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EP-4735053-A1 - REGULATORY ELEMENTS

EP4735053A1EP 4735053 A1EP4735053 A1EP 4735053A1EP-4735053-A1

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.

Inventors

  • MADILL, Martin
  • GRIFFITH, Alan

Assignees

  • Purespring Therapeutics Limited

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 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. 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. 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. 4. The regulatory element according to any preceding claim, wherein the branch point sequence comprises or consists of the nucleotide sequence YTNAY.
  5. 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. 6. The regulatory element according to any preceding claim, wherein the 3’ splice acceptor site comprises or consists of the nucleotide sequence YAGG.
  7. 7. The regulatory element according to any preceding claim, wherein the 5’ splice donor site comprises or consists of the nucleotide sequence GTRAGT.
  8. 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. 9. The regulatory element according to any preceding claim, wherein the regulatory element has a length of 120 nucleotides or less.
  10. 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. 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% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO: 5.
  12. 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. 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. 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. 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. 16. The regulatory element according to any preceding claim, wherein the regulatory element consists of the nucleotide sequence of SEQ ID NO: 6.
  17. 17. The regulatory element according to any preceding claim, wherein the regulatory element acts post-transcriptionally.
  18. 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. 19. An isolated polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 40 or 93.
  20. 20. An isolated polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 41.

Description

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 α345 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. 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. 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 of (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 a length of 120 nucleotides or less. The regulatory element may comprise or consist 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. 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, 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: 5. 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, 93% or more, 94%