals, and / or homology arms, is included in a recombinant nucleic acid molecule disclosed herein. In some aspects, the recombinant nucleic acid molecules disclosed herein are used to produce the transgenic animals disclosed herein by any one of the techniques described in the literature.

[0167] In some embodiments, a recombinant nucleic acid molecule disclosed herein comprises (i) a 5' homology arm, (ii) a GAA splice variant or fragment thereof, and (iii) a 3' homology arm. In some embodiments, the GAA splice variant or fragment thereof is a human GAA splice variant or fragment thereof. In some embodiments, the GAA splice variant or fragment thereof comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 or 2.

[0168] In some embodiments, the homology arms comprise a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a region of the Rosa26 locus in the mouse genome. In some embodiments, the homology arms comprise a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a region of the GAA locus in the mouse genome.

[0169] In some embodiments, a recombinant nucleic acid molecule disclosed herein comprising (i) a 5' homology arm, (ii) a GAA splice variant or fragment thereof, and (iii) a 3' homology arm is used to generate a transgenic animal disclosed herein by any one of the techniques described in the literature. In some embodiments, the genome of a non-human animal model (e.g., the genome of a mouse model) comprises a GAA splice variant or fragment thereof operably linked to an endogenous homologous promoter, polyadenylation signal, control region (e.g., and enhancer) of the transgenic mouse. In some embodiments, the endogenous homologous promoter, polyadenylation signal, or control region (e.g., and enhancer) is a mouse GAA or Rosa26 promoter, polyadenylation signal, control region (e.g., and enhancer). In some embodiments, the endogenous homologous promoter is a mouse GAA or Rosa26 promoter. In some embodiments, the endogenous homologous polyadenylation signal is a mouse GAA or Rosa26 polyadenylation signal. In some embodiments, the endogenous homologous control region is a mouse GAA or Rosa26 control region.

[0170] In some embodiments, the recombinant nucleic acid molecules disclosed herein further comprise a promoter, a polyadenylation signal, a regulatory region (e.g., and enhancer), or a combination thereof. For example, the recombinant nucleic acid molecules disclosed herein may comprise (i) a 5' homology arm, (ii) a polyadenylation signal, (iii) a GAA splice variant or fragment thereof, (iv) a promoter, and (v) a 3' homology arm.

[0171] In some embodiments, the promoter, polyadenylation signal, or control region (e.g., and enhancer) is a homologous (e.g., mouse promoter, polyadenylation signal, or control region) promoter, polyadenylation signal, or control region (e.g., and enhancer). In some embodiments, the homologous promoter, polyadenylation signal, or control region (e.g., and enhancer) is a mouse GAA or Rosa26 promoter, polyadenylation signal, or control region (e.g., and enhancer). In some embodiments, the promoter, polyadenylation signal, or control region (e.g., and enhancer) is a heterologous (e.g., non-mouse eukaryotic (e.g., human), bacterial, or viral promoter and / or control region in a transgenic mouse) promoter, polyadenylation signal, or control region (e.g., and enhancer). In some embodiments, the heterologous promoter, polyadenylation signal, or control region (e.g., and enhancer) is a human promoter, polyadenylation signal, or control region (e.g., and enhancer). In some embodiments, the human promoter, polyadenylation signal, or control region (e.g., and enhancer) is a GAA human promoter, polyadenylation signal, or control region (e.g., and enhancer). In some embodiments, the promoter is a GAA human promoter. In some embodiments, the polyadenylation signal is a GAA human polyadenylation signal. In some embodiments, the control region (e.g., and enhancer) is a GAA human control region (e.g., and enhancer).

[0172] In some embodiments, the promoter is selected from the group consisting of a CMV early enhancer / chicken beta actin (CBA) promoter, a CAG promoter, CMV, EF1α, EF1α with a CMV enhancer, a CMV promoter with a CMV enhancer (CMVe / p), and a CMV promoter with an SV40 intron. In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3. In some embodiments, the polyadenylation signal is an rGB-pA polyadenylation signal. In some embodiments, the polyadenylation signal comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:4.

[0173] In some embodiments, a recombinant nucleic acid molecule comprising (i) a 5' homology arm, (ii) a GAA splice variant or fragment thereof, and (iii) a 3' homology arm, and further comprising a promoter, a polyadenylation signal, a control region (e.g., and an enhancer), or a combination thereof, is used to produce a transgenic animal disclosed herein by any one of the techniques described in the literature.

[0174] In some embodiments, the genome of a non-human animal model (e.g., the genome of a mouse model) comprises a GAA splice variant or a fragment thereof operably linked to an exogenous promoter, polyadenylation signal, control region (e.g., and enhancer), or a combination thereof. In some embodiments, the exogenous promoter, polyadenylation signal, or control region (e.g., and enhancer) is a homologous (e.g., mouse promoter, polyadenylation signal, or control region) promoter, polyadenylation signal, or control region (e.g., and enhancer). In some embodiments, the homologous promoter, polyadenylation signal, or control region (e.g., and enhancer) is a mouse GAA or Rosa26 promoter, polyadenylation signal, or control region (e.g., and enhancer).

[0175] In some embodiments, the exogenous promoter, polyadenylation signal, or control region (e.g., and enhancer) is a heterologous (e.g., non-mouse eukaryotic (e.g., human), bacterial, or viral promoter and / or control region in a transgenic mouse) promoter, polyadenylation signal, or control region (e.g., and enhancer). In some embodiments, the heterologous promoter, polyadenylation signal, or control region (e.g., and enhancer) is a human promoter, polyadenylation signal, or control region (e.g., and enhancer). In some embodiments, the human promoter, polyadenylation signal, or control region (e.g., and enhancer) is a GAA human promoter, polyadenylation signal, or control region (e.g., and enhancer). In some embodiments, the promoter is a GAA human promoter. In some embodiments, the polyadenylation signal is a GAA human polyadenylation signal. In some embodiments, the regulatory region (eg, and enhancer) is a GAA human regulatory region (eg, and enhancer).

[0176] In some embodiments, the exogenous promoter is selected from the group consisting of a CMV early enhancer / chicken beta actin (CBA) promoter, a CAG promoter, CMV, EF1α, EF1α with a CMV enhancer, a CMV promoter with a CMV enhancer (CMVe / p), and a CMV promoter with an SV40 intron. In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3. In some embodiments, the polyadenylation signal is an rGB-pA polyadenylation signal. In some embodiments, the polyadenylation signal comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:4.

[0177] In some embodiments, the recombinant nucleic acid molecules disclosed herein further comprise a Neo (neomycin) resistance gene, an Amp (ampicillin) resistance gene.

[0178] Techniques for producing transgenic animals are described in the literature. See, for example, Houdebine, Transgenic animals—Generation and Use (Harwood Academic, 1997), Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 2nd ed., (Cold Spring Harbor Laboratory, 1994), Krimpenfort et al., Bio / Technology 1991, 9:844, Palmiter et al., Cell 1985, 41:343, Hammer et al., Nature 1985, 315:680, U.S. Patent Nos. 5,602,299, 5,175,384, 6,066,778 and 6,037,521, which are incorporated herein in their entireties. Techniques used to generate transgenic animals include intranuclear injection (Gordon, Proc. Nat. Acad. Sci. USA 1980, 77:7380-7384; U.S. Pat. No. 4,873,191), electroporation (Lo, Mol. Cell. Biol. 1983, 3:1803-1814), homologous recombination (Thompson et al., Cell 1989, 56:313-321; Hanks et al., Science 1995, 269:679-682), retroviral gene transfer into germ cell lines (Van der Putten et al., Proc. Nat. Acad. Sci. USA 1985, 82:6148-6152), and sperm-mediated gene transfer (Lavitrano et al., Cell 1989, 57:717-723), but are not limited to. In some embodiments, genome editing technologies, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided CRISPR-Cas nuclease systems, can be used to produce the transgenic animals of the present disclosure.In a preferred embodiment, the RNA-guided CRISPR-Cas9 nuclease system is used to generate the transgenic animals of the present disclosure.

[0179] The specificity of the Cas9 nuclease is determined by the 20-nt guide sequence within the sgRNA. For example, in the Streptococcus pyogenes (S. pyogenes) system, the target sequence (e.g., 5'-GTCACCTCCAATGACTAGGG-3') must immediately precede (i.e., 5') the 5'-NGG PAM, and the 20-nt guide sequence bases pair with the opposite strand to mediate Cas9 cleavage approximately 3 bp upstream of the PAM. The PAM sequence must immediately follow the target DNA locus but must not be part of the 20-nt guide sequence within the sgRNA. Bioinformatic CRISPR design tools can be used to design specific sgRNAs by taking the genomic sequence of interest and identifying appropriate target sites. In preferred embodiments, a particular sgRNA comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:18.

[0180] Off-target sites for each sgRNA can also be computationally predicted for each intended target using bioinformatics. To increase targeting specificity, an alternative strategy using the D10A nickase mutant of Cas9 (Cas9n) together with a pair of sgRNAs can be used. Because sometimes a particular sgRNA may not function for reasons that are still unclear, it is recommended to design at least two sgRNAs for each locus and test their efficiency in the intended cell type.

[0181] Depending on the desired application, sgRNAs can be delivered either as PCR amplicons containing expression cassettes or as sgRNA expression plasmids. In addition to PCR and plasmid-based delivery methods, Cas9 and sgRNAs can be introduced into cells as mRNA and RNA, respectively. Additionally, Cas9 can be delivered to cells as a polypeptide.

[0182] In some aspects, GAA splicing variants or wt-GAA, optionally operably linked to any promoter, and / or regulatory element, and / or polyadenylation signal, and linked to a nucleic acid sequence comprising homologous arms, are used as repair templates. In some embodiments, GAA splicing variants or wt-GAA, optionally operably linked to any promoter, and / or regulatory element, and / or polyadenylation signal, and linked to a nucleic acid sequence comprising homologous arms, are included in a plasmid, and this plasmid is introduced into cells. In some embodiments, GAA splicing variants or wt-GAA, optionally operably linked to any promoter, and / or regulatory element, and / or polyadenylation signal, and linked to a nucleic acid sequence comprising homologous arms, are delivered to cells as PCR amplicons.

[0183] In a preferred embodiment, Cas9 is delivered to the cell as a polypeptide, the sgRNA is delivered to the cell as RNA, and the repair template comprising GAA-IVS1-13T-G is delivered to the cell as a PCR amplicon.

[0184] In some embodiments, about 1 ng / μL, about 5 ng / μL, about 10 ng / μL, about 15 ng / μL, about 20 ng / μL, about 25 ng / μL, about 30 ng / μL, about 35 ng / μL, about 40 ng / μL, about 45 ng / μL, about 50 ng / μL, about 5 5 ng / μL, about 60 ng / μL, about 65 ng / μL, about 70 ng / μL, about 75 ng / μL, about 80 ng / μL, about 85 ng / μL, about 90 ng / μL, about 95 ng / μL, about 100 ng / μL of Cas9 polypeptide is delivered to the cell. In preferred embodiments, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, or about 50 ng / mL of Cas9 polypeptide is delivered to cells. In even more preferred embodiments, about 30 ng / mL of Cas9 polypeptide is delivered to cells.

[0185] In some embodiments, about 1 ng / μL, about 5 ng / μL, about 10 ng / μL, about 15 ng / μL, about 20 ng / μL, about 25 ng / μL, about 30 ng / μL, about 35 ng / μL, about 40 ng / μL, about 45 ng / μL, about 50 ng / μL, about 55 ng / μL, about 60 ng / μL, about 65 ng / μL, about 70 ng / μL, about 75 ng / μL, about 80 ng / μL, about 85 ng / μL, about 90 ng / μL, about 95 ng / μL, or about 100 ng / μL of a GAA splice variant or wt-GAA-containing repair template is delivered to cells. In preferred embodiments, about 1 ng / mL, about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, or about 40 ng / mL of amplicon corresponding to repair template comprising GAA splice variant or wt-GAA is delivered to cells.In even more preferred embodiments, about 15 ng / mL of amplicon corresponding to repair template comprising GAA splice variant or wt-GAA is delivered to cells.

[0186] In some embodiments, about 1 ng / μL, about 5ng / μL, about 10ng / μL, about 15ng / μL, about 20ng / μL, about 25ng / μL, about 30ng / μL, about 35ng / μL, about 40ng / μL, about 45ng / μL, about 50ng / μL, about 55n g / μL, about 60ng / μL, about 65ng / μL, about 70ng / μL, about 75ng / μL, about 80ng / μL, about 85ng / μL, about 90ng / μL, about 95ng / μL, about 100ng / μL of sgRNA is delivered to the cells. In preferred embodiments, about 1 ng / mL, about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, or about 40 ng / mL of RNA corresponding to the sgRNA is delivered to the cell. In even more preferred embodiments, about 15 ng / mL of RNA corresponding to the sgRNA is delivered to the cell.

[0187] In some embodiments, CRISPR / Cas9 gene editing method is used in combination with transfection of ES cells to generate transgenic mice. In a preferred embodiment, CRISPR / Cas9 gene editing method is used in combination with intranuclear injection to generate transgenic mice. In a more preferred embodiment, intranuclear injection is carried out on one-cell stage zygotes obtained by mating C57BL / 6N males (Charles River, China) with superovulated C57BL / 6N females (Charles River, China).

[0188] In some embodiments, injected embryos can be cultured in any suitable medium and then transferred to the oviducts of pseudopregnant females at any stage. In a preferred embodiment, injected embryos are cultured overnight in KSOM medium, and the injected embryos, which develop to the two-cell stage, are transferred to the oviducts of pseudopregnant females.

[0189] Transgenic animals can be screened for the presence and / or expression of the transgene and the presence and / or expression of splice variants of the transgene by any suitable method described in the literature. In some embodiments, screening is accomplished by in situ hybridization, Southern blot, or Northern blot analysis using an oligonucleotide probe complementary to at least a portion of the transgene DNA or RNA. In other embodiments, screening is accomplished by Western blot analysis using an antibody specifically binding to the protein encoded by the transgene. In some embodiments, whole transgenic animals and / or cells, tissues, or organs derived from transgenic animals are tested for the presence and expression of the transgene using in situ hybridization, PCR, Southern, Northern, or Western blot analysis. In some embodiments, DNA is prepared from tissues (e.g., tail, ear, muscle) of transgenic animals (e.g., transgenic mice) and analyzed by Southern blot or PCR for the transgene. In a preferred embodiment, animals can be screened for the presence of the transgene by PCR amplification. In an even more preferred embodiment, animals are screened for the presence of the transgene by PCR amplification using primers comprising a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 16, 17, 19-28.

[0190] In some embodiments, whole transgenic animals and / or cells, tissues, and organs derived from transgenic animals are tested for the presence and expression of splice variants of the transgene using in situ hybridization, PCR, Southern, Northern, or Western blot analysis. In some embodiments, cDNA is prepared from tissues (e.g., tail, ear, muscle) of transgenic animals (e.g., transgenic mice) and analyzed for the expression of splice variants of the transgene by Southern blot analysis or PCR. In a preferred embodiment, animals can be screened for the presence and expression of splice variants of the transgene by PCR amplification. In an even more preferred embodiment, animals are screened for the presence and expression of splice variants of the transgene by PCR amplification using primers comprising nucleic acid sequences that are at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 5-10.

[0191] In some embodiments, the sequence of the transgene can be verified by sequencing (e.g., Sanger sequencing). In some embodiments, PCR amplicons derived from DNA (e.g., genomic DNA or cDNA) from the transgenic animal can be sequenced by any one of the techniques described in the literature (e.g., Sanger sequencing). In some embodiments, specific primers can be used to sequence PCR amplicons derived from DNA (e.g., genomic DNA or cDNA) from the transgenic animal. In some embodiments, the primers used to sequence PCR amplicons derived from DNA (e.g., genomic DNA or cDNA) from the transgenic animal comprise nucleic acid sequences that are at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 29-31.

[0192] Founder animals can be crossed, inbred, outbred, or hybridized to produce a colony of the desired transgenic animals. Non-limiting examples of such breeding strategies include outbreeding founder animals with more than one integration site to establish distinct lines, inbreeding distinct lines to produce compound transgenics that express transgenes at higher levels due to the additive effects of each transgene, crossing heterozygous transgenic mice to increase transgene expression and / or produce mice homozygous for a given integration site, crossing distinct homozygous lines to produce compound heterozygous or homozygous lines, and crossing animals into different inbred genetic backgrounds to study the effects of allelic modifications on transgene expression and the physiological effects of transgene expression.

[0193] In some embodiments, a GAA splice variant is inserted into the non-human animal model genome. In some embodiments, wt-GAA is inserted into the non-human animal model genome. In some embodiments, a mutation (e.g., a TG mutation such as an IVS1-13T-G mutation) is introduced into the wild-type GAA gene by any one of the techniques described in the literature. In some embodiments, the mutation is introduced into the wild-type GAA gene prior to introduction of a transgene into the non-human animal model. In some embodiments, the mutation is introduced into the wild-type GAA gene simultaneously (i.e., simultaneously) with introduction of the transgene into the non-human animal model. In some embodiments, the mutation is introduced into the wild-type GAA gene after introduction of the transgene into the non-human animal model. In some embodiments, provided herein are non-human animal models generated by crossing a non-human animal model disclosed herein with a non-human animal model that lacks all copies of the GAA gene endogenous to the non-human animal model.

[0194] For example, a mouse model produced according to the methods disclosed herein that contains at least one copy of a GAA splice variant inserted into its genome (i.e., the genome of the mouse model's germ cells) and that contains at least one (e.g., two) copies of the GAA gene endogenous to the mouse model can be bred to a mouse model that lacks all copies of the GAA gene endogenous to the mouse model. The progeny of such a mating can be screened according to methods known in the published literature to select non-human animal models that contain the GAA splice variant and do not contain any copies of the GAA gene endogenous to the non-human animal model in their genome. Such non-human animal models are also referred to herein as fully humanized non-human animal models.

[0195] 4. Methods for Using Genetically Engineered Non-Human Animal Models of Pompe Disease The disclosed non-human animal models of Pompe disease can be used in a variety of studies.

[0196] In humans, the GAA gene is found on the long arm of chromosome 17 (17q25.2-q25.3) and consists of 20 exons. GAA pre-RNA undergoes alternative splicing, which generates mature RNA containing different exon combinations. The IVS1-13T-G (c.-32-13T>G) mutation weakens the splice acceptor of GAA exon 2, resulting in splicing defects and exon 2 skipping. Splicing defects and skipping of exon 2 result in low levels of active enzyme (12% of normal) resulting from leakage of normally spliced ​​mRNA.

[0197] There is a need in the art for splice modulators (e.g., antisense oligomers, antisense oligonucleotides, or small molecules) to enhance the inclusion of GAA exon 2 in the mature mRNA of patients with one c.-32-13T>G allele.

[0198] The genetically engineered non-human animal models of Pompe disease of the present invention, or cells, tissues, organs, or parts derived therefrom, can be used to test the efficacy of such splice regulators.

[0199] In some embodiments, the splice modulating agent may interact with or bind to one or more splicing proteins in a cell. In some embodiments, the splice modulating agent may activate one or more splicing proteins in a cell and / or inhibit one or more splicing proteins in a cell. In some embodiments, the splice modulating agent may interact with or bind to proteins that regulate one or more splicing proteins in a cell. In some embodiments, the splice modulating agent may activate one or more proteins that regulate one or more splicing proteins in a cell or inhibit one or more proteins that regulate one or more splicing proteins in a cell. In some embodiments, the splice modulating agent may interact with or bind to a target polynucleotide sequence of a partially processed mRNA transcript (i.e., pre-mRNA).

[0200] In some embodiments, the splice modulator may be an antisense oligomer (e.g., an antisense oligonucleotide) that binds to the target portion of a pre-mRNA. The ASO may have an exact sequence that is complementary or nearly complementary to the target sequence (i.e., sufficiently complementary to bind to the target). ASOs are designed to bind (hybridize) to a target nucleic acid (e.g., the target portion of a pre-mRNA transcript) under physiological conditions. Typically, when they hybridize to sites other than the intended (targeted) nucleic acid sequence, they hybridize to a limited number of sequences that are not the target nucleic acid (to a few sites other than the target nucleic acid). The design of the ASO can take into account the occurrence of the nucleic acid sequence of the target portion of the pre-mRNA transcript, or sufficiently similar nucleic acid sequences, in other locations in the genome or cellular pre-mRNA or transcriptome, so as to limit the possibility that the ASO will bind to other sites and cause "off-target" effects. Any antisense oligomer described in the literature can be used to perform the methods described herein.

[0201] In some embodiments, the ASO "specifically hybridizes" or is "specific" for the targeted portion of the target nucleic acid or pre-mRNA.

[0202] Oligomers, such as oligonucleotides, are "complementary" to one another when hybridization occurs between two single-stranded polynucleotides in an antiparallel configuration. A double-stranded polynucleotide can be complementary to another polynucleotide if hybridization can occur between one of the strands of a first polynucleotide and one of the strands of a second polynucleotide. Complementarity (the degree to which one polynucleotide is complementary to another) can be quantified in terms of the proportion (e.g., percentage) of bases on opposing strands that are expected to form hydrogen bonds with each other according to generally accepted base-pairing rules. The sequence of an antisense oligomer (ASO) need not be 100% complementary to the sequence of its target nucleic acid to hybridize. In certain embodiments, an ASO may contain at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence complementarity with a target region within the targeted nucleic acid sequence. For example, an ASO in which 18 out of 20 nucleotides of an oligomeric compound are complementary to a target region and therefore specifically hybridize represents 90% complementarity. In this example, the remaining non-complementary nucleotides may be clustered together or interspersed with complementary nucleotides, and do not need to be contiguous with each other or with complementary nucleotides. The percentage of complementarity of an ASO with a region of a target nucleic acid can be routinely determined using the BLAST program (basic local alignment search tool) and PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0203] An ASO need not hybridize to every nucleotide in a target sequence; the hybridizing nucleotides may be contiguous, consecutive, or non-contiguous. An ASO may hybridize to one or more segments of a pre-mRNA transcript such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop or hairpin structure may be formed). In certain embodiments, an ASO hybridizes to non-contiguous nucleotides in a target pre-mRNA transcript. For example, an ASO can hybridize to nucleotides in a pre-mRNA transcript that are separated by one or more nucleotide(s) to which the ASO does not hybridize.

[0204] The ASOs described herein may contain nucleotides complementary to nucleotides present in the target portion of a pre-mRNA. The term ASO embodies oligonucleotides and any other oligomeric molecules that contain nucleotides capable of hybridizing to complementary nucleotides on a target mRNA but do not contain sugar moieties, such as peptide nucleic acids (PNAs). ASOs may contain naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination thereof. In some embodiments, all of the nucleotides in an ASO are naturally occurring nucleotides. In some embodiments, all of the nucleotides in an ASO are modified nucleotides. In some embodiments, some of the nucleotides in an ASO are naturally occurring nucleotides and some of the nucleotides in an ASO are modified nucleotides. Chemical modifications of ASOs or ASO components that are compatible with the methods and compositions described herein will be apparent to those skilled in the art.

[0205] The nucleobase of the ASO may be any naturally occurring nucleobase or any synthetic or modified nucleobase.

[0206] The ASOs described herein also contain a backbone structure connecting the oligomeric building blocks. The backbone structure may include 3'-5' phosphodiester bonds connecting the sugar moieties of the oligomer. The backbone structures of the ASOs described herein may include (but are not limited to) phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoraniladates, phosphoramidates, and the like. In some embodiments, the backbone structure of the ASO does not contain phosphorus bonds but peptide bonds, for example, in peptide nucleic acids (PNAs), or linking groups including carbamate groups, amide groups, and linear and cyclic hydrocarbon groups. In some embodiments, the backbone modification is a phosphothioate bond. In some embodiments, the backbone modification is a phosphoramidate bond.

[0207] Any of the ASOs described herein may contain sugar moieties that include ribose or deoxyribose present in naturally occurring nucleotides, or modified sugar moieties or sugar analogs.

[0208] In some embodiments, each monomer of an ASO is modified in the same way. Such modifications present in each of the monomer components of an ASO are referred to as "uniform modifications." In some embodiments, a combination of different modifications may be desired; for example, an ASO may contain a combination of phosphorodiamidate linkages and sugar moieties containing morpholine rings (morpholinos). A combination of an ASO and different modifications is referred to as a "mixed modification" or "mixed chemistry."

[0209] In some embodiments, the ASO comprises one or more backbone modifications. In some embodiments, the ASO comprises one or more sugar moiety modifications. In some embodiments, the ASO comprises one or more backbone modifications and one or more sugar moiety modifications. In some embodiments, the ASO comprises a 2'MOE modification and a phosphorothioate backbone. In some embodiments, the ASO comprises a phosphorodiamidate morpholino (PMO). In some embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of the ASOs described herein, or any of the components thereof (e.g., nucleobases, sugar moieties), may be modified to achieve a desired property or activity of the ASO or to reduce an undesirable property or activity of the ASO. For example, the ASO, or one or more components of any ASO, may be modified to enhance binding affinity for a target sequence on a pre-mRNA transcript, reduce binding to any non-target sequence, reduce degradation by cellular nucleases (i.e., RNase H), improve uptake of the ASO into cells and / or the nucleus of a cell, alter the pharmacokinetics or pharmacodynamics of the ASO, or modulate the half-life of the ASO.

[0210] In some embodiments, ASOs are composed of 2'-O-(2-methoxyethyl) (MOE) phosphorothioate modified nucleotides. ASOs composed of such nucleotides are particularly suitable for the methods disclosed herein, as oligomers with such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable for oral delivery in some embodiments described herein. See, for example, Geary et al., J Pharmacol Exp Ther. 2001; 296(3): 890-7; Geary et al., J Pharmacol Exp Ther. 2001; 296(3): 898-904.

[0211] Methods for synthesizing ASOs will be known to those of skill in the art. Alternatively, or in addition, ASOs may be obtained from commercial sources.

[0212] Unless otherwise specified, the left-hand end of a single-stranded nucleic acid sequence (e.g., a pre-mRNA transcript, an oligonucleotide, an ASO, etc.) is the 5' end, and the left-hand direction of a single-stranded or double-stranded nucleic acid sequence is referred to as the 5' direction. Similarly, the right-hand end or direction of a nucleic acid sequence (single-stranded or double-stranded) is referred to as the 3' end or direction. Generally, a region or sequence 5' of a reference point in a nucleic acid is referred to as "upstream," and a region or sequence 3' of a reference point in a nucleic acid is referred to as "downstream." Generally, the 5' direction or end of an mRNA is where the initiation or start codon is located, and the 3' end or direction is where the termination codon is located. In some embodiments, nucleotides upstream of a reference point in a nucleic acid may be designated by negative numbers, while nucleotides downstream of the reference point may be designated by positive numbers. For example, a reference point (e.g., an exon-exon junction in an mRNA) may be designated as the "zero" site, and the nucleotide immediately adjacent to and upstream of the reference point is designated as "minus one," e.g., "-1," while the nucleotide immediately adjacent to and downstream of the reference point is designated as "plus one," e.g., "+1."

[0213] In other embodiments, the ASO is complementary to (and binds to) a target portion of the pre-mRNA that is downstream (3' direction) of the 5' splice site of an intron in the pre-mRNA (e.g., in the direction designated by a positive number relative to the 5' splice site) (Figure 1). In some embodiments, the ASO is complementary to a target portion of the pre-mRNA that is within the region +6 to +100 relative to the 5' splice site of the intron. In some embodiments, the ASO is not complementary to nucleotides +1 to +5 relative to the 5' splice site (the first five nucleotides located downstream of the 5' splice site). In some embodiments, the ASO may be complementary to a target portion of the pre-mRNA that is within the region +6 to +50 relative to the 5' splice site of the intron. In some embodiments, the ASO is complementary to a targeting portion within the region +6 to +90, +6 to +80, +6 to +70, +6 to +60, +6 to +50, +6 to +40, +6 to +30, or +6 to +20 relative to the 5' splice site of the intron.

[0214] In some embodiments, the ASO is complementary to a targeted region of the pre-mRNA that is upstream (5' relative) of the 3' splice site of an intron in the pre-mRNA (e.g., in the direction indicated by a negative number) (Figure 1). In some embodiments, the ASO is complementary to a targeted portion of the pre-mRNA that is within the region -16 to -100 relative to the 3' splice site of the intron. In some embodiments, the ASO is not complementary to nucleotides -1 to -15 (the first 15 nucleotides upstream of the 3' splice site) relative to the 3' splice site. In some embodiments, the ASO is complementary to a targeted portion of the pre-mRNA that is within the region -16 to -50 relative to the 3' splice site of the intron. In some embodiments, the ASO is complementary to a targeting portion within the region of -16 to -90, -16 to -80, -16 to -70, -16 to -60, -16 to -50, -16 to -40, or -16 to -30 relative to the 3' splice site of the intron.

[0215] In embodiments, the targeted portion of the pre-mRNA is within the region from +100 relative to the 5' splice site of the intron to -100 relative to the 3' splice site of the intron.

[0216] In some embodiments, the ASO is complementary to a target portion of a pre-mRNA within an exon adjacent to the 5' splice site (upstream) of an intron. In some embodiments, the ASO is complementary to a target portion of a pre-mRNA that is within a region +2e to -4e in the exon adjacent to the 5' splice site of the intron. In some embodiments, the ASO is not complementary to nucleotides -1e to -3e relative to the 5' splice site of the intron. In some embodiments, the ASO is complementary to a target portion of a pre-mRNA that is within a region of -4e to -100e, -4e to -90e, -4e to -80e, -4e to -70e, -4e to -60e, -4e to -50e, -4e to -40e, -4e to -30e, or -4e to -20e relative to the 5' splice site of the intron.

[0217] In some embodiments, the ASO is complementary to a target portion of a pre-mRNA within an exon adjacent to the 3' splice site (downstream) of an intron (Figure 1). In some embodiments, the ASO is complementary to a target portion of a pre-mRNA that is within the region +2e to -4e in the exon adjacent to the 3' splice site of the intron. In some embodiments, the ASO is not complementary to nucleotide +1e relative to the 3' splice site of the intron. In some embodiments, the ASO is complementary to a target portion of a pre-mRNA that is within the region +2e to +100e, +2e to +90e, +2e to +80e, +2e to +70e, +2e to +60e, +2e to +50e, +2e to +40e, +2e to +30e, or +2e to +20e relative to the 3' splice site of the intron. The ASO may be of any length suitable for specific binding and effective enhancement of splicing. In some embodiments, the ASO consists of 8 to 50 nucleotides. For example, the ASO may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleotides in length. In some embodiments, the ASO consists of more than 50 nucleotides.In some embodiments, the ASO may be 8 to 50 nucleotides, 8 to 40 nucleotides, 8 to 35 nucleotides, 8 to 30 nucleotides, 8 to 25 nucleotides, 8 to 20 nucleotides, 8 to 15 nucleotides, 9 to 50 nucleotides, 9 to 40 nucleotides, 9 to 35 nucleotides, 9 to 30 nucleotides, 9 to 25 nucleotides, 9 to 20 nucleotides, 9 to 15 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides, 10 to 35 nucleotides, 10 to 30 nucleotides, 10 to 25 nucleotides, 10 to 20 nucleotides, 10 to 15 nucleotides, 11 to 50 nucleotides, 11 to 40 nucleotides, 11 to 35 nucleotides, 11 to 30 nucleotides, 11 to 25 nucleotides, 11 to 20 nucleotides, 11 to 15 nucleotides, 12 to 50 nucleotides, 12 to 40 nucleotides, 12 to 3 ... The ASO may be 30 nucleotides, 12-25 nucleotides, 12-20 nucleotides, 12-15 nucleotides, 13-50 nucleotides, 13-40 nucleotides, 13-35 nucleotides, 13-30 nucleotides, 13-25 nucleotides, 13-20 nucleotides, 14-50 nucleotides, 14-40 nucleotides, 14-35 nucleotides, 14-30 nucleotides, 14-25 nucleotides, 14-20 nucleotides, 15-50 nucleotides, 15-40 nucleotides, 15-35 nucleotides, 15-30 nucleotides, 15-25 nucleotides, 15-20 nucleotides, 20-50 nucleotides, 20-40 nucleotides, 20-35 nucleotides, 20-30 nucleotides, 20-25 nucleotides, 25-50 nucleotides, 25-40 nucleotides, 25-35 nucleotides, or 25-30 nucleotides in length. In some embodiments, the ASO is 18 nucleotides in length. In some embodiments, the ASO is 15 nucleotides in length. In some embodiments, the ASO is 25 nucleotides in length.

[0218] In some embodiments, two or more ASOs are used that have different chemistries but are complementary to the same targeting portion of the pre-mRNA, hi some embodiments, two or more ASOs are used that are complementary to different targeting portions of the pre-mRNA.

[0219] In embodiments, the antisense oligonucleotides of the present invention are chemically linked to one or more moieties or conjugates, such as targeting moieties or other conjugates that enhance the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties, cholesteryl moieties, aliphatic chains, such as dodecanediol or undecyl residues, polyamine or polyethylene glycol chains, or adamantane acetic acid. Oligonucleotides containing lipophilic moieties and preparation methods have been described in the literature. In embodiments, the antisense oligonucleotides are conjugated to moieties, including, but not limited to, abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, such as N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), lipids, or polycarbonate compounds. Conjugates can be linked to one or more of the nucleotides comprising the antisense oligonucleotide at any of several positions on the sugar, base, or phosphate group, as understood in the art and described in the literature, for example, using linkers. The linker may comprise a bivalent or trivalent branched linker. In embodiments, the conjugate is attached to the 3' end of the antisense oligonucleotide. Methods for preparing oligonucleotide conjugates are described, for example, in U.S. Patent No. 8,450,467, "Carbohydrate conjugates as delivery agents for oligonucleotides," which is incorporated herein by reference.

[0220] In some embodiments, the nucleic acid to be targeted by the ASO is a pre-mRNA expressed in a cell, such as a eukaryotic cell. In some embodiments, the term "cell" may refer to a population of cells. In some embodiments, the cell is in a subject. In some embodiments, the cell is in vivo. In some embodiments, the cell is isolated from a subject. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vitro. In some embodiments, the cell is a cell or cell line associated with a condition or disease. In some embodiments, the cell is in vitro (e.g., in cell culture).

[0221] In some embodiments, the therapeutic agent may be a small molecule, for example, a small molecule may be a molecule less than 900 daltons.

[0222] In some embodiments, mRNA is extracted from cells, tissues, or organs derived from transgenic non-human animal models before and after administration of splice regulators.MRNA can be extracted from cells, tissues, or organs derived from transgenic non-human animal models by any means described in the literature.For example, mRNA can be extracted by organic extraction, such as phenol-guanidine isothiocyanate (GITC)-based solution, silica membrane-based spin column technology, and paramagnetic particle technology.

[0223] In some embodiments, mRNA is reverse transcribed into cDNA.MRNA can be reverse transcribed into cDNA by any means described in the literature.For example, any reverse transcriptase can be used, such as that contained in commercially available kit.

[0224] In some embodiments, the cDNA is processed by PCR. In some embodiments, specific pairs of primers can be used to perform PCR reactions, thereby detecting and / or measuring the presence and / or ratio of different splicing forms. In some embodiments, a pair of primers is used to amplify exon junctions that are not affected by the mutation and therefore amplify both the GAA gene without the splicing mutation and the GAA gene with the splicing mutation, and a pair of primers is used to amplify exon junctions that are only affected by the mutation and therefore do not contain the splicing mutation. For example, in one embodiment, a pair of primers is used to amplify 6-7 exon junctions that are not affected by IVS1-13T-G and therefore amplify both the wild-type and defectively spliced ​​c.-32-13T>G forms of the GAA gene, and a pair of primers is used to amplify 1-2 exon junctions that are affected by IVS1-13T-G and therefore amplify only the wild-type form of the GAA gene. In some embodiments, a pair of primers is used to amplify a region of the GAA gene that has a different length in mature mRNA derived from a GAA gene containing a splice mutation and a GAA gene that does not contain a splice mutation. For example, in one embodiment, a pair of primers is used to amplify exons 1-5, such that mature mRNA derived from a GAA gene containing an IVS1-13T-G mutation produces an amplicon of a different length than mature mRNA derived from a GAA gene that does not contain the IVS1-13T-G mutation. In another embodiment, the primers used for this PCR assay comprise nucleic acid sequences that are at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 11-12.In some embodiments, the protein product translated from the mature mRNA derived from the pre-mRNA transcribed from the mutGAA gene is extracted from cells, tissues, or organs derived from a transgenic non-human animal model before and after administration of a splice regulator. The protein product can be extracted from cells, tissues, or organs derived from a transgenic non-human animal model by any one of the means described in the published literature. Methods for detecting and analyzing protein products are also known in the published literature. For example, the protein content extracted from cells, tissues, or organs can be assayed by Western blot if a suitable antibody capable of recognizing a specific protein product is available. Many antibodies capable of recognizing the protein product of the GAA gene (i.e., the GAA protein encoded by the GAA gene) are known and commercially available. Nevertheless, most, if not all, of these antibodies are incapable of distinguishing between protein products of GAA genes from different species. For example, most, but not all, such antibodies are able to recognize (i.e., bind to) both human and mouse GAA gene protein products (i.e., bind to both the protein encoded by the human GAA gene and the protein encoded by the mouse GAA gene), a phenomenon also known as cross-reactivity.

[0225] In some embodiments, non-human animal models disclosed herein that comprise a GAA splice variant and lack all copies of the endogenous GAA gene are particularly useful for analyzing protein products translated from mature mRNA derived from pre-mRNA transcribed from the mutGAA gene before and after administration of a splice regulator. The absence of any copies of the endogenous GAA gene in the non-human animal model overcomes the cross-reactivity of most, if not all, available antibodies capable of binding to the protein product of the GAA gene (i.e., the GAA protein encoded by the GAA gene).

[0226] In some embodiments, the splice modifier alters the splicing of the pre-mRNA. In some embodiments, the splice modifier does not alter the splicing of the pre-mRNA. In some embodiments, the ratio of one variant of the target mRNA to another variant of the target mRNA is altered. In some embodiments, the ratio of one variant of the target mRNA to another variant of the target mRNA is not altered. In some embodiments, the ratio of one variant of the target protein to another variant of the target protein is altered. In some embodiments, the ratio of one variant of the target protein to another variant of the target protein is not altered. In some embodiments, the antisense oligomer increases the amount of functional protein (i.e., a protein with GAA activity) or RNA translated into functional protein. In some embodiments, the antisense oligomer does not increase the amount of functional protein (i.e., a protein with GAA activity) or RNA translated into functional protein.

[0227] In some embodiments, the amount of functional protein (i.e., protein with GAA activity) or the total amount of RNA translated into functional protein produced in cells contacted with a splice regulator is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, ...1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 3 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 3 to about 10-fold, about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold increase.

[0228] In some embodiments, the amount of functional protein (i.e., protein with GAA activity) or the total amount of RNA translated into functional protein produced in cells contacted with a splice regulator is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, ...1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 3 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 3 to about 10-fold, about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold increase.

[0229] In some embodiments, the amount of functional protein (i.e., protein with GAA activity) or the total amount of RNA translated into functional protein produced in cells contacted with a splice regulator is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, ...1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 3 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 3 to about 10-fold, about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold increase.

[0230] In some embodiments, the amount of functional protein (i.e., protein with GAA activity) or the total amount of RNA translated into functional protein produced in cells contacted with a splice regulator is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, ...1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 3 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 3 to about 10-fold, about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold increase.

[0231] The splice regulator may be delivered to the non-human animal model of Pompe disease of the present invention, or to cells, tissues, organs, or parts derived therefrom, by any means described in the literature. For example, the splice regulator may be administered to the non-human animal model via any suitable route. Cells, tissues, organs, or parts may be derived from the non-human animal model by any means described in the literature. Cells, tissues, organs, or parts derived from non-human animal models may be used as described in the literature (e.g., Parker (1961), Paul (1961), White (1963), and Merchant et al. (1964), White (1957) and Stevenson (1962), Stewart and Kirk (1954), Waymouth (1954, 1960, 1965), Hanks (1955), Biggers et al. (1957), Geyer (1958), Morgan (1958), Swim (1959), Paul (1960), Levintow and Eagle (1961), Murray and Kopech (1953), Murray and The splice regulator may be maintained and / or expanded in culture by any of the means described in Kopech (1965, 1966), Wolff (1952), Fell (1953, 1954, 1955, 1958, 1964), Gaillard (1942, 1948, 1953), Borghese (1958), Kahn (1958), Lasnitzki (1958, 1965), Trowell (1959, 1961b), and Grobstein (1962). The splice regulator may be administered (e.g., delivered) to cells, tissues, organs, or parts derived from non-human animal models by any of the means described in the literature. [Example]

[0232] Example 1: Generation of a Pompe Disease Mouse Model A LOPD knock-in mouse model was generated by inserting the human GAA gene into the Rosa26 locus of C57BL / 6 mice using CRISPR / Cas9 genome engineering (Cyagen Biosciences). First, a BAC clone containing human GAA (BAC clone RP11-75G22) was selected, and the IVS1 mutation was introduced using a positive / negative homologous recombination selection scheme. A synthetic fragment containing a zeomycin resistance cassette and a homologous arm containing the IVS1 mutation were introduced into the BAC containing the human GAA gene via homologous recombination. The zeomycin cassette was then removed by negative homologous recombination using a synthetic sequence that did not contain the zeomycin cassette. The modified gene was subcloned to introduce a CAG promoter and PolyA tail (rGB pA) into the PUC57 vector using the seamless cloning enzyme C115. The entire CAG-(IVS1)hGAA-polyA insert was sequenced and cloned in the reverse orientation into intron 1 of the Rosa26 locus (Figure 1). Correct insertion of the insert into the cloning vector was verified by restriction enzyme digestion (Figures 2 and 3). The Rosa26 homology arms were amplified by PCR, and the resulting targeting vector (PCR amplicon) along with Cas9 (polypeptide) and sgRNA (synthetic RNA) was co-injected into fertilized eggs. One-cell zygotes were obtained by mating C57BL / 6N males (Charles River, China) with C57BL / 6N females (Charles River, China) that had been superovulated by injection of pregnant male serum gonadotropin and human chorionic gonadotropin.

[0233] Knock-in mice were first genotyped for the presence of human GAA in the Rosa26 locus (F1 mice, Figure 4A-B) and then for the presence of the targeting vector to assess for potential random integration (F0 mice, Figure 5A-B). Mice determined to contain human GAA but not the targeting vector were confirmed by Sanger sequencing to carry the GAA IVS1 mutation and the correct Rosa26 insertion (Figure 6A-C) and then further bred to establish germline transmission. One F0 founder line with germline transmission of the human GAA insertion in the Rosa26 locus was identified.

[0234] Genotyping PCR conditions were as described in Table 1.

[0235] [Table 1]

[0236] Example 2: Determining the genomic copy number of the human GAA gene in a mouse model of Pompe disease 10–20 mg of fresh LOPD mouse tail or ear tissue was mechanically homogenized with a metal bead beater using Quick-DNA 96 Kit (Zymo Research) lysis buffer. Total DNA was isolated according to the kit's protocol and diluted 5–10-fold before use. Genotyping Master Mix (Thermo Fisher, Quantstudio 7 Pro) was used to amplify 1 μL of DNA in a PCR reaction containing an assay for human GAA detection on the VIC channel (Hs03961696_cn, Thermo Fisher) and mouse Tfrc on the VIC channel (Thermo Fisher). A relative standard curve for the double-stranded qPCR reaction was generated using a 1:1 mixture of mouse gDNA (Promega) and human gDNA (Promega). The relative amount of human GAA was calculated for each sample and normalized to Tfrc (Figure 7A). Multiplex qPCR analysis performed on genomic DNA from three F2 mice showed the insertion of one single copy of GAA into the genome.

[0237] Example 3: GAA TV expression analysis in a Pompe disease mouse model To determine the relative expression levels of the three GAA alternative splice forms (TV1, TV2, and TV3), qPCR analysis was performed on genomic DNA from F2 mice. cDNA from LOPD mouse quadriceps was amplified using GAA TV1, TV2, or TV3 primers with PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) on a Bio-Rad PCR thermocycler (Bio-Rad) according to the manufacturer's protocol. The relative abundance of each transcript variant was normalized to mouse Hprt (Mm.PT.39a.22214828, Integrated DNA Technologies) (Figure 7B).

[0238] Example 4: GAA expression analysis in a Pompe disease mouse model 10–20 mg of flash-frozen LOPD mouse tissue was mechanically homogenized with a metal bead beater, and RNA was extracted using the Chemagic 360 RNA System (Perkin-Elmer). RNA (0.5–1.0 μg) was reverse-transcribed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen) according to the manufacturer's protocol. Multiplex qPCR assays measuring GAA expression at the exon 1–2 locus (Hs00164635_m1, Thermo Fisher) on the FAM channel, GAA expression at the exon 6–7 locus (AR9HMGG, Thermo Fisher) on the VIC channel, and Hprt expression at the JUN channel (Mm03024075m1_qsy, Thermo Fisher) were performed on a Quantstudio 7 Pro PCR thermocycler (Thermo Fisher) with Multiplex Master Mix (Thermo Fisher). Analysis was performed on genomic DNA from F2 mice to determine the relative expression levels of the human and mouse GAA genes in different tissues (Figure 8A). Analysis was also performed on genomic cDNA derived from RNA extracted from the quadriceps muscles of three F2 mice to determine the relative amounts of correctly spliced ​​GAA RNA (exon 1-2 junction) and total GAA RNA (exon 6-7 junction). Results are consistent with missplicing at the exon 1-2 junction (Figure 8B). cDNA from the quadriceps muscles of LOPD mice was amplified by PCR. GAA exons 1-5 were amplified using primers GAA Ex1-5 Fwd and GAA Ex1-5 Rev with LA Taq polymerase and GC buffer (TaKaRa) in a Bio-Rad PCR thermocycler according to the manufacturer's protocol (PCR thermocycling conditions were 95°C for 5 min, (95°C for 30 s, 60°C for 30 s, 72°C for 2 min) × 44 cycles). PCR amplification products were visualized using a 2.2% agarose gel (Flashgel System, Lonza) (Fig. 9).End-point RT-PCR targeting exons 1-5 performed on RNA extracted from LOPD mice showed a missplicing pattern similar to that seen in LOPD patient cells.

[0239] Example 5: GAA splice variant analysis in a Pompe disease mouse model cDNA from the quadriceps muscle of LOPD mice was amplified by PCR. Exons 1 to 5 of human GAA were PCR-amplified using GAA Ex1-5 Fwd and Rev primers. PCR thermocycling conditions were 95°C for 5 minutes, followed by 44 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 2 minutes, using Takara LA Taq DNA polymerase with GC buffer II. Endpoint PCR reactions were purified using a QIAquick PCR Purification Kit (Qiagen), and the DNA concentration was normalized to 20 ng / µL. The mixture was subjected to Amplicon-EZ MiSeq 2x150bp sequencing (Azenta). More than 600,000 unfragmented primary reads (>75 bp) per sample were generated, which were mapped to human GAA using spliced ​​transcript alignments to the reference (STAR) alignment (GitHub). Junctions with over 6,000 exon-spanning reads were visualized using the Integrated Genome Viewer (Broad Institute). Total RNA-seq was performed from flash-frozen LOPD mouse quadriceps muscle tissue (Azenta). The resulting total reads of >100M were similarly mapped and visualized. MiSeq analysis of RT-PCR-amplified pools of exons 1–5 revealed several major GAA splice variants in LOPD mouse quadriceps muscle (Figure 10A). We observed similarities between the major GAA splice variants observed in LOPD mouse quadriceps muscle and those observed in LOPD patient cells (Figure 10B). All listed splice variants (SVs) are deleterious and are caused by IVS1 mutations. The only correctly spliced ​​transcript is the N (normal) variant. Each transcript variant appears to be affected by IVS1 mutations in the same manner.

[0240] Example 6: Pompe disease mouse model (GAA) LOPD(IVS1)+ / - Gaa + / + ) and the acid alpha-glucosidase mouse model (Gaa - / - ) comparison GAA, produced as described in Example 1LOPD(IVS1)+ / - Gaa + / + Mice were aged for 10 months with strain-matched wild-type animals (i.e., C57BL / 6N strain). - / - Mice (JAX 004154) were aged for 3 months. LOPD(IVS1)+ / - Gaa + / + , Gaa - / - GAA and wild-type animals were euthanized, and the quadriceps muscles were dissected and fixed in 10% neutral buffered formalin (NBF) solution. The fixed tissue (quadriceps) was sectioned and stained with hematoxylin and eosin (H&E) to visualize cellular and histological structures (i.e., histocellular analysis). Additionally, diaphragms were dissected from euthanized mice and fixed in 10% neutral buffered formalin (NBF) solution. The fixed tissue (diaphragm) was sectioned and stained with periodic acid-Schiff (PAS) to visualize lysosomes. The stained sections (H&E and PAS) were analyzed for cellular and histological structures and examined for the presence of enlarged lysosomes. LOPD(IVS1)+ / - Gaa + / + Quadriceps and diaphragm tissues dissected from Gaa mice (Figure 11B and Figure 11E) were comparable to the same tissues dissected from wild-type mice (Figure 11C and Figure 11F) and showed no signs of chronic inflammation, enlarged lysosomes, or myocyte degeneration. - / - Quadriceps and diaphragm tissues dissected from rats (Figures 11A and 11D) showed features of chronic inflammation, enlarged lysosomes, and myocyte degeneration. These results support the conclusion that GAA LOPD(IVS1)+ / - Gaa + / + This is consistent with the presence of the Gaa gene in the mouse model genome.

[0241] Example 7: Assaying splice regulators (GAA) in a Pompe disease mouse model LOPD(IVS1)+ / - Gaa + / + ). GAA LOPD(IVS1)+ / - Gaa + / +LOPD transgenic mice were injected with a single intravenous (IV) injection of PPMO (peptide-conjugated phosphorodiamidate morpholino) compounds targeting the IVS1-189 region (PPMO1-3, Table 2) or a single intravenous injection of PPMO compounds targeting the IVS1-69 region (PPMO4-5, Table 2). Control animals were injected with a single intravenous injection of a non-targeting PPMO compound (NTC, PPMO6, Table 2) or a single intravenous injection of sterile saline.

[0242] [Table 2]

[0243] After 7 days, PPMO1-injected and control mice were euthanized, and their quadriceps muscles were excised and snap-frozen in liquid nitrogen. 10–20 mg of snap-frozen LOPD mouse tissue was mechanically homogenized with a metal bead beater, and RNA was extracted using a Chemagic 360 RNA system (Perkin Elmer Chemagic). RNA (0.5–1.0 μg) was reverse-transcribed using the SuperScript VILO kit (Thermo Fisher) according to the manufacturer's protocol and subjected to PCR amplification of GAA exons 1–5. Amplicon sequencing of the amplified product pool was performed (Azenta), and splice junctions were analyzed as described in Example 5. The results demonstrate that the PPMO1 compound corrected LOPD splicing in vivo. Amplicon sequencing of PCR-amplified GAA exons 1–5 demonstrated a qualitative improvement in correctly spliced ​​GAA exons 1–2 after a single IV administration of the PPMO1 compound. Furthermore, PPMO1 treatment did not result in the production of any new splice junctions (FIG. 12).

[0244] The amplicon pools prepared as described above were separated by capillary electrophoresis (Perkin Elmer LabChip), and bands corresponding to mis-spliced ​​and correctly spliced ​​GAA were identified and quantified. PPMO compounds increased the amount of correctly spliced ​​GAA in vivo (Figure 13A) and restored all known GAA splice variants (Figure 13B-C).

[0245] A dose-response assay of PPMO compounds was performed. PPMO1-6 compounds were administered at 30 or 100 mg / kg to GAA. LOPD(IVS1)+ / - Gaa + / + Animals were injected IV (n=6). Control animals were injected with the same dose of NTC or sterile saline. Tissues were collected 7 days later, and quadriceps RNA was examined for GAA expression by qPCR as described in Example 4. All tested PPMO compounds increased the amount of correctly spliced ​​GAA in vivo (FIG. 14A). Furthermore, a dose-dependent increase in GAA was observed in the quadriceps muscle after administration of various doses of PPMO2 (FIG. 14B).

[0246] Example 8: Fully humanized LOPD animals (GAA LOPD(IVS1)+ / - Gaa - / - ) generation Fully humanized LOPD animals (GAA LOPD(IVS1)+ / - Gaa - / - ) to the GAA described in Example 1 LOPD(IVS1)+ / - Gaa + / + Animals, Gaa - / - (JAX 004154) It was produced by crossing with animals.

[0247] Genotyping PCR conditions were as described in Table 3.

[0248] [Table 3-1] [Table 3-2]

[0249] The resulting fully humanized GAA LOPD(IVS1)+ / - Gaa - / - The animals allow for protein expression analysis, for example, by Western blot, since the absence of the Gaa mouse gene avoids the issue of potential cross-reactivity of anti-GAA antibodies between mouse and human GAA proteins. LOPD(IVS1)+ / - Gaa - / - Animals were injected (IV) with PPMO2 compounds, and GAA protein was analyzed in the quadriceps muscle 7 days later by Western blot (ProteinSimple® Jes™ system) using a recombinant anti-GAA antibody (Abcam ab137068) and normalized to total protein. PPMO2 compounds increased GAA protein (primary translation) after a single intravenous dose of 150 mg / kg. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11]

Table 4-12

Table 4-13

Table 4-14

Table 4-15

Table 4-16

Table 4-17

Table 4-18

Table 4-19

Table 4-20

Table 4-21

Table 4-22

Table 4-23

Table 4-24

Table 4-25

Table 4-26

Table 4-27

Table 4-28

Table 4-29

Table 4-30

Table 4-31

Table 4-32

Table 4-33

Table 4-34

Table 4-35

Table 4-36

Table 4-37

Claims

1. 1. A transgenic non-human animal model comprising a nucleic acid sequence of an acid alpha-glucosidase (GAA) gene, or a fragment thereof, comprising a mutation, wherein the mutation causes defective splicing of a pre-mRNA transcribed from the nucleic acid sequence, and wherein the nucleic acid sequence comprising the mutation would encode a polypeptide having GAA activity if the nucleic acid sequence did not comprise the mutation.

2. The transgenic non-human animal model according to claim 1, wherein the mutation is a T-G mutation.

3. The transgenic non-human animal model according to claim 1 or 2, wherein the mutation is an IVS1-13T-G mutation.

4. 4. The transgenic non-human animal model of any one of claims 1 to 3, wherein the GAA gene, or a fragment thereof, containing the mutation, is transcribed into pre-mRNA.

5. 5. The transgenic non-human animal model of claim 4, wherein the pre-mRNA transcribed from the GAA gene, or a fragment thereof, containing the mutation, is processed by splicing into a mature mRNA.

6. 6. The transgenic non-human animal model of claim 5, wherein the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or a fragment thereof, containing the mutation differs from the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or a fragment thereof, that does not contain the mutation.

7. The transgenic non-human animal model of claim 6, wherein the mutation weakens the splice acceptor of GAA exon 2.

8. The transgenic non-human animal model of claim 7 , wherein the mutation results in skipping of exon 2.

9. 9. The transgenic non-human animal model of claim 8, wherein the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or a fragment thereof, containing the mutation does not contain exon 2.

10. 10. The transgenic non-human animal model of any one of claims 5 to 9, wherein the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or a fragment thereof, containing the mutation, is translated into a polypeptide having reduced GAA activity compared to a polypeptide translated from the mature mRNA transcribed from the GAA gene, or a fragment thereof, that does not contain the mutation.

11. 10. The transgenic non-human animal model of any one of claims 5 to 9, wherein the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or a fragment thereof, containing the mutation, is translated into a polypeptide that does not have GAA activity.

12. The transgenic non-human animal model according to any one of claims 1 to 11, wherein the non-human animal model is a model of Pompe disease.

13. The transgenic non-human animal model of claim 12, wherein the non-human animal model is a model of late-onset Pompe disease.

14. 14. The transgenic non-human animal model of any one of claims 1-13, wherein the nucleic acid sequence of the GAA gene, or a fragment thereof, comprising the mutation comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 or 2.

15. 15. The transgenic non-human animal model of any one of claims 1 to 14, wherein the nucleic acid sequence of the GAA gene, or a fragment thereof, comprising the mutation, is inserted into the Rosa26 locus.

16. 15. The transgenic non-human animal model of any one of claims 1 to 14, wherein the nucleic acid sequence of the GAA gene, or a fragment thereof, comprising the mutation, is inserted into the endogenous GAA locus.

17. 17. The transgenic non-human animal model of any one of claims 1 to 16, wherein the nucleic acid sequence of the GAA gene, or a fragment thereof, comprising the mutation, is operably linked to a heterologous promoter.

18. 18. The transgenic non-human animal model according to any one of claims 1 to 17, wherein the heterologous promoter is selected from the group consisting of CMV early enhancer / chicken beta actin (CBA) promoter, CAG promoter, CMV, EF1α, EF1α with CMV enhancer, CMV promoter with CMV enhancer (CMVe / p), and CMV promoter with SV40 intron.

19. The transgenic non-human animal model of claim 18, wherein the heterologous promoter is a CAG promoter.

20. 20. The transgenic non-human animal model of claim 18, wherein the heterologous promoter comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

3.

21. 21. The transgenic non-human animal model of any one of claims 1 to 20, wherein the nucleic acid sequence of the GAA gene, or a fragment thereof, comprising the mutation is operably linked to a heterologous polyadenylation signal.

22. The transgenic non-human animal model of claim 21, wherein the heterologous polyadenylation signal is the rGB-pA polyadenylation signal.

23. 23. The transgenic non-human animal model of claim 21, wherein the polyadenylation signal comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

4.

24. The transgenic non-human animal model according to any one of claims 1 to 23, wherein the transgenic non-human animal model is generated by an RNA-guided CRISPR-Cas nuclease system.

25. The transgenic non-human animal model according to any one of claims 1 to 24, wherein the non-human animal model is a mouse.

26. 26. The transgenic non-human animal model of claim 25, wherein the mouse is a C57BL / 6 mouse.

27. 1. A recombinant nucleic acid molecule comprising a nucleic acid sequence of an acid alpha-glucosidase (GAA) gene, or a fragment thereof, comprising a 5' homology arm, a polyadenylation signal, and a mutation that causes defective splicing of a pre-mRNA transcribed from the nucleic acid sequence, wherein the nucleic acid sequence comprising the mutation would encode a polypeptide having GAA activity if the nucleic acid sequence did not comprise the mutation, promoter, and 3' homology arm.

28. 28. The recombinant nucleic acid molecule of claim 27, further comprising a Neo (neomycin) resistance gene and an Amp (ampicillin) resistance gene.

29. 29. The recombinant nucleic acid molecule of claim 27 or 28, wherein the nucleic acid sequence of an acid alpha-glucosidase (GAA) gene, or a fragment thereof, comprising the mutation comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 or 2.

30. 30. The recombinant nucleic acid molecule of any one of claims 27 to 29, wherein the promoter is selected from the group consisting of CMV early enhancer / chicken beta actin (CBA) promoter, CAG promoter, CMV, EF1α, EF1α with CMV enhancer, CMV promoter with CMV enhancer (CMVe / p), and CMV promoter with SV40 intron.

31. 31. The recombinant nucleic acid molecule of claim 30, wherein the promoter is a CAG promoter.

32. 31. The recombinant nucleic acid molecule of claim 29 or 30, wherein the promoter comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

3.

33. 33. The recombinant nucleic acid molecule of any one of claims 27 to 32, wherein the polyadenylation signal is the rGB-pA polyadenylation signal.

34. 34. The recombinant nucleic acid molecule of any one of claims 27 to 33, wherein the polyadenylation signal comprises a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

4.

35. 35. The recombinant nucleic acid molecule of any one of claims 27 to 34, wherein the homologous arms comprise a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a region of the Rosa26 locus in the mouse genome.

36. 35. The recombinant nucleic acid molecule of any one of claims 27-34, wherein the homologous arms comprise a nucleic acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a region of the GAA locus in the mouse genome.

37. A method for producing a transgenic mouse, comprising delivering to a cell a recombinant nucleic acid molecule according to any one of claims 27 to 36.

38. 38. The method of claim 37, wherein the cell is a mouse embryonic stem cell or a one-cell mouse embryo.

39. 39. The method of any one of Claims 37 or 38, further comprising delivering an sgRNA and a Cas9 nuclease to the cell.

40. 40. The method of Claim 39, wherein the delivered sgRNA targets a locus in the mouse cell genome.

41. 42. The method of any one of claims 37 to 41, wherein the nucleic acid sequence of an acid alpha-glucosidase (GAA) gene, or a fragment thereof, comprising the polyadenylation signal, the mutation, and the promoter are stably integrated into a locus in the mouse genome.

42. 42. The method of any one of claims 38 to 41, wherein the nucleic acid sequence of an acid alpha-glucosidase (GAA) gene, or a fragment thereof, comprising the polyadenylation signal, the mutation, the promoter, the Neo (neomycin) resistance gene, and the Amp (ampicillin) resistance gene are stably integrated into a locus in the mouse genome.

43. 43. The method of any one of claims 40 to 42, wherein the locus is the Rosa26 locus.

44. 43. The method of any one of claims 40 to 42, wherein the locus is the GAA locus.

45. 27. A method for testing a splice modulator, comprising: (a) administering a splice modulator to a transgenic non-human animal model according to any one of claims 6 to 26; (b) obtaining a test sample from the non-human animal model; and (c) assaying for the presence of (i) mature mRNA derived from pre-mRNA transcribed from the GAA gene, or a fragment thereof, comprising the mutation, and / or (ii) mature mRNA derived from pre-mRNA transcribed from the GAA gene, or a fragment thereof, not comprising the mutation.

46. 46. ​​The method of claim 45, wherein the splice regulator is a small molecule.

47. 46. ​​The method of claim 45, wherein the splice regulator is an antisense oligonucleotide.

48. 48. The method of any one of claims 45 to 47, wherein the splice regulator is administered to the transgenic non-human animal model.

49. 49. The method of any one of claims 45 to 48, wherein the splice regulator is administered to a cell, tissue, or organ derived from the transgenic non-human animal model.

50. 50. The method of any one of claims 45-49, wherein the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or fragment thereof, containing the mutation, does not differ from the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or fragment thereof, not containing the mutation, after administration of the splice modulator.

51. 51. The method of any one of claims 45-50, wherein the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or a fragment thereof, containing the mutation, comprises exon 2 after administration of the splice modulator.

52. 52. The method of any one of claims 45-51, wherein the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or a fragment thereof, containing the mutation, is translated into a polypeptide having the GAA activity of a polypeptide translated from the mature mRNA transcribed from the GAA gene, or a fragment thereof, not containing the mutation, after administration of the splice regulator.

53. 50. The method of any one of claims 45-49, wherein the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or fragment thereof, containing the mutation, differs from the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or fragment thereof, not containing the mutation, after administration of the splice modulator.

54. 54. The method of any one of claims 45-49, or 53, wherein the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or a fragment thereof, containing the mutation, does not contain exon 2 after administration of the splice modulator.

55. 55. The method of any one of claims 45-49, 53, or 54, wherein the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or fragment thereof, containing the mutation, is translated into a polypeptide having reduced GAA activity after administration of the splice modulator, compared to a polypeptide translated from the mature mRNA transcribed from a GAA gene, or fragment thereof, that does not contain the mutation.

56. 56. The method of any one of claims 45 to 55, comprising extracting the mRNA from the cells, tissues or organs derived from the transgenic non-human animal model before administration of the splice regulator agent and after administration of the splice regulator agent.

57. 57. The method of claim 56, comprising reverse transcribing the extracted mRNA into cDNA.

58. 58. The method of claim 57, wherein the cDNA is amplified by PCR comprising a first pair of primers capable of amplifying an exon junction unaffected by the mutation and a second pair of primers capable of amplifying an exon junction affected by the mutation.

59. 59. The method of any one of claims 57 or 58, wherein the cDNA is amplified by PCR comprising a pair of primers capable of amplifying cDNA derived from an mRNA that contains an exon junction that is not affected by the mutation and cDNA derived from an mRNA that contains an exon junction that is affected by the mutation.

60. 60. The method of any one of claims 58 or 59, wherein the primers comprise a sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 11-12.

61. The transgenic non-human animal model of any one of claims 1 to 26, wherein the acid alpha-glucosidase (GAA) gene is a human acid alpha-glucosidase (GAA) gene.

62. 27. The transgenic non-human animal model of any one of claims 1 to 26, wherein at least one copy of an acid alpha-glucosidase gene endogenous to said non-human animal model is present in the genome of said transgenic non-human animal model.

63. 63. The transgenic non-human animal model of claim 62, wherein all copies of the acid alpha-glucosidase gene endogenous to the non-human animal model are present in the genome of the transgenic non-human animal model.

64. 27. The transgenic non-human animal model of any one of claims 1 to 26, wherein at least one copy of an acid alpha-glucosidase gene endogenous to said non-human animal model is absent from the genome of said transgenic non-human animal model.

65. 65. The transgenic non-human animal model of claim 64, wherein all copies of the acid alpha-glucosidase gene endogenous to the non-human animal model are absent from the genome of the transgenic non-human animal model.

66. 37. The recombinant nucleic acid molecule of any one of claims 27 to 36, wherein the acid alpha-glucosidase (GAA) gene is a human acid alpha-glucosidase (GAA) gene.

67. 45. The method of any one of claims 37 to 44, wherein the cell comprises at least one copy of an acid alpha-glucosidase gene endogenous to the cell genome.

68. 68. The method of claim 67, wherein the cell comprises all copies of the acid alpha-glucosidase gene endogenous to the cell genome.

69. 45. The method of any one of claims 37 to 44, wherein the cell lacks at least one copy of an acid alpha-glucosidase gene endogenous to the cell genome.

70. 70. The method of claim 69, wherein the cell lacks all copies of the acid alpha-glucosidase gene endogenous to the cell genome.

71. 69. A method of generating a transgenic mouse, comprising mating a first transgenic mouse generated by the method of any one of claims 67-68 with a second transgenic mouse that lacks all copies of the mouse GAA gene.

72. A method for testing a splice regulator, comprising: (a) administering a splice regulator to the transgenic non-human animal model of claim 65; (b) obtaining a test sample from the non-human animal model; and (c) assaying for the presence of (i) a protein product translated from a mature mRNA derived from a pre-mRNA transcribed from the GAA gene, or a fragment thereof, containing the mutation, and / or (ii) a protein product translated from a mature mRNA derived from a pre-mRNA transcribed from the GAA gene, or a fragment thereof, not containing the mutation.

73. 73. The method of claim 72, wherein the splice regulator is a small molecule.

74. 74. The method of claim 73, wherein the splice regulator is an antisense oligonucleotide.

75. 75. The method of any one of claims 72 to 74, wherein the splice regulator is administered to the transgenic non-human animal model.

76. 76. The method of any one of claims 72 to 75, wherein the splice regulator is administered to a cell, tissue, or organ derived from the transgenic non-human animal model.

77. 77. The method of any one of claims 72-76, wherein the protein product translated from a mature mRNA derived from a pre-mRNA transcribed from the GAA gene, or a fragment thereof, containing the mutation, does not differ from the protein product translated from a mature mRNA derived from a pre-mRNA transcribed from a GAA gene, or a fragment thereof, that does not contain the mutation, after administration of the splice modulator.

78. 78. The method of any one of claims 72-77, wherein the protein product translated from mature mRNA derived from pre-mRNA transcribed from the GAA gene, or fragment thereof, containing the mutation, comprises an amino acid sequence encoded by exon 2 after administration of the splice modulator.

79. 79. The method of any one of claims 72-78, wherein the protein product translated from a mature mRNA derived from a pre-mRNA transcribed from the GAA gene, or a fragment thereof, containing the mutation, has the GAA activity of a polypeptide translated from a mature mRNA transcribed from a GAA gene, or a fragment thereof, that does not contain the mutation, after administration of the splice modulator.

80. 77. The method of any one of claims 72-76, wherein the protein product translated from a mature mRNA derived from a pre-mRNA transcribed from the GAA gene, or a fragment thereof, containing the mutation, differs from the protein product translated from a mature mRNA derived from a pre-mRNA transcribed from a GAA gene, or a fragment thereof, that does not contain the mutation, after administration of the splice modulator.

81. 81. The method of any one of claims 72-76, or 80, wherein the protein product translated from mature mRNA derived from pre-mRNA transcribed from the GAA gene, or fragment thereof, containing the mutation, does not contain the amino acid sequence encoded by exon 2 after administration of the splice modulator.

82. 82. The method of any one of claims 72-76, 80, or 81, wherein the protein product translated from a mature mRNA derived from a pre-mRNA transcribed from the GAA gene, or a fragment thereof, containing the mutation, has reduced GAA activity compared to a polypeptide translated from a mature mRNA transcribed from a GAA gene, or a fragment thereof, that does not contain the mutation, after administration of the splice modulator.

83. 83. The method of any one of claims 72 to 82, comprising extracting protein content from said cells, tissues or organs derived from said transgenic non-human animal model before administration of said splice regulator agent and after administration of said splice regulator agent.

84. 84. The method of claim 83, wherein the protein content is analyzed by Western blot assay.

85. 85. The method of claim 84, wherein the Western blot assay comprises an anti-GAA antibody.

Images