Regulatory elements

Abstract

The present invention relates to a regulatory element comprising a fragment of a spliceosomal intron, wherein the regulatory element comprises a branch point sequence, a polypyrimidine tract, and a 3' splice acceptor site, and wherein the regulatory element does not comprise a 5' splice donor site.

Description

W O 2 0 2 s 2 5 / 0 0 3 7 0 9 A 1 (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 02 January 2025 (02.01.2025) WO 2025 / 003709 A1 WIPO PCT (51) International Patent Classification: A61K 48 / 00 (2006.01) C12N 15 / 86 (2006.01) C12N 15 / 85 (2006.01) (21) International Application Number: PCT / GB2024 / 051695 (22) International Filing Date: 28 June 2024 (28.06.2024) (25) Filing Language: English (26) Publication Language: English 29 June 2023 (29.06.2023) GB (30) Priority Data: 2309909.6 (71) (72) Applicant: PURESPRING THERAPEUTICS LIMIT- ED [GB / GB]; 8 Bloomsbury St, 2nd Floor, London WСІВ 3SR (GB). Inventors: MADILL, Martin; c / o Purespring Therapeu- tics Limited, 8 Bloomsbury St, 2nd Floor, London WCIB 3SR (GB). GRIFFITH, Alan; c / o Purespring Therapeutics Limited, 8 Bloomsbury St, 2nd Floor, London WCIB 3SR (GB). (74) Agent: D YOUNG & CO LLP; 3 Noble Street, London EC2V 7BQ (GB). (81) (84) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, AO. AT, AU. AZ, BA, BB, BG, BH. BN. BR. BW. BY, BZ. CA, CH, CL, CN. CO, CR, CU, CV. CZ, DE, DJ. DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT. HN, HR, HU, ID, IL, IN, IQ, IR, IS, IT, JM, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, MG, MK, MN, MU, MW, MX, MY, MZ, NА. NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO. RS. RU. RW. SA, SC, SD. SE, SG. SK, SL, ST. SV. SY, TH. TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, WS, ZA, ZM, ZW. Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, CV, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SC, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, ME, MK, MT, NL, NO, PL, PT, RO, RS, SE. SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN. GQ, GW, KM, ML, MR, NE, SN, TD, TG). Published: - with international search report (Art. 21(3)) (54) Title: REGULATORY ELEMENTS (57) Abstract: The present invention relates to a regulatory element comprising a fragment of a spliceosomal intron, wherein the regulatory element comprises a branch point sequence, a polypyrimidine tract, and a 3' splice acceptor site, and wherein the regulatory element does not comprise a 5' splice donor site. WO 2025 / 003709 PCT / GB2024 / 051695 REGULATORY ELEMENTS FIELD OF THE INVENTION The present invention relates to regulatory elements which are capable of increasing the expression of a protein-coding sequence in kidney cells (e.g. glomerular cells and podocytes). BACKGROUND TO THE INVENTION There are many diseases which affect kidney function by attacking the glomerulus. The glomerulus filters approximately 180 litres of plasma each day, and the healthy glomerular filtration barrier has an astonishing ability to retain about 99.9% of large proteins including albumin over our lifetimes without clogging. The glomerular filtration barrier (GFB) comprises 3 main layers: the glomerular endothelial cell, the glomerular basement membrane (GBM) and the podocyte. The GBM is made of a highly crosslinked macromolecular meshwork of type IV collagen, proteoglycans, and laminin. Genetic forms of glomerular disease can be caused by genetic defects in these molecular structures. For example, Alport syndrome is caused by pathogenic variants in the COL4A3, COL4A4 and COL4A5 genes, which result in abnormalities of the collagen IV a345 network of basement membranes. Alport syndrome affects approximately 1 in 5,000-10,000 of all individuals in continental Europe and the USA. The condition usually presents during childhood and is associated with a spectrum of phenotypes that include a progressive loss of kidney function, and can also include hearing loss and eye abnormalities. Other GBM-associated diseases include Pierson syndrome and Nail-patella syndrome (Chiang, C.K. and Inagi, R., 2010. Nature Reviews Nephrology, 6(9), p.539). The podocyte has also been implicated as a key cell in the progression of glomerular disease. Podocytes are mesodermally derived cells that are highly specialized and found only in the renal glomerulus. They exhibit unique characteristics such as foot processes and slit diaphragms, which are critical for glomerular filtration. Podocyte-associated genetic glomerular diseases include Nephrotic Syndrome, Frasier syndrome and Denys-Drash syndrome, Schimke immuno-osseous dysplasia, and Epstein and Fechtner syndrome. (Chiang, C.K. and Inagi, R., 2010. Nature Reviews Nephrology, 6(9), p.539). Accordingly, kidney cells (e.g. glomerular cells and podocytes) represent a potential target for gene therapy approaches. 1 WO 2025 / 003709 PCT / GB2024 / 051695 However, in order to maximise gene therapy potential, further regulatory elements which can increase expression of a protein-coding sequence in kidney cells (e.g. glomerular cells and podocytes) are required. SUMMARY OF THE INVENTION The present invention is based on the inventors surprising provision of a regulatory element which is capable of increasing expression of a protein-coding sequence in kidney cells, in particular glomerular cells, for example podocytes. In one aspect, the present invention provides a regulatory element comprising a fragment of a spliceosomal intron, wherein the regulatory element does not comprise a 5' splice donor site. The spliceosomal intron may be any naturally-occurring, chimeric, or variant spliceosomal intron. In some embodiments, the spliceosomal intron is a rabbit beta globin intron or a variant thereof having at least 80% sequence identity thereto. In some embodiments, the spliceosomal intron is the rabbit beta globin intron I or a variant thereof having at least 80% sequence identity thereto. of The regulatory element may comprise a branch point sequence, a polypyrimidine tract, and a 3' splice acceptor site. Suitably, the branch point sequence comprises or consists of the nucleotide sequence YTNAY. Suitably, the polypyrimidine tract comprises or consists (Y)n2, where n2=10 to 20. Suitably, the 3' splice acceptor site comprises or consists of the nucleotide sequence YAGG. Suitably, the 5' splice donor site comprises or consists of the nucleotide sequence GTRAGT. The regulatory element may be any suitable length, in particular any length suitable for use in gene therapy. Suitably, the regulatory element has a length of 25 nucleotides or more, 30 nucleotides or more, 35 nucleotides or more, 40 nucleotides or more, 50 nucleotides or more, 60 nucleotides or more, 70 nucleotides or more, 80 nucleotides or more, 90 nucleotides or more, or 100 nucleotides or more. Suitably, the regulatory element has length of 120 nucleotides or less. a The regulatory element may comprise or consist of the nucleotide sequence (N)xYTNAY(N)n1(Y)n2(N)n3 YAGG, wherein x = 10 to 100, n1 = 2 to 22, wherein n2 = 10 to 20, and wherein n1 + n2 + n3 = 15 to 40. In some embodiments, the regulatory element comprises or consists of a nucleotide sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 2 摘要 本發明涉及包含剪接體內含子的片段的調節元件，其中該調節元件包含分支點序 列、多聚嘧啶束和 3’剪接受體位點，並且其中該調節元件不包含 5’剪接供體位點。

Claims

CLAIMS 1. A regulatory element comprising a fragment of a spliceosomal intron, wherein the regulatory element comprises a branch point sequence, a polypyrimidine tract, and a 3’ splice acceptor site, and wherein the regulatory element does not comprise a 5’ splice donor site.

2. The regulatory element according to claim 1, wherein the spliceosomal intron is a rabbit beta globin intron or a variant thereof having at least 80% sequence identity thereto.

3. The regulatory element according to claim 1 or 2, wherein the spliceosomal intron is the rabbit beta globin intron I or a variant thereof having at least 80% sequence identity thereto.

4. The regulatory element according to any preceding claim, wherein the branch point sequence comprises or consists of the nucleotide sequence YTNAY.

5. The regulatory element according to any preceding claim, wherein the polypyrimidine tract comprises or consists of (Y)n2, where n2=10 to 20.

6. The regulatory element according to any preceding claim, wherein the 3’ splice acceptor site comprises or consists of the nucleotide sequence YAGG.

7. The regulatory element according to any preceding claim, wherein the 5’ splice donor site comprises or consists of the nucleotide sequence GTRAGT.

8. The regulatory element according to any preceding claim, wherein the regulatory element has a length of 25 nucleotides or more, 30 nucleotides or more, 35 nucleotides or more, 40 nucleotides or more, 50 nucleotides or more, 60 nucleotides or more, 70 nucleotides or more, 80 nucleotides or more, 90 nucleotides or more, or 100 nucleotides or more.

9. The regulatory element according to any preceding claim, wherein the regulatory element has a length of 120 nucleotides or less.

10. The regulatory element according to any preceding claim, wherein the regulatory element comprises or consists of the nucleotide sequence (N)xYTNAY(N)n1(Y)n2(N)n3YAGG, wherein x = 10 to 100, n1 = 2 to 22, wherein n2 = 10 to 20, and wherein n1 + n2 + n3 = 15 to 40.

11. The regulatory element according to any preceding claim, wherein the regulatory element comprises or consists of a nucleotide sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% ormore, 97% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO:

5.

12. The regulatory element according to any preceding claim, wherein the regulatory element comprises or consists of a nucleotide sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO:

6.

13. The regulatory element according to any preceding claim, wherein the regulatory element consists of a nucleotide sequence having 90% or more sequence identity to SEQ ID NO:

6.

14. The regulatory element according to any preceding claim, wherein the regulatory element consists of a nucleotide sequence having 95% or more sequence identity to SEQ ID NO:

6.

15. The regulatory element according to any preceding claim, wherein the regulatory element consists of a nucleotide sequence having 99% or more sequence identity to SEQ ID NO:

6.

16. The regulatory element according to any preceding claim, wherein the regulatory element consists of the nucleotide sequence of SEQ ID NO:

6.

17. The regulatory element according to any preceding claim, wherein the regulatory element acts post-transcriptionally.

18. The regulatory element according to any preceding claim, wherein the regulatory element increases expression of a protein from a protein-coding sequence downstream thereof.

19. An isolated polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 40 or 93.

20. An isolated polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:

41.

21. An isolated polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 42.

22. An isolated polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:

43.

23. The polynucleotide according to any of claims 19 to 22, wherein the nucleotide sequence is operably linked to a protein-coding sequence.

24. An isolated polynucleotide comprising the regulatory element according to any of claims 1 to 18.

25. The polynucleotide according to claim 24, wherein the regulatory element is operably linked to a protein-coding sequence.

26. The polynucleotide according to claim 24 or 25, wherein the regulatory element is operably linked to a promoter, preferably a kidney-specific promoter.

27. The polynucleotide according to any of claims 24 to 26, wherein the polynucleotide comprises a kidney-specific promoter, the regulatory element, and a protein-coding sequence.

28. An isolated polynucleotide comprising a kidney-specific promoter, a regulatory element, and a protein-coding sequence, wherein the regulatory element comprises a spliceosomal intron or a fragment thereof comprising a branch point sequence, a polypyrimidine tract, and a 3’ splice acceptor site.

29. The polynucleotide according to claim 28, wherein the spliceosomal intron is a rabbit beta globin intron or a variant thereof having at least 80% sequence identity thereto.

30. The polynucleotide according to claim 28 or 29, wherein the spliceosomal intron is the rabbit beta globin intron I or a variant thereof having at least 80% sequence identity thereto.

31. The polynucleotide according to any of claims 28 to 30, wherein the regulatory element is defined according to any of claims 1 to 18.

32. The polynucleotide according to any of claims 23 to 31, wherein the protein-coding sequence encodes a polypeptide associated with a kidney disease.

33. The polynucleotide according to any of claims 23 to 32, wherein the protein-coding sequence encodes a NPHS2, CFI, CFH, FHL-1, COL4A3, COL4A4, COL4A5, C1INH, C4BP, MASP2, C3, C5aR1, C5, C5a, CD55, CD35, CD46, CD59, vitronectin, clusterin, ADCK4, ALG1, ARHGAP24, ARGHDIA, CD151, CD2AP, COQ2, COQ6, DGKE, E2F3, EMP2, KANK2, LAGE3, LMNA, LMX1B, MAF B, NUP85, NUP93, NXF5, OSGEP, PAX2,PDSS2, PMM2, PODXL, SCARB2, SGPL1, Smad7, TP53RK, TPRKB, VDR, WDR73, WT1, ZMPSTE24, APOL1, NPHS1, TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2, ANKFY1, ANLN, CRB2, ITGA3, KANK1, KANK4, MAGI2, MYO1E, OCRL, PTPRO, SMARCAL1, SYNPO, TBC1D8B, XPO5, TNS2, NLRP3, or VEGFC polypeptide.

34. The polynucleotide according to any of claims 24 to 33, wherein the kidney-specific promoter is a podocyte-specific promoter.

35. The polynucleotide according to any of claims 24 to 34, wherein the kidney-specific promoter is a NPHS1 or a NPHS2 promoter.

36. The polynucleotide according to any of claims 24 to 35, wherein the kidney-specific promoter is a minimal NPHS1 promoter or a minimal NPHS2 promoter.

37. The polynucleotide according to any of claims 24 to 36, wherein the kidney-specific promoter comprises or consists of a nucleotide sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO:

35.

38. The polynucleotide according to any of claims 23 to 37, wherein the polynucleotide comprises a Woodchuck hepatitis post-transcriptional regulatory element (WPRE).

39. The polynucleotide according to any of claims 23 to 38, wherein the polynucleotide comprises a polyadenylation sequence.

40. The polynucleotide according to any of claims 23 to 39, wherein the polynucleotide comprises a 5’ ITR and / or a 3’ ITR, preferably wherein the 5’ ITR comprises or consists of a nucleotide sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO: 89; and / or the 3’ ITR comprises or consists of a nucleotide sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO:

90.

41. A vector comprising the polynucleotide according to any of claims 19 to 40.

42. The vector according to claim 41, wherein the vector is capable of transducing kidney cells, optionally wherein the vector is capable of specifically transducing kidney cells, further optionally wherein the vector is capable of specifically transducing podocytes.

43. The vector according to claim 41 or 42, wherein the vector is a viral vector, such as an adeno-associated virus (AAV) vector, a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a herpes simplex viral vector, an alphaviral vector, a flaviviral vector, a rhabdoviral vector, a measles viral vector, a Newcastle disease viral vector, a poxviral vector, and a picornaviral vector, preferably wherein the vector is an AAV vector.

44. The vector according to any of claims 41 to 43, wherein the vector is in the form of a viral vector particle, preferably wherein the viral vector is in the form of an AAV vector particle.

45. The vector according to any of claims 41 to 44, wherein the vector is the form of an AAV vector particle encapsidated by LK03, AAV3B, AAV9, ShH10, AAV-DJ, AAV2, AAV6.2, or AAV5 capsid proteins.

46. The vector according to claim 41, wherein the vector is a plasmid.

47. A kit for production of AAV vector particles comprising, a vector according to claim 46, and one or more helper plasmids encoding AAV replication and capsid proteins.

48. An isolated cell comprising the polynucleotide according to any of claims 19 to 40, or the vector according to any of claims 41 to 46.

49. The cell according to claim 48, wherein the cell is a packaging cell.

50. A pharmaceutical composition comprising the polynucleotide according to any of claims 19 to 40, the vector according to any of claims 41 to 45, or the cell according to claim 48.

51. A polynucleotide according to any of claims 19 to 40, a vector according to any of claims 41 to 45, a cell according to claim 48, or a pharmaceutical composition according to claim 50, for use as a medicament.

52. Use of a polynucleotide according to any of claims 19 to 40, a vector according to any of claims 41 to 45, a cell according to claim 48, or a pharmaceutical composition according to claim 50, for the manufacture of a medicament.

53. A polynucleotide according to any of claims 19 to 40, a vector according to any of claims 41 to 45, a cell according to claim 48, or a pharmaceutical composition according to claim 50, for use in preventing and / or treating a kidney disease.

54. Use of a polynucleotide according to any of claims 19 to 40, a vector according to any of claims 41 to 45, a cell according to claim 48, or a pharmaceutical composition according toclaim 50, for the manufacture of a medicament for preventing and / or treating a kidney disease.

55. A method of preventing and / or treating a kidney disease comprising administering a polynucleotide according to any of claims 19 to 40, a vector according to any of claims 41 to 45, a cell according to claim 48, or a pharmaceutical composition according to claim 50, to a subject in need thereof.

56. Use of a regulatory element according to any of claims 1 to 18 to increase expression of a polypeptide from a protein-coding sequence.

57. A method of increasing expression of a polypeptide from a protein-coding sequence, the method comprising introducing a regulatory element according to any of claims 1 to 18 upstream of the protein-coding sequence.

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