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US-20260125663-A1 - POLYNUCLEOTIDES

US20260125663A1US 20260125663 A1US20260125663 A1US 20260125663A1US-20260125663-A1

Abstract

The present invention relates to polynucleotides comprising a GBA nucleotide sequence that encodes a GCase protein or fragment thereof and wherein a portion of the coding sequence is not wild type. The present invention further relates to viral particles comprising a recombinant genome comprising the polynucleotide of the invention, compositions comprising the polynucleotides or viral particles, and methods and uses of the polynucleotides, viral particles or compositions.

Inventors

  • Amit Nathwani
  • Jenny McIntosh
  • Romuald CORBAU
  • Azadeh KIA
  • Carlos Miranda

Assignees

  • SPUR THERAPEUTICS LIMITED

Dates

Publication Date
20260507
Application Date
20250522
Priority Date
20190204

Claims (20)

  1. 1 .- 34 . (canceled)
  2. 35 . A non-wild type polynucleotide comprising a beta-glucocerebrosidase (GBA) nucleotide sequence, wherein the GBA nucleotide sequence encodes a β-Glucocerebrosidase (GCase) protein having GCase activity, wherein the GBA nucleotide sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 5, and wherein the GCase protein encoded by the GBA nucleotide sequence expresses in human liver cells at higher levels compared to a GCase encoded by a wild type GBA nucleotide sequence.
  3. 36 . The non-wild type polynucleotide of claim 35 , wherein the GBA nucleotide sequence is codon optimized for expression in human liver cells.
  4. 37 . The non-wild type polynucleotide of claim 35 , wherein the GBA nucleotide sequence comprises a reduced number of CpGs compared to a wild type GBA nucleotide sequence.
  5. 38 . The non-wild type polynucleotide of claim 35 , wherein the non-wild type polynucleotide further comprises a transcription regulatory element.
  6. 39 . The non-wild type polynucleotide of claim 35 , wherein the transcription regulatory element comprises a liver-specific promoter and/or an enhancer.
  7. 40 . A viral particle comprising a recombinant genome comprising the non-wild type polynucleotide of claim 35 .
  8. 41 . The viral particle of claim 40 , wherein the viral particle is an AAV, adenoviral, or lentiviral particle.
  9. 42 . The viral particle of claim 40 , wherein the viral particle comprises a liver-tropic or CNS-tropic capsid.
  10. 43 . A pharmaceutical composition comprising the non-wild type polynucleotide of claim 35 and a pharmaceutically acceptable excipient.
  11. 44 . A pharmaceutical composition comprising the viral particle of claim 40 and a pharmaceutically acceptable excipient.
  12. 45 . A method of treating a disease associated with GCase deficiency, the method comprising administering an effective amount of the polynucleotide according to claim 35 to a subject.
  13. 46 . The method of claim 45 , wherein the GCase deficiency diseases is Parkinson's disease or a synucleopathy, or a lysosomal storage disorder such as Gaucher disease.
  14. 47 . The method of claim 46 , wherein Gaucher disease is Gaucher disease type I, II, or III.
  15. 48 . A method of achieving a stable GCase activity in a subject, the method comprising expressing the GBA nucleotide sequence according to claim 35 in the subject.
  16. 49 . A non-wild type polynucleotide comprising a beta-glucocerebrosidase (GBA) nucleotide sequence, wherein the GBA nucleotide sequence encodes a β-Glucocerebrosidase (GCase) protein having GCase activity, wherein the polynucleotide comprises a variant of SEQ ID NO: 5, wherein the variant is identical to SEQ ID NO: 5 except that it comprises nucleotide substitutions such that the GCase protein has 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 amino acid substitutions relative to the wild type GCase amino acid sequence of SEQ ID NO: 25.
  17. 50 . A viral particle comprising a recombinant genome comprising the non-wild type polynucleotide of claim 49 .
  18. 51 . The viral particle of claim 50 , wherein the viral particle is an AAV, adenoviral, or lentiviral particle.
  19. 52 . The viral particle of claim 50 , wherein the viral particle comprises a liver-tropic or CNS-tropic capsid.
  20. 53 . A pharmaceutical composition comprising the non-wild type polynucleotide of claim 49 and a pharmaceutically acceptable excipient.

Description

REFERENCE TO A SEQUENCE LISTING XML This application contains a Sequence Listing which has been submitted electronically in XML format. The Sequence Listing XML is incorporated herein by reference. Said XML file, created on Sep. 5, 2025, is named 2967278-000009-US2_SL.xml and is 43,967 bytes in size. FIELD OF THE INVENTION The present invention relates to polynucleotides comprising a GBA nucleotide sequence encoding β-Glucocerebrosidase (GCase), viral particles comprising the polynucleotides and treatments utilising the polynucleotides. CROSS-REFERENCE TO RELATED APPLICATIONS The present application is a divisional of U.S. application Ser. No. 17/428,344, filed on Aug. 4, 2021, which is a National Stage Entry of International Application PCT/GB2020/050251, filed Feb. 4, 2020, which claims the priority to and the benefit of GB Patent Application No. 1901512.2, filed Feb. 4, 2019, and GB Patent Application No. 1917910.0, filed Dec. 6, 2019, the contents of each of which are incorporated herein by reference in their entireties. BACKGROUND TO THE INVENTION Gaucher disease (GD) is an autosomal recessive lipid storage disease characterised by the deposition of glucocerebroside in cells of the macrophage-monocyte system. GD is caused by mutations in the housekeeping GBA gene that impairs activity and/or production of the enzyme β-Glucocerebrosidase (GCase). There are three major types of GD which are characterised by the specific mutations which have been identified, and each type can display differing clinical symptoms. Type 1 GD has little or no involvement with the central nervous system but is mainly characterised by visceral manifestations such as enlarged spleen and liver, low blood cell counts, bleeding problems and bone disease. For the past 20 years, enzyme replacement therapy has emerged as the standard of care for type 1 GD. In addition to its high cost (˜$200,000 or ˜£150,000/patient/year), enzyme replacement therapy treatment in GD generally requires one or more injections every other week for life. This leads to a high proportion of GD patients displaying high levels of treatment burden. Accordingly, there is a need to provide an effective therapy vector for the treatment of GD, i.e. one that allows for a high level of GCase expression. The present application relates to a gene therapy approach for treating GD, involving administering a viral particle comprising a GBA polynucleotide encoding GCase. The polynucleotides and viral particles described herein can provide higher GCase expression compared to polynucleotides comprising a wild type GCase encoding polynucleotides. Such a gene therapy approach would avoid the need for frequent and lifelong intravenous injections of GCase. SUMMARY OF THE INVENTION The present application demonstrates that specific modifications to a GBA nucleotide sequence encoding for GCase can help to improve the expression level and the activity of the expressed GCase polypeptide in vitro and/or in vivo. For example, the present application demonstrates that using a codon-optimised GBA nucleotide sequence can improve the expression and/or activity of the encoded GCase protein. Such modified (i.e. non wild-type) and/or codon-optimised GBA nucleotide sequences may be further modified to provide further improvements in the expression and/or activity of the encoded GCase protein. Further modifications may include providing further modifications in the GBA nucleotide sequence such as the removal of CpG motifs, and/or the use of particular gene regulatory elements comprising specific promoter and/or enhancer sequences. It is believed that such improvements to a GBA nucleotide sequence can improve the efficacy of such a nucleotide in the treatment of GD. These modifications provide a GBA nucleotide sequence which is expressed highly, for example in the liver, and which encodes a GCase polypeptide or fragment thereof. As demonstrated in the Examples, the polynucleotides of the invention express GCase activity at higher levels than wild type GBA. Accordingly, in a first aspect of the invention, there is provided a polynucleotide comprising a GBA nucleotide sequence, wherein the GBA nucleotide sequence encodes a β-Glucocerebrosidase (GCase) protein or fragment thereof and wherein at least a portion of the GBA nucleotide sequence is not wild type. In a second aspect of the invention, there is provided a polynucleotide comprising a GBA nucleotide sequence, wherein the GBA nucleotide sequence encodes a GCase protein or a fragment thereof and comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 1000, at least 1200, at least 1300, less than 1494, less than 1611, between 1000 and 1494, between 1000 and 1611, between 1300 and 1494, between 1300 and 1611, or around 1494 nucleotides of SEQ ID NO: 1-8. In a third aspect of the invention, there is provided a viral particle com