Search

US-20260123610-A1 - POMPE DISEASE MOUSE MODEL GENERATION, CHARACTERIZATION AND METHODS OF USE

US20260123610A1US 20260123610 A1US20260123610 A1US 20260123610A1US-20260123610-A1

Abstract

Disclosed herein are transgenic non-human animal models of Pompe disease, methods of making, and methods of using the same. Disclosed herein are also nucleic acid molecules useful for making the non-human animal models of Pompe disease.

Inventors

  • Ryan Oliver
  • Kevin Kim

Assignees

  • SAREPTA THERAPEUTICS, INC.

Dates

Publication Date
20260507
Application Date
20230926

Claims (20)

  1. 1 . A transgenic non-human animal model, comprising a nucleic acid sequence of an acid alpha-glucosidase (GAA) gene, or fragment thereof, comprising a mutation, wherein the mutation causes a defective splicing of the pre-mRNA transcribed from the nucleic acid sequence, and wherein the nucleic acid sequence comprising the mutation would have encoded a polypeptide having a GAA activity, if the nucleic acid sequence would have not comprised the mutation.
  2. 2 . The transgenic non-human animal model of claim 1 , wherein the mutation is a T-G mutation.
  3. 3 . The transgenic non-human animal model of any one of 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 1-3 , wherein the GAA gene, or fragment thereof, comprising 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 fragment thereof, comprising the mutation is processed by splicing into 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 fragment thereof, comprising the mutation is different from the mature mRNA derived from a pre-mRNA transcribed from a GAA gene, or fragment thereof, not comprising the mutation.
  7. 7 . The transgenic non-human animal model of claim 6 , wherein the mutation weakens the splice acceptor of GAA exon 2.
  8. 8 . The transgenic non-human animal model of claim 7 , wherein the mutation leads to 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 fragment thereof, comprising the mutation does not comprise exon 2.
  10. 10 . The transgenic non-human animal model of any one of claims 5-9 , wherein the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or fragment thereof, comprising the mutation is translated into a polypeptide having reduced GAA activity compared to a polypeptide translated from the mature mRNA transcribed from the a GAA gene, or fragment thereof, not comprising the mutation.
  11. 11 . The transgenic non-human animal model of any one of claims 5-9 , wherein the mature mRNA derived from the pre-mRNA transcribed from the GAA gene, or fragment thereof, comprising the mutation is translated into a polypeptide not having GAA activity.
  12. 12 . The transgenic non-human animal model of any one of claims 1-11 , wherein the non-human animal model is a model of Pompe disease.
  13. 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 fragment thereof, comprising the mutation comprises a nucleic acid sequence which 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: 1 or 2.
  15. 15 . The transgenic non-human animal model of any one of claims 1-14 , wherein the nucleic acid sequence of the GAA gene, or fragment thereof, comprising the mutation is inserted in a Rosa26 locus.
  16. 16 . The transgenic non-human animal model of any one of claims 1-14 , wherein the nucleic acid sequence of the GAA gene, or fragment thereof, comprising the mutation is inserted in an endogenous GAA locus.
  17. 17 . The transgenic non-human animal model of any one of claims 1-16 , wherein the nucleic acid sequence of the GAA gene, or fragment thereof, comprising the mutation is operably linked to a heterologous promoter.
  18. 18 . The transgenic non-human animal model of any one of claims 1-17 , wherein the heterologous promoter is selected from the group consisting of a CV early enhancer/chicken β actin (CBA) promoter, CAGpromoter, CMV, EF1α, EF1α with a CMV enhancer, a CMV promoter with a CMV enhancer (CMVe/p), a CMV promoter with a SV40 intron.
  19. 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 any one of claim 18 or 19 , wherein the heterologous promoter comprises a nucleic acid sequence which 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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims the priority benefit of U.S. Provisional Application No. 63/377,516 filed Sep. 28, 2022, which is incorporated by reference herein in its entirety. RELATED INFORMATION The contents of any patents, patent applications, and references cited throughout this specification are hereby incorporated by reference in their entireties. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB The content of the electronically submitted sequence listing (Name: 4140_0610001_SequenceListing_ST26.xml: Size: 122,920 bytes; and Date of Creation: Sep. 5, 2023), filed with the application, is incorporated herein by reference in its entirety. FIELD OF DISCLOSURE The present disclosure pertains to the medical field including inherited genetic disorders and diseases, more specifically to the generation and methods of use of non-human animal models for the investigation of the etiology of, and for the investigation of therapy for, inherited genetic disorders and diseases. BACKGROUND Pompe disease (glycogen storage disease type II, OMIM #232300) is an inherited autosomal metabolic disorder due to deficiency of acid alpha-glucosidase (GAA) that acts within lysosomes and is responsible of glycogen breakdown to glucose (Hirschhorn R, Reuser A J J. Glycogen storage disease type II: Acid alpha-glucosidase (acid maltase) deficiency. In: Scriver C R, Beaudet A L, Sly W S, et al. (Eds.) The Metabolic & Molecular Bases of Inherited Disease, McGraw-Hill, New York, 2001:3389-420: van der Ploeg A T. Reuser A J. 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). Glycogen accumulates in the lysosome as well as in the cytoplasm leading to tissue damage both directly and by affecting different downstream metabolic pathways including the autophagic process. Although cardiac and skeletal muscles are the main tissues involved, 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 C I, et al. Childhood Pompe disease: clinical spectrum and genotype in 31 patients. Orphanet J Rare Dis 2016; 11:65). Pompe disease is a progressive disorder and based on the age at onset, Pompe disease can manifest as a severe infantile form (IOPD), presenting with cardiac hypertrophy, respiratory dysfunction and floppiness, and as a late onset form (LOPD), which is more benign and more heterogeneous with respiratory and skeletal muscles involvement (van der Ploeg A T, Reuser A J. Pompe's disease. Lancet 2008:372:1342-53). In LOPD, the first clinical manifestation can be either proximal muscle weakness or other complaints such as exercise intolerance, muscle pain or even isolated hyperCKemia. The clinical presentations are similar to those in other hereditary or acquired muscle disorders such as, for example, limb-girdle muscular dystrophies (LGMD), other muscle glycogenosis, and inflammatory mvopathies (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). 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 may be present in heterozvgosity or homozvgosity (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 of symptoms and clinical course of Pompe disease associated with the c.-32-13T-G variant. Molec. Genet. Metab. 126: 106-116, 2019). Several animal models exist which mimic the phenotypical manifestations of Pompe disease. For example, the acid maltase-deficient Japanese quails exhibit progressive myopathy and cannot lift their wings, fly, or right themselves from the 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 whom the GAA gene was disrupted by gene targeting in embryonic stem cells, homozygosity for the knock-out was associated with lack of enzyme activity and accumulation of glycogen in cardiac and skeletal muscle lvsosomes by 3 weeks of age, with a progressive i